Abstract Book Biotrans 2011

Biotrans 2011 Italy
10th International Symposium on Biocatalysis October 2 - 6, 2011 Giardini Naxos (ME), Sicily
Introduction
Dear Biotrans2011 Participants, On behalf of the Biotrans Steering and Scientific Committees and of the local Organizing Committee I wish to welcome you all to the 10th International Symposium on Biocatalysis and Biotransformations taking place from October 2nd to 6th, 2011, at Atahotel Naxos Beach in Giardini Naxos, Sicily. The Symposium will be attended by more than 640 industrial and academic participants, making Biotrans2011 probably the largest scientific event on Biocatalysis ever organized. This Meeting will continue the series of Symposia successfully held every two years since 1993. The first edition took place in Graz (Austria), following the brilliant intuition of Herfried Griengl to have also in Europe an international forum to discuss topics that nowadays are more and more related to sustainable chemistry, involving competences in the areas of Chemistry, Biochemistry, Biology and Engineering. More than 50 lectures and 400 poster communications will be presented, focusing on the following topics: Discovery and design of new biocatalysts Enzymes structure & mechanism, bioinformatics & modelling Biotransformations in organic synthesis Oxidative biocatalysis Biocatalysis for polymer and material chemistry Cascade chemo-enzymatic processes Biocatalysis and biorefineries Industrial processes research & development As in some of the previous editions, a COST Workshop related to the activities of the COST Action CM0701 CASCAT is fully embedded in the Conference Program. We thank the COST organization for the support given to our Conference and, similarly, we gratefully acknowledge the support by numerous Sponsors and Institutions, whose names are listed in this book. I wish all the participants a successful scientific meeting and an enjoyable stay in this wonderful part of Italy. Venue address
Atahotel Naxos Beach Via Recanati, 26 98035 Giardini Naxos (Messina) Italy
Sergio Riva
Chairman of the Biotrans2011 Symposium
Sponsor
Exhibitors
Permanent Steering Committee

Herfried GRIENGL Robert AZERAD Stefano SERVI Wolf-Dieter FESSNER Vladimir KREN Maurice FRANSSEN Vicente GOTOR Jean-Louis REYMOND Sergio RIVA
Scientific Committee
Sergio RIVA Yasuhisa ASANO Andrea BOMMARIUS Marco FRAAIJE Lucia GARDOSSI Marina LOTTI Mos ROSSI Georg SPRENGER Nicholas TURNER Mei-Xiang WANG (Italy) (Japan) (USA) (the Netherlands) (Italy) (Italy) (Italy) (Germany) (United Kingdom) (China)
Organizing Committee
Sergio RIVA Stefano SERVI Francesco MOLINARI Giovanni NICOLOSI Nicola DANTONA Paola DARRIGO Daniela MONTI Gianluca OTTOLINA Francesco SECUNDO Davide TESSARO
Under the patronage of
Biotrans 2011 - Italy
October 2-6, 2011
Conference Program
Conference Program
Sunday, October 2nd
Monday, October 3rd

Morning
10:30 - 12:30
Registration Chair:
9:00 - 9:45 9:45 - 10:15 PL-1 IL-1
Yasuhisa ASANO, Toyama Prefectural University, Toyama, Japan Enzymologists in wonderland: new biotech applications from evolved D-amino acid oxidases Loredano POLLEGIONI, Universit dell'Insubria, Varese, Italy Laccases in biocatalytic cascade reactions: regeneration of cofactors coenzymes Dietmar HALTRICH, BOKU University, Wien, Austria
14:00 - 18:00
Registration
10:15 - 10:45 18:00 - 18:15
Coffee Break
Opening Ceremony and Welcome Speech Sergio RIVA Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milano, Italy Chairs:
10:45 - 11:05 OC-1
Session 1: Oxidative Biocatalysts Marco FRAAIJE, University of Groningen, Groningen, The Netherlands Miguel ALCALDE, Departamento de Biocatlisis, CSIC, Madrid, Spain Regio- and stereo-selective biohydroxylations for organic synthesis: biocatalyst discovery and engineering Zhi LI, National University of Singapore, Singapore Enzymatic selective oxidation of alkanes under mild conditions Jullien DRONE, Institut Charles Gerhardt Montpellier, Montpellier, France Combining protein engineering with statistical modeling for the novel synthesis of hydroxytyrosol by toluene 4-monooxygenase Ayelet FISHMAN, Technion - Israel Institute of Technology, Haifa, Israel Engineering of oxidase enzymes for large scale production of APIs and intermediates Ee Lui ANG, Codexis Laboratories Singapore, Singapore From literature to industrial scale. Testosterone synthesis: a case history Riccardo MOTTERLE, F.I.S. S.p.A., Alte di Montecchio Maggiore (VI), Italy Recent developments in the application of oxidoreductases Stephan LTZ, Novartis Institute for BioMedical Research, Basel, Switzerland
18:15 - 19:15
KL
Key-note Lecture Protein engineering on the move from directed evolution to in silico approaches Uwe BORNSCHEUER Greifswald University, Greifswald, Germany Chair: Stefano SERVI, Milano Politecnico, Italy
11:05 - 11:25 11:25 - 11:45
OC-2 OC-3
11:45 - 12:05 19:30
OC-4 OC-5 IL-2
Welcome Reception
12:05 - 12:25 12:25 - 12:55
13:00 - 14:00
Lunch
14:00 - 16:00
Poster Session 1 (1-150)
October 2-6, 2011
Conference Program
Conference Program
Monday, October 3rd

Afternoon
Tuesday, October 4th

Morning
Session 2: Cascade Biocatalysis Chairs:

16:00 - 16:30 IL-3
Chair:
9:00 - 9:45 PL-2
Nicholas TURNER, University of Manchester, Manchester, UK Microbial enzymatic processes for the production of useful chemicals: screening and development of unique microbial enzymes and their industrial application Sakayu SHIMIZU, Toray Industries, Kamakura, Japan Modification of natural products using oxygenases Byung Gee KIM, Seoul National University, Seoul, South Korea
Andreas STOLZ, Stuttgart University, Stuttgart, Germany Laurence HECQUET, Universit Blaise Pascal, Clermont-Ferrand , France Combination of chemo- and biocatalysis in multi-step one-pot processes in aqueous reaction media Harald GRGER, Bielefeld University, Bielefeld, Germany Novel applications of ene-reductases in dynamic kinetic resolutions and redox-cascade biotransformations Marko MIHOVILOVIC, Wien University of Technology, Wien, Austria Enantiospecific photobiocatalytic oxyfunctionalization reactions Ekaterina CHURAKOVA, Delft University of Technology, Delft, The Netherlands A two-step, one-pot enzymatic synthesis of 2-substituted-1,3-diols Ioulia SMONOU, University of Crete, Heraklio, Greece
9:45 - 10:15
IL-5
16:30 - 16:50
OC-6
16:50 - 17:10 17:10 - 17:30
OC-7 OC-8
10:15 - 10:45
Coffee Break
Session 3: Biocatalysis and Natural Compounds Chairs:

10:45 - 11:05 11:05 - 11:25
Giovanni NICOLOSI, Istituto di Chimica Biomolecolare, CNR, Catania, Italy Liisa KANERVA, University of Turku, Turku, Finland
OC-12 Key building blocks for natural product synthesis via enzyme-mediated transformations
Jrg PIETRUSZKA, Dsseldorf Universitt and Forschungszentrum, Jlich, German

OC-13 Chorismatases in natural product biosynthesis
Jennifer ANDEXER, University of Cambridge, Cambridge, UK

17:30 - 18:00
Coffee Break
11:25 - 11:45 11:45 - 12:05 12:05 - 12:25
OC-14 Biocatalytic cyclization of citronellal
Gabriele SIEDENBURG, University of Stuttgart, Stuttgart, Germany

OC-15 A biotechnological process for the production of (+)-carvone
Maarten GROENEVELD, University of Groningen, Groningen, The Netherlands

OC-16 Berberine bridge enzyme: from biochemical studies to preparative-scale kinetic resolution
and beyond Joerg SCHRITTWIESER, University of Graz, Graz, Austria
12:25 - 12:55 18:00 - 18:30 18:30 - 18:50 18:50 - 19:10 I-4 OC-9
IL-6
Aldolases in cascade reactions for asymmetric synthesis Pere CLAPES, Instituto de Qumica Avanzada de Catalua, CSIC, Barcelona, Spain One-pot enzymatic synthesis of poly-N-acetyllactosamine oligosaccharides Lothar ELLING, RWTH Aachen University, Aachen, Germany amines Laszlo POPPE, University of Technology and Economics, Budapest, Hungary
13:00 - 14:00
Biocatalytic cuts & sutures of natural compounds Vladimir KREN, Institute of Microbiology, AVCR, Prague, Czech Republic
OC-10 Continuous-flow systems for synthesis, kinetic resolution and dynamic kinetic resolution of
Lunch
19:10 - 19:30
OC-11 Modeling framework for multi-enzyme in-pot processes applied to chiral amine production
Paloma SANTACOLOMA, Technical University of Denmark, Lyngby, Denmark
14:00 - 16:00
Poster Session 2 (151 - 300)
October 2-6, 2011
Conference Program
Conference Program
Tuesday, October 4th

Afternoon
Wednesday, October 5th
Session 4: New Enzymes for Biotransformations Chair:

16:00 - 16:30 16:30 - 16:50 16:50 - 17:10 17:10 - 17:30 IL-7
Chair:
9:00 - 9:45 9:45 - 10:15 PL-3 IL-9
Roger SHELDON, CLEA Technologies BV, Delft, The Netherlands Enzyme based nanocomposites: using nature to ward off emerging diseases Jonathan DORDICK, Rensselaer Polytechnic Institute, Troy (NY), USA Oxidative enzymes in lignin biorefinery Claudia CRESTINI, Universit di Tor Vergata, Roma, Italy
Andreas BOMMARIUS, Georgia Institute of Technology, Atlanta (GA),USA Industrial enzymatic C-C bond formation Martin SCHRMANN, DSM Pharma Chemicals, Geleen, NL Hiroyasu OGINO, Osaka Prefecture University, Osaka, Japan
OC-18 An expanded view of adaptation mechanisms in enzyme evolution
OC-17 Development of organic solvent-tolerant enzymes
Pietro GATTI-LAFRANCONI, University of Cambridge, Cambridge, UK

OC-19 Novel dioxygenases for hydroxy amino acid production
10:15 - 10:45
Coffee Break
Makoto HIBI, Kyoto University, Kyoto, Japan
Session 6: New Applications for Biocatalysis Chairs:

10:45 - 11:05 17:30 - 18:00
Maurice FRANSSEN, Wageningen University, Wageningen, NL Lucia GARDOSSI, Universit di Trieste, Trieste, Italy
OC-23 The enzyme refinery: functionalisation of lignin and lignocellulose
Georg GUEBITZ, Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria Coffee Break
11:05 - 11:25 11:25 - 11:45 OC-24 Release of phenolic monomers from complex lignins by an ether cleaving enzyme system
Volker SIEBER, TU Munich and Fraunhofer IGB Projektgruppe BioCat, Straubing, Germany
OC-25 Enzymatic synthesis of alkyl oligoxylosides from isolated xylans and from lignocellulosic
biomass Marjorie OCHS, INRA-Universit de Reims Champagne-Ardenne, Reims, France Antje SPIESS, RWTH Aachen University, Aachen, Germany
11:45 - 12:05 12:05 - 12:25
OC-26 Ionic liquid-assisted enzymatic depolymerisation of (ligno)-cellulose OC-27 Rationally engineered Candida antarctica lipase B as an efficient catalyst for poly(lactide)
Session 5: ThDP-Dependent Enzymes (sponsored by Thiamine consortium) Chair:

18:00 - 18:30 18:30 - 18:50 18:50 - 19:10 19:10 - 19:30 IL-8
synthesis Mats MARTINELLE, Royal Institute of Technology, Stockholm, Sweden
Georg SPRENGER, University of Stuttgart, Stuttgart, Germany Adventures with the ThDP enzyme transketolase Helen HAILES, University College London, London, UK Ayhan Sitki DEMIR, Middle East Technical University, Ankara, Turkey
OC-21 Conversion of pyruvate decarboxylase into an enantioselective carboligase
12:25 - 12:45
OC-28 In vitro protein and metabolic engineering of a biodegradation pathway
Pavel DVORAK, Institute of Microbiology, AVCR, Prague, Czech Republic
OC-20 Benzaldehyde lyase catalyzed direct amidation of aldehydes with nitroso compounds 13:00 - 14:00
Lunch
Kai TITTMAN, Georg-August-Universitt Gttingen, Gttingen, Germany

OC-22 Formation of tertiary alcohols by ThDP-dependent enzymes
Michael MLLER, University of Freiburg, Freiburg, Germany
14:30 - 20:00
Excursions
October 2-6, 2011
10
Conference Program
Conference Program
11
Thursday, October 6th

Morning
Thursday, October 6th

Afternoon
Chair:
9:00 - 9:45 PL-4
Martina POHL, Forschungszentrum Jlich, Jlich, Germany Engineering enzyme-catalyzed unnatural reactions, (sponsored by BioNoCo) Romas KAZLAUSKAS, University of Minnesota, Saint Paul (MN), USA Session 7: Enzymes structure Chair:
16:00 - 16:20
Session 9: Standards and Bioinformatics Vytas SVEDAS, Lomonosov Moscow State University, Moscow, Russia
OC-36 Standards are boring ... but better data reporting would substantially increase the value of
work in biocatalysis Peter HALLING, University of Strathclyde, Glasgow, UK
Chair:
9:50 - 10:10
Jennifer LITTLECHILD, University of Exeter, Exeter, UK

OC-29 How does the protein structure control the chirality of hydroxylation in mononuclear
16:20 - 16:40
OC-37 Bioinformatic analysis of function-related variable amino acid residues responsible for
nonheme iron dioxygenases? Grit STRAGANZ, Graz University of Technology, Graz, Austria Gideon GROGAN, University of York, York, UK
functional divergence of enzyme families Dmitry SUPLATOV, Lomonosov Moscow State University, Moscow, Russia
16:40 - 17:00
OC-38 The Laccase Engineering Database (LccED) as a tool for understanding the classification
10:10 - 10:30
OC-30 Structure and mechanism of bacterial maleate isomerase
of actinobacterial laccases Marilize LE ROESHILL, Cape Peninsula University of Technology, Belleville, South Africa
10:30 - 11:00
Coffee Break Session 8: Biotransformations (sponsored by Evocatal GmbH)
17:00 - 17:30
Coffee Break
Chairs:
11:00 - 11:20 11:20 - 11:40
Wolf-Dieter FESSNER, Technische Universitt Darmstadt, Darmstadt, Germany Vicente GOTOR, University of Oviedo, Oviedo, Spain
OC-31 Friedel-Crafts alkylation catalyzed by methyltransferases
Session 10: Scaling-up Biotransformations Chair:

17:30 - 17:50
Robert DICOSIMO, DuPont Central Research and Development, Wilmington (DE), USA
OC-39 Progress on Amoxicillin synthesis by selecting the best acylase, expressing it in Pichia pastoris
Mandana GRUBER-KHADJAWI, Austrian Centre of Industrial Biotechnology, Graz, Austria

OC-32 Chemo-enzymatic approaches towards both enatiomers of dehydroxymethylepoxy-quinomicin
(DHMEQ) Takeshi SUGAI, Keio University, Tokyo, Japan
and immobilising it on ReliZyme Ulrich GIESECKE, W42 Consulting GmbH, Penzberg, Germany biocatalysts for the bio-based chemistry Loris SINIGOI, SPRIN S.p.A., Trieste, Italy
17:50 - 18:10
OC-40 How to transform a good enzyme in an efficient biocatalyst: formulation and application of
11:40 - 12:00
OC-33 Production, characterization and synthetic application of a purine nucleoside phosphorylase
from Aeromonas hydrophila Daniela UBIALI, Pavia University, Pavia, Italy
18:10 - 18:30
OC-41 Integration of biotransformation and SMB separation for the high-yield production of fine
12:00 - 12:20
OC-34 Chemoenzymatic syntheses of selected biologically active chiral heteroorganic compounds
Piotr KIELBASINSKI, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, d, Poland
12:20 - 12:40 OC-35 Arzetta: rapidly identifying and customizing enzyme activity
chemicals Mattias BECHTOLD, ETH, Zurich, Switzerland
18:30 - 19:00
IL-10
A green process for Boceprevir-T based on amine oxidase mediated desymmetrization Tao LI, Merck & Co., Rahway (NJ) USA
Alexandre ZANGHELLINI, Arzeda Corporation, Seattle (WA), USA

19:00 - 19:15
Conclusions
13:00 - 14:00 14:00 - 16:00
Lunch Poster Session 3 (301 - 438)

20:30
Conference Dinner and Poster Awards
October 2-6, 2011
12
Conference Program KL
13
Protein engineering on the move from directed evolution to in silico approaches

Uwe T. Bornscheuer Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany E-mail: uwe.bornscheuer@uni-greifswald.de Protein engineering has developed in the past decade to a highly important technology[1,2] as it is a useful tool to create enzymes with desired properties (with respect to e.g., substrate specificity, stereoselectivity or thermostability), but also helps to understand how proteins evolved and how they function. Whereas initially rational protein design based on detailed analysis of three-dimensional structures was the method of choice, directed evolution in essence a random mutagenesis followed by screening or selection of desired mutants became an important alternative. More recently, researchers used combinations of both methods. In this lecture, the principle strategies and current challenges in protein engineering will be highlighted. Examples will include the creation of an epoxide hydrolase from an esterase scaffold within the /-hydrolase fold enzyme family[3] and the inversion of enantioselectivity of an esterase active towards sterically demanding tertiary alcohols.[4] The vast number of protein sequences available from databases substantially facilitates protein engineering. We used this plethora of information to develop a method for an in silico neutral drift based on the analysis of >2.800 sequences of enzymes from the /-hydrolase fold family using the 3DM database.[5] This resulted in small, but smart focused protein libraries, from which enzyme variants with substantially enhanced thermostability, enantioselectivity and altered substrate range could be identified.[6] Finally, a detailed in silico analysis enabled the identification of a toolbox of novel (R)-selective transaminases.[7]
KL - Keynote Lecture PL - Plenary Lecture IL - Invited Lecture OC - Oral Communication PC - Poster Communication
[1] [2] [3] [4] [5] [6] [7] Biotrans 2011 - Italy
Kazlauskas, R.J., Bornscheuer, U.T. (2009) Nature Chem. Biol., 5, 526-529. Lutz, S., Bornscheuer, U.T. (Eds.) (2009) Protein Engineering Handbook, Wiley-VCH, Weinheim Jochens, H., Stiba, K., Savile, C., Fujii, R., Yu., J.-G., Gerassenkov, T., Kazlauskas, R.J., Bornscheuer, U.T. (2009) Angew. Chem. Int. Ed., 48, 3532-3535. Bartsch, S., Kourist, R., Bornscheuer, U.T. (2008) Angew. Chem. Int. Ed., 47, 1508-1511. Kourist, R., Jochens, H., Bartsch, S., Kuipers, R., Padhi, S.K., Gall, M., Bttcher, D., Joosten, H.-J., Bornscheuer, U.T. (2010), ChemBioChem, 11, 1635-1643. Jochens, H., Bornscheuer, U.T. (2010), ChemBioChem, 11, 1861-1866; Jochens, H., Aerts, D., Bornscheuer, U.T. (2010), Protein Eng. Des. Sel., 23, 903-909. Hhne, M., Schtzle, S., Jochens, H., Robins, K., Bornscheuer, U.T. (2010), Nature Chem. Biol, 6, 807-813.
October 2-6, 2011
14
Monday, October 3 PL-1
15
Enzymologists in wonderland: new biotech applications from evolved D-amino acid oxidases
Loredano Pollegioni The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano (via Mancinelli 7, Milano) and Universit degli Studi dellInsubria (via Dunant 3, Varese), Italy E-mail: loredano.pollegioni@uninsubria.it D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxidative deamination of amino acids D-isomers resulting in -keto acids, ammonia and hydrogen peroxide.[1] Owing to the absolute stereoselectivity (L-amino acids are neither substrates nor inhibitors) and broad substrate specificity, in past years DAAO has been investigated for use as an industrial biocatalyst - the most important application being the two-step conversion of cephalosporin C into 7-amino cephalosporanic acid. [2] Most recently has light been shed on the extraordinary functional plasticity of this enzyme, mainly because of detailed investigations into structure-function relationships on yeast DAAO (i.e. elucidating the 3D structure and performing site-directed mutagenesis) and by identifying novel DAAOs. These investigations showed that the active site residues of DAAO are not directly involved in catalysis, but operate by maintaining the proper conformation and a favorable microenvironment for optimal catalysis.[2] The extraordinary functional plasticity of DAAO makes this flavoenzyme a suitable scaffold for developing new properties. These findings have boosted research on DAAO: by applying the most advanced techniques in protein engineering, substrate specificity, oxygen affinity, cofactor binding, and oligomeric state of the enzyme have been redesigned.[3] Recent developments devoted to produce improved DAAO variants for established applications, including as a biocatalyst for resolving racemic amino acid mixtures (even unnatural ones) and as a tool for biosensing (i.e. for the rapid and reliable detection of the neurotransmitter D-serine in the brain or the total D-amino acid content in food specimen). Furthermore, DAAOs were also developed for novel areas of research, including within medical (an enzyme variant most active at low oxygen concentration allows a higher toxic effect on cancer cells), environmental and agricultural fields (i.e. as a new mechanism of herbicide resistance). In the next future, new concepts and fields of research/application of DAAO will further develop mainly by synthesizing creative thinking and classical scientific analysis based on the solid foundation of previous studies.
[1] [2] [3]
Pollegioni, L. et al. (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell. Mol. Life Sci. 64, 1373-94 Pilone, M.S. and Pollegioni, L. (2010) Enzymes, D-amino acid oxidases. In Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology (Flickinger, M.C., ed.), pp. 1-11, John Wiley & Sons Pollegioni, L. and Molla, G. (2011) New biotech applications from evolved D-amino acid oxidases. Trends Biotechnol., in press
October 2-6, 2011
16 IL-1
Lectures
Monday, October 3 OC-1
17
Laccases in biocatalytic cascade reactions: regeneration of cofactors coenzymes

Dietmar Haltrich, Roman Kittl, Christoph Sygmund, Roland Ludwig Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU University of Natural Resources and Life Sciences, 1190 Vienna, Austria E-mail: dietmar.haltrich@boku.ac.at Laccases belong to a diverse superfamily of multi-copper oxidases (MCOs) that catalyse one-electron transfer oxidations of various phenolic compounds, complexed metal ions and dye molecules, with transfer of these electrons to the four copper centers of laccase. These electrons are then passed on to dioxygen, resulting in its reduction to water rather than hydrogen peroxide. Laccases are widespread in nature, and depending on their origin they can show widely varying properties including redox potential, substrate specificity, stability and catalytic optima. Laccases have found increased interest and application in organic synthesis in recent years.[1,2] We have shown that laccases can be used to efficiently regenerate flavin-containing oxidoreductases, both dehydrogenases and oxidases, by reoxidising artificial redox mediator used by these oxidoreductases as electron acceptors in reactions that are of biocatalytic interest. This can enable the use of flavin-dependent dehydrogenases in biocatalysis since then the electron acceptor can be added in minute, stoichiometric amounts. When using flavin-containing oxidases, this approach of laccase regeneration can contribute to the stabilisation of the enzyme during turnover, since the formation of highly reactive hydrogen peroxide can be greatly reduced when using alternative electron acceptors that are continuously regenerated by laccase. This concept employing laccase can be even further expanded to continuous in situ regeneration of NAD+ and NADP+ from NADH and NADPH, respectively, in coenzyme-dependent, dehydrogenase-catalyzed reactions. Here, the enzymatic regeneration system uses laccase in combination with redox mediators to reoxidise NADH or NADPH by concomitant reduction of oxygen to water. We tested several redox mediators under different conditions, and Meldolas blue appeared to be the most promising candidate as a redox mediator for these processes. Laccases from the Japanese lacquer tree Rhus vernificera, the basidiomycete Trametes pubescens, and the ascomycete Melanocarpus albomyces were tested with Meldolas blue as redox mediator in different dehydrogenase-catalyzed conversions to corroborate the application potential. One of these reactions employed NAD(P)-dependent glucose dehydrogenase for the oxidation of glucose, and the redox mediator Meldolass blue, laccase and oxygen as regenerative system. For both coenzymes turnover numbers of >1000 were obtained in 0.5-L batch biocatalytic experiments.
Regio- and stereo-selective biohydroxylations for organic synthesis: biocatalyst discovery and engineering
Zhi Li Department of Chemical & Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576 E-mail: chelz@nus.edu.sg Regio- and stereo-selective biohydroxylations are important reactions for the syntheses of enantiopure alcohols that are useful synthons, aroma materials, and pharmaceutical intermediates. One of the key challenges for the practical application of this type of transformation in organic synthesis is the development of efficient biocatalysts. We recently identified Cellulosimicrobium cellulans EB-8-4 for the allylic hydroxylation of Dlimonene with >99% regio- and stereo-selectivity to give (+)-trans-carveol, a useful and valuable fragrance and flavor compound.[1] This biotransformation afforded 42-times higher product concentration[2] than the best known result. Similarly, Pseudomonas monteilii TA-5 was developed as a powerful biocatalyst for the enantioselective benzylic hydroxylation to give several (R)-benzylic alcohols containing reactive functional groups in 9399% ee as the only products with 56-66% isolated yield.[3] This strain catalyzed also the hydroxylation of indan and tetralin, giving (R)-1-indanol and (R)-1-tetralol in 99% ee and 6267% yields.[4] We also engineered a recombinant Escherichia coli expressing the P450pyr monooxygenase from Sphingomonas sp. HXN-200 for the regio- and stereo-selective hydroxylation.[5] Biohydroxylation of N-benzylpyrrolidin-2-one with the E. coli cells gave (S)-N-benzyl-4-hydroxypyrrolidin-2-one (a useful intermediate for preparing oral carbapenem antibiotic CS-834) in >99% ee, with 2.6-fold increase of product concentration in comparison with the wild type strain. The recombinant biocatalyst demonstrated also excellent regio- and stereo-selectivity for the hydroxylation of (-)--pinene giving the valuable (1R)-trans-pinocarveol in 82% yield, with 200-fold increase of the product concentration compared with the best reported biosystem for the same transformation. The enantioselectivity of P450pyr monooxygenase for the biohydroxylation of N-benzyl pyrrolidine was improved by directed evolution.[6] After three generations of evolution, the best mutant 1AF4A was able to produce the product in 83% (R) ee compared to the wild types 43% ee (S). This demonstrated the first example of evolution of a P450 monooxygenase with inverted enantioselectivity. To facilitate the evolution of enantioselective enzyme, a generally useful high-throughput method for determining the enantioselectivity of biohydroxylations was developed by the use of isotopically labeled enantiopure substrates and MS analysis.[7] Based on this assay and the structure information of the P450pyr hydroxylase, directed evolution led to the discovery of a triple mutant with excellent enantioselectivity to produce (S)-N-benzyl 3-hydroxypyrrolidine (an useful pharmaceutical intermediate) in >98% ee.[8]
[1] [2]
Riva, S. Laccases: blue enzymes for green chemistry. Trends Biotechnol. (2006) 24:219226 Kunamneni, A., Camarero, S., Garca-Burgos, C., Plou, F.J., Ballesteros, A., Alcalde, M. Engineering and applications of fungal laccases for organic synthesis. Microb. Cell Fact. (2008) 7:32
[1] [2] [3] [4] [5] [6] [7] [8]
Wang, Z.; Lie, F.; Lim, E.; Li, K.; Li, Z. Adv. Synth. Catal. 2009, 351, 1849 1856. Wang, Z.; Wu, J.; Li, Z. Manuscript in preparation. Chen, Y., Lie, F.; Li. Z. Adv. Synth. Catal. 2009, 351, 2107 2112. Lie, F.; Chen, Y.; Wang, Z.; Li, Z. Tetrahedron: Asymmetry 2009, 20, 12061211. Zhang, W.; Tang, W. L.; Wang, Z.; Li, Z. Adv. Synth. Catal. 2010, 352, 3380-3390. Tang, W.; Li, Z.; Zhao, H. Chem. Commun. 2010, 46, 54615463. Chen, Y.; Tang W. L.; Mou, J.; Li, Z. Angew. Chem. Int. Ed. 2010, 49, 5278 5283. Pham Q. S.; Pompidor, G.; Li, X.; Li, Z. Manuscript in preparation.
October 2-6, 2011
18 OC-2
Lectures
19
Enzymatic selective oxidation of alkanes under mild conditions

Mlanie Bordeauxa, Nakry Pena, Anne Galarneaua, Franois Fajulaa, Jullien Dronea a Institut Charles Gerhardt Montpellier UMR 5253 CNRS/ENSCM/UM2/UM1,8 rue de lEcole Normale 34296 Montpellier, France E-mail: jullien.drone@enscm.fr The selective catalytic oxidation of alkanes under mild conditions remains a major challenge in industrial and synthetic chemistry because these routes are generally energy intensive and environmentally unfriendly. [1,2] As a result, much research is conducted on finding and engineering new catalysts to approach the ideal catalyst for alkane hydroxylation.[3] Biocatalysts that oxidize alkanes allow organisms to use hydrocarbons as a source of energy and cellular building blocks are theorically approaching the features of the ideal biocatalyst.[4] The recent discovery of a new and promising P450 alkane hydroxylase family (the CYP153 family) has attracted attention.[5] We have used molecular engineering to successfully convert a low-activity octane hydroxylase (CYP153A13a from Alcanivorax borkumensis) into a fast and regioselective medium-chain terminal alkane hydroxylase we named P450 A13-red [6] (Figure 1).
Combining protein engineering with statistical modeling for the novel synthesis of hydroxytyrosol by toluene 4-monooxygenase
Moran Brouka, Yuval Novb, Ayelet Fishmana Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa, 32000, Israel; b Department of Statistics, University of Haifa, Haifa 31905, Israel E-mail: afishman@tx.technion.ac.il
a
Figure 1. The regioselective medium-chain terminal alkane hydroxylation by P450 A13-red. We will present our results concerning the construction and the characterization of A13-red as well as our efforts to develop a sustainable, continous-flow process based on this potent biocatalyst.
Hydroxytyrosol (HTyr), one of the most important phenols present in olives, stands out as a compound of high added value due to its exceptional antioxidant, antimicrobial and anti-carcinogenic activities. It is believed to be the antioxidant with the highest free radical scavenging capacity: double that of quercetin and more than three times that of epicatechin. It has been demonstrated that HTyr inhibits human low-density lipoprotein (LDL) oxidation, scavenges free radicals, inhibits platelet aggregation and confers cell protection. The vast amount of data accumulated regarding the benefits of HTyr, together with its high bioavailability in human, make it a good candidate to serve as an antioxidant for either pharmaceutical or food preparations (i.e. functional foods). Despite the great potential of HTyr, its commercial availablity is limited, therefore a biotechnological process is highly desired. The goal of this research is to engineer toluene monooxygenases (TMOs) for the biosynthesis of commercially-valuable HTyr, from a cheap and abundant substrate, 2-phenylethanol (PEA). This enzymatic hydroxylation is a novel and promising method to synthesize HTyr in a low cost single-step reaction, with high selectivity while utilizing an environmentally friendly process. Escherichia coli cells manipulated to express TMOs are capable of oxidizing a wide range of substituted aromatic and phenolic compounds with high regiospecificity. Despite the resemblance of PEA to the natural substrate, toluene, it was found to be a very poor substrate for the wild-type enzymes. In this research, by employing several protein engineering approaches, the substrate specificity and oxidation activity were dramatically improved. The non-rational approach of directed evolution, led to the discovery of a distant residue from the active site, S395, which affects the enzymes activity.[1] Another valuable residue, D285, located at the entrance of the channel leading to the active site, was found based on rational design.[2] Furthermore, a statistical model was developed to give predictions to which mutations should be combined to give further rise in activity. One triple mutant suggested by this model, had a 200-fold improvement in activity compared to the wild-type enzyme.[3] It was concluded that increasing the size of the active site pocket and the channel entrance, enables for the first time HTyr formation, which the wild-type enzyme was not capable of producing.
[1] [2] [3] [4] [5] [6]
Labinger, J. A.; Bercaw, J. E. Nature 2002, 417, 507-514. Punniyamurthy, T.; Velusamy, S.; Iqbal, J. Chem. Rev. 2005, 105, 2329-2364. Kamata, K.; Yonehara, K.; Nakagawa, Y.; Uehara, K.; Mizuno, N. Nat Chem 2010, 2, 478-483. Beilen, J. B. v.; Funhoff, E. G. Curr. Opin.Biotechnol. 2005, 16, 308-314. Funhoff, E. G.; Salzmann, J.; Bauer, U.; Witholt, B.; van Beilen, J. B. Enz. Microbiol. Technol. 2007, 40, 806-812. Bordeaux, M.; Galarneau, A.; Fajula, F.; Drone, J. Angew. Chem. Int. Ed. 2011, 50, 2075-2079.
[1] [2] [3]
Food Chem. 116:114 (2009) J. Mol. Catal. B:Enzymatic. 66:72 (2010) Appl. Environ. Microbiol. 76:6397 (2010)
October 2-6, 2011
20 OC-4
Lectures
21
Engineering of oxidase enzymes for large scale production of APIs and intermediates
Ee Lui Ang Codexis Laboratories Singapore Pte Ltd, 61 Science Park Rd #03-15/24, The Galen, Singapore Science Park II, Singapore 117525, Singapore E-mail:eelui.ang@codexis.com This presentation focuses on the recent work by Codexis to develop oxidase enzymes, namely mono-oxygenases and amine oxidases, to accept a range of substrates with high enantioselectivity for large scale production of specific APIs and intermediates. To engineer mono-oxygenase enzymes to work as biocatalysts for scalable and economically feasible industrial sulfoxidation processes, significant changes had to be made to various properties of the wild type enzyme. Using directed evolution, we were able to reverse the enanatioselectivity of the mono-oxgenase enzyme, and suppress the formation of over-oxidation by-products. We also made significant improvements in multiple parameters such as activity and thermostability, as well as substrate and product tolerance to deliver a high productivity enzyme suitable for large scale biocatalysis. The development of amine oxidases for large-scale chiral synthesis of important drug intermediates with marked advantages over current chemical process routes will also be discussed.
From literature to industrial scale. Testosterone synthesis: a case history

Riccardo Motterlea, Stefano Fogalb, Giancarlo Arvottia, Chiara Bezzea, Elisabetta Bergantinob, Andrea Castellina. a F.I.S. Fabbrica Italiana Sintetici S.p.A., Viale Milano 26, Alte di Montecchio Maggiore, Vicenza, Italy; b Dipartimento di Biologia, Universit degli Studi di Padova, Viale G. Colombo 3, Padova, Italy E-mail: riccardo.motterle@fisvi.com A chemo-enzymatic process for testosterone (TS) synthesis from commercially available androstendione (AD) is presented (see fig. 1). Stereo- and regio-selective reduction of AD cheto group in 17 position afforded the corresponding alcohol (TS).
CH3 CH3 H O CH3 H CH3 OH
KRD-FIS 001 O
O Androstendione Gluconic Acid NAD(P)H NAD(P)
Testosterone
GDH
Glucose
Figure 1. Testosterone synthesis from androstendione. With the purpose of developping a sustainable process to be implemented at industrial level, we selected the chemo-enzymatic approach as the most appropriate.[1,2] According to literature, no commercial enzymes were available to efficiently catalyze this transformation. Crossing bibliographic and bioinformatics data, we assumed that murine 17HSD (17-hydroxysteroid dehydrogenase) type 5[3] could represent a good candidate. The enzyme was genetically modified and then cloned for recombinant expression in E. coli[4] (KRD-FIS 001). After verification of the ability of the enzyme to specifically catalyze the desired transformation, fermentation and isolation processes were optimized and scaled-up to few m3 bioreactor level. At the same time, the development of the chemo-enzymatic process was carried out. Since the enzyme KRDFIS 001 can use both NADH and NADPH as cofactors, a glucose dehydrogenase (GDH) was coupled for cofactor regeneration. Moreover, due to the extremely low water solubility of both AD and TS, the stability of the enzyme to organic co-solvent presence was investigated and methanol was found to be the best choice. Optimal pH and temperature for the biotransformation were set up and kinetic parameters were measured. From an initial dilution of more than 30.000 L/Kg, unfeasible and useless for industrial implementation, the process was further optimized by stabilizing the enzyme to work at highly concentrated conditions. The resulting engineered chemo-enzymatic process was claimed in a patent application and verified at Kilo level. Optimization is still ongoing, with the aim to scale-up at industrial level for multiton production campaign.
[1] [2] [3] [4] Cremonesi, P., Carrea, G., et al. 1973, Arch. Biochem. Biophys., 159, 1, 7-10. Donova, M.V., Egorova, O.V. et al. 2005, Process Biochem., 40, 7, 2253-2262. Rheault, P. and Charbonneau, A. 1999, Biochim. Biophys. Acta, 1447, 17-24. Fogal S., Motterle R. et al. 2010. Chem. Eng. Transaction, 20, 61-66.
October 2-6, 2011
22 IL-2
Lectures
Monday, October 3 IL-3
23
Recent developments in the application of oxidoreductases

Falk Hildebranda, Christina Kohlmanna, Kirsten Schroera,b, Matthias Kittelmannb, Stephan Ltza,b a Institute of Biotechnology 2, Reserach Centre Jlich, Germany b Novartis Institutes for BioMedical Research, Basel, Switzerland E-mail: stephan.luetz@novartis.com Oxidoreductases are valuable tools for the synthesis of chiral builduing blocks as well as drug metabolites on preparative scale, e.g.[1-3] They are especially appealing biocatalysts for several reasons, e.g. their ability to be used in asymmetric synthesis reactions (100% yield) rather than kinetic resolutions or their ability to catalyze reaction which are difficult to achieve chemically, e.g. selective C-H-bond oxidation. Several recent developments in the application of Oxidoreductases will be given, including the use of alternative reaction media (e.g. ionic liquids) to solubilize poorly water-soluble substrates [4] as well as the engineering of whole cell catalysts for redox reactions will be presented. Limitations and opportunities will be discussed from an application point of view.
Harald Grger Faculty of Chemistry, Bielefeld University, Universittsstr. 25, 33615 Bielefeld, Germany E-mail: harald.groeger@uni-bielefeld.de
Combination of chemo- and biocatalysis in multi-step one-pot processes in aqueous reaction media
Multi-step one-pot processes represent an attractive synthetic concept for the improvement of overall process efficiency by decreasing the required number of work up and purification steps. By avoiding such time-, capacity- and solvent-intensive process steps, multi-step one-pot syntheses contribute to a significantly improved process economy as well as to more sustainable synthetic routes. A key criterion for multistep one-pot processes is the compatibility of the individual reaction steps with each other. Accordingly, most of todays known multi-step one-pot processes are based on either chemocatalytic multi-step reactions or pure biotechnological processes such as, e.g., fermentation. In contrast, successful combinations of chemo- and biocatalytic reactions, in particular in aqueous reaction media, are much less widely known. In this contribution strategies for the combination of chemo- and biocatalysts towards the development of multi-step one-pot processes in aqueous reaction media are presented. Since palladium-catalyzed cross-coupling reactions are of particular importance in the field of metal catalysis, as enzymatic reductions are in the field of biocatalysis, we were interested in the investigation of the compatibility of these types of reactions with each other in water. As an example for such a one-pot process the synthesis of chiral biaryl-containing alcohols via Suzuki-cross-coupling reaction and subsequent asymmetric enzymatic reduction is discussed. [1,2] Very recently we could also demonstrate the compatibility of a metal-catalyzed cross-metathesis reaction with a biotransformation. A further research focus has been on the combination of an organo- and biocatalytic reaction sequence. It turned out that the reaction mixture resulting from an asymmetric organocatalytic aldol reaction is compatible with a direct subsequent enzymatic reduction without the need for a work-up step of the aldol reaction. [3,4] Furthermore, a combination of non-catalyzed Wittig reactions with biocatalytic reductions of C=O and C=C double bonds, respectively, in aqueous reaction media has been achieved.[5,6]
[1] [2] [3] [4]
K. Goldberg, K. Schroer, S. Ltz, A. Liese Applied Microbiology & Biotechnology 76 (2007), 237-248, DOI: 10.1007/ s00253-007-1002-0 K. Goldberg, K. Schroer, S. Ltz, A. Liese, Applied Microbiology & Biotechnology 76 (2007), 249-255, DOI: 10.1007/ s00253-007-1005-x K. Schroer, M. Kittelmann, S. Ltz, Biotechnology&Bioengineering 106 (2010), 699-706, DOI: 10.1002/bit.22775 C. Kohlmann, L. Greiner, W. Leitner, C. Wandrey, S. Ltz, Chemistry A European Journal 15 (2009), 11692-11700, DOI: 10.1002/chem.200901046
[1] [2] [3] [4] [5] [6]
E. Burda, W. Hummel, H. Grger, Angew. Chem. 2008, 120, 9693; Angew. Chem. Int. Ed. 2008, 47, 9551. E. Burda, W. Bauer, W. Hummel, H. Grger, ChemCatChem 2010, 2, 67-72. K. Baer, M. Krauer, E. Burda, W. Hummel, A. Berkessel, H. Grger, Angew. Chem. 2009, 121, 9519; Angew. Chem. Int. Ed. 2009, 48, 9355. G. Rulli, N. Duangdee, K. Baer, W. Hummel, A. Berkessel, H. Grger, Angew. Chem. 2011, in press; Angew. Chem. Int. Ed. 2011, in press. M. Krauer, W. Hummel, H. Grger, Eur. J. Org. Chem. 2007, 5175. M. Krauer, T. Winkler, N. Richter, S. Dommer, A. Fingerhut, W. Hummel, H. Grger, ChemCatChem 2011, 3, 293-296.
October 2-6, 2011
24 OC-6
Lectures
25
Novel applications of ene-reductases in dynamic kinetic resolutions and redox-cascade biotransformations

Marko D. Mihovilovica a Vienna University of Technology, Institute of Applied Synthetic Chemistry, Getreidemarkt 9/163-OC, A-1060 Vienna, Austria E-mail: mmihovil@pop.tuwien.ac.at Novel applications of ene-reductases for the preparation of chiral compounds will be discussed. The bioreduction of Baylis-Hillman reaction products was studied; in this context, a partial dynamic kinetic resolution process was observed yielding syn-products, predominantly (X = OH). The conversion of substrates containing protected amines (X = NHCOOR) was found to proceed to anti-products, exclusively. This represents a novel approach to non-natural -amino acids.
Enantiospecific photobiocatalytic oxyfunctionalization reactions

E. Churakova, I.W.C.E. Arends, F. Hollmann Department of Biotechnology, Delft University of Technology, Julianalaan 136, Delft, The Netherlands E-mail: e.churakova@tudelft.nl Heme peroxidases widely considered as versatile biocatalysts. Unlike the P450 monooxygenases, hemeperoxidases do not rely on expensive NADH cofactor and catalyze a broad range of oxidation/oxyfunctionalization reactions simply utilizing hydrogen peroxide (H2O2) as oxidant with high degrees of chemo-, regio- and stereo-specificity. But the prosthetic heme group can be easily inactivated even by low H2O2 concentrations.[1] To avoid this chemical degradation of enzyme active site we have developed an alternative, light-driven approach for in situ H2O2 generation. Visible-light exited flavins such as flavin adenine mononucleotide (FMN) can oxidize simple and abundant electron donors such as ascorbic acid. The resulting reduced flavins (FMNH2) quickly react with molecular oxygen to eventually yield the re-oxidized flavins and hydrogen peroxide. Promising preliminary results for photoenzymatic approach have been obtained with chloroperoxidase-catalyzed sulfoxidation reactions.[2] Here, we report a practical and simple photoenzymatic oxyfunctionalization method by combining the photocatalytic approach for the controlled in situ H2O2 generation method from O2 to a novel and highly promising haloperoxidase from Agrocybe aegerita (AaP) (Figure 1). We have investigated the applicability of AaP as catalyst for photobiocatalytic epoxidation and hydroxylation reactions. The proposed in situ generation of H2O2 proved to be a suitable approach to achieve stable and robust oxyfunctionalization activity (more than 40-fold increase of productivity time as compared to the stoichiometric use of H2O2). High productivities and excellent enantiomeric excesses (>97%) were obtained for the majority of studied substrates.
Recently, we discovered regiodivergent Baeyer-Villiger oxygenations of various enantio- and regioisomeric terpenones. By combining this biotransformation with a preceeding ene-reductase reaction, troublesome chemical hydrogenation of easily available enone precursors can be avoided. This three enzyme redox cascade process (including a suitable dehydrogenase for cofactor regeneration) represents a novel combination of redox biocatalysts in a modular fashion aiming at the formation of potential fragrance compounds.
Figure 1. Photoenzymatic oxyfunctionalization by combining in situ H2O2 generation with AaP-catalyzed hydroxylation/epoxidation
[1] [2]
Van Rantwijk, F. and Sheldon, R.A., Curr. Opin. Biotechnol. 11 (2000) 554 Perez, D.I., Mifsud Grau M., Arends I.W.C.E. and Hollmann F., Chem. Commun. 44 (2009) 6848
October 2-6, 2011
26 OC-8
Lectures
Monday, October 3 IL-4
27
Ioulia Smonou*, Kalaitzakis Dimitris Department of Chemistry, University of Crete, 71003, Heraklio, Crete, Greece smonou@chemistry.uoc.gr
A two-step, one-pot enzymatic synthesis of 2-substituted-1,3-diols
Aldolases in cascade reactions for asymmetric synthesis

Pere Claps Instituto de Qumica Avanzada de Catalua-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain. E-mail: pere.clapes@iqac.csic.es Carbon-carbon bond formation is a cornerstone reaction in synthetic organic chemistry and a pivotal process in the construction of the skeletal framework of complex molecular targets. Among the methods available, the aldol addition reaction is a powerful strategy that enables the concomitant funcionalization and creation of stereogenic centers.[1] Asymmetric direct aldol additions mediated by aldolases are finding increasing acceptance in chemical research and production of asymmetric compounds due to the high selectivity and catalytic efficacy.[2] Enzymatic cascade reactions as the consecutive series of biocatalytic reactions, including sequential process in combination with organic reactions, have undeniable benefits namely atom economy, time labor, resource management and waste generation. In this paper, the application of biocatalytic cascade reactions involving the use of aldolases will be discussed. Particularly, D-fructose-6-phosphate aldolase from E. coli (FSA)[3] was found to catalyze stereoselectively aldol additions of dihydroxyacetone, hydroxyacetone and hydroxybutanone to a variety of aldehyde acceptors.[4] Recently it was uncovered that FSA has the ability to catalyze the homo- and cross-aldol additions of glycolaldehyde.[5] This property has expanded its synthetic potential in cascade reactions by the virtue to generate an aldehyde, which may be use as acceptor in another consecutive aldol addition. Different redesigns of FSA wild-type may allow to control its selectivity towards donors and acceptors and therefore to modulate its reactivity in front of different substrates.
Optically active 1,3-diols are important targets for many synthetic methodologies, since they are high value chiral synthons or building blocks for the synthesis of many natural products and pharmaceuticals. They have frequently been used as valuable intermediates in the synthesis of drugs and natural products with important biological activity.[1] Stereoselective ketoreductase-catalysed reductions[2-4] of a-substituted-bhydroxy ketones will be described. The stereoisomeric syn or anti -substituted 1,3-diols were prepared in high optical purities (>99% de, >99% ee) and chemical yields. This is a simple, highly stereoselective and quantitative method for the synthesis of different diastereomers of chiral diols. Furthermore, we will present a biocatalytic cascade reaction, which was designed for the stereoselective synthesis of optically pure 2-alkyl-1,3-diols employing two enzymes.[5] The cascade process consists of two consecutive steps: a stereoselective diketone reduction and a hydroxy ketone reduction. Chiral diols were formed by the addition of ketoreductases in the same vessel, in high stereoselectivity and chemical yield, without the isolation of the intermediate -hydroxy ketones.
Figure 1. Example of in situ product utilization of FSA-catalyzed cross-aldol addition of glycolaldehyde.
[1] [2] [3] [4] [5]
R. D. Norcross and I. Paterson, Chem. Rev. 1995, 95, 2041 K. Nakamura, T. Matsuda, in Enzyme Catalysis in Organic Synthesis, 2002, vol.3, Edited by Drauz K, Waldmann H. Weinheim: Wiley-VCH, pp.991-1047. D. Kalaitzakis, J. D. Rozzell, S. Kambourakis, I. Smonou, Org. Lett. 2005, 7, 4799-4801. D. Kalaitzakis, J. D. Rozzell, S. Kambourakis, I. Smonou, Adv. Synth. Catal. 2006, 348, 1958-1969. D. Kalaitzakis and I. Smonou, J. Org. Chem., 2010, 75 (24), 86588661.
[1] [2] [3] [4]
[5] Biotrans 2011 - Italy
S. Mukherjee, J. W. Yang, S. Hoffmann, B. List, Chem. Rev. 2007, 107, 5471-5569. P. Claps, W.-D. Fessner, in Stereoselective Reactions of Carbonyl and Imino Groups, Vol. 2 (Ed.: G. A. Molander), Georg Thieme Verlag KG, Suttgart (Germany), 2011, pp. 677-734. M. Schrmann, G. A. Sprenger, J. Biol. Chem. 2001, 276, 11055-11061. J. A. Castillo, J. Calveras, J. Casas, M. Mitjans, M. P. Vinardell, T. Parella, T. Inoue, G. A. Sprenger, J. Joglar, P. Claps, Org. Lett. 2006, 8, 6067-6070; M. Sugiyama, Z. Hong, P. H. Liang, S. M. Dean, L. J. Whalen, W. A. Greenberg, C.-H. Wong, J. Am. Chem. Soc. 2007, 129, 14811-14817; A. L. Concia, C. Lozano, J. A. Castillo, T. Parella, J. Joglar, P. Claps, Chem. Eur. J. 2009, 15, 3808-3816. X. Garrabou, J. A. Castillo, C. Gurard-Hlaine, T. Parella, J. Joglar, M. Lemaire, P. Claps, Angew. Chem. Int. Ed. 2009, 48, 5521-5525.
October 2-6, 2011
28 OC-9
Lectures
29
One-pot enzymatic synthesis of poly-n-acetyllactosamine oligosaccharides

Claudia Rech, Ruben P. Rosencrantz, and Lothar Elling Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany E-Mail: l.elling@biotec.rwth-aachen.de The glycan poly-N-acetyllactosamine (poly-LacNAc) is a major constituent of glycoproteins and glycolipids of the outer cellular glycocalix and plays an essential role in galectin-mediated cell-cell and cell-matrix recognition. Poly-LacNAc is therefore a target molecule for the biofunctionalisation of biomaterial surfaces. It consists of a repeating unit of Gal(1-4)GlcNAc (type 2 LacNAc). Multi-step sequential enzymatic synthesis combining human 1-4galactosyltransferase (4GalT) and 1-3N-acetylglucosaminyltransferase (3GlcNAc) from Helicobacter pylori was previously established.[1] Poly-LacNAc oligomers with up to three LacNAc units were obtained. We here demonstrate for the first time the combination of two glycosyltransferases in a one-pot synthesis resulting in the synthesis of defined mixtures of poly-LacNAc oligomers (Figure 1). Parameters such as reaction time, enzyme ratios, length and concentration of the starting acceptor substrate were optimized. Mixtures of poly-LacNAc oligomers ranging from tri- to trideca-saccharides were obtained as analysed by MALDI-TOF. After a final galactosylation step single LacNAc oligomers ([LacNAc]1-6) could be isolated by preparative HPLC. Work is in progress to analyse the binding specificity of galectins to defined mixtures poly-LacNAc oligomers and to explore their potential use for the biofunctionalization of biomaterial surfaces.
HO OH O HO OH HO O AcHN O N H S N H H N O O
Continuous-flow systems for synthesis, kinetic resolution and dynamic kinetic resolution of amines
Zoltn Borosa,b, Pter Falusa, Gbor Hornynszkya,b, Jzsef Nagya,b, Lszl rgec, Ferenc Darvasc, Lszl Poppea,b a Department of Organic Chemistry and Technology, and Research Group for Alkaloid Chemistry of the Hungarian Academy of Sciences; Budapest University of Technology and Economics, Megyetem rkp. 3., H-1111 Budapest, Hungary; bSynBiocat Ltd, Lzr dek u 4/1, H-1173 Budapest, Hungary; cThalesNano Inc., Graphisoft Park, Zhony u. 7., H-1031 Budapest, Hungary. E-mail: poppe@mail.bme.hu Amines are indispensable building blocks in numerous drugs, pesticides and color pigments. Thus, development of general and efficient methods to prepare enantiopure amino compounds is still required. First, novel reductive amination of ketones with ammonium formate and various metals was developed in onepot and one-step reactions using batch and continuous-flow methods (Fig. 1a).[1a] Next, lipase-catalyzed kinetic resolutions in continuous-flow reactors were studied in between 0-70 C (Fig. 1b). Depending on the substrate, maximum of enantiomer selectivity (E) at certain temperatures or even continuous increase of E with increasing temperature has been found (Fig. 1c). A chemoenzymatic cascade system was used for continuous-flow dynamic kinetic resolutions.[1b]
OH
UDP-GlcNAc 3GlcNAc-T Uridine + 2 Pi AP UDP
UDP-Gal 4Gal-T1 UDP
UDP-Glc 4-Epimerase
UDP-Glc
AP
Uridine + 2 Pi
HO HO OH HO OH O HO OH HO O
AcHN O
HO OH O O OH HO O OH
AcHN O N H 1-5 AcHN O OH N H
S N H
H N O
AcHN O OH
HO OH O 1-5 O OH HO O
S N H
H N O
Figure 1. One-pot enzymatic synthesis of poly-LacNAc oligomers.

We thank Prof. Dr. V. Ken (Academy of Sciences of the Czech Republic) for the synthesis of linker-modified GlcNAc and NMR analysis, Prof. B. Ernst (University of Basel) for the gift of UDP-Gal., Dr. W.W. Wakarchuck (National Research Council of Canada) for 3GlcNAcT. We also thank Prof. Dr. F.-G. Hanisch (Cologne University) for MALDI-TOF analysis. Financial support by the DFG within the Research Training Group 1035 Biointerface, by the EU-COST action CM07010, and by the excellence initiative of the German federal and state governments through ERS@RWTH Aachen University is gratefully acknowledged.
[1]
(a) Falus P., Boros Z., Hornynszky G., Nagy J., Darvas F., rge L., Poppe L. Tetrahedron Lett., 2011, 52, 1310-1312; (b) Poppe L., Tomin A., Boros Z., Varga E., rge L., Darvas F. Novel dinamic kinetic resolution process, Hung. Pat. P0900720, 2009.
[1]
B. Sauerzapfe et al., Glycoconjugate J. 2009, 26, 141.
October 2-6, 2011
30 OC-11
Lectures
Monday, October 4 PC-2
31
Modeling framework for multi-enzyme in-pot processes applied to chiral amine production
Paloma A. Santacolomaa, Kresimir Janesa, Pr Tufvessona, Grkan Sinb, Krist V. Gernaeya, John M. Woodleya a PROCESS; bCAPEC; Department of Chemical and Biochemical Engineering, Technical University of Denmark, Sltofts Plads, Building 229,DK-2800 Kgs. Lyngby E-mail: psa@kt.dtu.dk Nowadays multi-enzyme processes are seen as an alternative to assist in the synthesis of complex compounds of industrial interest.[1] In general, a multi-enzyme in-pot process is characterized by the mixture of enzymes that catalyze several reactions in a single pot. In this manner, purification steps of intermediate products may be eliminated. Consequently, it potentially leads to considerable process improvements like increases in the process yield, and reduction in downstream processing and operating costs. In the analysis of these types of processes, mathematical models and computational tools enable a systematic development of multi-enzyme in-pot processes.[2] A systematic methodology is exemplified stepwise through the production of chiral amines by combining the action of three enzymes in a single reactor.[3] In the applied method, chiral amines are synthesized from ketones by using the first enzyme (transaminase, E.C. 2.6.1.2) together with L-alanine which provides the amino donor (see figure 1). The generated pyruvate, from the first reaction, is reduced by the second enzyme (lactate dehydrogenase, LDH, E.C. 1.1.1.27) to L-lactate. Removing the pyruvate contributes to both driving the reaction to completion and eliminating pyruvate inhibition of the transaminase. The third enzyme (glucose dehydrogenase, GDH, E.C. 1.1.1.47) is added to the system in order to recycle the NADH cofactor. [4] The aim of this contribution is to present a methodological framework for modeling multi-enzyme in-pot processes in order to formulate reliable models for further applications such as optimization, control and prediction.
Microbial and enzymatic processes for the production of useful chemicals: screening and development of unique microbial enzymes and their industrial applications
Sakayu Shimizu New Frontiers Research Laboratories, Toray Industries, Inc., Kamakura, Kanagawa 248-8555; Department of Bioscience and Biotechnology, Kyotogakuen University, Kameoka, Kyoto, 621-8555, Japan. E-mail: Sakayu_Shimizu@nts.toray.co.jp; sshimizu@kyotogakuen.ac.jp Over the past decade, the industrial use of microbial functions, such as their unique enzyme systems, catalysis and so on, has developed rapidly and is gathering increasing attention, particularly their use in solving environmental problems. Here, several unique microbial enzymes or reactions recently discovered by us and now used industrially are introduced, through which I will emphasize importance of screening for potential microorganisms and mutual collaboration between academia and industries. Screening is a key step in process development, because, in many cases, the substrates in industrial processes are artificial compounds, and, in many cases, enzymes to catalyze suitable reactions for such processes are still unknown. Therefore, screening for novel enzymes that are capable of catalyzing new reactions is constantly needed. In addition, the discovery of new enzymes sometimes provides clues for designing new enzymatic processes. One of the most efficient and successful means of finding new enzymes is to screen a large number of microorganisms, because of their characteristic diversity and versatility. However, it is obviously very difficult to propose rational method of screening for novel enzymes; it sometimes involves something like midnight-walk without moonlight. There are three important stages in a general strategy: (1) designing the process and deciding the type of enzymatic activity desired; (2) deciding which groups of microorganisms are to be selected and screened; and (3) designing an appropriate, convenient and sensitive assay that will allow as many microorganisms as possible to be screened. It is also important, during the course of screening, to observe the functions of microorganisms carefully in order to obtain the desired (but serendipitous or random) results. Here, several unique microbial enzymes or reactions recently discovered by us and now used industrially are introduced (single cell oil production by oleaginous microorganisms, reductive transformation of unsaturated fatty acids, lactonase process for the optical resolution of lactones, bioreduction system for chiral alcohol synthesis, hydroxylation of amino acids, novel membrane reactor system for the continuous fermentaion, etc.), through which I will emphasize importance of the followings: i) screening for potential microorganisms from our rich resources, ii) thoughtful use of new technologyies, iii) mutual collaborations between academia and industries, and iv) rational and strategic support by the government.
Figure 1. Reaction scheme for the synthesis of chiral amines by applying a three-enzymatic in-pot process (transaminase, LDH and GDH)
[1] [2] [3] [4] Bruggink, A., Schoevaart, R. and Kieboom, T. Org. Process Res. Dev. 2003, 4, 622 Santacoloma, P.A., Sin, G., Gernaey, K.V. and Woodley, J.M. Org. Process Res. Dev. 2011, 15, 203 Koszelewski, D., Lavandera, I., Clay, I., Guebitz, G.M., Rozzell, D. and Kroutil, W. Adv. Synth. Catal. 2008, 350 (17), 2761 Truppo, M.D., Rozzell, J.D. and Turner, N.J. Org. Process Res. Dev. 2010, 14, 234
October 2-6, 2011
32 IL-5
Lectures
Tuesday, October 4 OC-12
33
Modification of natural products using oxygenases

Byung-Gee Kim 1,2, Nahum Lee, Kwon-Young Choi, Bishnu Pandey, EunOk-Jung 1 School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744, Korea 2 Bioengineering Institute and Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-744, Korea Phone: +82-2-880-8945 Fax: +82-2-872-7528 E-mail: E-mail: byungkim@snu.ac.kr Natural products are mainly synthesized through secondary metabolite pathways. After the synthesis, they are further modified using tailoring enzymes to generate their diversities. Among them, the most interesting and prevalent enzyme reaction systems are the ones for hydroxylation, methylation, and glycosylation. Here, we will talk on several examples of monooxygenase reactions for such products which can enhance and/or change their biological functions: various hydroxylations of i) (iso)flavonoids(daidzein, genistein, apigenin, etc), ii) lignan and iii) stilbenoids using P450s and tyrosinase from Actinomycetes(Streptomyces, Norcadia etc.), and how we can develop self-sufficient P450s of Streptomyces in E.coli system as a successful substitute for the P450 reactions.
Key building blocks for natural product synthesis via enzyme-mediated transformations
Jrg Pietruszkaa, Martina Bischopa, Thomas Classena, Nils Eichenauera, Thomas Fischera, Margarete Korpaka, Irene Kullartza, Katharina Neufelda, Anja C. M. Nordschilda, Melanie Schlzela, and Diana Sandkuhla a Heinrich-Heine-Universitt Dsseldorf im Forschungszentrum Jlich, Jlich, Germany E-mail: j.pietruszka@fz-juelich.de Biocatalytic approaches towards new building blocks in organic synthesis have increasingly emerged as an important tool in recent years. Nowadays, a number of biotransformations are primarily applied in the chemical and pharmaceutical industries delivering fine chemicals, e.g. for drugs. The mild reaction conditions - also triggering high stereo-, regio-, and chemoselectivity - and the often elegant short-cuts in synthetic endeavours lead to economic and ecological advantages of the enzymatic conversions.[1] The focus of our projects is on natural product syntheses and the development of new synthetic methods (see e.g. ref. [2]). The synthesis of the highly selective antitumor reagent psymberin (Figure 1), isolated from the sponge Psammocinia spp.[3], is one of the more recent target molecules in our group, but also marine oxylipins such as the constanolactones caught our attention. The progress on a chemoenzymatic approach towards key building blocks for organic synthesis will be presented. Enzymes utilized range from hydrolases and (ene)reductases to monooxygenases.
Figure 1. Selected natural target compounds. Figure 1. Daidzein Hydroxylation
[1] Bishnu Prasad Pandey, et. al, Enzyme and Microbial Technology, in press, 2011 [2] Bishnu Prasad Pandey, et. al , Biotechnology & Bioengineering, 105(4):697 - 704, 2010
[1] [2] [3]
T. Fischer, J. Pietruszka, Top. Cur. Chem. 2010, 297, 1-43. (a) M. Bischop, V. Doum, A. C. M. Nordschild (ne Rieche), J. Pietruszka, D. Sandkuhl, Synthesis 2010, 527-537; (b) J. Pietruszka, R. C. Simon, Eur. J. Org. Chem. 2009, 3628-3634; (c) M. Korpak, J. Pietruszka, Adv. Synth. Catal. 2011, in press. (a) R. H. Cichewicz, F. A. Valeriote, P. Crews, Org. Lett. 2004, 6, 19511954; (b) G. R. Pettit, J. Xu, J. Chapius, R. K. Pettit, L. P. Tackett, D. L. Doubek, J. N. A. Hooper, J. M. Schmidt, J. Med. Chem. 2004, 47, 11491152.
October 2-6, 2011
34 OC-13
Lectures
35
Chorismatases in natural product biosynthesis

Jennifer N. Andexera,b, Steven G. Kendrewc, Orestis Lazosb, Mohammed Nur-e-Alamc, Anna-Sophie Zimmermannb, Steven Mossc, Barrie Wilkinsonc, Peter F. Leadlayb a Pharmaceutical and Medicinal Chemistry, University of Freiburg, Albertstr. 25, 79104 Freiburg, Germany; b Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom; c Biotica Technology Ltd., Chesterford Research Park, Cambridge CB10 1XL, United Kingdom E-mail: jennifer.andexer@pharmazie.uni-freiburg.de Ascomycin (FK520, 1) is a macrocyclic polyketide produced by Streptomyces hygroscopicus var. ascomyceticus with potent antifungal and immunosuppressive properties.[1]
a
Biocatalytic cyclization of citronellal

Gabriele Siedenburga, Michael Breuerb, Bernhard Hauerc, Dieter Jendrosseka Inst. f. Microbiology, University Stuttgart, bBASF-SE, Ludwigshafen, cInst. f. Technical Biochemistry, University Stuttgart, Germany; E-mail: imbgsi@imb.uni-stuttgart.de; imbdj@imb.uni-stuttgart.de
Key enzyme of hopanoid biosynthesis is squalene-hopene cyclase (SHC) which catalyzes the polycyclization reaction of squalene to the pentacyclic triterpene hopene - the precursor of all hopanoids. Hopanoids stabilize the cytoplasm membrane of many bacteria similar to the function of sterols in eukarotes. The SHCcatalyzed reaction is one of the most complex biochemical reactions and involves the formation of 5 ring structures, the alteration of 13 covalent bonds, and the formation of 9 stereo centers. Zymomonas mobilis an important ethanol producing bacterium - harbours two SHC-encoding genes (ZMO872, ZMO1548) that were cloned and over-expressed in E. coli. Hopene-forming activity was confirmed for both SHCs. Interestingly, ZMO1548 was able to cyclise monoterpene citronellal to isopulegol (see figure). This finding is contrary to former results using the model SHC from Alicyclobacillus acidocaldarius[1,2] and several other SHCs cloned from different organisms in this study. Isopulegol is used as a flavor in different products and is an important inter-mediate in the production of menthol. Our finding is remarkable because cyclization of mono- sesqui- and diterpenes normally requires activation of the linear precursor by diphosphate.[3,4] Depending on the stereo-configuration of the substrate [(S)- or (R)-citronellal] different isopulegol stereoisomers were formed. Cyclization of citronellal by SHC is the first example of an enzyme-catalyzed cyclization of a not-activated linear monoterpene.
Figure 1. Structures of ascomycin (1)/ brasilicardin (2) and chorismatase-catalysed reactions. As for the closely related rapamycin and FK506, the unusual 3,4- dihydroxycyclohexane carboxylic acid starter unit has been shown to be derived from shikimate metabolism.[2] However, the exact nature of the genes and enzymes involved in this feeder pathway has never been established. Here, we have examined the hypothesis that the FkbO gene product catalyses a key enzymatic step linking the shikimic pathway to the starter unit. FkbO was expressed in Escherichia coli, purified, and tested for its ability to catalyse the direct hydrolysis of chorismate (4) (Figure 1). It was found to be an efficient catalyst for this reaction.[3] The only precedent for such a chorismatase activity is in the side reaction of known isochorismatases such as EntC from E. coli.[4] A homologous enzyme (Hyg5) is found in a less characterised cluster in the rapamycin-producing strain. This enzyme was shown to carry out the hydrolysis of chorismate as well, but leads to 3-hydroxybenzoic acid (5) instead of 3.[3] Bra8, an enzyme from the brasilicardin cluster[5] exhibits very high similarity to Hyg5 and is suggested to be responsible for the biosynthesis of the 3-hydroxybenzoate moiety in brasilicardin (4).[3] Our results show that FkbO and Hyg5 are genuine chorismatase and reveal another metabolic fate for chorismate apart from the classic pathways leading to aromatic amino acids, isochorismate or ubiquinones.
[1] [2] [3] [4] [5] M. A. Gregory et al., Angew. Chem. Int. Ed. 2005, 44, 4757. N. L. Paiva, M. F. Roberts, A. L. Demain, J. Ind. Microbiol. 1993, 12, 423. J. Andexer et al., Proc. Natl. Acacd. Sci. USA 2011, 108, 4776. F. Rusnak et al., Biochemistry 1990, 29, 1425. Y. Hayashi et al., J. Antibiot. 2008, 61, 164. [1] [2] [3] [4] Hoshino, T.; Ohashi, S. Org. Lett. 2002, 4, 2553-2556. Wendt, K. U.; Lenhart, A.; Schulz, G. E. J Mol Biol 1999, 286, (1), 175-87. Bohlmann, J.; Meyer-Gauen, G.; Croteau, R. Proc Natl Acad Sci U S A 1998, 95, (8), 4126-33. Davis, E. M.; Croteau, R. Top. Curr. Chem. 2000, 209, 53-95.
October 2-6, 2011
36 OC-15
Lectures
37
A biotechnological process for the production of (+)-carvone

Maarten Groeneveld1, Wouter Duetz2 and Marco Fraaije1 1 Department of biochemistry, University of Groningen, Groningen, The Netherlands; 2 EnzyScreen BV, Leiden, The Netherlands (+)-Carvone is a naturally occurring terpenoid with various interesting industrial applications. It is used as a reversible anti-sprouting agent and can be used as precursor for synthesis of enantiopure compounds. Furthermore, carvone displays antimicrobial activity. (+)-Carvone is one of the main constituents of caraway seed oil or dill seed oil, but high costs of cultivation and extraction of the product have thus far hampered a wider application in agriculture. In this project we aim to develop a biotechnological process for the production of enantiopure (+)-carvone, using D-limonene, an inexpensive waste product from orange juice industry, as starting substrate. Various bacterial strains have been identified previously that can perform either the conversion of D-limonene into the intermediate alcohol ((+)-trans-carveol), or the conversion of this intermediate into the end product, but the enzymes involved have yet to be identified. In our research the initial focus has been on the identification of the enzyme responsible for the conversion of D-limonene to (+)-trans-carveol. Previous screening efforts of various soil isolates have yielded strains that were capable of this conversion, when grown on toluene as the sole carbon source.[1] Toluene degradation is often initiated by a dioxygenase. Genome sequencing of one of the strains with a high specific activity on D-limonene resulted in a small set of putative dioxygenase genes of which one system was closely related to known toluene and biphenyl dioxygenases. We demonstrate functional expression of this dioxygenase system in Pseudomonas putida. The recombinant strain shows specific activity on D-limonene, with transcarveol as only product. Details on the expression of and biotransformations by this newly identified dioxygenase system will be presented. Efforts in establishing the subsequent oxidation of (+)-trans-carveol into (+)-carvone will also be discussed.
Berberine bridge enzyme: from biochemical studies to preparative-scale kinetic resolution and beyond
Joerg H. Schrittwiesera, Verena Rescha, Johann H. Sattlera, W.-D. Lienharta, E.-M. Fischeredera, Silvia Wallnerb, Peter Macherouxb, Wolfgang Kroutila a Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz Heinrichstrasse 28, 8010 Graz, Austria; b Institute of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria E-mail: joerg.schrittwieser@edu.uni-graz.at Berberine Bridge Enzyme (BBE) is a flavoprotein involved in the biosynthesis of protopine, protoberberine and benzophenanthridine alkaloids in plants. Its natural role is the conversion of (S)-reticuline to (S)-scoulerine by an intramolecular oxidative cyclisation reaction. This transformation represents a CH activation at an N-methyl group by molecular oxygen, a reaction unparalleled in organic synthesis (Scheme 1).[1]
MeO HO N OH O2 OMe (S)-reticuline (S)-scoulerine H2 O2 OMe BBE cat. MeO HO N OH
Scheme 1. Natural reaction of Berberine Bridge Enzyme (BBE). Fascinated by its extraordinary reactivity, we have employed BBE for biotransformations on a preparative scale for the first time.[2] Optimisation studies on the reaction conditions have revealed that the enzyme is highly tolerant towards organic co-solvents, elevated temperatures and basic pH values.[3] Furthermore, BBE was shown to convert various non-natural benzyl-isoquinoline substrates at satisfactory rates and with high enantioselectivity, allowing efficient kinetic resolution on a preparative scale (0.5 g). Thus, the produced (S)-berbine derivatives could be isolated along with the remaining (R)-enantiomers of the substrates in good to excellent yield and enantiomerically pure form (Scheme 2).[2]
R1 R2 R3 N OH O2 R4 ra c-benzylisoquinolines (S)-berbines H2 O2 R4 (R)-benzylisoquinolines R4 BBE cat. R1 R2 R3 N OH R1 R2 R3 N OH
Scheme 2. Kinetic resolution of benzylisoquinoline alkaloids catalysed by BBE. Recently we have focussed on the development of extended reaction systems that allow for recycling of the non-converted (R)-benzylisoquinolines, as well as on the investigation of novel substrates and the elucidation of the enzymes regioselectivity.
[1] [2] [3] A. Winkler, A. Lyskowski, S. Riedl, M. Puhl, T. M. Kutchan, P. Macheroux, K. Gruber, Nat. Chem. Biol. 2008, 4, 739741. J. H. Schrittwieser, V. Resch, J. Sattler, W.-D. Lienhart, K. Durchschein, A. Winkler, K. Gruber, P. Macheroux, W. Kroutil, Angew. Chem. Int. Ed. 2011, 50, 10681071. V. Resch, J. H. Schrittwieser, S. Wallner, P. Macheroux, W. Kroutil, submitted.
Figure 1. Conversion of D-limonene into trans-carveol and (+)-carvone
[1]
Duetz, W.A., Fjllman, A.H.M., Ren, S., Jourdat, C., Witholt, B. (2001) Biotransformation of D-limonene to (+)-transcarveol by toluene-grown Rhodococcus opacus PWD4 cells. Appl Environ Microbiol 67, 2829-2832.
October 2-6, 2011
38 IL-6
Lectures
Tuesday, October 4 IL-7
39
Biocatalytic cuts and sutures of natural compounds

V. Ken Institute of Microbiology, Center of Biocatalysis and Biotransformations, Academy of Sciences of the Czech Republic, Vdesk 1083, CZ-14220 Praha 4, Czech Republic, E-mail: kren@biomed.cas.cz Chemists are like surgeons: They have to make precise cuts in the molecules followed by delicate sutures. Natural products are often very complex compounds with inherent sensitivity to unnatural e.g. harsh chemical modification methods. Biocatalytic methods often mimicking natural enzymatic processes can be effectively used in the selective modifications of these complex molecules. Sequential or cascade reactions are often of a great advantage. We demonstrate this approach on two topics: The one belonging to a classics in the natural products, e.g. ergot alkaloids, and another being a very lively area of chemoprotective flavonoids. Ergot alkaloids are natural compounds with very long and often picturesque history. They underwent a dynamic development and are still being used in numerous pharmaceutical preparations. They cover a broad range of therapeutic uses as e. g., uterine atonia, postpartum bleeding, migraine, orthostatic circulatory disturbances, senile cerebral insufficiency, hypertension, hyperprolactinemia, acromegaly, and parkinsonism. Unfortunately, in present time the use of ergot alkaloids declines mainly because they are not quite selective and are considered to be dirty drugs.[1] Selective oxidations accomplished by various bio-systems, as e.g., enzymes, microorganisms and plant cell cultures afforded in our hands a broad variety of new compounds, some of which having direct application in pharma industry. Microbial amidases enabled direct preparation of optically pure lysergic acid from ergopeptides keeping high stereoselectivity. Silybin is a major component of Silymarin - flavonolignan complex from Silybum marianum (milk thistle). It has been used mostly as hepatoprotectant but recently it proved to be very effective in prostatic cancer treatment.[2] Natural silybin is equimolar mixture of two hardly separable diastereoisomers having substantially different biological activities. Preparatory production of optically pure silybins A and B in a d.e. over 98 % was accomplished using lipases.[3] Lipases served also for selective protection/deprotection methods of this polyol (5 OH groups). Alternative method for diastereomeric separation employing glycosidases was developed. Multienzyme systems with integrated cofactor regeneration for preparation of silybin metabolites, such as glucuronides and sulfates, will be presented as well. Acknowledgement: Support by the Czech Science Foundation (P207/11/0629), Ministry of Education of the Czech Republic (LC06010, OC09045, and AV0Z50200510), and ESF COST Chemistry project CASCAT CM0701 is acknowledged.
Industrial enzymatic C-C bond formation

Michael Wolberg1,2, Martin Schrmann1, Stefan Jennewein3, Daniel Mink1 1 DSM Pharma Chemicals Advanced Synthesis, Catalysis and Development, P.O.Box 18, NL-6160 MD Geleen, The Netherlands; 2DSM Pharma Chemicals ResCom, Regensburg, Germany; 3present adress: Fraunhofer Institut, Aachen, Germany In the past decades biocatalysis has developed from an interesting academic research area to a valuable synthetic tool for the large-scale production of bulk chemicals, pharmaceutical and agro intermediates[1]. As a leading company in the production of pharmaceutical intermediates, DSM has developed and brought to the plant more than 25 biocatalytic processes for the cost-effective manufacturing of homochiral compounds. In order to reduce development time and to further strengthen and expand our position in our key competence biocatalysis, the pro-active development of proprietary biocatalyst platforms is one of our key success factors. Recently, carbon-carbon bond forming enzymes like (R)- and (S)-hydroxynitrile lyases, threonine aldolases and 2-deoxy-D-ribose 5-phosphate aldolases (DERA) were added to our biocatalytic toolbox. At DSM DERA has been successfully applied in the enantioselective syntheses of several versatile chiral compounds on commercial scale. These compounds represent advanced intermediates for pharmaceuticals. The key step in their synthesis is the consecutive aldol reaction of an acceptor aldehyde with two molecules of acetaldehyde yielding a deoxy-sugar derivative catalyzed by DERA. In this step the complete carbon chain and two chiral centres are built up. The DERA reaction products can than further be reacted chemically to a large variety of chiral synthons.
O Cl H + O H + O H O O Cl O NR2 O Cl O CN Cl O SR O Cl O O Cl O O O
DERA
O Cl OH
TsOH (3 mol%) toluene,
Figure 1. DERA reaction products.
[1] [2] [3]
Ken V., Cvak L. (eds.): Ergot - Genus Claviceps, Medicinal & Aromatic Plants - Industrial Profiles, Harwood Academic Publ., Amsterodam, London (1999). Gak, R.; Walterov, D.; Ken, V.: Silybin and silymarin new and emerging applications in medicine. Curr. Med. Chem. 14, 315-338 (2007). Gak R., Marhol P., Purchartov K., Monti D., Biedermann D., Riva S., Cvak L., Ken V.: Large-scale separation of silybin diastereoisomers using lipases. Proc. Biochem. 45, 1657-1663 (2010).
[1] [2]
Schmidt, A. et al., Nature 409, 258 (2001). Schoemaker, H. et al., Science 299, 1694 (2003).
October 2-6, 2011
40 OC-17
Lectures
41
Development of organic solvent-tolerant enzymes

Hiroyasu Ogino Osaka Prefecture University, Sakai, Osaka 599-8531, Japan E-mail: ogino@chemeng.osakafu-u.ac.jp Enzymes catalyze specific reactions without by-product under mild conditions. It is possible to construct environmentally-friendly sustainable bioprocesses, when enzymes are used as catalysts for chemical processes. A number of commercially important organic products are water-insoluble and as such should be synthesized in organic solvents. However, most enzymes are instable and lose their catalytic activities in the presence of organic solvents. By the addition of organic solvents to the enzymatic reaction solution, the solubility of substrates and the reaction rate can often be markedly increased. In the presence of organic solvents hydrolytic enzymes can also perform synthetic reactions, which are the reverse reactions of hydrolysis. This happens because the molar fraction of water is reduced by the addition of an organic solvent to the reaction mixture, thus shifting the reaction equilibrium towards synthetic catalysis. A further advantage of nonaqueous reactions is that the risk of unwanted microbial contamination is normally very low. Then, organic solvent-tolerant enzymes were developed.[1] Lipase is one of the most useful enzymes as an industrial catalyst. Substrates of lipase, such as lipids and esters, are usually water-insoluble and lipase catalyzes not only ester hydrolysis but also ester synthesis and ester transesterifications in the presence of organic solvents. Then lipase is often used in the presence of organic solvents. An organic solvent-stable lipase, LST-03 lipase, from an organic solvent-tolerant microorganism has been developed.[2] Protease catalyzes hydrolysis of peptide bonds in the presence of water. However, in the presence of organic solvents it also can catalyze synthesis of peptide bonds. An organic solvent-stable protease, PST-01 protease, from an organic solvent-tolerant microorganism has been also developed. The reason of organic solvent-stability of enzymes has been investigated by monitoring the conformational transitions of enzymes in the presence of organic solvents, site-directed mutagenesis, and random mutagenesis.[3] And enzymatic synthesis of aspartame, a low-calorific artificial sweetener, was investigated using the PST-01 protease in the presence of organic solvents. The synthesis yield was improved by investigation of reaction condition and attained 83% in the presence of 50% (v/v) DMSO. Furthermore, the improvement of reaction rate of aspartame precursor was attained by changing amino acid residues which were in close vicinity of catalytic center of the PST-01 protease to be like those of thermolysin. The mutation made it possible that the reaction rate increases about ten times.[4]
An expanded view of adaptation mechanisms in enzyme evolution.

Pietro Gatti-Lafranconia, Thomas Shafeea and Florian Hollfeldera a Dept. of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB1 1GA, UK. E-mail: pg356@cam.ac.uk Directed evolution [1, 2] is arguably surpassing rational approaches towards creating improved biocatalysts and theories are emerging as to how to go about it.[3, 4] Successful examples usually involve relatively stable model enzymes that have been studied for a long time, providing in-depth knowledge of homology, ancestry, stability, structure and mechanism. Structure influences enzymatic activity to a much larger extent than by simply providing a stable scaffold for catalytic activity. Studies on intrinsic disorder [5], promiscuity [6] and cold-adapted enzymes [7] indicate that activity and structure often evolve jointly and that properties of a catalyst are a compromise between multiple selective pressures. Several factors are thus likely to be relevant for the evolution of new functions and activities. This paradox is the basis for the idea that robustness towards mutation is encompassing factors in addition to structural stability. To address the general question what makes a protein evolvable? we study viral proteases as model system for the evolution of hydrolytic enzymes and of their structural properties. Viruses are subject to high mutation rates and their enzymes are expected to retain the original function despite such low conservation. [8] Striking evidence for this thesis is a comparison of the mutation rate in humans, eukaryotes and bacteria (~108 per base pair per generation) that is increased in viral systems by 103-105-fold [9]. Despite this dramatic difference, viral protein evolution has been successful, suggesting a robustness able to deal with high mutation rates without being necessarily more stable (e.g. against thermal denaturation). Our studies are aimed at testing this hypothesis and provide clues on the robustness/functionality trade-off. Possible pathways of evolution will be addressed for a cystein and an aspartic protease, using an in vivo screening system. Specificity changes and catalytic rate increases are pursued by exerting selective pressure in different ways. The properties of selected mutants are then analysed by a combination of biochemical and structural techniques to identify and characterise evolutionary patterns (and whether they depend on the protein to be evolved or on the nature of the pressure applied). I will specifically address how the likelihood and speed of adaptation can be increased (e.g. the influence of intrinsic structural properties such as stability, robustness, and tolerance to mutations) and analyse efficient evolutionary routes with regard to the adaptation of the catalytic machinery and to the role of structure, oligomerisation and symmetry in evolution.
[1] [2] [3] [4]
H. Ogino, Organic solvent-stable enzymes, in: K. S. Siddiqui, T. Thomas (Eds.), Protein Adaptation in Extremophiles, Nova Science Publishers, Inc., New York, 193-236 (2008). T. Kawata and H. Ogino, Amino acid residues involved in organic solvent-stability of the LST-03 lipase, Biochem. Biophys. Res. Commun. 400, 384388 (2010). H. Ogino et al., Effect of exchange amino acid residues of the surface region of the PST-01 protease on its organic solventstability, Biochem. Biophys. Res. Commun. 358, 1028-1033 (2007). H. Ogino et al., Enhancement of the aspartame precursor synthetic activity of an organic solvent-stable protease, Protein Eng. Des. Sel. 23, 147-152 (2010).
[1] [2] [3] [4] [5] [6] [7] [8] [9]
Shivange, A. V., et al. (2009) Advances in generating functional diversity for directed protein evolution Curr Opin Chem Biol. 13, 19-25 Turner, N. J. (2009) Directed evolution drives the next generation of biocatalysts Nat Chem Biol. 5, 567-573 Tokuriki, N., et al. (2008) How protein stability and new functions trade off PLoS computational biology. 4, e1000002 Dougherty, M. J., et al. (2009) Directed evolution: new parts and optimized function Curr Opin Biotechnol. 20, 486-491 Uversky, V. N., et al. Understanding protein non-folding Biochim Biophys Acta. 1804, 1231-1264 Babtie, A. et al. (2010) What makes an enzyme promiscuous?, Curr Opin Chem Biol 14: 200-207. Gatti-Lafranconi, P., et al. (2010) Evolution of stability in a cold-active enzyme elicits specificity relaxation and highlights substrate-related effects on temperature adaptation J Mol Biol. 395, 155-166 Tokuriki, N., et al. (2009) Do viral proteins possess unique biophysical features? Trends Biochem Sci. 34, 53-59 Sanjuan, R., et al. (2010) Viral mutation rates J Virol. 84, 9733-9748
October 2-6, 2011
42 OC-19
Lectures
Tuesday, October 4 IL-8
43
Novel dioxygenases for hydroxy amino acid production

Makoto Hibia, Sakayu Shimizub, Kenzo Yokozekia,c, Jun Ogawab a Industrial Microbiology and bDivision of Applied Life Sciences, Grad. Sch. Agric., Kyoto University, Japan; c Research Institute for Bioscience Products & Fine Chemicals, Ajinomoto Co., Inc., Suzuki-cho, Kawasakiku, Kawasaki 210-8681, Japan E-mail: mhibi@kais.kyoto-u.ac.jp -Ketoglutarate-dependent dioxygenase family is an important class of hydroxylating catalyst, because the enzymes belonging to the family are soluble protein and catalyze irreversible hydroxylation with high specific activities. Among them, amino acid dioxygenases are useful for the production of hydroxy amino acids, some of which exert worthwhile physiological activities on the mammals, and also are components of peptide antibiotics and chiral precursors in the chemical synthesis of other compounds. So far, we have obtained several novel amino acid dioxygenases and analyzed their characteristics toward developing effective production processes of various hydroxy amino acids. L-Isoleucine-4-dioxygenase (IDO) was found as responsible enzyme for the generation of (2S,3R,4S)4-hydroxyisoleucine from L-isoleucine in Bacillus thuringiensis 2e2 [1,2]. As well as L-isoleucine, IDO stereoselectively hydroxylates several branched-chain L-amino acids and produced, 4-hydroxy-L-leucine, 4-hydroxy-L-norvaline, 4-hydroxy-L-norleucine, 5-hydroxy-L-norleucine, and 3-hydroxy-L-allo-isoleucine (Fig. 1). Interestingly IDO also catalyzes sulfoxidation of some sulfur-containing L-amino acids such as L-methionine and L-ethionine, and generates the corresponding sulfoxides (Fig. 1). Moreover we found two dioxygenases of Nostoc punctiforme and Burkholderia ambifaria also had the activities to hydroxylate some kinds of amino acids and amino acid derivatives, and these substrate specificities and regio selectivity for the hydroxylation are different from IDO. Then by using these novel dioxygenases as catalysts in the biotransformation system developed for IDO [3], it would be possible to produce various hydroxy amino acids industrially as well as 4-hydroxyisoleucine.
Adventures with the ThDP Enzyme Transketolase

Helen C. Hailes a Department of Chemistry, University College London, 20 Gordon Street, London, WC1H 0AJ, UK E-mail: h.c.hailes@ucl.ac.uk ,-Dihydroxy ketones 1 are important synthetic intermediates used for the synthesis of a range of compounds such as ketosugars. They can also be aminated to the 2-amino-1,3-diol functionality, present in many natural and synthetic biologically active molecules including antibiotics, alkaloids and amino sugars. We have been working towards the engineering and directed evolution of novel transketolase (TK) (E.C. 2.2.1.1) mutants[1] capable of converting a multitude of aliphatic, cyclic and aromatic aldehyde substrates into chiral ,-dihydroxy ketones and their subsequent transformation into aminodiols using transaminases (TAm) (Scheme 1). This presentation will focus on the ThDP enzyme TK and the development of tools to identify TK mutants active against selected substrates. This will include a colorimetric assay suitable for high-throughput usage and several strategies to establish ees and absolute stereochemistries of the products formed.[2,3] Substrate preferences and stereoselectivities achieved will be presented with a range of substrates. Notably several active site single-point mutations have proven to be particularly valuable for enhancing and reversing the stereoselectivity of E. coli TK with aliphatic non -hydroxylated aldehydes.[4] In addition, cyclic aliphatic aldehydes have been readily accepted by some TK variants to give products in >99% ee,[5] and high enantioselectivities observed when using chiral cyclic aldehydes. Several mutants have also been observed to accept aromatic aldehydes such as benzaldehyde.[6] Finally, use of the TK products in TAm reactions to convert ,-dihydroxyketones to the corresponding amines will be highlighted.
O R O H + O OLi+ OH Transketolase (TK) R ThDP, Mg2+ OH O OH Transaminase (TAm) R PLP, amine donor NH2 OH
,'-dihydroxy ketone
OH 2-amino-1,3-diol
Scheme 1. Use of TK to generate ,-dihydroxy ketones and conversion to the useful 2-amino-1,3-diols using TAm This work is part of BiCE, which in parallel is establishing tools for the rapid characterisation and scale-up of these bioconversions.
[1] [2] [3]
Kodera, T., et al. (2009) Biochem. Biophys. Res. Commun. 390(3), 506-510 Ogawa, J., et al. (2011) Appl. Microbiol. Biotechnol. 89(6), 1929-1938 Smirnov, S. V., et al. (2010) Appl. Microbiol. Biotechnol. 88(3), 719-726
[1] [2] [3] [4] [5] [6]
E. G. Hibbert, T. Senussi, S. J. Costelloe, W. Lei, M. E. B. Smith, J. M. Ward, H. C. Hailes and P. A. Dalby, J. Biotechnology, 2007, 131, 425. M. E. B. Smith, U. Kaulmann, J. M. Ward, H. C. Hailes, Bioorg. Med. Chem., 2006, 14, 7062. J. L. Galman, H. C. Hailes, Tetrahedron: Asymm., 2009, 20, 1828. M. E. B. Smith, E. G. Hibbert, A. B. Jones, P. A. Dalby, H. C. Hailes, Adv. Syn. Cat., 2008, 350, 2631. A. Czares, J. L. Galman, L. G. Crago, M. E. B. Smith, J. Strafford, L. Ros-Sols, G. J. Lye, P. A. Dalby, H. C. Hailes, Org. Biomol. Chem., 2010, 8, 1301. J. L. Galman, D. Steadman, S. Bacon, P. Morris, M. E. B. Smith, J. M. Ward, P. A. Dalby, H C. Hailes, Chem. Commun., 2010, 46, 7608.
October 2-6, 2011
44 OC-20
Lectures
45
Benzaldehyde lyase catalyzed direct amidation of aldehydes with nitroso compounds

Peruze Ayhan and Ayhan S. Demir Department of Chemistry, Middle East Technical University, 06531 Ankara, Turkey asdemir@metu.edu.tr Benzaldehyde lyase (BAL), a novel thiamine diphosphate (ThDP) dependent enzyme from Ps. Fluorescens Biovar I, is reported to perform the enantioselective formation of (R)- and (S)-benzoins and (R)-2-hydroxypropiophenone ((R)-2-HPP) derivatives via C-C bond cleavage and C-C bond formation. (R)-2-HPP derivatives are formed in preparative scale by benzaldehyde lyase (BAL) catalyzed C-C bond formation from aromatic aldehydes and acetaldehyde, methoxy- and dimethoxyacetaldehyde in a buffer/DMSO solution with remarkable ease in high chemical yields and optical purities.[1] Nitroso compounds exhibit high reactivity.[2] The polarization of the nitrogen-oxygen bond, resembling that of the carbon-oxygen bond in a carbonyl group, makes the nitroso group succeptible to addition of nucleophiles. We suggest a possibility that involved the reaction of an enamine-carbanion intermediate with nitrosobenzene forming hydroxamic acid instead of acyloin. The chemistry and biochemistry of hydroxamic acids are well documented.
O H NO O N OH
Conversion of pyruvate decarboxylase into an enantioselective carboligase

Danilo Meyera, Cindy Wechslera, Martina Pohlb, Michael Mllerc, Kai Tittmanna a Georg-August-Universitt Gttingen, Justus-von-Liebig-Weg 11, 37077 Gttingen, Germany; bForschungszentrum Jlich, Jlich, Germany; cUniversitt Freiburg, Freiburg i. B., Germany E-mail: ktittma@gwdg.de Pyruvate decarboxylase (PDC) catalyzes the nonoxidative conversion of pyruvate into acetaldehyde and carbon dioxide and requires the cofactors thiamin diphosphate (ThDP, the vitamin B1 coenzyme) and a divalent metal ion for activity. PDCs are catalytically promiscuous as they catalyze carboligation side reactions with externally added aldehydes. This reactivity is exploited for the asymmetric synthesis of 2-hydroxy ketones such as (R)-phenylacetyl carbinol, the precursor of (-)-ephedrine. The products yields of these side reactions are, however, limited since PDCs heavily favor the native reaction that is aldehyde formation over any offpathway reaction. We were able to identify and characterize an active site variant (Glu473Gln) in which partitioning between aldehyde release versus carboligation is inverted with an up to 100-fold preference for the latter pathway.[1] Due to a defective protonation of the central reaction intermediate, turnover stalls at this highly reactive catalytic stage, and addition of aldehydes leads to a quantitative and enantioselective formation of 2-hydroxy ketones as exemplified shown for (R)-phenylacetyl carbinol, which is afforded with unmatched yields, rates, and purity.[2] This protein variant thus constitutes an example for the rational design of biocatalysts with greatly enhanced accidental catalytic promiscuity by selective blockage of the native reaction and accumulation of reactive intermediates under steady-state turnover conditions.
NO
O N
OH
OH
OH
Figure 1. BAL catalysed reactions The method described herein presents the first enzyme catalyzed highly selective synthesis of N-arylhydroxamic acids via C-N bond linkage. The reaction works in organic-aqueous medium, overcomes the solubility problem with organic substrates, and may pave the way for large-scale preparations. A further improvement can be obtained with the use of immobilized enzyme to utilize lower enzyme activities. The products are obtained in high yields starting from simple, easily available aromatic aldehydes, benzoins, and nitrosobenzene via C-N bond formation and C-C bond cleavage and followed by C-N bond formation reaction.
[1] [2] A. S. Demir, M. Pohl, E. Janzen, M. Mller, J. Chem. Soc., Perkin Trans. 1 2001, 633-635; b) P. Dnkelmann, D. KolterJung, A. Nitsche, A. S. Demir, P. Siegert, B. Lingen, M. Baumann, M. Pohl, M. Mller, J Am. Chem. Soc. 2002, 124, 1208412085. J. Kulys, H.-J. Deussen, K. Krikstopaitis, R. Lolck, P. Schneider, A. Ziemys, Eur. J. Org. Chem. 2001, 18, 3475-3484; b) P. F. Santos, A. M. Lobo, S. Prabhakar, Synth. Commun. 1995, 25, 3509-3518. [1] [2] Meyer et al., Biochemistry 49, 8197-8212 (2010) Meyer et al., J. Am. Chem. Soc. 133, 3609-3616 (2011)
October 2-6, 2011
46 OC-22
Lectures
Tuesday, October 4 PL-3
47
Formation of tertiary alcohols by ThDP-dependent enzymes

Patrizia Lehwalda, Maryam Beigia, Michael Richtera,b, Anja Kurutscha, G. A. Sprengerc, M. Mllera a Inst. of Pharmaceutical Sciences, University of Freiburg, 79104 Freiburg, Germany, b Laboratory for Biomaterials, Empa - Swiss Federal Laboratories for Materials Testing and Research, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland, c Inst. of Microbiology, University of Stuttgart, Allmandring 31,70569 Stuttgart, Germany. E-mail: michael.mueller@pharmazie.uni-freiburg.de
Enzyme-based nanocomposites: using nature to ward off emerging diseases

Jonathan S. Dordicka a Center for Biotechnology & Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA E-mail: dordick@rpi.edu Nature is unparalleled in its structural and functional diversity. Living organisms make fantastic materials under myriad conditions with properties we cannot emulate today using conventional approaches. In many cases, nature has provided us with a blueprint to design and assemble both natural and synthetic building blocks to create a new generation of functional, organized, and responsive materials. We have taken cues from nature to design materials with unique structural and functional properties, along with new process technologies with the ability to produce a wide range of biomimetic structures.[1-3] Specifically, we have focused on the generation of nanostructures that are functionalized with and in some cases constructed from biological molecules, complete with tailored selectivities and biocatalytic activities. For example, these nanostructures have been exploited in the generation of biocatalytically functional polymeric films, coatings, and paints that kill bacteria, prevent biofilm formation, and reduce fouling by bioorganic molecules.[4] In this talk I will highlight our recent efforts to exploit the interface of biology with materials science, with a particular focus on enzyme-nanomaterial composites with a wide range of activities that endow surfaces with self-cleaning properties. In particular, surfaces have been generated with tailored activity against hospital-acquired infections (e.g., MRSA) and spores.[5,6] Such activity provides a safe and potentially broadly applicable route to eliminating toxic compounds and pathogenic microorganisms from common surfaces.
Thiamine diphosphate (ThDP)-dependent enzymes are well-established catalysts in the field of asymmetric synthesis.[1] One reaction type catalyzed by various of these enzymes covers C-C-bond formations. Enzymes like benzaldehyde lyase (BAL) from Pseudomonas fluorescens or pyruvate decarboxylase from Saccharomyces cerevisiae (ScPDC) and Zymomonas mobilis (ZmPDC), catalyze the coupling of two aldehydes to chiral 2-hydroxy ketones with high enantioselectivity.[2] Our investigation of a ThDP-dependent enzyme from Yersinia pseudotuberculosis O:VI (YerE), first described by Liu et al. in 1998[3], revealed, that this enzyme plays a unique role amongst ThDP-dependent enzymes accepting not only aldehydes as acceptor substrates but also ketones. Moreover it is the first enzyme known so far, that catalyzes an intermolecular enantioselective crossed aldehyde-ketone carboligation giving chiral tertiary alcohols as products.[4]
O H3C HO CH3
O COO
-
O COO
-
H3C
O H3C COOpyruvate
R1 R2 ketones YerE
O H3C HO
R2 R1
(S)-acetolactate
AHAS YerE
tertiary alcohols
Next to this unique activity YerE is able to catalyze a diversity of carboligation reactions that have been described for other ThDP-dependent enzymes as well. For example, the formation of acetolactate is common for many ThDP-dependent enzymes. Detailed investigation of MenD-catalyzed transformations leading to regioisomeric 2-hydroxy ketones[5] showed that the outcome can not solely be attributed to the donor-acceptor characteristics of the substrates. Instead, in a side reaction the acetolactate derivative (with a tertiary hydroxyl group) can be decarboxylated to the respective isomeric secondary alcohol.
[1] [1] [2] [3] [4] [5] M. Pohl, G. A. Sprenger, M. Mller, Curr. Opin. Biotechnol. 2004, 15, 335. M. Pohl, B. Lingen, M. Mller, Chem. Eur. J. 2002, 8, 5288. H. Chen, Z. Guo, H.-W. Liu, J. Am. Chem. Soc. 1998, 120, 11796. P. Lehwald, M. Richter, C. Rhr, H.-w. Liu, M. Mller, Angew. Chem. 2010, 122, 2439. A. Kurutsch, M. Richter, V. Brecht, G. A. Sprenger, M. Mller, J. Mol. Catal. B: Enzym. 2009, 61, 56. [2] [3] [4] [5] [6] Biotrans 2011 - Italy
G. John, G. Zhu, J. Li, and J.S. Dordick (2006), Enzymatically Derived Sugar-Containing Self-Assembled Organogels with Nanostructured Morphologies, Angew. Chem. Int. Ed. Engl. 45, 4772-4775. C.Z. Dinu, S.S. Bale, D.B. Chrisey, and J.S. Dordick (2009), Manipulation of Individual Carbon Nanotubes by Reconstructing the Intracellular Transport of a Living Cell, Adv. Mater. 21, 1182-1186. G.E. Sroga and J.S. Dordick (2006), Controlled Hierarchical Assembly of Switchable DNA-Multiprotein Complexes, Biotechnol. Bioeng. 94, 312-321. P. Asuri, S.S. Karajanagi, R.S. Kane, and J.S. Dordick (2007), Polymer-Nanotube-Enzyme Composites as Active Antifouling Films, Small 3, 50-53. C.Z. Dinu, G. Zhu, S.S. Bale, G. Anand, P.J. Reeder, K. Sanford, G. Whited, R.S. Kane, and J.S. Dordick (2009), EnzymeBased Nanoscale Composites for Use as Active Decontamination Surfaces, Adv. Funct. Mater. 20, 392-398. R.C. Pangule, S.J. Brooks, C.Z. Dinu, G. Zhu, S.S. Bale, S. Salmon, D.W. Metzger, R.S. Kane, and J.S. Dordick (2010), Antimicrobial Nanocomposite Films Based on Nanotube-Enzyme Conjugates, ACS Nano 4, 3993-4000.
October 2-6, 2011
48 IL-9
Lectures
Wednesday, October 5 OC-23
49
Oxidative enzymes in lignin biorefinery

Raffaele Saladinoa, Federica Meloneb, Silvia Decinab Claudia Crestinib. a Dipartimento ABAC University of Tuscia, via San Camillo de Lellis, 01100, Viterbo, Italy; bDipartimento di Scienze e Tecnologie Chimiche, Tor Vergata University, via della Ricerca Scientifica, 00133, Roma, Italy. E-mail: crestini@uniroma2.it Today the rising energy consumption and the depletion of fossil fuel feedstocks have focused the attention on the use of alternative renewable materials and on the development of environmentally friendly processes that operate in mild reaction conditions. Lignin is the second most abundant organic polymer in plant kingdom and constitutes up to 30% of wood. It constitutes to date the bottleneck to the development of integrated biorefinery processes since it is the residue of modern saccharification processes. Current processes of bioethanol production from wood originate about 500g of lignin each liter of bioethanol. From this viewpoint the development of processes of lignin upgrade through oxidative depolymerization or functionalisation is mandatory. In Nature the selective oxidation of lignin is carried out by white rot basidiomycetes fungi that produce a pool of extracellular ligninolytic enzymes such as laccases and peroxidases. In particular laccases can easily oxidise phenolic groups and in presence of radical mediator, such as 1-Hydroxybenzotriazole, their reactivity can be extended towards other functional groups as phenyl-aryl ethers. Mnperoxidase and lignin peroxidases are able to oxidize lignin at the phenolic and nonphenolic aryl-ether positions respectively. Laccases and peroxidases constitute an interesting tool for the development of alternative oxidative processes due to their low substrate specificity and relatively wide pH of action. In the last years both laccases and peroxidases have been used in several biotechnological applications such as oxidation of organic pollutants, pulp delignification, bleaching and development of biosensors or biofuel cells. Unfortunatly, the exploitation of their potentiality is prevented, specially in the case of peroxidases, by their low stability. The basic requirement for the development of economically sustainable enzymatic processes are the possible recycle of the catalyst and a high stability of the enzyme. As such a number of different immobilization techniques have been developed. Recently, significant efforts have been made to develop organo-catalytic cascade reactions with the objective to mimic the biosynthetic strategy, but to date the innovative potential of biocatalysis by promoting the multistep catalytic concept by multienzyme systems has not been fully explored. The use of oxidative enzymes in lignin upgrade, the design and development of oxidative multienzyme biocatalysts for lignin selective oxidation will be reported.
a
The enzyme refinery: functionalisation of lignin and lignocellulose

Gibson S. Nynhongoa, Endry Nugroho Prasetyoa, Tukayi Kudangaa, Georg M. Guebitza,b Institute of Environmental Biotechnology, Graz University of Technology, bAustrian Centre of Industrial Biotechnology ACIB, Petergasse 14, 8010 Graz E-mail: guebitz@tugraz.at Within the wood biorefinery approach enzymatic functionalisation of lignin and lignocellulose shows increasing potential. Covalent grafting of functional phenolic molecules onto lignocellulose materials such as MDF, pulp fibres or veneers was demonstrated by using sophistical surface analytical tools such as XPS leading to increased hydrophobicity, antimicrobial properties or enhanced strength.[1-6] Novel mechanistic insights in the grafting reactions and targeted polymerisation of lignin sulfonates were obtained by using lignin model molecules.[2] For guaiacylglycerol -guaiacyl ether (erol), syringylglycerol -guaiacylether and dibenzodioxocin reaction pathways (e.g. 4-O-5 versus 5-5 linking) of laccase catalysed coupling functional phenolic molecules (e.g. fluorophenols, aromatic amines) were established. These models also acted as electron mediators oxidizing non-laccase substrates. Apart from lignocellulose materials, oxidoreductases seem to have a potential to increase the molecular weight of calcium lignosulfonates leading e.g. to improved dispersion properties.[6] Thereby, FTIR spectroscopy, 13C NMR and Py-GC/MS analysis demonstrated no substantial changes in the aromatic signal of the lignosulfonate indicating targeted enzymatic oxidation of functional groups without degrading the lignin backbone.
Figure 1. Hydrophobic functionalization of lignocellulose
[1] [2] [3] [4] [5] [6]
Kudanga et al: Bioresource Technology 2010, 101:2793-2799. Kudanga et al: Journal of Molecular Catalysis B: 2009, 61:143149 Kudanga et al: J. Biotechnol, 2011, in press Kudanga et al: Enzyme Microbial Technology 2010, 46:272-280. Widsten et al: Process Biochemistry, 2010, 45:10721081 Nugroho Prasetyo et al: Bioresource Technology 2010, 101(14):50545062
October 2-6, 2011
50 OC-24
Lectures
51
Release of phenolic monomers from complex lignins by an ether cleaving enzyme system
J. Reitera, H. Strittmatterb, M. Kolba, D. Schiedera, L.O. Wiemannb, V. Siebera,b a TU Mnchen, Lehrstuhl fr Chemie Biogener Rohstoffe, Schulgasse 16, 94315 Straubing; Germany; b Fraunhofer IGB Projektgruppe BioCat, Wissenschaftszentrum Straubing, Schulgasse 16, 94315 Straubing; Germany E-mail: sieber@tum.de The increasing interest towards greener production processes in chemistry leads to increasing research for new ways to degrade the complex lignin-backbone by means of biocatalysis and combined chemoenzymatic catalysis. Lignin is regarded as a potential substitute for phenolic and other aromatic, oil-based chemicals in the post oil age. The cleavage of the -O-4 -aryl ether linkage is the most favoured, since it accounts for approximately 50% of all ether linkages in lignin. The enzymatic cleavage was proposed to be part of the lignin catabolism in the proteobacterium Sphingobium sp.[1] There three enzymes LigD, a C-dehydrogenase, LigF, a -etherase and LigG, a glutathione lyase are supposed to be involved in lignin degradation. However so far, this enzyme system has only been analyzed in crude extracts and only with model substrates, such as guaiacylglycerol--guaiacyl ether (GGE). Lig D oxidizes GGE to -(2methoxyphenoxy)--hydroxypropiovanillone (MHPV). The ether linkage of MHPV is then cleaved by Lig F to glutathione--hydroxypropiovanillone (GS-HPV) and guaiacol, while GS-HPV is further processed by the glutathione lyase into hydroxypropiovanillone. We cloned and recombinantly expressed the three genes in E. coli and did a detailed characterization of the enzymes. We showed the activity on the lignin model substrate guaiacylglycerol--guaiacyl ether (GGE). In addition using the recombinant enzymes we could show for the first time the efficiency of this enzyme system to release lignin monomers from real complex lignin structures coming from different technical processes. Using the etherase system a more stable product formation is achieved compared to using the conventional laccase and peroxidase based enzymatic lignin cleavage, where radical formation leads to in situ repolymerization of released monomers.
Enzymatic synthesis of alkyl oligoxylosides from isolated xylans and from lignocellulosic biomass
Marjorie Ochs1, Murielle Muzard2, Richard Plantier-Royon2, Boris Estrine3, Caroline Rmond1 1 UMR FARE, INRA- Universit de Reims Champagne-Ardenne, 51688 Reims Cedex 2, France 2 Institut de Chimie Molculaire de Reims ICMR, CNRS UMR 6229, Universit de Reims ChampagneArdenne, 51687 Reims Cedex 2, France 3 Agro-Industrie Recherches et Dveloppement Green Chemistry Department, 51110 Pomacle, France E-mail: Marjorie.Ochs@reims.inra.fr The development of economically viable biorefineries from lignocelluloses requires to be based on multiproducts concept aiming to produce biomaterials, bioethanol and bio-based molecules.[1] In this context, the use of arabinoxylans in addition to the cellulosic fraction represents a major improvement to assure the sustainability of biorefineries. Alkyl glycosides are sugar-based nonionic surfactants, mainly synthesized by chemical route (Fischer glycosidation or Koenigs-Knorr reaction). These methods require hard conditions such as high temperature and acid pH. An alternative route is possible through glycoside hydrolases. In presence of water molecules acting as acceptors, these enzymes cleave glycosidic bonds by an acid-base catalysis. Besides, in the presence of acceptors different from water, they can also catalyze transglycosylation reactions.[2] Xylanases can catalyze the fractionation and the functionalization of xylans into alkyl oligoxylosides when an aliphatic alcohol is added in the reaction media (Figure 1). The produced pentose-based surfactants present higher degrees of polymerization compared to molecules produced by classical chemistry. These new products can exhibit different behaviors as surfactants regarding to the monomers. In our study, we have investigated numerous parameters in order to improve yields of synthesis of n-pentyl and n-octyl oligoxylosides from isolated xylans and two different xylanases (Figure 1). Furthermore, synthesis of octyl xylosides was directly obtained from wheat bran, an abundant lignocellulosic biomass coming from milling industries. Yields of synthesis, composition and structures of the products as well as their properties as surfactants will be presented.
xylans
xylanase H2O / ROH 60 C
HO HO
O OH
OR
O HO
O OH n = 1 to 6
O HO n
O OH
OR
Figure 1. Transglycosylation is performed by a xylanase when R is an alkyl group.
[1]
Masai E, Ichimura A, Sato Y, Miyauchi K, Katayama Y, Fukuda M., (2003) Roles of the enantioselective glutathione Stransferases in cleavage of beta-aryl ether. J.Bacteriol., 185(6):1768-75.
[1] [2]
J. H. Clark, V. Budarin, F. E. I. Deswarte, J. J. E. Hardy, F. M. Kerton, A. J. Hunt, R. Luque, D. J. Macquarrie, K. Milkowski, A. Rodriguez, O. Samuel, S. J. Tavener, R. J. White and A. J. Wilson, Green Chem. 2006, 8, 853-860. a) B. M. de Roode, M. C. R. Franssen, A. van der Padt and R. M. Boom, Biotechnol. Prog. 2003, 19, 1391-1402; b) F. van Rantwijk, M. W. V. Oosterom and R. A. Sheldon, J. Mol. Catal. B: Enzym. 1999, 6, 511-532.
October 2-6, 2011
52 OC-26
Lectures
53
Ionic liquid-assisted enzymatic depolymerisation of (ligno)-cellulose

Antje Spiess , Philip Engel , Helene Wulfhorst , Gernot Jger , Benjamin Bonhage , Michele Girfoglio Uli Commandeurb, Raffaele Cannioc a RWTH Aachen University, AVT Enzyme Process Technology, Worringerweg 1, 52074 Aachen, Germany; b RWTH Aachen University, AVT Biochemical Engineering, Worringerweg 1, 52074 Aachen, Germany; c RWTH Aachen University, Molecular Biotechnology, Worringerweg 1, 52074 Aachen, Germany; cInstitute of Protein Biochemistry, National Research Centre, Via P. Castellino 111, 80131 Naples, Italy E-mail: antje.spiess@avt.rwth-aachen.de
a a a b a b,c,
Rationally engineered Candida antarctica lipase B as an efficient catalyst for poly(lactide) synthesis
Mohamad Takwa, Marianne Wittrup Larsen, Karl Hult and Mats Martinelle Royal Institute of Technology, School of Biotechnology, Department of Biochemistry, AlbaNova University Centre, 106 91 Stockholm, Sweden. E-mail: matsm@biotech.kth.se The stable enzyme Candida antarctica lipase B (CALB) was rationally redesigned as an efficient catalyst for the ring opening polymerization (ROP) of D,D-lactide with a 90-fold improved activity as compared with the wild-type enzyme. No efficient lipase is available to date for lactide polymerization and huge amount of enzyme is needed for the process.[1] Poly(lactides) are usually synthesised via polycondensation of lactic acid monomers or via ring opening polymerization (ROP) of lactides using metal catalysts.[2] An available efficient enzyme catalyst would be of great interest for the synthesis and development of new functional poly(lactide) based materials.[3] Wild-type CALB has a good activity for the initiation of D,D-lactide, indicating that the low activity displayed by CALB towards the ROP of lactide occurs during the propagation step (deacylation with the growing polymer as acyl acceptor).[1,4] Based on molecular modelling simulations of the tetrahedral intermediate of the propagation step, three amino acids in the active site were changed into alanines. A kinetic study showed that two mutants had 90 fold increased activity as compared to the wild-type CALB. In a preparative synthesis of poly(D,D-lactide) the mutants greatly improved the rate and the degree of polymerization.
A
R-OH + O O O O
The recently discovered solubility of (ligno)-cellulose in some ionic liquids is exploited as an alternative pretreatment method for the conversion of wooden biomass. After dissolution, there are two alternative routes for the depolymerisation: either cellulose is precipitated to yield amorphous small cellulose particles with enhanced degradability, or cellulases are needed that work in neat ionic liquids. First, a high-throughput screen for suitable ionic liquids was established based on scattered light measurement, and a number of ionic liquids capable of dissolving cellulose were identified.[1] After precipitation, ionic liquid adheres to the cellulose particle surface. Already at 10 % (v/v) residual ionic liquid content in an aqueous system, which is realistic for unwashed precipitated cellulose, the residual activity of a commercial cellulase preparation drops to between 10 and 30 % due to the viscosity and ionic strength of the solvent.[2] However, this is compensated by up to an 18-fold increased initial reaction rate activity of cellulases toward the resulting amorphous precipitate. For full degradation of the resulting amorphous cellulose, the ratio of exo- and endo-active glucanases has to be optimised.[3] For that purpose, the above-mentioned high-throughput screening system was adapted for following the particle erosion during enzymatic cellulose degradation.[4,5] The ionic liquid pretreatment with subsequent precipitation has also been shown to be fully effective for wooden biomass. For the alternative approach of hydrolysing dissolved cellulose in ionic liquid, an endoglucanase from a thermophilic organism, S. solfataricus, was evaluated for enzymatic activity in 80 90 % (v/v) ionic liquid, namely EMIM Ac, EMIM Et2PO4, and MMIM Et2PO4. For that purpose, both viscosity reduction and sugar release in the ionic liquid were quantitatively evaluated. To our knowledge, we thus identified the first endoglucanase acting in pure ionic liquid. Summarising, we explored ionic liquids as a viable pre-treatment of ligno-cellulosic biomass and could show how the cellulolytic enzymes can be favourably used in these novel reaction systems.
CALB
Q157A, I189A, L278A
O R O O O O n O O O O H
Figure 1. A Enzymatic ring opening polymerization of D,D-lactide. B - Structure of the Q157A, I189A, L278A mutant with the tetrahedral intermediate representing the propagation step where a D,D-lactide unit is the acyl donor and benzyl D,D-dilactate is the acyl acceptor.
This work is part of the IRENE project that has received funding from the European Community's Seventh Framework Programme under the FP7-KBBE-2008-2B grant agreement n 227279.
[1] [2] [3] [4] [5]
Zavrel M, Bross D, et al. Biores. Technol., 100: 2580-2587 (2008). Engel P, Mladenov R, et al. Green Chem. 12: 1959-1966 (2010). Rinaldi R, Engel P, et al. ChemSusChem 3: 1151-1153 (2010). Jger G, Wu Z, et al. Biotechnol. Biofuels 3: 18 (2010). Jger G, Wulfhorst H, et al. Biotechnol. J. 6: 1-12 (2011).
[1] [2] [3] [4]
M. Hans, H. Keul and M. Moeller, Macromol. Biosci., 2009, 9, 239. O. Dechy-Cabaret, B. Martin-Vaca and D. Bourisso, Chem. Rev., 2004, 104, 6147. R. Auras, B. Harte and S. Selke, Macromol. Biosci., 2004, 4, 835. N. Y. Jeon, S.-J. Ko, K. Won, H.-Y. Kang, B. T. Kim, Y. S. Leea and H. Lee, Tetrahedron Lett., 2006, 47, 6517.
October 2-6, 2011
54 OC-28
Lectures
Thursday, October 6 PL-4
55
In vitro protein and metabolic engineering of a biodegradation pathway

Pavel Dvoraka, Zbynek Prokopa, Jan Brezovskya, David Bednara, Sarka Bidmanovaa and Jiri Damborskya a Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 5/A13, 625 00 Brno, Czech Republic E-mail: paveld@chemi.muni.cz Metabolic engineering has recently attracted a lot of attention in biotechnology community for its ability to create synthetic systems empowering engineered microorganisms to acquire new properties. Due to complexity of life, however, many limitations persist for detailed understanding of engineered in vivo systems. Besides engineered microorganisms, recent attempts of metabolic engineering aims also at constructing in vitro systems.[1] In vitro systems are simpler, better predictable, can be tightly controlled and do not suffer from unwanted changes caused by natural evolution. Progress in techniques of enzyme immobilization and development of appropriate analytical tools enable detailed studies of entire metabolic pathways reconstructed out of the living cells. Furthermore, development of protein engineering tools makes possible to overcome the limitations of wild-type enzymes isolated from natural sources. Combination of protein and metabolic engineering is attractive concept for both basic research and biotechnology applications. Here we demonstrate application of such approach for engineering of catabolic pathway for degradation of toxic environmental pollutant. Target compound can be converted by the five-step reaction of three enzymes to the harmless product. We decided to prove and study the multi-enzyme conversion in vitro. The genes of three enzymes were synthesized, corresponding enzymes were expressed and purified and steady-state kinetic parameters with target metabolites were determined. Multi-enzyme reaction was run in a buffer solution. Degradation of target compound and its intermediates by respective enzymes was followed by gas chromatography. Two bottlenecks of the pathway were identified: (i) poor catalytic efficiency of the first enzyme of the pathway has been improved by computer-assisted design and focused directed evolution[2], and (ii) enantioselectivity of the second enzyme of the pathway is currently being engineered by the same approach. Immobilization of optimized enzymes in cross-linked enzyme aggregates and their entrapment in polyvinyl alcohol particles is in progress. Resulting immobilized multi-enzyme biocatalyst will be used in waste sites treatment and developed concept should be applicable also to other systems.
This work was financially supported by the grants LC06010, MSM0021622412 and CZ.1.05/2.1.00/01.0001 from the Czech Ministry of Education and by the biotechnology company LentiKats.
Engineering enzyme-catalyzed unnatural reactions

Romas Kazlauskas University of Minnesota, Department of Biochemistry, Molecular Biology & Biophysics and The Biotechnology Institute, 1479 Gortner Avenue, Saint Paul, MN 55108 USA E-mail: rjk@umn.edu Although enzymes catalyze a wide range of reactions, there are many synthetically useful reactions where no enzyme catalyst exists. These enzymes do not exist because 1) biology has alternative pathways to the product, 2) the product is too reactive for biology, or 3) biology had no need for the product. Our goal is to create enzymes that catalyze new reactions by engineering existing enzymes. One example is the direct condensation of carboxylic acids and alcohols or amines to make esters or amides. This direct react is most useful to chemists, but the reaction is thermodynamically unfavorable in water, so biology uses and alternative pathway. Biology couples ester or amide formation to ATP hydrolysis to drive the reaction. Chemists can create an unnatural reaction by using a hydrolase in an organic solvent where ester formation is favorable. New types of organic solvents, called deep eutectic solvents, extend the range of these reactions to include polar substrates. A second example is perhydrolysis of carboxylic acids and esters to form peroxycarboxylic acids. These peracids are effective disinfectants because they oxidize most biomolecules. There are a few enzymes that catalyze perhydrolysis, but this ability is likely a side product of another function like lactone hydrolysis and perhydrolysis does occur under natural conditions. Since peracids are effective oxidants for organic chemistry, we have engineered hydrolases to be more effective perhydrolases. Two key strategies were distinguishing between water and hydrogen peroxide and minimizing the hydrolysis of the product peracid. A third example is the nitro-aldol addition or Henry reaction, where nitromethane adds to an aldehyde, forming a carbon-carbon bond. Hydroxynitrile lyases catalyze a similar reaction, addition of cyanide to an aldehyde, but they are poor catalysts for the nitro-aldol addition. Instead of directly engineering a hydroxynitrile lyase for this new reaction, we engineered a related esterase. This approach created a generalist enzyme that catalyzes several additions to the aldehyde including the nitro-aldol addition.
[1] [2]
C. Hold et S. Panke, J. R. Soc. Interface 6 (2009) S507-S521 M. Pavlova et al., Nat. Chem. Biol. 5 (2009) 727-733
October 2-6, 2011
56 OC-29
Lectures
Thursday, October 6 OC-30
57
How does the protein structure control the chirality of hydroxylation in mononuclear nonheme iron dioxygenases?
Sarah Prattera, Cornelia Konstantinovicsa, Cristiana di Giuroa, Grit Straganza a Institute of Biotechnology and Biochemical Engineering, Graz University of Techology, Petersgasse 12, A-8010 Graz, Austria Country E-mail: grit.straganz@tugraz.at Enzymatic mononuclear nonheme iron (II) dioxygenases (MNHEs) are key enzymes in O2 metabolism, which perform a stunning diversity of O2 dependent reactions, among the hydroxylation of non-activated carbon atoms with high stereo- and enantio-specificity.[1] These complex, highly specific reactions cannot be achieved by synthetic chemistry and are therefore potentially interesting biocatalysts. But how are these high enantiospecificities obtained? We have studied the structural features that control the chirality of hydroxylation in p-S-Hydroxy-Mandelate Synthase (HMS).[2] The impact of substrate structure on the reactivity and enantio-specificity of HMS is characterized. Our results show a remarkable impact of substrate structure on enatiospecificity. Based on these results and assisted by computational analysis, structural features in the proteins active site are identified, which account for the high enantiospecificity of HMS and subjected to mutational analysis. Based on these results a HMS variant with inverted enantio-specificity is rationally designed.
Structure and mechanism of bacterial maleate isomerase

Florian Fischa,b, Marie Delenneb, Neil C. Brucea, Gideon Groganb Department of Biology, University of York, Heslington, York, YO10 5YW U.K.; bDepartment of Chemistry, University of York, Heslington, York, YO10 5DD U.K E-mail: grogan@ysbl.york.ac.uk
a
The Aspartate-Glutamate Racemase superfamily is a group of sequence-diverse enzymes of interest to applied biocatalysis it has members that catalyse reactions including the racemisation of amino acids, hydantoins and also the stereoselective decarboxylation of aryl malonates.[1] As part of a genome-mining project designed to unearth novel members of this superfamily, a gene from Nocardia farcinica was cloned, expressed and the enzyme assayed for activity. Although the enzyme possessed neither amino acid racemase nor decarboxylase activity, it was observed to catalyse the cis-trans isomerisation of the double bond of maleate (Figure 1) to yield fumarate, and was hence termed maleate isomerase (MIase) The structure of an inactive mutant of the enzyme complexed with maleate has revealed a mechanism distinct from that of other members of the Asp-Glu racemase superfamily, in which a covalent succinyl-cysteine-like intermediate is recruited as part of the reaction coordinate.[2] (Figure 1). This is the first structural evidence for the succination of cysteine in an enzyme, and also hints at a catalytic plasticity within the superfamily that is wider than previously envisaged.
Figure 1.
Figure 1. Structural model of the product ligated HMS active site, based on the crystal structure of HMS from Amycolatopsis orientalis.[3]
[1] for review see: M. Costas, M. P.Mehn, M. P.Jensen, L.Que Jr., (2004) Chem. Rev. 104, 939-986. [2] Choroba, O.W., Williams, D. H., Spencer, J. B. (2000) J. Am. Chem. Soc., Vol. 122, pp. 5839-5390. [3] Brownlee, J., He, P., Moran, G.R. and Harrison, D.H. (2008), Biochemistry, Vol. 47, No. 7, pp. 2002-2013. [1] [2] K. Okrasa et al. (2009) Angew. Chem. Int. Ed., 41, 7691-7694. F. Fisch et al. (2010) J. Am. Chem. Soc., 132, 1145511457.
October 2-6, 2011
58 OC-31
Lectures
59
Friedel-Crafts alkylation catalyzed by methyltransferases

Mandana Gruber-Khadjawi, Harald Stecher, Martin Tengg ACIB GmbH, Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria E-mail: mandana.gruber@acib.at During the last decades biocatalysis has grown to a strong discipline in applied life sciences. A new field in biocatalysis is the enzymatic Friedel-Crafts alkylation. Two major goals are targeted here, namely a novel enzymatic C-C bond formation and the possibility of using modified cofactors for cofactor dependent enzymes. Till recently biocatalytic C-C bond formations were applied for stereoselective synthesis. The creation of chirality with high selectivity was the advantage of biocatalysts compared to chemo- and/or metal catalysts. Other selectivities of enzymes, in this case methyltransferases (MTase), are addressed for Friedel-Crafts alkylation. The S-Adenosyl-L-methionine dependent methyltransferases investigated could overcome all the disadvantages, which accompany a classical Friedel-Crafts alkylation, namely poor selectivity regarding regioisomers and multiple substitutions. The second goal was to show the ability of cofactor dependent enzymes to accept non-natural cofactors. In general cofactors are highly conserved mediators of biological processes and the cofactor-dependence of enzymes is thus highly restricted. We show that chemically modified (artificial) cofactors could successfully be applied in biocatalysis. Modified cofactors decorated with allyl, propargyl and benzyl residues were used for Friedel-Crafts alkylation and extended the formation of aromatic compounds beyond the natural methylation reaction. These enzymes are not as restricted as believed. The selective alkylation of aromatics under mild reaction conditions can be performed for the synthesis of (fine)chemicals as well as bioactive intermediates, most prominent the pharma-metabolites.
MTase HOOC NH2 NH2 N N O H OH H OH N N R R1
Chemo-enzymatic approaches towards both enatiomers of dehydroxymethylepoxyquinomicin (DHMEQ)

Takeshi Sugaia, Chihiro Hiraokab, Manabu Hamadab, Yukihiro Niitsua, Ryohei Kobayashia, Kaoruko Nakagawaraa, Takakazu Sekia, Masashi Suzukia, Yasunobu Yamashitaa, Toshinori Higashia, Mitsuru Shojia, Kazuo Umezawab a Department of Pharmaceutical Science, Keio University, Tokyo 105-8512, Japan; bDepartment of Applied Chemistry, Keio University, Yokohama 223-8522, Japan E-mail: sugai-tk@pha.keio.ac.jp DHMEQ is unique artificial molecule. (2S,3S,4S)-1a works as NF-B inhibitor while (2R,3R,4R)-1a activates Nrf2 in Figure 1, and their antiinflammatory and antitumor activities are promising. To secure (2S,3S,4S)-1a, Burkholderia cepacia lipase-catalyzed enantioselective hydrolysis was very effective as shown in Scheme 1.[1] Figure 1. Both enantiomer of DHMEQ.
R1
Scheme 1. B. cepacia lipase-catalyzed enantioselective hydrolysis of diacyl DHMEQ. This protocol was not effective for (2R,3R,4R)-1c because it decomposed during chromatographic separation. We overcame the situation by applying substrate molecular technology with a handstand substrate 2b in Scheme 2, so that the enantiomeric preference would be reversed from that in Scheme 1. The product, (1S,2S,3S)-2a in Scheme 2 was efficiently derived to (2R,3R,4R)-1a by chemical transformations.[2]
R = methyl (natural) R = allyl, propargyl, benzyl (non-natural)
SAM and analogs
Figure 1. Enzymatic Friedel-Crafts alkylation catalyzed by methyltransferases Scheme 2. Reversal of enantiomeric preference by application of handstand substrate.
H. Stecher, M. Tengg, B. J. Ueberbacher, P. Remler, H. Schwab, H. Griengl, M. Gruber-Khadjawi, Angew. Chem. Int. Ed. 2009, 48, 9546-9548. [1] [2] Tetrahedron, 2010, 66, 7083. Org. Biomol. Chem., 2011, in press.
October 2-6, 2011
60 OC-33
Lectures
61
Production, characterization and synthetic application of a purine nucleoside phosphorylase from Aeromonas hydrophila
Daniela Ubialia,d, Carla D. Serrab,d, Immacolata Serraa,d, Carlo F. Morellib,d, Giuseppe Amati,c Marco Terrenia,d, Alessandra M. Albertinic,d, Paolo Manitto*b,d, Giovanna Speranza*b,d a Dipartimento di Scienze del Farmaco, Universit degli Studi di Pavia, Italy; bDipartimento di Chimica Organica e Industriale, Universit degli Studi di Milano, Italy; cDipartimento di Genetica e Microbiologia, Universit degli Studi di Pavia, Italy; dItalian Biocatalysis Center, Pavia, Italy E-mail: daniela.ubiali@unipv.it Nucleoside phosphorylases (NPs; E.C. 2.4.2) catalyze the reversible cleavage of the glycosidic bond of (deoxy)ribonucleosides in the presence of inorganic orthophosphate (Pi) to generate the nucleobase and -D-(deoxy)ribose-1-phosphate (R-1-P) (Eq. 1).[1]
Chemoenzymatic syntheses of selected biologically active chiral heteroorganic compounds

Magorzata Kwiatkowska, Jerzy uczak, Piotr ywa and Piotr Kiebasiski Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Department of Heteroorganic Chemistry, Sienkiewicza 112, 90-363 d, Poland E-mail: piokiel@bilbo.cbmm.lodz.pl As continuation of our investigations on the use of enzymes in the synthesis of chiral non-racemic heteroorganic compounds[1], some attempts were made at the application of this methodology to prepare derivatives of desired structure. In this way, a series of enantiomerically pure tridentate ligands were prepared and used as very efficient catalysts in various reactions of the asymmetric carbon-carbon bond formation.[2-4] Recently we have focused our attention on the preparation of drugs and biologically active compounds, both natural and their synthetic analogs. In this communication the partial outcomes of our recent studies will be discussed, which will comprise chemoenzymatic syntheses of the following compounds: Both enantiomers of an analeptic drug - modafinil 1. Unknown, enantiomerically pure fluorinated analogs of the natural product - sulforaphane 2, which exhibits anticancer, antidiabetic, and antimicrobial properties. Unknown phosphoryl analogs of emeriamine (an inhibitor of fatty acid oxidation) both enantiomers of phosphoemeriamine 3.
(deoxy)ribonucleoside + Pi
NP
nucleobase + R-1-P
Eq. 1
If a second nucleobase is added to the reaction medium, the formation of a new nucleoside can result (transglycosylation). Thus, NPs can be used for chemoenzymatic synthesis of both natural and unnatural nucleosides, as an alternative to conventional chemical methods which are generally plagued by low stereoselectivity, multi-step procedures and modest yields. Starting from the results of a microbiological screening[2] which highlighted the broad specificity of Aeromonas hydrophila, also towards 6-modified purine nucleosides with antiviral and antitumor activity, we decided to investigate the potential of A. hydrophila purine phosphorylases PNPs (E.C. 2.4.2.1) as biocatalysts for practical uses. We have cloned and over-expressed one of A. hydrophila PNPs, tested its substrate specificity, and used it for synthetic applications, also at a preparative scale. To this aim, PNP encoded by deoD gene was expressed as a fusion protein (His6 tag) and recovered in high purity and concentration. Substrate specificity was firstly assayed towards natural nucleosides (according to Eq. 1) revealing that this PNP catalyzes both the phosphorolysis of 6-oxo and 6-amino purine (deoxy)ribonucleosides. Then, a library of nucleoside analogs (Figure 1) was synthesized and then submitted to enzymatic phosphorolysis as well. This assay revealed that 1-, 2-, 6- and 7-modified nucleosides are accepted as substrates, whereas 8-substituted nucleosides are not. To explore the synthetic potential of PNP, a few transglycosylations were carried out using 7-methylguanosine iodide as a D-ribose donor and 6-substituted purines as acceptor. This procedure takes advantage of both the irreversible phosphorolysis of the 7-methyl riboside[3] and the ready preparation of this nucleoside from guanosine. Following this approach, 6-chloro- and 6-methoxy-purine riboside were synthesized in quantitative yield and high purity.
Figure 1. This work was partially funded by Regione Lombardia (MD 2007, ID 4165). We thank Prof. A. Iribarren (University of Quilmes, Buenos Aires) for providing A. hydrophila strain. [1] [2] [3] M. Pugmire and S. E. Ealick, Biochem. J. 2002, 361, 1-25 J. A. Trelles et al., Biotechnol. Lett. 2005, 27, 759-63 W. J. Hennen and C. Wong, J. Org. Chem. 1989, 54, 4692-95
Ph Ph
O S 1
O NH2 RF
O S 2
NCS
O HO
O H2N H P 3
NMe3
The use of a variety of biocatalysts: whole cells and enzymes lipases, nitrilases and oxidoreductases will be presented.
Acknowledgements: Financial support by the Polish Ministry of Science and Higher Education, grant No N209 454039 (for P. K.), is gratefully acknowledged.
[1] [2] [3] [4]
Kiebasiski, P.; Mikoajczyk, M., Heteroatom-containing Compounds in Future Directions in Biocatalysis, Matsuda, T. (Ed.), Elsevier, 2007, pp. 159-203. Rachwalski, M; Kwiatkowska, M; Drabowicz, J; Kos, M.; Wieczorek, W. M.; Szyrej, M.; Siero, L.; Kiebasiski, P., Tetrahedron: Asymmetry 2008, 19, 2096-2101. Rachwalski, M.; Leniak, S.; Sznajder, E.; Kiebasiski, P., Tetrahedron: Asymmetry 2009, 20, 1547-1549. Rachwalski, M.; Leniak, S.; Kiebasiski P., Tetrahedron: Asymmetry 2010, 21, 1890-1892; Tetrahedron: Asymmetry 2010, 21, 2687-2689.
October 2-6, 2011
62 OC-35
Lectures
63
Arzetta: rapidly identifying and customizing enzyme activity

Yih-En Andrew Bana, Eric Althoffa, Daniela Grabsa and Alexandre Zanghellinia a Arzeda Corp., 2722 Eastlake Ave E. Ste 150, Seattle WA 98101 E-mail: alexandre.zanghellini@arzeda.com Computational enzyme design is a new field that, along with more established enzyme engineering techniques, holds great promise to probe both the most fundamental aspects of enzyme catalysis and to help address our most pressing societal needs. Recent successes achieved by the scientific team at Arzeda have included the design de novo of three families of around 100 new enzymes catalyzing a retro-aldol reaction[1,2], a Kemp elimination reaction[3,4] and a Diels-Alder reaction[5]. To complement this successful technique of de novo enzyme design[6], we have developed an innovative approach Enzyme Identification - that leverages computational enzyme design to rapidly engineer enzymes with known catalytic mechanisms for unnatural substrates and increasingly demanding transformations and reaction environments. Coupled with traditional enzyme optimization techniques such as directed evolution, this technique opens new avenues for enzyme discovery. Arzetta Enzyme Identification is a two-pronged approach. First, by screening in-silico the large amounts of structural and sequence data available in public databases, it allows the fast discovery of existing enzymes that possess both the necessary catalytic machinery and the substrate binding pocket to catalyze the desired reaction. Second, for those enzymes that possess the necessary catalytic machinery but lack an active site compatible with the substrate(s) of interest, Arzedas Enzyme Identification automatically redesigns and remodels the active site pocket enabling catalysis of the desired reaction. The method screens both the entire space of existing protein structures (from the Protein Data Bank) as well as homologous sequences for which no structural information is available. Using large scale cloud computing (500s to 1000s CPU cores), we are able to screen libraries on the order of 1037, as opposed to the typical 1014 (100 rounds of 1012 library size) for directed evolution within 2 to 4 weeks. Ranking our predicted candidates, we only select ~100-200 different sequences to be assayed experimentally for activity. In our lecture, we will present the developed technology and its application to various enzyme design challenges.
Standards are boring . but better data reporting would substantially increase the value of work in biocatalysis
Peter J Halling WestCHEM, Department of Pure & Applied Chemistry, Univ Strathclyde, Glasgow G1 1XL, UK E-mail: P.J.Halling@strath.ac.uk Too much published work in biocatalysis is of limited value to readers because the papers lack essential information about the conditions of the experiments or the results obtained. We have probably all experienced finding that a paper forgets to state the substrate concentration, or gives a rate with incomplete units. Recently a working group of the European Federation of Biotechnology Section on Applied Biocatalysis (ESAB) has compiled a set of guidelines for the reporting of biocatalysis experiments.[1] This presentation would:a) further publicise the guidelines and invite inputs on how they can be improved (http://www.esabweb.org/ Forum/Guidelines+for+reporting+biocatalytic+reactions.html) b) present in more detail some of the less well known issues covered in the guidelines, which are unfamiliar to some researchers in this interdisciplinary field. This will draw on examples taken from published papers. c) promote an initiative that has developed a pilot electronic tool that ensures all necessary data is recorded, initially focussed on pure enzymology (http://www.beilstein-institut.de/en/projects/ strenda/e-form/) d) present a proposal to extend a tool of this type to more complicated systems encountered in biocatalysis, and invite comments on the functionality useful. For example, the tool might be used right from the initial recording of experiments in the lab. Then it might automatically output summary information for local discussions, and later for publication. After publication, full details of the experiments could be transferred to suitable database(s) of biocatalysis results.
[1] [2] [3] [4] [5] [6]
L. Jiang and E. Althoff et al., Science, 2008, 319(5868), 1387 E. Althoff et al., submitted, 2011 D. Roethlisberger et al., Nature, 2008, 453(7192), 190 O. Khersonsky, J.Mol.Biol, 2011, 407(3), 391 J. Siegel and A. Zanghellini et al., Science, 2010, 329(5989), 309 A. Zanghellini and L. Jiang et al., Protein Science, 2006, 15, 2785 L.
[1]
L. Gardossi, P. B. Poulsen, A. Ballesteros, K. Hult, V. K. vedas, . Vasi-Raki, G. Carrea, A. Magnusson, A. Schmid, R. Wohlgemuth and P. J. Halling (2010). "Guidelines for reporting of biocatalytic reactions." Trends in Biotechnology 28(4): 171-180.
October 2-6, 2011
64 OC-37
Lectures
65
Bioinformatic analysis of function-related variable amino acid residues responsible for functional divergence of enzyme families
Dmitry Suplatova,b, Daria Shalaevaa, Vladimir Arzhanika, Evgeny Kirilina, Vytas vedasa,b a Faculty of Bioengineering and Bioinformatics and bBelozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Vorobjev hills 1-73, Moscow 119991, Russia E-mail: d.a.suplatov@belozersky.msu.ru Enzymes within single family usually share a common function but differ in more specific features and can be divided into subfamilies with different specificity, enantioselectivity, stability, etc. New bioinformatic analysis methodology has been developed to identify function-related variable residues in protein structures that are responsible for functional divergence within families of homologous enzymes. We suggest to use a term subfamily-specific position(s) or SSP(s) to outline those residues to be conserved within subfamilies of enzymes, but different between subfamilies:
The Laccase Engineering Database (LccED) as a tool for understanding the classification of actinobacterial laccases
Marilize Le Roes-Hilla, Nuraan Khana, Demet Sirimb, Jrgen Pleissb, Stephanie Burtona a Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa; bInstitute for Technical Biochemsitry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany E-mail: leroesm@cput.ac.za The Laccase Engineering Database (LccED) was developed with the aim to understand the classification and distribution of laccases and related multicopper oxidases found in nature.[1] Multicopper oxidases consist out of a large group of enzymes including, but not limited to, bilirubin oxidases, laccases, ascorbate oxidases, ferroxidases and ceruloplasmin. These enzymes play important roles in nature, varying from lignin depolymerization, pathogenicity, antibiotic production to pigmentation. The LccED allows for the classification of these oxidases into 11 superfamilies which can further be grouped into 56 homologous families, all based on the protein sequence and structure information currently available. Using this database as a tool, all actinobacterial multicopper oxidases were analysed for their distribution among the different families and their potential inferred function in the producing strains. Protein sequence alignments and phylogenetic studies confirmed the distribution of actinobacterial MCOs among these families and presented a means of distinguishing between two major types of enzymes often considered to be actinobacterial laccases: phenoxazinone synthases[2] and small laccase (SLAC) homologues. Degenerate primers were specifically designed for the different actinobacterial sequence groupings using CODEHOP[3] and used as a screening tool to search for novel phenoxazinone synthase and SLAC homologue sequences in our actinobacterial culture collection. The results obtained allowed for a new classification of actinobacterial laccases based on protein sequence information.
Specificity scores are ranked taking into account structural information alongside with sequences, hits are chosen based on assigned statistical significance. Bioinformatic analysis of alpha-beta hydrolase enzyme superfamily was performed. Multiple structure-guided sequence alignment was created based on 238 alphabeta hydrolase PDB structures. Bioinformatic analysis revealed SSPs responsible for discrimination between lipase-amidase activities and esterase-hydroxynitrile lyase activities within alpha-beta hydrolase fold. Common structural organization of totally conserved positions of catalytic residues and oxyanion holes was observed among serine carboxypeptidase, lipase B and hydroxynitrile lyase amid significant difference of functional properties and ability to catalyze distinct chemical transformations. Developed methodology was applied also to study evolution of structure-functional relationship in other enzyme families: Ntn-hydrolases, penicillin-binding proteins, etc. It was shown, that patterns of SSPs can be effectively used to design enzyme mutants with improved catalytic properties and to predict functional properties of newly discovered enzymes.
This work was supported by the European Commission (grant agreement n 227279) and the Russian Ministry for Science and Education (contract n. # 02.527.11.0001) as a joint EC-Russian FP7-KBBE-2008-2B project IRENE. [1] [2] [3] Sirim, D., Wagner, F., Wang, L., Pleiss, J. (2011). The laccase engineering database: a classification and analysis system for laccases and related multicopper oxidases. Accepted for publication in Database. Le Roes-Hill, M., Goodwin, C., Burton, S. (2009). Phenoxazinone synthase: whats in a name? Trends in Biotechnology 27:248-258. Rose, T.M., Henikoff, J.G., Henikoff, S. (2003). CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic Acids Research 31:3763-3766.
October 2-6, 2011
66 OC-39
Lectures
67
Progress on Amoxicillin synthesis by selecting the best acylase, expressing it in Pichia pastoris and immobilising it on ReliZyme
Ulrich Giesecke1, Ansgar Stratmann2, Enrico Mercalli3 1 CEO, W42 Consulting GmbH, Sdstrasse 19, 82377 Penzberg, Germany; 2CEO, W42 Industrial Biotechnology GmbH, Science to Business Center, Lipperweg 192, 45772 Marl, German; 3Technical & Marketing Manager, Resindion S.r.l., Via Roma 55, 20082 Binasco, Italy E-mail: ulrich.giesecke.w42@t-online.de Amoxicillin is enzymatically synthesised from 6-aminopenicillinic acid (6-APA) and p-hydroxy-phenylglycine (HPH) in activated form e.g. as methyl- (HPM) or hydroxyethylester (HPG). The conversion is influenced by the concentrations of educts, the activation of the side chain, pH and temperature, but also the enzyme source, which affects the ratio of -lactam synthesis to hydrolysis of activated side chain and by that yield. Good enzymes show less inhibition by phenylacetic acid, which is a by-product of penicillin G hydrolysis to 6-APA. For evaluation Pichia pastoris strains are constructed for expressing several wild-type or evolved penicillin G amidases (PGA) from different organisms. As genes are stable integrated in the Pichia genome, no resistance marker is required during fermentation. W42s advanced Pichia expression platform ensures high titres. Furthermore the use of synthetic media simplifies isolation. The co-operation with Resindion/Mitsubishi Chemicals provides access to newest developments on resins for protein or metabolite purification and in particular on carriers for enzyme immobilisation. The well introduced Sepabeads EC and the recently launched ReliZyme are offered in different particle size, porosity and functionality for meeting the requirements of the enzyme and the application. Amoxicillin precipitates during synthesis, which causes a special challenge in separating it from the biocatalyst. The problem is addressed by larger carrier size and the corresponding filter screen, which allows retaining the biocatalyst, while the dense product slurry passes through the filter without blocking. The resulting increased diffusion limitation is partly balanced by larger pore size. As best carrier selected is a prototype of ReliZyme EP113/MA.
How to transform a good enzyme in an efficient biocatalyst: formulation and application of biocatalysts for the bio-based chemistry
Loris Sinigoia, Diana Fattorb, Patrizia Spizzoa, Alessandra Bassoa, Paolo Braiucaa, Lucia Gardossia,b a SPRIN S.p.A., Technologies for Sustainable Chemistry, c/o BIC Incubatori FVG, via Flavia 23/1, 34148 Trieste, Italy. b Dipartimento di Scienze Chimiche e Farmaceutiche, Universit degli Studi di Trieste, Piazzale Europa 1, 34127 Trieste, Italy. E-mail: sinigoi@sprintechnologies.com Nowadays, biocatalysis using immobilized enzymes has achieved a position of steadily increasing importance for the biotechnological production of food additives, agrochemicals, cosmetics, flavors, and, in particular, for pharmaceuticals. The increasing market for these compounds has resulted in a growing demand to identify enzymes with novel and specifics properties. Although a wide number of enzymes have been discovered and/or engineered, there is still a gap between their large availability at lab level and their full exploitation in industrial processes. The presentation will point up how a biocatalyzed processes at industrial scale can be accelerated by a multidisciplinary approach that combines the extraction of information from data and rationalization of the experimental practice, cutting time and costs. Examples of rational approach to the design of biocatalysed processes for the for industrial conversion of oils, fats, carbohydrates and other key compounds will be presented. Specific focus will be given to enzyme screening, design of immobilized enzyme, process development, reactor configuration, scale up and regulatory aspects. This comprehensive approach will be discussed in the perspective of a more rational and effective exploitation of technological and scientific advances in the field of Industrial Biotechnology, which is a pre-requisite for overcoming the long time-to-market for biocatalysis products and making them competitive with highly optimized chemical processes.
[1] [2] [3] [4] [5] Biotrans 2011 - Italy
Hanefeld U., Gardossi L., Magner E., Understanding Enzyme Immobilisation, Chem. Soc. Rev., 2009, 38, 453. Eriksson M., Boyer A., Sinigoi L., Johansson M., Malmstrom E., Hult K., Trey S., Martinelle M., One-Pot Enzymatic Route to Tetraallyl Ether Functional Oligoesters: Synthesis, UV Curing, and Characterization, J. Polym. Sci., Part A: Polym. Chem.,2010, 48, 5289. Ricca E., Calabr V., Curcio S., Iorio G., Gardossi L., Basso A., Fructose production by inulinase covalently immobilized on Sepabeads in batch and fluidized bed bioreactor, Int. J. Mol. Sci.,2010, 11, 1180. Basso A., Spizzo P., Ferrario V., Knapic L., Savko N., Braiuca P., Ebert C., Ricca E., Calabr V., Gardossi L., Endo and exoinulinases: enzyme-substrate interaction and rational immobilisation, Biotechnol. Progr., 2010, 26, 397. Braiuca P., Knapic L., Ferrario V., Ebert C., Gardossi L., A 3D-QSAR Model for Predicting the Enantioselectivity of Candida antarctica Lipase B, Adv. Synth. Catal., 2009, 351, 1293.
October 2-6, 2011
68 OC-41
Lectures
Thursday, October 6 IL-10
69
Integration of biotransformation and SMB separation for the high-yield production of fine chemicals
M.Bechtold, M.Freder, N. Wagner, S.Panke Bioprocess Laboratory, ETH Zurich, Switzerland E-mail: matthias.bechtold@bsse.ethz.ch Despite the unique ability of enzymes to catalyze reactions to difficult-to-synthesize molecules with almost absolute stereoselectivity, application on industrial scale is often prevented by constraints like inhibition, a readily degrading product and an unfavorable position of the thermodynamic equilibrium. Integration of product formation and separation (also referred to as in-situ product removal (ISPR)) constitutes an efficient solution for such constrained biotransformations. ISPR should enable rapid selective product recovery and transfer of product to an auxiliary phase and thus provides control of the products residence time and concentration in the reaction space.[1] As integration of separation and reaction limits the available phyisco-chemical space for separation methods, and due to the frequently large similarity between substrate and product in biotransformations an exceptionally powerful separation strategy is required. Chromatography based on aqueous eluents is in fact a promising option for the separation step. Here, we demonstrate the benefit of integrating biotransformations with continuous chromatography realized as simulated moving bed (SMB) technology - for thermodynamically limited reactions, in particular isomerisations and C-C bond formations. Such reactions are intrinsically equilibrium-limited, but a large variety of interesting molecules such as unusual saccharides or sialic acids can be potentially obtained that are rather difficult to produce by organic chemistry. Using representative isomerisations kinetics and SMB data from literature in model-based simulations of such an integrated process (Fig.) highly competitive productivities well above 100 g L-1 (operating volume of SMB and EMR) day-1 with chemical yields of >95% are obtained.[2] In practice, we explore the potential of SMB-based ISPR for the threonine-aldolase catalyzed synthesis of L-allothreonine from glycine and acetaldehyde and the D-tagatose epimerase catalyzed production of psicose from fructose or glucose. Cheap stationary phase materials were identified for both separation problems with little screening effort that indeed allowed for efficient SMB separation using aqueous solutions at ambient pH. The full process was successfully implemented over several days for psicose production (see Fig.) that was obtained in >99.5 % purity with respect to the substrate.[3] Efficient operation of such a process with several unit operations requires model-based design tools based on Continuous integrated operation of enzyme membrane reactor (EMR), SMB and nanofiltration (NF) . a comprehensive characterization of the involved elements, The reaction mixture formed in the EMR is fed to the from enzyme kinetics and stability to adsorption isotherms. SMB for substrate-product separation. Unconverted Therefore model-based experimental analysis procedures substrate is recycled to the reactor after balancing the [4] inherent dilution of the SMB by a nanofiltration con- amenable to high-throughput were developed.
centration step.
A green process for Boceprevir-T based on amine oxidase mediated desymmetrization

Tao Lia, Alexey Zaksb, Azzeddine Lekhala, Tim Brennanc, Alexandre Ambrogellya, Guy Gloord, and George Wonga a Merck & Co., Inc. 126 E. Lincoln Ave. Rahway NJ 07065, USA; bAkermin, Inc. 4633 World Parkway Circle, St. Louis, MO 63134, USA; cHospira, Lake Forest, IL 60045, USA; d Chesilton, Colts Neck, NJ 07722,USA E-mail: tao.li@merck.com A practical, highly efficient method was invented for asymmetric oxidation of 6,6-dimethyl-3-azabicyclo[3.1.0]hexane. This reaction involves an amine oxidase mediated desymmetrization, and a concurrent bisulfite trapping to give the sulfonate adduct. Based on this reaction, a process was developed to prepare Boceprevir T. This process has been scaled up to 500 kg production. The production cost has been substantially reduced due to significant improvement in yield as well as green chemistry performance.
Amine oxidase N H N O2 H2O2 Catalase H2O + 1/2 O2
NaHSO3 N H SO3COOH N H HCl 60% yield > 99% ee Boceprevir-T
Sulfonic acid
[1] [2] [3] [4]
M. Bechtold, S. Panke, Chimia 63 (2009) 345. M. Bechtold, et al., Chem. Ing. Tech. 82 (2010) 65. N. Wagner, et al., Org. Process Res. Dev. submitted. M. Bechtold, et al., Biotechnol. Bioeng. 98 (2007) 812.
[1] [2]
Carr, R.; Alexeeva, M.; Dawson, M. J.; Gotor-Fernandez, V.; Humphrey, C. E.; Turner, N. J. ChemBioChem.2005, 6(4), 637-639. Sablin, S. O.; Yankovskaya,V.; Bernard, S.; Cronin, C. N.; Singer, T. P. Eur. J. Biochem. 1998, 253(1), 270-279.
October 2-6, 2011
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Cost CM0701 & Cascade Chemo Enzymatic processes 1

New substances for effective electron transfer in bioconversion reactions catalyzed by oxidoreductases
Julija Razumienea, Vidute Gurevicienea, Rolandas Meskysa, Valdemaras Razumasa a VilniusUniversity Institute of Biochemistry, Moksliniku str. 12, Vilnius LT-08662, Lithuania E-mail: julija.razumiene@bchi.vu.lt The implementation of industrially promising biocatalysts especially oxidoreductases tackles a problem of cofactor regeneration or availability of an efficient redox mediator. A greet number of electron donors or acceptors are known and used in chemical technologies however there still is a lack of cheap but efficient redox mediators applicable for enzyme catalyzed redox reactions. Bioelectrosynthesis requires mediating systems both in oxidized and in reduced forms which are stable during multiple rounds of bioelectrocatalytic reactions. In this work new materials demonstrating appropriate redox properties and able to guaranty effective electron exchange between the enzyme and the electrode have been synthesized. In parallel with experimental approach, quantum chemical calculations have been applied for the evaluation of molecular properties (structure, energy, etc.) of mediating materials acting in bioelectroconversion systems. Fungal laccase from Coriolopsis byrsina has been employed to couple primary and secondary amines with hydroquinone. Laccase-catalyzed amination represents an efficient method for the synthesis of biologically important C-N bonds and allows the use of mild reaction conditions, aqueous solvent system, room temperature and normal pressure. The use of laccases offers an opportunity to synthesize various substituted quinones, potential redox mediators for bioelectrocatalytic processes. Bioelectrocatalytic systems using the synthesized electron transfer mediators have been investigated on a base of glucose oxidation reaction catalysed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH). This type of bioelectrodes exhibited very high sensitivity due to synergy of electron acceptors conversion. The efficiency of biocatalytic systems as a factor of the electron transfer rate between the active site of enzyme and electrode surface via redox conversions of mediator varied from 0.08(0.01)105 A M-1 of the enzyme active centres for 2-[(2-aminophenyl)disulfanyl]aniline to 2.29(0.17) 05 A M-1 for 2-(Nmethylanilino)-1,4-benzoquinone, that was higher than efficiency of biocatalysis based on PQQ-GDH and various mediators DCPIP, PMS, metal complexes and ferrocene derivatives. The action of the bioelectrodes was characterized by experimental kinetic analysis and quantum chemical calculations using density functional theory with Beckes threeparameter exchange hybrid functional combined with the Lee-Yang-Parrs correlation functional (B3LYP). In the calculations, the high level basis set 6-311++G** was used.
________________ [1] Razumiene, J. et al., 2005, Talanta 67, 783790. [2] Tetianec, L. et al., 2011, Appl. Biochem. Biotechnol. 163, 404414.
71 2
Synthesis of quinoid compounds catalyzed by fungal laccase
Daiva Taurait, Marius Morknas, Rolandas Mekys Institute of Biochemistry, Vilnius University, Mokslinink 12, LT-08662 Vilnius, Lithuania E-mail: daiva.tauraite@bchi.vu.lt Laccases are copper-containing phenol oxidases, widely distributed in plants and fungi. High stability of these enzymes in solution, the mild reaction conditions used in laccasecatalyzed reactions and their selectivity for phenolic structures make laccases attractive for chemical synthesis. Laccases is of great interest in the enzyme-catalyzed production of new biologically active compounds via phenolic oxidation, phenolic oxidative coupling and oxidation coupled with nuclear amination [1-3]. Recently, biotransformation using laccase has been applied to synthesize novel penicilins and cephalosporins [4]. The synthesis of potentially new antibiotic agents is an important topic in chemical and medical research. In the present study we have employed fungal laccase from Coriolopsis byrsina (this new laccase was isolated in the Institute of Biochemistry) to couple various primary and secondary amines with hydroxybenzenes and hydroxypyridines. Laccase-catalyzed amination represent an efficient method for the construction of biologically important C-N bonds and allows for the use of mild reaction conditions, aqueous solvent system, no toxic reaction solvents, room temperature and normal pressure.
References: [1] Mikolasch A., Schauer F. Appl. Microbiol. Biotechnol. 2009, 82, 605. [2] Witayakran S., Zettili A., Ragauskas A. Tetrahedron Lett. 2007, 48, 2983. [3] Wellington K.W, Steenkamp P., Brady D. Bioorg. Med. Chem. 2010, 18 1406. [4] Mikolasch A., Niedermeyer T.H.J., Lalk M., Witt S., Seefeldt S., Hammer E., Schauer F., Gesell M., Hessel S., Julich W.D., Lindequist U. Chem. Pharm. Bull. 2007, 55, 412.
POSTER SESSION
1. Cost CM0701 & Cascade Chemo Enzymatic processes 2. Oxydative biocatalysis 3. Biotransformation in organic synthesis 4. Discovery and design of new biocatalysts 5. Enzyme structure & mechanism, bioinformatics & modelling 6. New applications of biocatalysis 7. Industrial processes research & development 8. BIONOCO & THDP dependent enzymes p 71 p 85 p 96 p 124 p 145 p 158 p 172 p 178
3
Baeyer-Villiger monooxygenases in aroma compound synthesis
Michael J. Fink Vienna University of Technology, Getreidemarkt 9 / 163, A-1060 Vienna, Austria E-mail: mfink@ioc.tuwien.ac.at Chiral lactones represent a major structural class of flavor and fragrance compounds and in this function they mostly exhibit enantiodivergent behavior in terms of odor thresholds and properties. Consequently a stereoselective synthesis of such compounds is of great importance.[1] In this study we exploited for the first time the well-established and efficient biocatalytic toolbox of Baeyer-Villiger monooxygenases (BVMOs)[2] for the synthesis of chiral lactones as flavor and fragrance compounds. First, we will present a facile protocol for the preparation of Aerangis lactone 3 combining continuous flow chemistry with biocatalysis. (S,S)-3, main component of the scent of African white-flowering orchids, was synthesized in a single operation reaction cascade starting from cheap dihydrojasmone 1:
4
Saccharomyces cerevisiae in directed evolution: an efficient tool to improve enzymes
Eva Garcia-Ruiz, Diana Mate, David Gonzalez-Perez, Alina Roman, Patricia Molina, Pamela Torres, Miren Zumarraga, Susana Camarero, Antonio Ballesteros, Francisco J. Plou & Miguel Alcalde. a Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain; bCentro de Investigaciones Biolgicas, CSIC, 28040 Madrid, Spain E-mail: malcalde@icp.csic.es. For most of us, the budding yeast Saccharomyces cerevisiae represents an efficient tool in the directed evolution scenario. The eukaryotic machinery of S. cerevisiae offers an array of possibilities to construct mutant libraries or to recombine (shuffle) DNA fragments. Unlike other heterologous host used for directed evolution, the high frequency of homologous recombinations in S. cerevisiae favors its use to clone eukaryotic proteins and in new in vivo protocols aimed at generating diversity. This communication summarizes our experience working with yeast for directed enzyme evolution. Examples of fungal oxidoreductases (laccases and peroxidases) engineered by in vitro evolution in our lab using S. cerevisiae as host are provided. Different biomolecular tools based on S. cerevisiae physiology for the generation of diversity are described (IvAM, IVOE, in vivo DNA shuffling). __________________
Figure 1 Exemplary process for the synthesis of (S,S)-3 in a flow reactor-biotransformation cascade
By fine-tuning the catalytic hydrogenation of 1 in a continuous flow reactor to high diastereoselectivity and high product titer in a hydrophobic solvent it was possible to directly introduce the reactor efflux into the biotransformation vessel. The subsequent BaeyerVilliger oxidation produces the naturally occurring (S,S)-3 with perfect selectivity within 25 min resulting in a calculated space-time yield of > 400 g per liter and day.
Figure 2 Kinetic resolution towards -cis-Jasmine lactone (R)-5 with excellent stereoselectivity
Secondly, four Jasmine lactones, constituents of the natural odor of Jasminium sp. were prepared by kinetic resolution of the racemic ketone precursors with excellent enantioselectivity.[3] Preliminary organoleptic evaluation shows significant differences in odor perception and threshold between racemic and optically pure samples. In summary we conclude that the chosen examples represent a strong demonstration of the proficiency of BVMOs for applications in aroma compound synthesis. _______________________
[1] E. Brenna, C. Fuganti, S. Serra, Tetrahedron: Asymmetry 2003, 14, 1-42. [2] G. de Gonzalo, M. D. Mihovilovic, M. W. Fraaije, ChemBioChem 2010, 11, 2208-2231. [3] Fink, M.J.; Rudroff, F.; Mihovilovic, M.D.; submitted.
1.-Mate, D., Garca-Ruiz, E., Camarero, S. and Alcalde, M. (2011). Directed Evolution of Fungal Laccases. Current Genomics, 12: 113-122. 2.-Alcalde, M. (2010). Mutagenesis protocols in Saccharomyces cerevisiae by in vivo overlap extension. In: In vitro mutagenesis Protocols, 3rd ed. Methods in Molecular Biology 634. (Bramman J. ed) Totowa, New Jersey: Springer-Humana Press. 3-15. ISBN: 978-1-60761-651-1. 3.-Mate, D., Garca-Burgos, C., Garca, E., Ballesteros, A., Camarero, S. and Alcalde, M. (2010). Laboratory evolution of high redox potential laccases. Chemistry & Biology, 17: 1030-1041. 4.-Garca, E., Mat, D., Ballesteros, A., Martnez, A.T. and Alcalde, M. (2010). Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast. Microbial Cell Factories, 9: 17. 5.-Zumarraga, M., Camarero, S., Martinez-Arias, A., Ballesteros, A., Plou, F.J. and Alcalde, M. (2008). Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra. Proteins: Structure, function and bioinformatics, 71: 250-260. 6.-Zumarraga, M., Bulter, T., Shleev, S., Polaina, J., Plou, F.J., Ballesteros, A. and Alcalde, M. (2007). In vitro evolution of a fungal laccase in high concentrations of organic cosolvents. Chemistry & Biology, 14: 1052-1064. 7.-Alcalde, M., Zumarraga, M., Polaina, J., Ballesteros, A. and Plou, F.J. (2006). Combinatorial saturation mutagenesis by in vivo overlap extension for the engineering of fungal laccases. Combinatorial Chemistry and High-throughput Screening, 9: 719-727. 8.-Bulter, T., Alcalde, M., Sieber, V., Meinhold, P., Schlachtbauer, C., and F.H. Arnold. (2003). Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Applied Environmental Microbiology, 69: 987-995.
October 2-6, 2011
72 5
Apo-avoprotein preparation and reconstitution
Stefano Gandolfi, Anita Larovere, Gianluca Ottolina Istituto di Chimica del Riconoscimento Molecolare CNR Via Mario Bianco 9, 20131 Milano, Italy E-mail: gianluca.ottolina@icrm.cnr.it The routing of electron from redox enzymes and direct contact of enzyme redox center with electrodes are one of the main goals in electroenzymatic processes. Reconstitution of enzymes or proteins with semi artificial cofactors yields electrically contacted enzymes electrodes. Flavoenzymes catalyze a wide range of biochemical reactions. They are involved in the dehydrogenation of a variety of metabolites, in one- and two-electron transfer from and to redox centres, in light emission, in the activation of oxygen for oxidation and hydroxylation reactions. Catalysis by flavoprotein involves a reductive half-reaction, where the enzyme-bound flavin is reduced, and an oxidative half-reaction, where the reduced flavin is re-oxidized. The catalysis involve the consumption of NAD(P)H or the production of hydrogen peroxyde such as with monooxygenases or oxidases, recently some flavoenzymes were coupled to an electric reductions [1,2]. We focus on the synthesis of FAD derivatives at N6 position, preparation of apo-flavoenzymes and subsequent reconstitution with modified FAD to recovery the holo-enzymes. Some preliminary data on the effect of the FAD modification with phenylacetone monooxygenase (PAMO) from Thermobifida fusca and glucose oxidase (GOx) from Aspergillus niger are reported.

Encapsulation of Baeyer-Villiger monooxygenases in polyelectrolyte complex capsules
Marek Bukoa, Andrea Schenkmayerova, Peter Gemeinera, Alica Vikartovska, Marko D. Mihovilovib, Igor Lackc a Institute of Chemistry, Slovak Academy of Sciences, Dbravsk cesta 9, SK-845 38 Bratislava, Slovakia; bInstitute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, A-1060 Vienna, Austria; cPolymer Institute, Slovak Academy of Sciences, Dbravsk cesta 9, SK-845 38 Bratislava, Slovakia E-mail: Marek.Bucko@savba.sk Baeyer-Villiger biooxidations catalysed by Baeyer-Villiger monooxygenases (BVMOs, EC 1.14.13.xx) are attractive for industrial biotechnology processes since they enable biocatalytic production of chiral precursors for subsequent syntheses of bioactive compounds [1]. BVMOs cyclopentanone monooxygenase from Comamonas sp. NCIMB 9872 (CPMO, EC 1.14.13.16) and cyclohexanone monooxygenase (CHMO, EC 1.14.13.22) from Acinetobacter calcoaceticus NCIMB 9871, both overproduced in recombinant Escherichia coli cells, are intensively exploited due to their remarkable substrate profile and stereoselectivity [2]. Controlled encapsulation of cells in polyelectrolyte complex (PEC) capsules made by reaction of oppositely charged biopolymers using an air-stripping nozzle and multiloop reactor [1,2] provides an universal immobilization technique for BVMOs. Encapsulation of E. coli overexpressing CPMO preserved the viability of the cells during encapsulation process for effective Baeyer-Villiger biooxidation of synthetic 8-oxabicyclo[3.2.1] oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one with high enantioselectivity and improved the enzyme storage stability [3]. Encapsulated E. coli with CHMO tested in newly developed continuous reactor connected with flow calorimeter and integrated with bubble-free oxygenation [4], exhibited high operational stability within 14 repeated continuous Baeyer-Villiger biooxidations of model ketone rac-bicyclo[3.2.0]hept-2-en6-one to (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0] oct-6-en-3-one, and showed high enzyme stability during 91 days of storage. Importantly, continuous mode of the latter Baeyer-Villiger biooxidation appears to be an appropriate configuration with convenient mechanical conditions for optimal performance of CHMOPEC capsules. Additionally, an original approach for determination of kinetic properties of Baeyer-Villiger biooxidations by flow calorimetry was introduced. It is evident that investigation of the whole-cell CHMO encapsulated in PEC capsules and used in the form of continuous packed-bed minireactor is an easy-to-use tool for further development of immobilized BVMOs.
________________ [1] Mihovilovic, M.D. Curr. Org. Chem. 10 (2006) 1265-1287. [2] de Gonzalo, G. et al. ChemBioChem 11 (2010) 2208-2231. [3] Huck, M., Buko, M. et al. Biotechnol. Lett. 32 (2010) 675-680. [4] Buko, M. et al. Enzyme Microb. Technol (2011), doi:10.1016/j.enzmictec.2011.05.013 Acknowledgements. This work was supported by the Slovak Grant Agency for Science VEGA 1/0335/10, the COST Action CM0701 and the Slovak Research and Development Agency under the contract APVV51-033205.

Synthesis of Enantiomerically Enriched Dimers of Vinylphenols by Tandem Action of Laccases and Lipases
a
73 10
Exploitation of a new laccase/mediator system for NAD(P)+ cofactor regeneration in dehydrogenase-catalyzed oxidations
Daniela Montia, Erica E. Ferrandia, Ilabahen A. Patelb, Sergio Rivaa, Dietmar Haltrichb, Roland Ludwigb aICRM-CNR, via Mario Bianco 9, 20131 Milano, Italy; bUniv Nat Resources & Appl Life Sci, Dept Food Sci & Technol, Muthgasse 11, A-1190 Vienna, Austria E-mail: daniela.monti@icrm.cnr.it To be economically sustainable, biocatalytic oxido-reductions catalyzed by NAD(P)Hdependent dehydrogenases must be coupled to efficient cofactor regeneration systems. A new regeneration approach for NAD(P)+ cofactors using a laccase/ABTS system and molecular oxygen as terminal electron acceptor, has been recently suggested [1]. Other chemical mediators, such as quinones, have been used with laccases for the oxidative regeneration of flavine dependent oxidases [2]. Meldolas blue has been widely used for the electrochemical regeneration of nicotinamide cofactors [3]. In this work we investigated the possibility of using this dye as a mediator in laccase-promoted NAD(P)+ regeneration (Figure 1). As a model reaction, the g-scale oxidations of cholic acid or its methyl ester to the corresponding 7-keto derivatives catalyzed by the NADH-dependent 7-hydroxysteroid dehydrogenase (7-HSDH) from Bacteroides fragilis were carried out in aqueous solution and in a biphasic system, respectively. NAD+ was used in catalytic amounts and in situ regenerated by the Meldolas blue / Trametes pubescens laccase system. Reactions were performed in a batch reactor up to 1 L total volume.
P. Gavezzotti,a C. Navarra,a S. Caufin,a B. Danieli,b P. Magrone,a D. Monti,a S. Riva.a Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy; E-mail: paolo.gavezzotti@gmail.com. b Dipartimento di Chimica Organica e Industriale, Universit di Milano, Via Venezian 21, 29133 Milano, Italy. The tandem use of laccases and lipases has been exploited for the simple preparative synthesis of enantiomerically enriched dimeric phenols. Laccase-catalyzed oxidation of isoeugenol (1) and vinyl guaiacol (2) in biphasic systems gave as main products the racemic compounds (3) and (4), possessing structures similar to the -5 dimers found in lignin. The synthesis of enantiomerically enriched (3) and (4) could be obtained by alcoholysis reactions catalyzed by commercial preparations of lipases in organic solvents. Albeit the E-values were quite low (due to the remote stereocenters to be discriminated), the enantiocomplementary behavior of the tested lipases allowed the simple isolation of the target compounds with e.e. up to 90 %. It is noteworthy that these results have been achieved in vitro exploiting commercially available enzymes, without using the so-called dirigent proteins evolved by nature to direct the enantioselective coupling of phenols in vivo.
________________ [1] Ruinatscha, R, C Dusny, K Buehler, A Schmid. Adv. Synth. Catal. 2009 351 2505. [2] Xiao, Y, F Patolsky, E Katz, JF Hainfeld, I Willner. Science 2003 299 1877.
Figure 1. Meldolas blue/laccase regeneration system.

________________ [1] Aksu S. et al. (2009) Adv. Synth. Catal. 351:1211-1216 [2] Van Hecke et al. (2009) Biores. Technol. 100:5566-5573 [3] Gorton et al. (2001) J. Electroanal. Chem. 161:103-1220
______________ [1] P. Gavezzotti, C. Navarra, S. Caufin, B. Danieli, P. Magrone, D. Monti, S. Riva, submitted to Adv. Synth. Catal
7
Transglycosylation with Fungal -N-Acetylhexosaminidase Yields Charged Immunoactive Glycosides
Kristna Slmova, Pavla Bojarova, Karel Keneka, Radek Gaka, Sergio Rivab, Karel Bezoukac and Vladimr Kena a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vdesk 1083, CZ-142 20 Praha 4, Czech Republic; bIstituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, I-20131 Milano, Italy; cDepartment of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-128 40 Praha 2, Czech Republic E-mail: slamova.kristyna@gmail.com -N-Acetylhexosaminidases (EC 3.2.1.52, CAZy GH 20) have recently gained a lot of attention, not only due to their implication in human physiology and disease, but also due to their great potential in the enzymatic synthesis of carbohydrates and glycomimetics. Here, we present a screening of a set of C-6 modified derivatives of pNP-GlcNAc as potential substrates of a library of 24 fungal -N-acetylhexosaminidases. -NAcetylhexosaminidase isolated from the filamentous fungus Talaromyces flavus exhibited extraordinary tolerance to N-acetylglucosaminides bearing various oxidized or charged moieties (aldehyde 1, carboxylate 2, sulphate 3, phosphate 4) in the substrate pyranose C-6 position both in hydrolytic and transglycosylation reaction modes. Oligosaccharides carrying a negatively charged moiety are strong ligands of the activation receptors of natural killer cells, particularly of CD69 protein, which results in immunostimulation in vivo [1]. Transglycosylation reactions catalyzed by -N-acetylhexosaminidase from T. flavus with substrates 1, 2 and 3 afforded four novel disaccharides bearing either carboxylate or sulphate moiety. The products were isolated and characterized and their affinity towards the NK cell activating receptors, and thus their immunostimulating potential, was assessed.
8
Identification and characterization of a novel uronate dehydrogenase for the production of hexaric acids
Martina Andberga, Harry Boera, Tarja Parkkinenb, Hannu Maaheimoa, Peter Richarda, Juha Rouvinenb, Anu Koivulaa, Merja Penttila a VTT Technical Research Centre of Finland, P.O. Box 1000, FI-02044 VTT, Finland; b Department of Chemistry, University of Eastern Finland, P.O.Box 111, FI-80101 Joensuu, Finland E-mail: martina.andberg@vtt.fi D-Galacturonic acid is the main component of pectin and an important carbon source for microorganisms living on decaying plant material. Pectin is likely to become important in biotechnology since pectin-rich cheap raw materials can be converted to fuels and chemicals. At least three microbial pathways for D-galacturonic acid catabolism have been described [1]. In the oxidative pathway, described for some prokaryotic species, Dgalacturonic acid is first oxidized to meso-galactaric acid and then in the following steps to -ketoglutarate. In this work the D-galacturonic acid dehydrogenase from Agrobacterium tumefaciens was purified and the corresponding gene, udh, identified. It codes for a protein with 267 amino acids that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily which are NAD(P)-dependent enzymes. The udh gene was functionally expressed in Saccharomyces cerevisiae and a His-tagged version of the protein was purified and used for characterization and structure determination [2]. Agrobacterium tumefaciens uronate dehydrogenase (Udh, EC 1.1.1.203) is specific for NAD+ as cofactor, and accepts D-galacturonic acid and D-glucuronic acid as substrates with similar affinities. The crystal structure of an apo form of Udh, and a complex structure with NADH and product (substrate soaked) as well as the 3D structure of an inactive mutant in complex with NAD+, have been determined recently [3]. This structure suggested Udh to exist as a homo-hexamer, which was also the major form in solution. Furthermore, the structural data showed that Udh accepts the -pyranosic form of D-galacturonic acid as a substrate, and the reaction product is D-galactaro-1,5-lactone (6-membered ring) bound in the complex structure above the nicotinamide ring. The product rearranged non-enzymatically to D-galactaro-1,4-lactone (5-membered ring), which is the product observed in vitro by NMR analysis of the reaction catalysed by Udh. The udh gene has also been introduced into engineered fungal strains with modified metabolic pathways for the conversion of D-galacturonic acid into galactaric acid. In these strains the D-galacturonic acid reductase was disrupted and the expressed udh gene expressed converts D-galacturonic acid to galactaric acid, which has applications in food, cosmetics, and pharmaceuticals and as a platform chemical [4]. This work has been carried out within Finnish Centre of Excellence in White BiotechnologyGreen Chemistry (decision number 118573).
________________ [1] Richard, P., and Hilditch, S. (2009) Appl. Microbiol. Biotechnol. 82, 597-604. [2] Boer, H., Maaheimo, H., Koivula, A., Penttil, M., and Richard, P. (2010) Appl. Microbiol. Biotechnol. 86, 901-909. [3] Parkkinen, T., Boer, H., Jnis, J., Andberg, M., Penttil, M., Koivula, A., and Rouvinen, J. J. Biol. Chem., in press. [4] Mojzita, D., Wiebe, M., Hilditch, S., Boer, H., Penttila, M., and Richard, P. (2010) Appl. Environ. Microbiol. 76, 169-175.
11
Deracemization of non-natural Amino Acids based on DKR of the corresponding N-protected Thioesters
Davide Tessaroa,b, Lorenzo Ceriolia, Paola DArrigoa,b, Giuseppe Pedrocchi-Fantonic, Stefano Servia,b, Fiorenza Vianic Politecnico di Milano, Department of Chemistry, Materials, and Chemical Engineering, piazza Leonardo da Vinci 32, 20133 Milano, Italy b The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universit degli Studi dellInsubria, via Mancinelli 7, 20131 Milano, Italy c ICRM-CNR, via Mancinelli 7, 20131 Milano, Italy E-mail: davide.tessaro@polimi.it
a
12
From S to R: key residues controlling enantiomer Preference and activity in -transaminase
Maria Svedendahl Humblea, Karim Engelmark Cassimjeea, Vahak Abedib, Hans-Jrgen Federselb, Per Berglunda a KTH Royal Institute of Technology, Division of Biochemistry, AlbaNova University Center, SE-106 91 Stockholm, Sweden; bAstraZeneca R&D, SE-151 85 Sdertlje, Sweden E-mail: perbe@kth.se Transaminases (EC 2.6.1.X) are attractive biocatalysts for synthesis of chiral amines and -amino acids. These enzymes catalyse the interchange of an amino and a keto group, commonly from an amino acid to a ketoacid using the cofactor pyridoxal-5-phoshate (PLP). However, enzymes that accept a variety of amines and ketones have also been identified and are called -transaminases. These enzymes inherently show high stereoselectivity and thereby provide a valuable tool for producing pharmaceutically interesting chiral amines. The -transaminases are employed in industrial-scale production of both R- and S- amines of high enantiomeric purity. We previously used an S-selective -transaminase variant from Arthrobacter citreus and created an R-selective variant using a rational design approach based on a homology enzyme model.1 This homology modelling/rational design approach was recently explored on an -transaminase from Chromobacterium violaceum. A homology structure guided the creation of an -transaminase variant where the activity was increased and the enantioselectivity reversed. This work led to identification of key amino acid residues that control the activity and enantiomer preference. Both stereoselective synthesis with isopropyl amine, a cost efficient choice as amino donor in industry, and kinetic resolution will be discussed. In addition, a novel active-site quantification method based on a half transamination reaction will be presented.
Enzymatic kinetic resolution of racemic mixtures has multiple applications in the preparation of single enantiomers from racemates. When kinetic resolution is coupled with an in situ racemization, chemical or enzymatic, the yield limitation can be overcome leading to a much more efficient process: a deracemization based on a dynamic kinetic resolution (DKR). Requisites for a successful DKR are an enzyme selective for one form of the racemic mixture, a racemizing system acting on the substrate but not on the product, and a rate of racemization higher than the enzymatic reaction. These conditions require the design of suitable substrates. Since in thioesters the acidity of the hydrogen in -position is higher in comparison to the corresponding oxo-esters, amides or acids, we attempted to submit a number of N-protected--aminoacid thioesters to enzymatic hydrolysis (subtilisin) in the presence of a base-catalyzed continuous substrate racemization. In a preliminary work, complete deracemization of a number of -aryl-substrates has been effected, giving quantitative yields of L-amino acids in enantiomerically pure form [1]. The application of this technology to non-aromatic substrates required a careful design of the reaction conditions, in terms of solvent, racemizing agent, temperature, and enzyme form. In particular, suitable racemization conditions have been studied also with the aid of computational means [2]. A set of conditions appropriate for a one-pot DKR was found, and several aromatic as well as aliphatic N-Boc-amino acids have been obtained with high conversion and in enantiopure form. Cost Action CM0701-CASCAT Cascade Chemo-Enzymatic Processes: New Synergies between Chemistry and Biochemistry
Figure 1. Modified substrates of -N-acetylhexosaminidases. Acknowledgement: Support from grants FP7 NOVOSIDES (contract KBBE-4-265854), Czech Science Foundation (305/09/H008, P207/11/0629) and ESF COST Chemistry CASCAT CM0701 is gratefully acknowledged.
________________ [1] Bojarov P. et al., 2008, J. Mol. Catal. B: Enzymatic 50, 69.
________________ [1] Svedendahl, M.; Branneby, C.; Lindberg, L.; Berglund, P. ChemCatChem 2010, 2, 976-980. [1] Arosio D.; et al. Adv. Synth. Catal. (2007), 349, 1345 1348 [2] DArrigo, P.; et al. Tetrahedron: Asymmetry (2011), doi:10.1016/j.tetasy.2011.5.005
October 2-6, 2011
74 13
Diketopiperazines as novel substrates for hydantoinases
a

Oxidation of organophosphorous pesticides by myeloperoxidase for application in AchE based bioanalytical methods
Tamara Lazarevi Pati, Vesna Vasi Vina Institute of Nuclear Sciences, PO Box 522, University of Belgrade, 11001 Belgrade, Republic of Serbia E-mail: evasic@vinca.rs The aim of the work was to improve the sensitivity of acethylcholinesterase (AChE) bioanalytical set-ups for determination of organophosphorous pesticides (OPs), based on immobilized enzyme[1]. For this purpose, the possibility of enhancing the sensitivity of the sensor by converting OPs from thio- to oxo-forms[2], which are more potent AChE inhibitors, enzyme myeloperoxidase (MPO) isolated from human blood was used as an oxidant. The flow injection-type biosensor (FIA) consisting of a reactor with immobilized acethylcholinesterase (AChE) and spectrophotometric detector ( = 412 nm) was used for determination of OPs. The influence of MPO concentration, pH, temperature and incubation time between MPO and OPs on OPs (concentration from 1x10-4 1x10-6 M) was investigated, in order to find out the experimental conditions for the most efficient OPs oxidation. The aqueous solution samples of spiked water with some model thioorganophosphorous compounds (diazinon, malathion, chlopyrifos, phorate, azynofos methyl and commercial OPs control mixture) were used. All oxidation experiments were verified using UPLC and GC-MS analysis. The products were identified as oxon derivatives (phosphates), where the sulfur atom from thioate group was substituted by an oxygen atom. No hydrolysis products were detected after enzymatic oxidation of these pesticides was stopped by caltalase. The formed oxo-analogs were also detected using two AChE bioassays native enzyme and FIA manifold. In FIA system inhibition of AChE was determined by comparing the enzyme activity before and after passage of an oxidized pesticide solution through the reactor for a given period of time. The calibration curves for OPs was constructed. While the lower detection limit using native enzyme (10% AChE inhibition, 10 min incubation time) before oxidation was ca. 1x10-5 M, the detection limit after oxidation was below 1x10-7 M. However, the detection limits for FIA system depended on the flow rate, yielding the value below 1x10-9 M flow rate below 0.5 ml/min. Therefore, it was safely concluded that, due to the greater inhibitory power of OPs after oxidation, MPO may be regarded as an excellent oxidant for improving sensitivity of bioanalytical methods for OPs determination in water. _____________________
[1] L.Poganik, M.Franko, Biosens.Bioelectron. 18 (2003) 631-641 [2] H.S.Lee, Y.A.Kim, Y.A.Cho, Y.T.Lee, Chemosphere 46 (2002) 571-576

A variation of the "Hydantoinase Process for -L- and -amino acids production
A.I. Martnez-Gmez, M.J. Rodrguez-Alonso, S. Martnez-Rodrguez, P. Soriano-Maldonado J.M. Clemente-Jimnez, F. Rodrguez-Vico, F.J. Las Heras-Vzquez. Dpto. Qumica Fsica, Bioqumica y Qumica Inorgnica, Edificio CITE I. Universidad de Almera, Carretera de Sacramento s/n. 04120. La Caada de San Urbano. Almera. Spain. E-mail: fjheras@ual.es L-amino acids are used as additives in animal and human feed, in the pharmaceutical and cosmetics industries, and as chiral synthons in organic synthesis [1]. -amino acids have unique pharmacological properties, and their use as building blocks of -peptides, pharmaceutical compounds, and natural products is of growing interest [2]. Biotechnological production using the Hydantoinase Process is an inexpensive and environment-friendly enzymatic method, mainly applicable to the production of optically pure D-amino acids [3]. However, this enzymatic cascade has hardly been used to produce -L-amino acids and has not previously been used to obtain -amino acids. The aim of this work is to produce optically pure -L-amino acids from a wide spectrum of D,L-5-monosubstituted hydantoins used as substrates, by the combination of hydantoin-metabolizing enzymes. A second variation of this process lets the hydrolysis of 5- or 6-substituted dihydrouracils and the decarbamoylation of the intermediates to produce -amino acids. Both cascade chemoenzymatic processes enhance the versatility of the Hydantoinase Process to the production of more pharmacological compounds than those described to date.
a
75 18
Nitrile effect on amidase activity in nitrile hydratase-amidase sequential reaction
Cantarella M.a, Cantarella L.b, Gallifuoco A.a, Spera A.a, Martinkov L.c Department of Chemistry, Chemical Engineering and Materials, University of L'Aquila, via Campo di Pile Zona industriale di Pile, 67100, L'Aquila, Italy, E-mail: maria. cantarella@.univaq.it b Department of Industrial Engineering, University of Cassino,03043 Cassino (FR), Italy, c Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, CZ-142 20 Prague 4, Czech Republic. As we already showed, the possibility to realize high yield of nicotinic acid is effective through the use of CSMRs (continuous stirred tank membrane reactors) operated in series, employing the catalytic cascade system nitrile hydrates-amidase, NHase/AMase, present in resting cells of Microbacterium imperiale CBS 489-74 [1].
M. Perzborna, F. Strhlea, C. Syldatka, J. Rudata KIT Institute of Process Engineering in Life Sciences, Section II: Technical Biology, Engler-Bunte-Ring 1, 76131 Karlsruhe, Germany E-mail: mareike.perzborn@kit.edu Diketopiperazines (DKPs) are the smallest possible cyclic peptides composed of two -amino acids. They are abundant natural compounds produced by a variety of bacteria and fungi as secondary metabolites. Certain DKPs possess antitumor, antiviral, antifungal and antibacterial functions and are described as a new class of quorum sensing molecules. [1; 2] Despite their abundance in nature, little is known about the biodegradation of DKPs and only for few strains hydrolysis of DKPs is reported. In this work two different approaches are under way to investigate the enzymatical hydrolysis of DKPs: Certain strains with hydantoinase activity are tested for hydrolysis of DKPs. Recently we have shown that these cyclic amidases are able to cleave different substituted dihydropyrimidines [3]. Due to the structural similarity between hydantoins, dihydropyrimidines and DKPs (see figure 1), a selective hydrolysis of DKPs by hydantoinases seems possible and is studied in this work. Besides the proven hydrolysis of certain hydantoin and dihydropyrimidine derivatives, first results also indicate activity against different DKPs for some of these strains. Paenibacillus chibensis (DSM 329) and Streptomyces flavovirens (DSM 40062) are described for the hydrolysis of an aspartame derivative cyclo(L-Asp-L-Phe) [4]. It was shown that also other DKPs can be hydrolyzed by the two mentioned strains. The activity of DSM 40062 can be increased by enzyme induction with cyclo(L-Asp-L-Phe) during cultivation and the activity is decreased by the serine peptidase inhibitor PMSF. For DSM 329 the DKP hydrolysis is reduced by the metallopeptidase inhibitor EDTA. The purification and further characterization of the two responsible enzymes are in progress.
During this study it became evident that 3-cyanopyridine (3-cnp) was able to modulate the AMase activity. The focus of this paper is on the combined effect of 3-cnp concentration and temperature on the amidase activity. To this end use was made of continuous stirred tank membrane reactor fed with buffered solution AMase substrate (100 mM nicotinamide) added with 3-cnp at various concentration. The investigation was performed at 40C and 50C as at these temperatures, the inactivation constant (evaluated with a firstorder inactivation mechanism hypothesis) is negligible. Interestingly, this study provides evidence on a synergistic effect between temperature and 3-cnp concentration.
[1]Cantarella L., Gallifuoco A., Malandra A., Martnkov L., Spera A., Cantarella M. High-yield continuous production of nicotinic acid via nitrile hydrataseamidase cascade reactions using cascade CSMRs Enzyme Microb Technol 48 (2011) 345350doi:10.1016/j.enzmictec.2010.12.010
Figure 1. Enzymatical hydrolysis of cyclic amides. Hydantoins are cleaved to N-carbamoyl--amino acids, whereas dihydropyrimidine derivatives react to N-carbamoyl--amino acids. Depending on the substituent, both reactions can be performed by cyclic amidases (hydantoinases) [3]. An analogous hydrolysis of diketopiperazines would lead to dipeptides and is investigated in this work.
________________ [1] Martins MB, Carvalho I (2007) Tetrahedron: 63, 9923-9932. [2] Campbell J, Lin Q, Geske GD, Blackwell HE (2009) ACS Chem Biol: 4, 1051-1059. [3] Bretschneider U, Syldatk C, Rudat J (2010) Chem Ing Tech: 82, 161-165. [4] EP 0 220 028 - B1 (1990) AJINOMOTO CO.
________________ [1] Leuchtenberger, W., Huthmacher, K. and Drauz, K. Appl. Microbiol.Biotechnol. 2005, 69: 1-8. [2] Juaristi, E. and Lopez-Ruiz, H. Curr. Med. Chem. 1999, 6:983-1004. [3] Clemente-Jimenez, J. M., Martinez-Rodriguez, S., Rodriguez-Vico F. and Las Heras-Vazquez, F. J.. Recent Patents Biotechnol. 2008, 2:35-46.
15
Immobilization of beta-galactosidase on membranes for process intensification
Peter Jochemsa,b, Yamini Satyawalia, Sandra Van Roya, Ludo Dielsa,b, Winnie Dejonghea a Flemish Institute for Technological Research (VITO nv), Separation & Conversion Technology (SCT), Mol, Belgium b University of Antwerp, Department of Bioscience Engineering, Antwerp, Belgium E-mail: winnie.dejonghe@vito.be Beta-galactosidase has two important applications i.e. the removal of lactose from milk and the production of galacto-oligosaccharides (GOS). The immobilization of this enzyme on a membrane enables the integration of conversion and separation. Especially during the production of GOS this is important, because of the equilibrium between GOS production and GOS hydrolysis by beta-galactosidase. In the present study, the enzyme, beta-galactosidase, was immobilized on mixed-matrix membranes, which are capable of adsorbing enzymes (proteins) on their surface. These membranes act as a support and a barrier and in such a case a membrane is called a biocatalytic membrane. The aim of the study was to immobilize beta-galactosidase in an optimal way on a membrane and to investigate the effect of immobilization on enzyme as well as membrane characteristics. After immobilization, the enzymes remained active however their apparent characteristics were affected and changes in the pH and temperature optima were observed. In addition, the membrane characteristics such as molecular weight cut-off, permeability, etc. were monitored before and after immobilization. Observing these alterations in enzyme and membrane properties is essential for optimizing the overall system and realizing maximal productivity. The focus here was on achieving optimal activity per square meter membrane, high stability of the immobilized enzymes, etc. Until now our main focus is on absorption of enzymes on mixed-matrix membranes, but we are also exploring the possibilities of applying an entrapment technique, which was developed at VITO, to immobilize enzymes on a membrane. The main reason for this is the current lack of general immobilization techniques, as well as a rational approach to immobilize enzymes. These adsorption and entrapment techniques will be applied on other enzymes to better understand the limits of our different techniques. The economical relevance of biocatalytic membranes will largely depend upon their stability. However, the potential of the combination of biotechnology and membrane technology is huge.
16
Amidation of rac-N-Boc amino acid thioesters under DKR conditions
Lorenzo Ceriolia, Paola DArrigoa,b, Giuseppe Pedrocchi-Fantonic, Stefano Servia,b, Davide Tessaroa,b, Fiorenza Vianic a Politecnico di Milano, Department of Chemistry, Materials, and Chemical Engineering, piazza Leonardo da Vinci 32, 20133 Milano, Italy b The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universit degli Studi dellInsubria, via Mancinelli 7, 20131 Milano, Italy cICRM-CNR, via Mancinelli 7, 20131, Milano, Italy E-mail: lorenzo.cerioli@chem.polimi.it Amide bond formation is a labour intensive process often involved in the synthesis of fine chemicals; esters and thioesters aminolysis is a practical method of amide synthesis avoiding inconvenient carboxylate activation. In recent evaluations of chemical processes trying to comply to the rule of GMF and green chemistry, amide bond formation was identified as one of the most utilized and problematic synthetic step in the pharmaceutical industry. From a detailed study it was found that N-acylation reactions for amide formation were used in more than 50% of current synthesis of drug candidates [1]. We have recently developed an efficient methodology for the hydrolysis of N-protected amino acid thioesters under Dynamic Kinetic Resolution (DKR) conditions, where the relatively high acidity of the -hydrogen was exploited in a base-catalysed substrate racemization occurring in the same pot with a selective, protease-mediated, hydrolysis. As a result, several amino acid derivatives have been obtained with high conversions and enantiomeric excesses [2]. We now report on a similar procedure aimed at the deracemization of a group of rac-N-BocAas-thioesters with N-protected amino acid amides formation. Starting with the relative thioester, in presence of a racemizing strong organic base (DBU) and a nitrogen nucleophile in t-ButOH with controlled water activity, ammoniolysis or aminolysis catalyzed by CLEAAlcalaseTM yield the corresponding amide with high optical purity and high chemical yields. The reaction is carried out under kinetic control, and a number of parameters (amine concentration, base ratio, water content, enzyme amount) are critical for the successful DKR process. Here we present some results so far obtained employing this methodology [3]. Cost Action CM0701-CASCAT Cascade Chemo-Enzymatic Processes: New Synergies between Chemistry and Biochemistry
[1] Constable, D.J.C. et al. Green Chem. 2007, 9, 411-420. [2] Arosio, D. et al. Adv. Synth. Catal. 2007, 349, 1345-1348 [3] Tessaro, D. et al., submitted for publication.
19
Heterologous expression and characterization of novel fungal arylacetonitrilases
A.B.Vesel1, P. Weyrauch2, A. Petkov1, A. Ringelov1, L. Martnkov1 1 Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vdesk 1083, CZ-142 20 Prague, Czech Republic 2 Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms-University Mnster, Correnstrasse 3, D-48149 Mnster, Germany The aim of this work is the search for new arylacetonitrilases, which are enzymes with a high potential in the production of enantiomerically enriched -substituted carboxylic acid or their amides. Using genome mining with a sequence of a characterized arylacetonitrilase from Neurospora crassa as the template, two sequences of potential arylacetonitrilases were selected. Sources of the corresponding genes are filamentous fungi Nectria haematococca (teleomorph of Fusarium solani) and Arthroderma benhamiae. Synthetic genes were prepared with optimized codon frequency and expressed in E. coli to give functional enzymes. The enzymes are distantly related (with amino acid identities below 40 %) to the well-known bacterial arylacetonitrilases from Pseudomonas fluorescens [1] and Alcaligenes faecalis [2] and share 66-67 % amino acid identities with the enzyme from N. crassa. Nitrilase activity with phenylacetonitrile per 1 L of E. coli culture achieved up to 69,000, 28,000 and 6,900 U for enzymes for N. crassa, N. haematococca and A. benhamiae, respectively. The enzymes were purified to near homogeneity. They all exhibited preference for phenylacetonitrile and (R,S)-mandelonitrile as substrates. The major reaction product of the latter substrate was enantiomerically enriched R-mandelic acid; all enzymes produced minor amounts of mandelic acid amide. Financial support from projects P504/11/0394, 305/09/H008 (Czech Science Foundation), IAA500200708 (Grant Agency of the AS CR, Czech Republic), LC06010, OC09046 (Ministry of Education of the Czech Republic), and Institutional Research Concept AV0Z50200510 (Institute of Microbiology).
20
Kinetics of biocatalytic asymmetric phosphorylation of mevalonate by 31P-NMR
Rie Matsumia, Christine Hellriegelb, Bernhard Schoenenbergerb, Thomas Milesib, John van der Oosta, Roland Wohlgemuthb a Laboratory of Microbiolog, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands; bSigma-Aldrich, Research Specialties, Industriestrasse 25, CH9470 Buchs, Switzerland, E-mail: roland.wohlgemuth@sial.com Synthetic routes that include asymmetric phosphorylation reactions are of considerable interest. Biocatalytic enantiospecific phosphorylations in aqueous solutions are complement-tary synthetic tools and are suitable for the synthesis of water-soluble target molecules with high step economy and avoiding the use of protecting groups. Numerous wellknown bio-catalytic phosphorylation reactions are key to central metabolic pathways and a variety of enzymes are available for catalyzing these phosphate transfer reactions. In our endeavour to synthesize enantiomerically pure metabolites of the mevalonate pathway, the direct biocatalytic phosphorylation of mevalonate seemed attractive, because (3R)meva-lonate or racemic mevalonate could be phosphorylated and both (3R)-mevalonate5-phos-phate and (3S)-mevalonate would be accessible.
OH
+ -
O P O
OH COO-X+
XO XO
+ -
(3R)-Mevalonate-5-phosphate
R-mevalonate kinase (MK) ATP, MgCl2 pH 8

OH COO-X+ HO O O OH
+
OH COO-X+ HO
racemic mevalonate
(3S)-mevalonate
[1] Kiziak, C., Conradt, D., Stolz, A., Mattes, R., Klein, J.: Nitrilase from Pseudomonas fluorescens EBC191: Cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology 151, 3639-3648 (2005) [2] Nagasawa, T., Mauger, J., Yamada, H.: A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization. Eur. J. Biochem. 194, 765-772 (1990)
We describe a simple one-step phosphorylation reaction using a cloned archaeal mevalonate kinase and its mevalonate substrate as starting material. In order to accelerate the development of a robust synthetic procedure, a continuous and quantitative 31P-NMR method has been developed for the analysis of mevalonate-5-phosphate formation. The accurate product quantitation was performed with a calibration curve of different mevalonate-5-phosphate concentrations using the absolute concentration determined by quantitative 1H-NMR. The results clearly show that the kinetic resolution of racemic mevalolactone can be used for the preparation of S-mevalonate and that the production of R-mevalonate-5-phosphate is best prepared by starting from R-mevalolactone. This continuous 31P-NMR methodology can now be used to characterize the enantioselectivity of the various mevalonate kinases available and determine their utility for the synthesis of both enantiomerically pure as well as racemic mevalonate-5-phosphates.
October 2-6, 2011
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Activity of recombinant Escherichia coli whole cell catalysts synthesizing hydroxynitrile lyase and nitrilase activities in ionic liquids
Stefanie Bauma, Fred van Rantwijkb, and Andreas Stolza* a Institut fr Mikrobiologie, Universitt Stuttgart, Allmandring 31, 70569 Stuttgart, Germany b Laboratory of Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands * E-mail: Andreas.Stolz@imb.uni-stuttgart.de (Chiral) 2-hydroxynitriles, 2-hydroxycarboxamides and 2-hydroxycarboxylic acids are interesting products for the chemical industry and also can be used as building blocks for various follow-up reactions. We are currently analysing the synthesis of chiral (S)2-hydroxycarboxylic acids and (S)-2-hydroxycarboxamides from aldehydes and cyanide by combining a (S)-selective hydroxynitrile lyase (oxynitrilase) from the cassava plant (Manihot esculenta) with a nitrilase from the bacterium Pseudomonas fluorescens EBC191 in a bienzymatic cascade reaction [1,2]. The conversion of benzaldehyde plus cyanide into mandelic acid and mandeloamide by a recombinant Escherichia coli whole cell catalyst which simultaneously expressed the (S)-oxynitrilase from cassava and the arylacetonitrilase from P. fluorescens EBC191 was studied. Benzaldehyde exhibited a stroner inhibitory effect than cyanide on the nitrilase activity and significantly inhibited the reaction in concentrations 25 mM. In order to reduce the toxic effects of the substrates, it was tested if two-phase systems consisting of a buffered aqueous phase and the ionic liquids 1-n-butyl-1-pyrrolidiniumbis(trifluoromethylsulfonyl)imide (BMpl NTf2) or 1-n-butyl-3-methylimidazolium-hexafluorophosphate (BMim PF6) could be used for the intended biotransformation of benzaldehyde plus cyanide into (S)-mandelic acid and/or (S)-mandeloamide. The distribution coefficients of the substrates, intermediates and products of the reaction were determined and it was found that BMpl NTf2 and BMim PF6 were highly efficient as substrate reservoirs for benzaldehyde and that also HCN was soluble to a certain extent in the ionic liquids. The recombinant whole cell catalyst was active in the presence of BMpl NTf2 or BMim PF6 phases and converted benzaldehyde plus cyanide into mandelic acid and mandeloamide. The two-phase systems allowed the conversion of benzaldehyde dissolved in the ionic liquids to a concentration of 700 mM with product yields (=mandelic acid plus mandeloamide) of 82- 96 %.The cells were slightly more effective in the presence of BMpl NTf2 than in the presence of BMim PF6. In both two-phase systems benzaldehyde plus cyanide were converted into (S)-mandeloamide and (S)-mandelic acid with enantiomeric excesses 94. The recombinant E. coli cells formed in the two-phase systems with ionic liquids and increased substrate concentrations higher relative amounts of mandeloamide than in purely aqueous system with lower substrate concentrations.
________________ [1] Sosedov et al., 2009, Adv. Synth. Catal. 351:1531-1538 [2] Sosedov et al., 2010, Appl. Environ. Microbiol. 76:3668-3674

Enzymatic Nitrile Reduction using wild type and mutant Nitrile Reductase QueF
Birgit Wildinga, Norbert Klempiera, Barbara Petschacherb, Margit Winklerb, Sigrid Eggerc, Regina Kratzerc, Bernd Nidetzkyc, Georg Steinkellnerd, Andrzej Lyskowskid, Karl Gruberd a ACIB c/o Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria; bACIB, c/o Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria; cACIB c/o Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria; dACIB Core Facility Structural Biology c/o Institute of Molecular Biosciences, University of Graz, Humboldtstrae 50/III, 8010 Graz, Austria E-mail: birgit.wilding@acib.at Novel enzymatic activity has been reported recently as part of the biosynthetic pathway to the hypermodified nucleoside queuosine.[1] The enzyme QueF is so far the only enzyme known capable of reducing a nitrile group to its corresponding primary amine and thus would be a valuable contribution to the present spectrum of nitrile transforming enzymes. A substrate screening of different aliphatic, aromatic and heterocyclic nitriles employing purified wild type nitrile reductases from Escherichia coli (type I QueF), Bacillus subtilis (type II QueF) and Geobacillus kaustophilus (type II QueF) has revealed the enzymes high substrate specificity towards its natural substrate 7-cyano-7-deazaguanosine. We have synthesized various compounds derived from 7-cyano-7-deazaguanosine, including different substituted deazapurines, to start exploring the scope of nitrile reductase in a twofold approach: investigation of the substrate specificity by screening these substrate derivatives using wild-type enzymes, as well as redesign of the enzymes active site and screening mutant E. coli and G. kaustophilus nitrile reductases towards both, the natural substrate and the substrate derivatives. Our design of active site mutants of G. kaustophilus (type II QueF) is guided by a recent report of a QueF model based on B. subtilis QueF[2] and with respect to E. coli (type I QueF) by a three-dimensional active site model based on the high resolution crystal structure of Vibrio cholerae QueF[3]. Thus, by using complementary NADPH-absorption and HPLC-MS screening methods it could be shown that non-natural substrates, derived from 7-cyano-7-deazaguanosine can be reduced to their corresponding amines.

Enzymatic C-C Bond Formation: New Insights into the Transaldolase Enzyme Family
Shiromi Baiera, Tatjana Sandalovab, Melanie Schrmannc, Tomoyuki Inouea, Sabine Huba, Gunter Schneiderb, Georg A. Sprengera, c, Anne K. Samlanda a Institute of Microbiology, Universitt Stuttgart, Allmandring 31, 70550 Stuttgart, Germany; bDivision of Molecular Structural Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden; cInstitute for Biotechnology 1, Forschungszentrum Jlich, 52405 Jlich, Germany E-mail: anne.samland@imb.uni-stuttgart.de Transaldolases (Tal) catalyse the reversible transfer of a dihydroxyacetone moiety from a ketose donor, e.g. D-fructose-6-phosphate, onto an aldehyde acceptor, e.g. D-erythrose4-phosphate.[1] Upon this transfer a new C-C bond is formed with a strict stereo selectivity for the 3S,4R-stereo configuration. The reaction proceeds via a Schiff base intermediate and no cofactors are required. Besides its physiological substrates Tal accepts a variety of non-phosphorylated donors and acceptors as substrate.[2] These properties make Tals interesting enzymes for biocatalytic syntheses of sugar derivatives. Despite its nearly ubiquitous distribution the members of the Tal enzyme family show a high degree of variation in their primary sequence. By sequence comparison the Tals were grouped into five different subfamilies.[1] We wondered how this variation in sequence is reflected in the structure and biochemical properties of different Tals from different subfamilies. Especially, how the variation in sequence affects properties important for biocatalytic applications, such as substrate specificity and stability. Here, we compare the Tals of Bacillus subtilis, Escherichia coli and Corynebacterium glutamicum. The biochemical properties, e.g. substrate specificity, and the (/)8-barrel fold of the subunits are rather conserved but the oligomerisation state differs. There are monomeric, dimeric and decameric enzymes known now. The decameric enzymes exhibit high thermostability which makes them highly attractive for application in biocatalysis. Monomeric enzymes are interesting as they are easier to manipulate.
77 26
Recent advances with transketolase : development of a new assay and an efficient immobilisation procedure
Nadia Touisnia,b, Karima Benaissia,b, Franck Chamantraya, Virgil Helainea, Laurence Hecqueta, Christine Moustyb, Vanessa Prevotb, Claude Foranob a Laboratoire de Synthse et Etude de Systmes Intrt Biologique, UMR 6504, bLaboratoire des Matriaux Inorganiques, UMR 6002, Universit Blaise Pascal, Clermont-Ferrand, F-63000, France. E-mail: Laurence.Hecquet@univ-bpclermont.fr Transketolase (TK) is a cytosolic and key enzyme of the non-oxidative branch of the pentose phosphate pathway. TK has been used as a biocatalyst for ketose synthesis and currently the modification of the substrate specificity of TK by mutagenesis is of great interest to broaden its application range [1]. In addition, some recent studies have shown that human TK is a target in cancer and neurodegenerative diseases offering medical applications [2]. Considering this context, we have recently explored two fields of research focused on Layered Double Hydroxydes as a very convenient support for enzymes. In the field of the development of TK assays, we have recently proposed the electrochemical detection of TK activity using a tyrosinase biosensor[3,4]. Tyrosinase was immobilized on Layered Double Hydroxydes on the surface of the biosensor and the conditions were optimized for enhancing the sensitivity and specificity of the TK activity detection. The co-immobilization of TK on this biosensor will be investigated for biomedical applications. To improve TK efficiency for biocatalytic applications, we have also investigated Layered Double Hydroxydes as support. We have developed an easy, cheap, efficient and biocompatible procedure [5] revealing no activity loss, high immobilization rate, excellent re-usability and storage stability. This process offers promising potentials for TK and for a wide range of enzyme-catalysed reactions.
___________________________ [1] a) M. E. B. Smith, E.G. Hibbert, A.B. Jones, P. A. Dalby and H. C. Hailes, Adv. Syn. Catal., 2008, 350, 2631-2638. b) Czares, A., Galman, J.L., Crago, L.G., Smith, M.E.B., Strafford, J., Ros-Sols, L., Lye, G.J., Dalby, P.A., Hailes, H.C. Org. Biomol. Chem. 2010, 8, 1301-1309. [2] a) Zhao, J., Zhong, C.-J., Neurosci. Bull. 2009, 25, 94-99. b)Le Huerou, Y., Gunawardana, I., Thomas, A.A., Boyd, S.A., de Meese, J., Dewolf, W., Gonzales, S.S., Han, M., Hayte, L., Kaplan, T., Lemieux, C., Lee, P., Pheneger, J., Poch, G., Romoff, T.T., Sullivan, S., Weiler, F., Wright, S.K., Lin, J., Bioorg. Med. Chem. Lett. 2008. 18, 505-508. [3] a) Charmantray, F., Helaine, V., Lasikova, A., Legeret, B., Hecquet, L., 2009. Tet. Lett. 49, 3229-3233. b) M. Sanchez-Paniagua Lopez, F. Charmantray, V. Hlaine, L. Hecquet, C. Mousty. Biosens. and Bioelectron. , 2010, 26, 139-143 [4] G. Simon, M. Bouzon, F. Charmantray, V. Hlaine, B. Legeret, P. Marlire, L. Hecquet. Bioorg. Med. Chem. Lett., 2009, 19, 3767-3770. [5] K. Benaissi, V. Hlaine, V. Prvot, C. Forano and L. Hecquet. Adv. Syn. Catal., 2011, in press ((doi: 10.1002/adsc.201000925).
________________ [1] (a) VanLanen, S.G.; Reader, J.S.; Swairjo, M.A.; de Crcy-Lagard, V.; Lee, B.; Iwata-Reuyl, D. Proc. Natl. Acad. Sci. U. S. A. 2005, 102 (12), 4264-4269. (b) Lee, B.W.K.; Van Lanen, S.G.; Iwata-Reuyl, D. Biochemistry 2007, 46, 12844-12854. [2] Swairjo, M.A.; Reddy, R.R.; Lee, B.; Van Lanen, S.G.; Brown, S.; de Crcy-Lagard, V.; Iwata-Reuyl, D.; Schimmel, P. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2005, F61, 945-948. [3] Kim, Y.; Zhou, M.; Moy, S.; Morales, J.; Cunningham, M.A.; Joachimiak, A. J. Mol. Biol. 2010, 404 (1), 127-137.
Figure 1. reduction of 7-cyano-7-deazaguanosine in the biosynthetic pathway of queuosine [1]
Figure 1. Overall structure of monomeric, dimeric and decameric transaldolases. Of the decameric structure of Tal from B. subtilis only one of the two pentameric rings is shown for clarity.
________________ [1] A. K. Samland, G. A. Sprenger, Int J Biochem Cell Biol 2009, 41, 1482-1494. [2] G. A. Sprenger, U. Schrken, G. Sprenger, H. Sahm, J Bacteriol 1995, 177, 5930-5936.
23
Preparation of an enantioenriched taxol sidechain precursor by nitrilase and nitrile hydratase
Birgit Wildinga, Carina Hasenhrla, Gary Blackb, Justin Perryb, Margit Winklerc, Norbert Klempiera a ACIB, c/o Institute of Organic Chemistry, Graz University of Technology, Stremayrgasse 9, 8010 Graz, Austria; bSchool of Applied Sciences, University of Northumbria, Newcastle upon Tyne, NE1 8ST, United Kingdom; cACIB c/o Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria. E-mail: birgit.wilding@acib.at Taxol (paclitaxel) is a widely used drug in cancer chemotherapy. In recent years, the synthesis of the taxol sidechain has attracted considerable interest. Several precursors of the taxol sidechain were prepared by chemo-enzymatic approaches using acylases, lipases, and reductases.[1] We have recently reported on the enantioselective synthesis of - and -aminoacids[2] by employing nitrilases and nitrile hydratases. During this work the taxol sidechain precursor trans-2,4-diphenyl-4,5-dihydrooxazole-5-carbonitrile was also discovered to be a suitable substrate.[3] The present work is devoted to the enantioselective transformation of the taxol side chain precursor trans-2,4-diphenyl-4,5-dihydrooxazole-5-carbonitrile as well as its cis-isomer. A set of nitrilases and nitrile hydratases were screened for their specificity and selectivity towards the two isomers. Most interestingly, all nitrilase-catalyzed reactions did not yield the carboxylic acid, but rather the pure amide. Work to convert the amide into the carboxylic acid by amidase from different bacterial strains is ongoing.
24
Novel chemoenzymatic synthesis of bioactive cyclophellitol and epi-cyclophellitol
Nicola DAntonaa, Raffaele Morronea, Paolo Bovicellib, Giovanni Gamberaa, Daniela Biondia, Giovanni Nicolosia, David Kubacc, Ludmila Martinkovac a Istituto di Chimica Biomolecolare del CNRUOS Catania, Via P. Gaifami 18, I-95126 Catania, Italy; bIstituto di Chimica Biomolecolare del CNRUOS Sassari, Traversa La Crucca 3-Baldanica, I-07040 Sassari, Italy; cLaboratory of Biotransformation, Institute of Microbiology of the Academy of Sciences of the Czech Republic, Vdensk 1083, CZ-142 20 Prague 4, Czech Republic E-mail: nicola.dantona@icb.cnr.it The development of molecules with enhanced biological activity is a continuing primary goal in biomedical research. The need for compounds interacting with biological receptors characterized by a high grade of asymmetry leads researchers to synthesize molecules possessing well defined stereochemical patterns, with the declared aims of obtaining better substrate-receptor interactions, minimizing collateral effects due to different isomeric forms, and reducing the amount of administered drug. In last decades there has been considerable interest in the synthesis and testing of glycosidases inhibitors; this interest is based not only upon the inherent usefulness of these compounds as probes of mechanism and active site structure of glycosidases, but also upon their utility in probing mechanisms of glycoprotein processing and their therapeutic potential. (+)-Cyclophellitol, is a highly specific and effective irreversible inactivator of -glucosidases and also inhibit the human glucocerebrosidase. Moreover since glucosidase inhibitors have the interesting property to inhibit HIV infection and metastasis formation, cyclophellitol represents a promising antiHIV agent. Interestingly the unnatural (+)-1,6-epi-isomer, bearing an -epoxide configuration, displays a potent -glucosidase inhibitory activity. To date most syntheses of cyclophellitol start with enantiomerically pure natural products, most notably carbohydrates, and require long synthetic sequences due to the need to manipulate functionality largely by the use of protecting groups. Only two preparations of (+)-cyclophellitol, based on typical asymmetric synthesis methods, starting from achiral precursors are reported in literature [1-2]. A possible alternative route for the synthesis of enantiopure cyclophellitols, not yet explored, is the biocatalysis. We present hereby a novel chemoenzymatic synthesis of both enantiomeric series of cyclophellitol and epi-cyclophellitol in optically active form, and more in general the first example in literature of a synergic use of lipase, nitrile hydratase and amidase is described [3].
27
Remote Enantioselectivity of Transketolase in the Synthesis of Non-Natural Sugars
Titu Devamani,a Dong Yi,a Juliane Abdoul-Zabar,b Virgil Hlaine,b Franck Charmantray,b Laurence Hecquet,b Wolf-Dieter Fessnera* a Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersenstrae 22, D-64287 Darmstadt, Germany; bUniversit Blaise Pascal, Laboratoire SEESIB-CNRS UMR 6504, BP 10448, F-63177 Aubire, France E-mail: devamani@mail.oc.chemie.tu-darmstadt.de Transketolase (TK; EC 2.2.1.1) catalyses the stereospecific transfer of a two carbon ketol unit to the carbonyl terminus of a variety of aldehydes.[1] The use of TK for C-C bond formation is receiving increased attention for asymmetric synthesis because of the highly stereocontrolled formation of an (S)-configurated chiral center with efficient concomitant chiral resolution for (2R)-hydroxyaldehydes.[2] Recent attempts are directed at broadening the substrate specificity of TK mutants to -unsubstituted acceptors for the synthesis of non-natural sugars.[3] We have developed a novel, sensitive high-throughput colorimetric assay for TK activity based on the pH change upon conversion of the irreversible donor substrate hydroxypyruvate, which also allows rapid screening of substrate tolerance of TK variants.
28
Recombinant transketolases from geobacillus strains: study of thermostability and catalytic properties
Juliane Abdoul-Zabar,a Franck Charmantray,a Virgil Hlaine,a Laurence Hecquet,a* I. Sorel,b D. Louis,b P. Marlire,c Titu Devamani,d Dong Yi,d Wolf-Dieter Fessnerd a Universit Blaise Pascal, Laboratoire SEESIB-CNRS UMR 6504, BP 10448, F-63177 Aubire, France; bCEA, DSV, IG, Genoscope, Laboratoire des Applications, 2 rue Gaston Crmieux, CP5706, F-91057 Evry, France; cIsthmus SARL, 81 rue Raumur, F-75002 Paris, France; dInstitut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersenstrae 22, D-64287 Darmstadt, Germany E-mail:laurence.hecquet@univ-bpclermont.fr Transketolase (TK) is a thiamine diphosphate (ThDP)-dependent enzyme that is extremely efficient for asymmetric synthesis of enantiopure building blocks by carboligation. TK from yeast[1] and E. coli[2] is used with hydroxypyruvate (HPA) as donor substrate, thereby render-ing the ketol transfer irreversible. TK is highly enantioselective for (2R)-configurated hydroxyaldehyde acceptor substrates, consequently producing ketose structures having a (3S,4R) configuration. The synthetic potential of TK has been applied to various bioactive products such as the pheromone -exo-brevicomin,[3a] as well as to 6-deoxysugars,[3b] aza-sugars,[3c] thiosugars,[3d] phosphorylated sugars,[3e,f] and aminoalcohols.[3g]
Figure 1. Transketolase catalyzed synthesis of non-natural sugars Using this technology we have evaluated the behaviour of TK enzymes from different sources towards various acceptor substrates. In particular, we have investigated the selectivity of various TK in their capacity to resolve remote chiral centers. The stereoselectivity of TK catalysis is demonstrated by the preparative synthesis of non-natural deoxysugar derivatives, which are important chiral precursors for the synthesis of various bioactive molecules.
________________ [1](a) Kobori, Y.; Myles, D. C.; Whitesides, G. M. J. Org. Chem. 1992, 57, 5899-5907. (b) Morris, K. G.; Smith, M. E. B.; Turner, N. J.; Lilly, M. D.; Mitra, R. K.; Woodley, J. M. Tetrahedron Asymmetry 1996, 7, 2185-2188. (c) Zimmermann, F. T.; Schneider, A.; Schorken, U.; Sprenger, G. A.; Fessner, W.D. Tetrahedron Asymmetry 1999, 10, 1643-1646. (d) Hecquet, L.; Demuynck, C.; Schneider, G.; Bolte, J. J. Mol. Catal. B: Enzym. 2001, 11, 771-776. [2](a) Turner, N. J. Curr. Opin. Biotechnol. 2000, 11, 527-531. (b) Fessner, W.-D.; Helaine, V. Curr. Opin. Biotechnol. 2001, 12, 574-586. (c) Wohlgemuth, R. J. Mol. Catal. B: Enzym. 2009, 61, 23-29. [3]Cazares, A.; Galman, J. L.; Crago, L. G.; Smith, M. E. B.; Strafford, J.; Rios-Solis, L.; Lye, G. J.; Dalby, P. A.; Hailes, H. C. Org. Biomol. Chem. 2010, 8, 1301-1309.
To improve synthetic processes based on TK catalysis, we have investigated new TK sources from thermophilic microorganisms. TK genes from Geobacillus stearothermophillus and Geobacillus thermodenitrificans were cloned and overexpressed, and the enzymes were purified. Here we report results of our studies on thermostability and substrate specificities at different temperatures in comparison with TK from yeast and E. coli. These new recombinant TKs offer interesting prospects for synthetic applications.
________________ [1] Nilsson, U.; Meshalkina, L.; Lindqvist,; Y. Schneider, G. J. Biol. Chem. 1997, 272, 1864-1869. [2] P. Asztalos, C. Parthier, R. Golbik, M. Kleinschmidt, G. Hbner, M. S. Weiss, R. Friedemann, G. Wille, K. Tittmann, Biochemistry 2007, 46, 12037-12052. [3] (a) Myles, D. C.; Andrulis, P. J.; Whitesides, G. M. Tetrahedron Lett. 1991, 32, 4835-4838. (b) Hecquet, L.; Bolte, J.; Demuynck, C. Tetrahedron 1996, 52, 8223-8232. (c) Ziegler, T.; Straub, A.; Effenberger, F. Angew. Chem. Int. Ed. 1988, 27, 716-717. (d) Charmantray, F.; Dellis, P.; Helaine, V.; Samreth, S.; Hecquet, L. Eur. J. Org. Chem. 2006, 5526-5532. (e) Zimmermann, F. T.; Schneider, A.; Schorken, U.; Sprenger, G. A.; Fessner, W.-D. Tetrahedron: Asymmetry 1999, 10, 1643-1646. (f) Charmantray, F.; Helaine, V.; Legeret, B.; Hecquet, L. J. Mol. Catal. B: Enzym. 2009, 57, 6-9. (g) Ingram, C. U.; Bommer, M.; Smith, M. E. B.; Dalby, P. A.; Ward, J. M.; Hailes, H. C.; Lye, G. J. Biotechnol. Bioeng. 2007, 96, 559-569.
Figure 1: Conversion of trans-2,4-diphenyl-4,5-dihydrooxazole-5-carbonitrile using nitrilase and nitrile hydratase

________________ [1] (a) Koskinen, A.M.P.; Karvinen, E.K. In Enantioselective Synthesis of -Amino Acids; Juaristi, E., Ed.; Wiley-VCH: New York, 1997; p 423-431. (b) Sih, C.J. In Enantioselective Synthesis of -Amino Acids; Juaristi, E., Ed.; Wiley-VCH: New York, 1997; p 433-441. (c) Cardillo, G.; Tolomelli. A.; Tomasini, C. Eur. J. Org. Chem. 1999, 155-161. (d) Gou, D.-M.; Liu, Y.-C.; Chen, C.-S. J. Org. Chem. 1993, 58, 1287-1289. (e) Feske, B.D.; Kaluzna, I. A.; Stewart, J. D. J. Org. Chem. 2005, 70, 9654-9657. [2] (a) Winkler, M.; Martnkov, L.; Knall, A.-C.; Krahulec, S.; Klempier, N. Tetrahedron, 2005, 61, 4249-4260. (b) Winkler, M.; Knall, A.-C.; Kulterer, M.R.; Klempier, N. J. Org. Chem. 2007, 72 (19), 7423-7426. [3] Winkler, M.; Glieder, A.; Klempier, N. Chem. Commun. 2006, 1298-1300. [4] commercial enzyme preparations, Codexis Inc., CA, and Prozomix Limited, UK.
_____________ [1] Schlessinger, R.H.; Bergstrom, C.P. J. Org. Chem. 1995, 60, 16-17. [2] Trost, B.M.; Patterson, D.E.; Hembre, E.J. Chem. Eur. J. 2001, 7, 3768-3775. [3] D'Antona, N.; Morrone, R.; Bovicelli, P.; Gambera, G.; Kubc, D.; Martnkov, L. Tetrahedron: Asymmetr 2010, 21, 2448-2454.
October 2-6, 2011
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Sialyltransferase Catalyzed Runaway Block Polymerizationone-pot Synthesis of neo-Glycopolymers Containing 2,6-Sialyllactose Repeating Units
Ning He, Wolf-Dieter Fessner* Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersenstrasse 22, D-64287 Darmstadt, Germany E-mail: he@mail.oc.chemie.tu-darmstadt.de Sialic acids, a diverse family of acidic 9-carbon monosaccharides,[1] represent one of the most important constituents of cell-surface glycoconjugates. Sialic acids usually occupy the outer-most position on glycolipids and glycoproteins of vertebrates or on surface polysaccharides of pathogenic bacteria.[2] Recently it has been found that capsular and cellwall polysaccharides of certain pathogenic microorganisms also contain internal sialic acid residues as part of their oligosaccharide repeating units. These motifs are believed to play important roles as patho-genic virulence factors and might constitute specific therapeutic targets.

Chemoenzymatic synthesis of neo-sialoconjugates using bacterial sialyltransferases
Dong Yi, Ning He, Wolf-Dieter Fessner* Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersenstrae 22, 64287 Darmstadt, Germany E-mail: yi@mail.oc.chemie.tu-darmstadt.de Bacterial -2,3/6-sialyltransferases (SiaT) are useful tools for the efficient regio- and stereo-specific synthesis of sialosides and for the modification of glycoproteins in vitro. We have developed a new assay for determining SiaT activity and have investigated various enzymes such as 2,3SiaT from Photobacterium phosphoreum (2,3SiaTpph)[1] or Campylobacter jejuni (CstII),[2] as well as 2,6SiaT from Photobacterium leiognathi JT-SHIZ-145 (2,6SiaTple)[3] for their substrate specificity towards sialyl donors and acceptors. Various sialyllactose and sialyl-LacNAc derivatives could be produced from combinations of donor and acceptor derivatives, by suitable coupling the SiaT reaction with CMP-sialate synthetase from Neisseria meningitidis (CSS)[4] for in situ nucleotide activation of sialic acids, and with galactosyltransferase from Helicobacter pylori (GalT)[5] for synthesis of LacNAc precursors, respectively.

FSA-Catalyzed Carboligation: New Synthetic Opportunities from an Ancient Enzyme Scaffold
Sebastian A. Junker,a Madhura V. Rale,a Anna Szekrnyi,b Pere Claps,b Wolf-Dieter Fessnera* a Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersen-strae 22, 64287 Darmstadt, Germany; bCatalonia Institute for Advanced Chemistry, CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain E-mail: junker@mail.oc.chemie.tu-darmstadt.de Aldol reactions constitute a powerful methodology for carbon-carbon bond formation in synthetic organic chemistry. Biocatalytic carboligation offers a green, uniquely regio- and stereoselective tool to perform this transformation. New enzymes from the transaldolase scaffold, such as fructose-6-phosphate aldolase (FSA) from E. coli, were recently shown to be unusually flexible in their substrate scope,[1] which renders them particularly valuable for the synthesis of complex polyfunctional targets.[2] So far, wild-type FSA has been demonstrated to utilize dihydroxypropanone, hydroxypropanone, 1-hydroxybutanone, and hydroxyethanal as aldol donor components for highly stereoselective carboligation reactions.
79 34
Organocatalytic synthesis of D-glucose via aldol chemistry under enzyme-like conditions
Jean-Louis Reymonda, Tamis Darbre a University of Berne, Department of Chemistry and Biochemistry, Freiestrasse 3, 3012 Berne, Switzerland E-mail: jean-louis.reymond@ioc.unibe.ch Prebiotic carbohydrate synthesis is believed to involve spontaneous or catalytic aldol condensations with formaldehyde and/or similar C1, C2 or C3 building blocks. We showed recently that Zn(Pro)2 promotes the formation of C4 and C6 carbohydrates from glycolaldehyde under neutral conditions [1]. We subsequently showed through mechanistic investigation of the aldol reaction of Zn(Pro)2 that this catalysts exhibits a substrate dependent mechanism using an enamine intermediate with acetone but an enolate intermediate with dihydroxyacetone [2]. The same chemistry was then used in the study of peptide dendrimer aldolase enzyme models with multiple N-terminal prolines, using an umbelliferone-based fluorogenic assay for enolization [3]. In a related study, we herein report an investigation of the formation of D-glucose from glycolaldehyde and dihydroxyacetone under aqueous conditions using various organocatalysts, including typical enamine-specific catalysts such as D-Proline, mixed mechanism catalysts such as Zn(Pro)2, and pure general base catalysts such as N-methyl morpholine. The formation of D-glucose was followed using an enzyme assay [4] based on GOX and HRP-ABTS as the colorimetric system. Iterative microtiterplate screening and engineering of the conditions revealed optimal catalytic conditions for the formation of D-glucose from these simple building blocks in a single step. The assay offers the opportunity to further optimize the reaction to reach the one-step quantitative conversion that would be the hallmark of a perfect organic process.
Figure 1. Synthesis of neo-sialopolysaccharides by one-pot, two enzymes reaction. Polysaccharides containing internal sialic acid residues are extremely difficult to obtain by isolation from natural sources or by chemical synthesis. We report the first successful example of glycosyltransferase-catalyzed polymerization yielding structurally defined polysaccharides with internal sialic acid residues. We have discovered that sialic acids with N-acyl-linked galactosyl or lactosyl units can be efficiently activated by the CMPsialate synthetase (CSS) from Neisseria meningitidis[3] and that such conjugates can be transferred repetitively by the 2,6-sialyltransferase (SiaT) from Photobacterium leiognathi JT-SHIZ-145.[4] Such cascades yield neo-glycopolysaccharides containing internal sialyllactoside or sialyl-galactoside repeating units, respectively, thus mimicking naturally occurring polysaccharides.
________________ [1](a) Schauer, R. Glycoconj. J. 2000, 17, 485-499; (b) Angata, T.; Varki, A. Chem. Rev. 2002, 102, 439-469. [2]Stanley, P.; Cummings, R. D. in Essentials of Glycobiology, 2nd ed (Varki A, et al., Eds), Cold Spring Harbor Laboratory Press, New York 2009, Chapter 13. [3]Knorst, M.; Fessner, W.-D. Adv. Synth. Catal. 2001, 343, 698-710. [4]Yamamoto, T.; Hamada, Y.; Ichikawa, M.; Kajiwara, H.; Mine, T.; Tsukamoto, H.; Takakura, Y. Glycobiology 2007, 17, 1167-1174.
Figure 1. Aldol formation catalyzed by variant fructose-6-phosphate aldolases (FSA*). In this work we demonstrate the utility of rational protein engineering to design variant FSA mutant proteins (FSA*) that offer an extended tolerance for donor substrate modifications towards the creation of novel products that are of commercial interest.[3] Figure 1. Chemoenzymatic synthesis of neo-sialoconjugates
________________ [1] Tsukamoto, H.; Takakura, Y.; Yamamoto, T. J. Biol. Chem. 2007, 282, 29794-29802. [2] Gilbert, M.; Brisson, J.-R.; Karwaski, M.-F.; Michniewicz, J.; Cunningham, A.-M.; Wu, Y.; Young, N. M.; Wakarchuk, W. W. J. Biol. Chem. 2000, 275, 3896-3906. [3] Yamamoto, T.; Hamada, Y.; Ichikawa, M.; Kajiwara, H.; Mine, T.; Tsukamoto, H.; Takakura, Y. Glycobiology 2007, 17, 1167-1174. [4] Knorst, M.; Fessner, W.-D. Adv. Synth. Catal. 2001, 343, 698-710. [5] Endo, T.; Koizumi, S.; Tabata, K.; Ozaki, A. Glycobiology 2000, 10, 809-813. ________________ [1] Rale, M.; Schneider, S.; Sprenger, G. A.; Samland, A. K.; Fessner, W.-D. Chem. Eur. J. 2011, 17, 2623-2632 [2] Samland, A. K.; Rale, M.; Sprenger, G. A.; Fessner, W.-D. ChemBioChem 2011, 14, in press (DOI: 10.1002/chem.201002942) [3] (a) Castillo, J. A.; Calveras, J.; Casas, J.; Mitjans, M.; Vinardell, M. P.; Parella, T.; Inoue, T.; Sprenger, G. A.; Joglar, J.; Claps, P. Org. Lett. 2006, 8, 6067-6070; (b) Garrabou, X.; Castillo, J. A.; Guerard-Helaine, C.; Parella, T.; Joglar, J.; Lemaire, M.; Clapes, P. Angew. Chem., Int. Ed. 2009, 48, 5521-5525; (c) Castillo, J. A.; Guerard-Helaine, C.; Gutierrez, M.; Garrabou, X.; Sancelme, M.; Schuermann, M.; Inoue, T.; Helaine, V.; Charmantray, F.; Gefflaut, T.; Hecquet, L.; Joglar, J.; Clapes, P.; Sprenger, G. A.; Lemaire, M. Adv. Synth. Catal. 2010, 352, 1039-1046.
Figure 1. Catalytic formation of D-glucose ________________

[1] Prebiotic carbohydrate synthesis: zinc proline catalyzes direct aqueous aldol reactions of -hydroxy aldehydes and ketones. J. Kofoed, J.-L. Reymond, T. Darbre, Org. Biomol. Chem. 2005, 3, 1850-1855. [2] Dual Mechanism of Zinc-Proline Catalyzed Aldol Reactions in Water. J. Kofoed, T. Darbre, J.-L. Reymond, Chem. Commun. 2006, 1482-1484. [3] Artificial aldolases from peptide dendrimer combinatorial libraries. J. Kofoed, T. Darbre, J.-L. Reymond, Org. Biomol. Chem. 2006, 3268-3281. [4] Enzyme Assays. J.-L. Reymond, V. S. Flux, N. Maillard, Chem. Commun. 2009, 34-46.
31
Sialic acid synthases from Campylobacter jejuni for the enzymatic synthesis of sialoconjugates
Mark Schnellbcher, Wolf-Dieter Fessner* Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersenstrae 22, 64287 Darmstadt, Germany E-mail: schnellbaecher@mail.oc.chemie.tu-darmstadt.de Sialic acids are a family of acidic 9-carbon monosaccharides that represent one of the most important constituents of cell-surface glycoconjugates in vertebrates, where they mediate recognition phenomena in physiological events, including cell-cell adhesion and intercellular communication processes, blood coagulation, fertilization, or cellular development. However, they are also of importance in the pathogenesis of a variety of diseases, including inflammatory disease, cancer metastasis, and bacterial or viral infection. Sialylated glycoconjugates are commerically significant for the synthesis of novel pharmaceuticals. The pathogenic microorganism Campylobacter jejuni has three genes encoding for PEPdependent sialic acid synthases (SiaS).[1,2] NeuB1-3 genes from C. jejuni coding for SiaS were cloned and heterologously expressed in E. coli. The substrate specificity of these SiaS enzymes was investigated using a broad range of sugar acceptors.[3] The synthetases have been applied to the stereoselective preparation of novel sialic acid analogs, and the latter can be activated using the CMP-sialate synthetase (CSS) from Neisseria meningitidis[4] and transferred to furnish the corresponding sialylated glycoconjugates in high yields.
32
Straightforward enzymatic construction of various aldoses from the most simple building blocks
Anna Szekrnyia, Xavier Garraboua, Alejandro Caicedob, Jordi Bujonsa, Pere Clapsa a Catalonia Institute for Advanced Chemistry, CSIC, Jordi Girona 18-26, 08034, Barcelona Spain bUniversity of Barcelona, Gran Via de les Corts Catalanes 585, 08007 Barcelona Spain E-mail: aszqbm@cid.csic.es In nature the sugar formation is achieved by a diversity of enzymes and often, chiral building blocks, however in preparative chemistry obtaining the same carbohydrates is also expeditious. Here we present a straightforward alternative, for the preparation of unprotected aldoses of various size by biocatalytic aldol reactions. In recent years preparation of carbohydrates by aldol reactions have gained considerable attention. The first protected hexoses were prepared by consecutive organocatalytic and metal-catalyzed aldol additions [1]. Recently our group discovered that D-fructose-6-phosphate aldolase (FSA) readily uses glycolaldehyde as donor substrate for the aldol reaction thus, it is available to synthesise aldose sugars [2]. During our most recent work with FSA, a number of mutants were obtained and with this library and using achiral building blocks, three- to six-carbon aldoses were synthesised. The selection of the enzyme plays a crucial role in the control of the number of glycolaldehyde additions, obtaining L-glyceraldehyde and D-threose by the wild-type FSA and L-xylose and D-idose by the engineered enzymes FSA A129G and FSA A129G S166G. Owing to the stereochemical promiscuity at the C3 of the mutant FSA A165G it was also possible prepare the L-glucose. Interestingly it is not only possible to vary the size but also the stereochemistry of the obtained carbohydrates.
35
Application of transaminases for the synthesis of pharma intermediates and materials science products
Martin Schrmanna, Stefan Turkb, Axel Trefzerb, Monika Mllera, Elly Raemakersa, Oliver Maya a DSM Innovative Synthesis, Urmonderbaan 22, NL-6167 RD Geleen, The Netherlands; b DSM Biotechnology Center, Alexander Fleminglaan 1, NL-2600 MA Delft , The Netherlands E-mail: martin.schurmann@dsm.com Royal DSM N.V. is a global life science and material science company actively developing sustainable processes for production of chemical building blocks and intermediates from renewable resources. As an industry leader in sustainability DSM takes a multidisciplinary approach including chemo- and biocatalysis, organic synthesis and metabolic engineering. According to this approach, the sustainability of process concepts and designs are evaluated early on to focus development on the most cost-efficient and sustainable option. In this presentation we will highlight aspects of biocatalyst identification, strain development and reaction engineering in the field of transaminase technology (figure 1) will be highlighted. This will be illustrated with examples of typical active pharmaceutical ingredients intermediates and using nylon-6 as an example. Caprolactam is the single building block for nylon-6 and does not occur in nature. We will share our results on identification and application of transaminases in routes towards the fermentative production of 6-amino-caproic acid as an alternative monomer for the production of nylon-6. The identification of novel (R)-selective transaminases, screening and application of transaminases for the synthesis of chiral amines as intermediates of active pharmaceutical ingredients will be exemplified in the second part of this presentation.
36
Regeneration of oxidized form of coenzyme with addition of co-substrate
Martina Sudar, Zvjezdana Findrik, Ana Vrsalovi Preseki and ura Vasi-Raki University of Zagreb, Faculty of Chemical Engineering and technology, Savska c. 16, 10000 Zagreb, Croatia, E-mail: dvracki@fkit.hr Dehydrogenases are enzymes, which need so called "free" coenzyme for their catalytic activity. The coenzyme acts as a second substrate in the catalytic reaction [1]. These biocatalysts are used to obtain chiral products such as amino acids [2] or alcohols [3], from their non-chiral precursors, as well as to obtain aldehydes in the first step of the cascade reactions to obtain chiral 2-hydroxy ketones or precursors for iminocyclitols. To use the coenzyme economically and successfully in the biocatalytic reaction, which also implies shifting of the reaction equilibrium towards formation of the product; it is necessary to regenerate the coenzyme in situ. From the literature [1] that describes a range of methods of coenzyme regeneration, it is clear that the problem of regeneration of oxidized form of coenzyme (NAD+) is still not successfully resolved. This study used a method of regeneration of NAD+ by adding co-substrate in the reaction medium. Acetone was used as a cosubstrate. As a model reaction oxidation of hexanol was carried out.
Figure 1.Biocatalytic cascade for the efficient synthesis of novel sialylated glycoconjugates (SiaS = sialic acid synthase; CSS = CMP-sialate synthetase; SiaT = sialyltransferase)
________________ [1] Linton, D.; Karlyshev, A. V.; Hitchen, P. G.; Morris, H. R.; Dell, A.; Gregson, N. A.; Wren, B. W. Mol. Microbiol. 2000, 35,1120-1134. [2] Chou, W. K.; Dick, S.; Wakarchuk, W. W.; Tanner, M. E. J. Biol. Chem. 2005, 280, 35922-35928. [3] Sundaram, A. K.; Pitts, L.; Muhammad, K.; Wu, J.; Betenbaugh, M.; Woodard, R. W. Biochem. J. 2004, 383, 83-89. [4] Knorst, M.; Fessner, W.-D. Adv. Synth. Catal. 2001, 343, 698-710.
Figure 1. General scheme of transaminase reactions.
Alcohol dehydrogenase (YADH), which was isolated from baker's yeast, was used as a biocatalyst. This enzyme needs coenzyme: nicotinamide adenine dinucleotide NAD(H) for its catalytic activity. The enzyme was isolated by the method described in the literature [4] and kinetically characterized by initial reaction rate method. The kinetic parameters were estimated from the experimental data for this enzyme using nonlinear regression. According to the estimated kinetic parameters, it can be concluded that the reaction product: hexanal significantly inhibits the reaction rate and acts as inhibitor of the enzyme. Using experimental data, a formal kinetic model and the model of batch reactor were developed. Using the model, the process in the batch reactor was simulated, and the amount of biocatalyst required for the implementation of reaction was estimated. Experimental results of the implementation of biocatalytic oxidation of hexanol are in good agreement with the results of simulation of processes by model, which indicates the validity of the model and estimation of kinetic parameters. Oxidation of hexanol was carried out in a reactor volume of 10 ml, temperature 30 C and pH 8.8 in 0.1 M pyrophosphate buffer.
________________ References: [1] Wichmann R., Vasic-Racki D., Cofactor Regeneration at the Lab scale, Adv. Biochem. Biotechnol, 2005, 92: 225-260. [2] Findrik Z., Vasic-Racki D., Biotechnol. Bioeng., 2007, 98: 956-967. [3] Findrik Z., Vasi-Raki D., Ltz S., Daumann T., Wandrey C., Biotechnol. Lett., 2005, 27: 10871095. [4] Racker E., J. Biol. Chem., 1950, 184: 313-319
Figure 1. Aldoses prepared by FSA variants

________________ [1] Northup, A. B.; Macmillan, D. W. C. Science 2004, 305, 1752. [2] Garrabou, X.; Castillo, J. A.; Gurard-Hlaine, C.; Parella, T.; Joglar, J.; Lemaire, M.; Claps, P. Angew. Chem. Int. Ed. 2009, 48, 5521.
October 2-6, 2011
80 37
Economic Evaluation of -transaminase-based processes
Joana Lima-Ramos, Pr Tufvesson and John M. Woodley Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU). DK-2 800 Kgs. Lyngby, Denmark E-mail: jlr@kt.dtu.dk Several biocatalytic processes, such as those catalysed by -transaminases (-TAm), have emerged as an attractive alternative for chemical synthesis, due to high enantioselectivity, the possibility to tailor the biocatalyst properties using molecular biology tools and a potential green profile [1]. Nevertheless, as a relatively new technology, many of the proposed transaminase processes do not fulfil the required economic metrics [1, 2]. In fact, many of the potentially attractive biocatalytic processes entail one or more challenges that need to be overcome to make the technology more widely applicable. While several different solutions have been suggested to overcome these challenges [1], the success of a biocatalytic process ultimately depends on the economic profile of the process. Among the challenges encountered for the biocatalytic synthesis of chiral amines using transaminases, the most important ones that might strongly determine process success are: unfavourable equilibrium, low substrate solubility (affecting the substrate and downstream cost), low activity, and low stability of the biocatalyst as well as product (affecting the downstream, substrate, reactor and catalyst cost), and substrate and product inhibition (affecting the catalyst cost). Based on the process requirements and constraints, it is possible to build operating windows, which represent information concerning the operating conditions. Early stage economic analysis is a powerful tool to evaluate and select the most suitable solution(s) dependent on the desired product and the corresponding challenges. The work presented here shows how economic analysis can be used for selection of technologies and configurations in the development of biocatalytic transamination processes. An economic analysis of the different solutions (that are able to meet the defined operating conditions) will determine the optimal process strategy/design. This approach allows a fast and effective comparison of different process choices, in addition to process debottlenecking, based on an iterative approach.
________________ [1] Tufvesson P, Lima-Ramos J, Jensen JS, Al-Haque N, Neto W, Woodley JM. (2011) Process considerations for the asymmetric synthesis of chiral amines using transaminases. Biotech Bioeng 108 (7): 1479-1493. [2] Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM. (2011). Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Proc Res Dev 15(1): 266274

Immobilization of Acid Phosphatase (PhoN-Sf) and Aldolase (RAMA) and Use in a Two-Enzyme Cascade.
A. F. Hartog, L. Babich and R. Wever Vant Hoff Institute for Molecular Sciences, University of Amsterdam, Nieuwe Achtergracht 129,1018 WS Amsterdam, The Netherlands. PhoN-Sf and RAMA-aldolase (Sigma) were immobilized on Immobead-150 (Sigma) or Sepabead-EC-EP (gift from Resindion s.r.l.) and the beads were compared for their binding efficiency, stability of enzymes and use in synthesis. The rate of binding of the PhoN-Sf to the two types of beads differed but after 24 hours the immobilization was complete. More than 98% was bound and the activity on the beads was about 70% compared to that of the free enzyme. Beads remained active for many months. A small column (0.5 ml) was packed with the beads and it was used for the continuous production of glucose-6-phosphate (G6-P) from glucose and pyrophosphate. It was possible to isolate G-6-P (32 g from 800 ml containing 120 mM G-6-P ) as a barium salt [1]

Combined metal and enzyme catalyzed transformations of thioesters to alcohol in water
Moumita Koley, Micheal Schnuerch, Marko D. Mihovilovic Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, 1060 Vienna, Austria. mkoley@ias.tuwien.ac.at Enzyme and metal catalyzed reactions are two very important transformations in synthetic chemistry.1,2 Bioctalysis often offers an environmentally friendly alternative to chemical transformations in combination with excellent chemo-, regio- and stereoselectivity. Transition metal catalyzed cross-coupling reactions had an immense impact on C-C bond formation in organic synthesis which was recently recognized with the noble prize in chemistry 2010. Palladium (0) catalyzed, copper(I)-mediated desulfitative LiebeskindSrogl (L-S) cross-coupling reactions are one of the recent addition to the long list of transition metal catalyzed reactions.2 The combination of both in a one pot protocol is often limited due to complementary reaction conditions, inhibitory effects of metal catalysts and poor solvent stability of enzymes. In this study we present the formation of benzyl alcohols starting from thioesters by a Liebeskind-Srogl (L-S) cross coupling reaction and a subsequent enzymatic reduction (Scheme1).
O Ar/R + S Ar/R X B(OH)2 Pd(PPh3)4 + CuTc water, 50oC, 18h O Ar/R OH Enzyme X Ar/R X
81 42
Chemo-enzymatic synthesis of new chiral C2-symmetric bipyridyls and bipyridyls N,N-dioxide alcohols
Claudia Sanfilippo and Giovanni Nicolosi Istituto di Chimica Biomolecolare del CNR, Via Paolo Gaifami 18, I-95127 Catania, Italy E-mail: claudia.sanfilippo@icb.cnr.it Optically active C2-symmetric 2,2-bipyridines and their related N,N-dioxides bearing stereogenic carbons in the side chains have recently attracted great interest in organic and coordination chemistry.[1] The access to such derivatives in optically active form has been mostly achieved by chiral pool approach or asymmetric synthesis, but a valuable alternative route could rely on kinetic resolution of racemic bipyridine frameworks. In this context, lipase catalysed acylation could provide useful tool for the preparation of enantiopure bipyridine alcohols, as an extension of well-known biocatalysed resolution of phenyl substituted alkanols. However, few data are available on resolution or desymmetrization processes applied to racemic or meso- bipyridine having secondary hydroxy groups in side chain. As part of our continuous interest in the study of lipase behaviour concerning the stereorecognition of bipyridines and phenanthroline derivatives with central and/or axial chirality,[2] we have focused our attention on the lipase catalysed kinetic resolution of racemic C2-symmetric 6,6-bis(2-hydroxypropyl)-2,2-bipyridine 2, obtained as an inseparable 1:1 mixture of dl- and meso-forms by metallation and treatment with acetaldehyde of commercially available 3,3-dimethyl-2,2-bipyridine. The esterification of the whole stereoisomeric mixture with vinyl acetate as acyl donor in presence of immobilised Mucor Miehei lipase (Lipozyme IM) has resulted in the selective transformation of the alcoholic functions on the stereogenic carbon having R configuration. This selective chiral recognition gave the simultaneous resolution of ()-2 and the desymmetrization of meso isomer, allowing the recovery of the diacetate (R,R)-4, monoacetyl derivative (R,S)-3 and unreacted alcohol (S,S)-2a, in high optical purities. The absolute configuration was confirmed by NMR spectroscopy. The enantiopure products obtained were then converted into the corresponding N,N-dioxide derivatives, whose stereochemistry and axial stability were investigated by dynamic circular dichroism spectroscopy.
One Pot Protocol
Scheme1: Combine metal and enzyme catalyzed transformation Figure 1. RAMA was also immobilized on the 3 different sorts of beads. In the assay with fructose-1,6-bisphosphate as substrate all 3 types of beads showed less than 1% of the original activity. To our surprise and in line with Suau et al. the immobilized aldolase was still active in the coupling of an aldehyde to dihydroxyacetone phosphate although the activity was decreased to about 30-40% of that of the soluble enzyme. Beads were used successfully in a fed-batch setup and in column setup at room temperature.
References 1. T. van Herk, A. F. Hartog, A. M. Van der Burg, R. Wever, Adv. Synth. Catal. 2005, 347, 1155-1126 2. T.Suau, G.Alvaro, M. Dolors Benaiges and J. Lopez-Santin. Biocatal.Biotransform. 2009, 27, 136-142
We will demonstrate the first Liebeskind-Srogl cross coupling reaction in water instead of THF or dioxane as solvent under mild reaction conditions. Furthermore we will show yeast a reductase (PIK3) for the enzyme catalyzed transformation of ketones to the corresponding alcohols that gave good yields and enantioselectivities. The dichloromethane extract of the L-S reaction, subsequent dilution with EtOH and water was used directly for the enzymatic reduction without further purification and gave full conversion to the corresponding alcohol. ______________
[1] Bommarius, A. S. and B. R. Riebel-Bommarius. Biocatalysis. Weinheim, Wiley-VCH. 2004. [2]. Diederich, F., Stang, P. J., Eds. Metal-Catalyzed Cross-Coupling Reactions; Wiley-VCH: Weinheim, 1998. [3] Liebeskind, L. S.; Srogl, J. J. Am. Chem. Soc. 2000, 122, 1126011261 [4] Kaluzana, I. A.; Feske, B. D.; Wittayanan, W.; Ghiviriga, I.; Stewart, J. D. J. Org. Chem. 2005, 70, 342-345.
____________
[1] H.-L. Kwong, H.L. Yeung, C.-T. Yeung, W.-S. Lee, C.-S. Lee, W.-L. Wong Coord. Chem. Rev. 2007, 251, 2188-2222 [2] Sanfilippo, G. Nicolosi Tetrahedron:Asymm 2008, 19, 2171-2176.
39
High-Throughput Screening of Transaminases
Simon Williesa, Kirk Malonea, Rehanna Aslama, Nick Turnera a School of Chemistry, Manchester Interdisciplinary Biocentre, 131 Princess Street, Univerisity of Manchester, M1 7DN, UK E-mail: Simon.Willies@manchester.ac.uk Transaminase (TA) enzymes are of great interest due to their potential for the synthesis of chiral amines, as pharmaceutical intermediates or high value chemicals. However, many of these products are not accepted by any known TA and the substrate specificity has to be altered via a process of directed evolution. Very few TAs have been evolved towards non-natural substrates mainly due to the lack of high-throughput screens available. The screens available are mainly based on liquid phase assays with a limited throughput. We have developed a solid phase screen able to assay thousands of TA mutants at once in a high-throughput manner, allowing directed evolution of transaminases to be carried out efficiently. Our screen is based on a liquid phase assay [1], utilising an amino acid oxidase (AAO). A D-AAO is co-expressed along with our TA mutant library and can be used to screen for (R)-selective TA activity (Fig. 1). To circumvent the problems associated with coexpressing an L-AAO (toxicity), we have also co-expressed a racemase with the D-AAO to allow (S)-selective TA libraries to be screened (Fig. 2).
40
Productivity enhancement of C=C bioreductions: coupling of isolated enoate reductases with the in situ substrate feeding product removal technology
Elisabetta Brennaa, Francesco G. Gattia, Daniela Montib, Fabio Parmeggiania, Alessandro Sacchettia a Politecnico di Milano, Dipartimento CMIC Giulio Natta, Via Mancinelli, 7, 20131, Milano, Italy; bIstituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco, 9, 20131, Milano, Italy. E-mail: fabio.parmeggiani@chem.polimi.it The bioreduction of prochiral activated C=C bonds is a valuable synthetic tool for the generation of optically pure synthones. The very first examples of such a reaction were carried out using Saccharomyces cerevisiae resting cells (bakers yeast).1 In the last decade, a great improvement in terms of conversion and simplicity of the work-up has been achieved using isolated enoate reductases (ERs), such as the OYEs (Old Yellow Enzymes), instead of whole microbial cells.2 However, these reactions typically suffer of low productivity, mainly due to the low substrate concentration employed in most experimental set-ups. We developed the combination of the in situ substrate feeding product removal (SFPR) concept with the use of isolated OYEs: when applied to the reduction of a set of different prochiral ,-unsaturated aldehydes 1 to the corresponding saturated products 2 (e.g. precursors of the active pharmaceutical ingredients Robalzotan, Rotigotine and Navaglitazar), this strategy leads to an outstanding enhancement of productivity (over two orders of magnitude). Moreover, even though aldehydes such as 1 and 2 are known to be intrinsically unstable compounds, 3 our system allows to preserve both the optical purity of the products and the chemical stability of the reagents. In the toughest case, the strategy has been further improved employing a cascade system with a selected alcohol dehydrogenase (ADH). The latter chemoselectively reduces the saturated aldehyde to the corresponding alcohol 4 in the presence of the unsaturated starting material, thus completely suppressing the spontaneous racemization.
b.y. or ERs+ADHs O H X ADH MeO OH X 3 X 4 MeO OH 1 ADH ERs MeO OYE2/OYE3 X 2 O H MeO
43
Cascade Systems in -Transaminase Reactions
Kreimir Janea, Krist V. Gernaeya, Pr Tufvessona, John M. Woodleya a Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), DK-2800, Kgs. Lyngby, Denmark; E-mail: kreja@kt.dtu.dk Production of chiral amines using transaminases has recently been reported as an interesting alternative to present organic synthesis methods. The chiral amines are building blocks for many new pharmaceuticals and two biocatalytic methods of production have been demonstrated: kinetic resolution and asymmetric synthesis. The latter approach has the advantage over existing biocatalytic and chemical methods that the theoretical yield is 100% compared to 50%. A major challenge of this approach is the unfavourable thermodynamic equilibrium which needs to be improved in order to meet the criteria of technical feasibility of biocatalytic process. This can be achieved by removing the product or coproduct formed. For example, if 2-propilyamine is used as amine donor, evaporation of formed acetone can be used. However, this approach is limited by the selectivity of the separation method. An alternative for shifting equilibrium is by degrading or recycling one of the reaction coproducts in-situ using additional enzymatic reactions. These cascade systems have been shown to be feasible at micro-litre scale, but still systems need to be characterized and be shown to be scalable and economically feasible in order to be industrially implemented. The four selected cascades systems to be investigated are lactate dehydrogenase (EC 1.1.1.27)/glucose dehydrogenase (EC 1.1.99.10), alanine dehydrogenase (EC 1.4.1.1)/glucose dehydrogenase (EC 1.1.99.10), pyruvate decarboxylase (EC 4.1.1.1) and acetolacetate synthase (EC 2.2.1.6). The aim of this work is to determine the effectiveness and constrains of these co-product degradation systems at process relevant conditions. An interaction matrix is suggested to describe the effects of the compounds involved in the process on the enzymes present in the reaction mixture. Further, the determination of kinetic parameters and establishment of kinetic mathematical model will help in future work of reactor selection and overall better understanding of this one-pot multi-enzyme system.
44
Lipase Catalysis in the Formation of Aromatic Heterocyclic Cyanohydrin Esters - a Dynamic Combinatorial Library
Riku Sundell, Michaela C. Turcu, Liisa T. Kanerva Institute of Biomedicine/Department of Pharmacology, Drug Development and Therapeutics/ Laboratory of Synthetic Drug Chemistry, University of Turku, FIN-20014, Turku, Finland E-mail: rhjsun@utu.fi The application of dynamic combinatorial chemistry (DCC) in the generation of dynamic combinatorial libraries (DCL) of small molecules has proved to be a valuable tool for research.1 In the present study, a DCL of aromatic heterocyclic thiophen-2-aldehyde cyanohydrins was exposed to a kinetic resolution resulting in an one-pot dynamic combinatorial resolution (DCR)2 as presented in figure 1. Dynamic kinetic resolution (DKR) for the formation of heteroaromatic cyanohydrin esters is thoroughly studied starting from the corresponding aldehydes and cyanide source in the presence of a base, an acyl donor and a lipase.3 We now describe the formation of a DCL based on the above mentioned DKR system staring with a mixture of five aldehydes 1(a-e), Amberlite IRA-904 as a base, acetone cyanohydrin as a source of cyanide and isopropenyl acetate in the presence of one of the lipases, Candida antarctica lipase B (Novozym 435), Pseudomonas fluorescens lipase (IMMAPF-T2-150) or Burkholderia cepacia lipase (lipase PS-D). A mixture of enantiomerically enriched cyanohydrin acetates was formed as the final products (e.e. 25-99 % depending on the structure of the cyanohydrin). The DCL was followed by chiral GC giving information about the conversions of the aldehydes to cyanohydrins and then to the acylated products while e.e.s of the intermediates and products could also be observed at the same time.
Figure 1. D-AAO linked transaminase screen for Rselective activityd
Figure 2. Racemase and D-AAO linked assay for detection of Sselective tansaminase activity
Figure 1. The DCL of aromatic heterocyclic cyanohydrin esters.

_________________ [1] Lehn, J.-M., Chem. Eur. J., 1999, 5, 2455-2463. [2] Sakulsombat, M.; Vongvilai, P. and Ramstrm, O. Org. Biomol. Chem., 2011, 9, 1112-1117. [3] Paizs, C.; Toa, M.; Majdik, C.; Thtinen, P.; Irimie, F.-D. and Kanerva, L. T. Tetrahedron: Asymmetry, 2003, 14, 619-627.; Paizs, C.; Thtinen, P.; Lundell, K.; Poppe, L.; Irimie, F.-D. and Kanerva, L. T., Tetrahedron: Asymmetry, 2003, 14, 1895-1904.; Paizs, C.; Thtinen, P.; Toa, M.; Majdik, C.; Irimie, F.-D. and Kanerva, L. T. Tetrahedron, 2004, 60, 10533-10540.
________________ [1] Hopwood et al. A fast and sensitive assay for measuring the activity and
________________ [1] (a) Servi, S. Synthesis 1990, 1; (b) Csuk, R.; Glnzer, B.I. Chem. Rev. 1991, 91, 49. [2] Toogood, H.S.; Gardiner, J. M.; Scrutton, N.S. ChemCatChem 2010, 2, 892. [3] Stueckler, C.; Mueller, N.J.; Winkler C.K.; Glueck S.M.; Gruber K.; Steinkeller G.; Faber K. Dalton Trans. 2010, 39, 8472.
October 2-6, 2011
82 45
Reactor selection for multi-enzymatic processes
Rui Xuea, Jrn D. Mikkelsenb, Anne S. Meyerb and John M. Woodleya Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark. Sltofts Plads Building 229, Kgs. Lyngby, Denmark b Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark. Sltofts Plads Building 229, Kgs. Lyngby, Denmark rxue@kt.dtu.dk
a

Stereoselective synthesis of sugar cyanohydrins by cascade chemoenzymatic oxidation-hydrocyanation process
Ari Hietanena, Liisa T. Kanervaa a Institute of Biomedicine/Department of Pharmacology, Drug Development and Therapeutics/Laboratory of Synthetic Drug Chemistry, University of Turku, FIN-20014 Turku, Finland E-mail: armahi@utu.fi Chiral cyanohydrins represent an important class of intermediates toward a diverse set of pharmaceuticals and other high-valued products while carbohydrates have emerged as invaluable moieties in biologically active compounds. Due to their high functional group and stereochemical density new and improved methods for stereo- and regioselective modification of such scaffolds are constantly needed. Our approach towards this end is based on the effective combination of chemo- and biocatalysis in a cascade-type reaction resulting in the selective hydrocyanation of carbohydrate derived aldehydes. Recently, we have published our results on the hydrocyanation of monosaccharide derivatives containing carbaldehyde moities at the anomeric center [1]. Oxidation of the allylated precursor and biocatalytic hydrocyanation were highly efficient in yield, whereas stereoselectivity was obtained via diastereoselective lipase catalyzed kinetic resolution (Scheme 1a). The whole process was performed without intermediate purifications, making the overall synthetic sequence efficient, high-yielding and selective. We have now engaged into developing a similar synthesis toward the hydrocyanation of the non-reducing end of selected monosaccharides (Scheme 1b). The close proximity of the reaction site with the sugar ring and its acetal group provides unique challenges and opportunities compared with our previous project and a further development of chemo- and biocatalytic methods needs to be succeeded in to obtain the target compounds.

In situ aldehyde generation for aldol addition reactions catalyzed by Dfructose-6-phosfate aldolase
Maria Mifsuda, Anna Szekrnyia, Jesus Joglara, Pere Clapsa a Biotransformation and Bioactive Molecules Group. Instituto de Qumica Avanzada de Catalua (IQAC),Consejo Superior de Investigaciones Cientficas (CSIC), Jordi Girona 18-26, 08034, Barcelona, Spain. E-mail: mmgqbm@cid.csic.es The need of alternative processes for the industrial synthesis of chemicals that are cleaner, safer and environmentally friendly represents one of the major challenges of this century. Particularly in the production of pharmaceuticals or agrochemicals, where the waste generation can surpass 100kg/kg product.[1] In this regard one-pot procedures involving multiple catalytic events have the great advantage that the desired product can be prepared in a single reaction solvent, workup procedure and purification step.[2] Enzymatic reactions are carried out under mild conditions in aqueous media. For that reason the combination of both concepts cascade and biocatalysts will have an environmentally benefit in the chemical production methodology. Synthetic applications of FSA were recently reported demonstrating that it is a robust and synthetically useful catalyst with a great potential for highly steroselective aldol additions of dihydroxyacetone (DHA), hydroxyacetone (HA), hydroxybutanone (HB), and glycolaldehyde (GO) to a large variety of aldehydes. [3] [4]
83 50
From alcohols to amines employing cascade-reactions
Katharina Taubera, Michael Fuchsa, Johann Sattlera, Jan Pfefferb, Wolfgang Kroutila a University of Graz, Heinrichstra 28, 8010 Graz, Austria; bEvonik Degussa GmbH, PaulBaumann-Strae 1, 45772 Marl, Germany E-mail: katharina.tauber@edu.uni-graz.at Optically pure amines are key intermediates required for the preparation of a broad range of biologically active compounds. For example (R)-4-phenylbutan-2-amine is a precursor for dilevalol [1], an anti-hypertensive, while 1-phenyl-1-propylamine is the precursor of a potent anti-depressive agent [2]. Although the formation of chiral amines can be accomplished by several chemical routes, enzymatic pathways offer many advantages such as mild reaction conditions, high enantiomeric excess and economical viability. So far amines have often been prepared from ketones applying -transaminase (-TA) and different cofactor recycling systems [3]. Herein we are starting from racemic alcohols thereby combining the alcohol dehydrogenase (ADH) catalysed formation of ketones with the amination catalysed by -TAs in a cascade reaction. For this cascade (figure 1) only three enzymes are required. The NAD+ consumed by the ADH in the oxidation step is directly regenerated by the alanine dehydrogenase (Ala-DH), which recycles the formed pyruvate to the L-alanine co-substrate and thereby consuming ammonium. Since no redox-reagents are consumed this represents a redox-neutral cascade. If NADP+ is the preferred cofactor for the ADH, two separate cofactor regeneration systems were applied. For the oxidation step the flavincontaining oxidoreductase YcnD from Bacillus subtilis [4] was used for NADP+ recycling and for the transamination the Ala-DH in combination with formate/FDH- recycling system was employed.
In recent years, biocatalysis has been providing a unique stereoselective and green tool in synthetic organic chemistry. Single enzymes, either soluble or immobilized, have been used in many different reactions [1]. Currently, the idea of using multi-enzymatic systems for industrial production of chemical compounds becomes more and more attractive [2]. Multi-enzymatic processes use two or more enzymes to catalyse reactions in a defined pathway via a cascade, a parallel, or a network configuration [3]. Such schemes overcome many of the conventional problems with integrating biocatalysis such as media, temperature and pH swaps. One of the key issues in the development of an enzymatic process is the selection of the reactor(s) [4, 5]. Multi-enzymatic process contains more than one reaction steps. Thus, the choices and combinations of types of catalyst and reactor are much more than those for a single enzymatic process. It is necessary therefore to attempt a classification and analysis of these reactor types to facilitate the selection of the correct type of the reactor for a sucessful multi-enzymatic process. In this presentation, an analysis applying the approach of reaction engineering to evaluate reactors for multi-enzymatic processes has been carried out. The evaluation considers the kinetic constraints, the catalyst constraints and the reaction medium. These factors determine the type and the operational mode of the reactor. The results indicate the optimal choices of the reactors which can make the best use of the available enzymes.
________________ [1] P. Claps, W.-D. Fessner, G.A. Sprenger, A.K. Samland, Recent progress in stereoselective synthesis with aldolases, Current Opinion in Chemical Biology, 14 154-167. [2] P.A. Santacoloma, G. Sin, K.V. Gernaey, J.M. Woodley, Multienzyme-Catalyzed Processes: NextGeneration Biocatalysis, Organic Process Research & Development, 15 (2010) 203-212. [3] A. Cornish-Bowden, Fundamentals of Enzyme Kinetics, 3rd Edition ed., Portland Press 2004. [4] J.M. Woodley, M.D. Lilly, Biotransformation reactor selection and operation, in: J.M.S. Cabral, D. Best, L. Boross, J. Tramper (Eds.) Applied Biocatalysis, Harwood Academic, 1994, pp. 371. [5] M.D. Lilly, P. Dunnill, Biochemical reactors, Process Biochemistry, (1971) 29-32.
Figure 1. Synthetic iminosugars strategy Usually, the aldehyde required for the asymmetric aldol reaction was produced in previous steps. The aim of this work is develop an useful oxidation method for the in situ generation of the acceptor aldehyde required for the aldol reaction. Scheme 1. Chemoenzymatic synthesis of the diastereomers of monosaccharide derived cyanohydrins functionalized at a) the reducing (R = allyl) and b) non-reducing end (R = Me).
________________ [1] A. Hietanen, F. S. Ekholm, R. Leino, L. T. Kanerva, Eur. J. Org. Chem. 2010, 6974-6980. This work was performed in the frame of the COST Action CM0701. ________________ [1] R. A. Sheldon, Journal of Molecular Catalysis A: Chemical 1996, 107, 75. [2] K. C. Nicolaou, D. J. Edmonds, P. G. Bulger, Angewandte Chemie International Edition 2006, 45, 7134. [3] A. L. Concia, C. Lozano, J. A. Castillo, T. Parella, J. Joglar, P. Claps, Chemistry A European Journal 2009, 15, 3808. [4] X. Garrabou, J. A. Castillo, C. Gurard-Hlaine, T. Parella, J. Joglar, M. Lemaire, P. Claps, Angewandte Chemie International Edition 2009, 48, 5521.
Figure 1. Formation of amines via biocatalytic cascade.

________________ [1] J.E. Clifton, I. Collins, P. Hallett, D. Hartley, L.H.C. Lunts, P.D. Wicks, J. Med. Chem. 1982, 25, 670 679. [2] J.W. Corbett, M.R. Rauckhorst, F. Qian, R.L. Hoffman, C.S. Knauer, L.W. Fitzgerald, Bioorg. Med. Chem. Lett. 2007, 17, 6250 6256. [3] D. Koszelewski, I. Lavandera, D. Clay, G.M. Guebitz, D. Rozzell, W. Kroutil, Angew. Chem., Int. Ed. 2008, 47, 9337 9340. [4] A. Morokutti, A. Lyskowski, S. Sollner, E. Pointner, T. B. Fitzpatrick, C. Kratky, K. Gruber, P. Macheroux, Biochemistry 2005, 44, 13724-13733.
47
Smart design of solid multi-enzymatic systems for cofactor recycling
Fernando Lpez-Gallegoa, Javier Rocha-Martna* and Jos Manuel Guisana. a Biocatalysis department. Institute of Catalysis (CSIC). *javirocha@icp.csic.es The assembling of enzymes applied to step-wise catalysis is one of the most attractive topics for the production of many compounds at industrial level [1]. However, ex-vivo implementation of assembled multi-enzyme system is much more challenging. The major difficulty to make multi-enzyme systems to work lies on the different reaction conditions of each catalytic step which often are incompatible. The nature has solved this issue by compartmentalizing reaction pathways alongside the exquisite selectivity of the enzymes. However, efficient in vitro compartmentalization is not such trivial, thus researchers are encouraged to find new ways of assembling different catalytic modules in the same biocatalyst. Here, protein immobilization, in addition to its well known advantages for improving enzyme performance [2], may play a crucial role to open a new avenue aiming the design of smartly controlled immobilization patterns for enzyme assembling. The nanotechnology has been developing this concept in nanostructures; nevertheless they present enormous limitations for their industrial application in synthetic processes. Solid porous support like agarose, epoxy-acrylic resins or chitosans, would be a promising alternative since their surface can be tailor-made functionalized and unlike nanostructures they are much more cost-effective and manageable at industrial scale. Here, as a proof of the concept we present the smart co-localization by tailor-made immobilization of three different biological redox systems with their corresponding nicotinamide cofactor recycling. In all cases, the co-immobilization strategy was specifically designed in base to activity and stability requirements of each system. In all cases, the co-immobilized catalysts under very low cofactor concentration (0,5M) recycled the cofactor up to 15-fold more efficient than when the two enzymes were separately immobilized in two catalysts. It would mean that the immobilized system in two catalysts would need 40 times more reclycing enzyme per mg of main enzyme to achieve the same recycling efficiency of a co-imobilized system with the same amount of both main and recycling enzyme. Finally, we have also demonstrated with labeling studies and confocal microscopy analysis that protein distribution inside the support pores affected to the recycling efficiency, being observed that more homogeneous distribution of both main and recycling enzymes was 1.6-fold more efficient that heterogeneous distributions. Therefore, we have experimentally proofed that the coordinated spatial assemble of several catalytic domains can increase the efficiency of synthetic routes. ______________________
[1]. Lpez-Gallego, F. and Schmidt-Dannert, C. Curr. Op. Chem Biol. 2010. 14.174-183. [2]. Mateo et al. Enzyme Microb. Technol. 2007. 40. 1451-1463.
48
Application of alcohol dehydrogenases in chemoenzymatic tandem processes starting from ,-dihalogenated acetophenones
Kinga Kdziora,a Fabricio R. Bisogno,a Barbara Grischek,b Ivn Lavandera,a Vicente Gotor-Fernndez,a Wolfgang Kroutil,b Vicente Gotora
a b
51
One-pot enzymatic oxidation and aldol addition for the synthesis of aminopolyols from aminoalcohols: peroxidase vs. alcohol dehydrogenase approaches
Milja Pei, Rossmery A. Rodrguez-Hinestroza, Marta Enrich, M. Dolors Benaiges, Carmen Lpez, Gregorio lvaro, Josep Lpez-Santn Department of Chemical Engineering, Applied Biocatalysis Unit associated to IQAC (UAB-CSIC). School of Engineering. Universitat Autnoma de Barcelona. 08193-Bellaterra (Cerdanyola del Valls), Catalunya, Spain E-mail: gregorio.alvaro@uab.es Aminopolyols are precursors of iminocyclitols, which have high therapeutic interest as glycosidase inhibitors. Those compounds can be synthesized from commercially available aminoalcohols by means of two sequential enzymatic reactions. The enzymes chloroperoxidase from Caldariomyces fumago (CPO) and horse liver alcohol dehydrogenase (HLADH) are able to oxidize aminoalcohols [1,2]. The enzyme rhamnulose-1-phosphate aldolase (RhuA) catalyzes the aldol addition between different aminoaldehydes and dihydroacetone phosphate (DHAP) to obtain the corresponding aminopolyols [3]. The aim of this work is the multienzymatic synthesis of aminopolyols from the aminoalcohol Cbz-ethanolamine by two sequential oxidation and aldol addition reactions and the comparison of the results obtained when oxidation is performed by chloroperoxidase or alcohol dehydrogenase. One key point is to direct oxidation to aminoaldehyde avoiding subsequent oxidation to non desired aminoacids. Before optimizing the coupled reaction catalyzed by CPO and RhuA, the individual reactions were studied separately in discontinuous operation, using both soluble and immobilized enzymes. The continuous addition of tert-butyl hydroperoxide was required to obtain a high yield of oxidation of Cbz-ethanolamine (over 70%) while preserving the enzymatic activity. The operational conditions for the one-pot multienzymatic reaction were selected as a compromise between the best operational conditions for both individual reactions. The use of stable immobilized instead of soluble enzymes allowed to increase the aminopoyol production from 22 to 76%, and to reuse the enzymatic derivatives. Moreover, different batch reactor configurations were studied, from a stirred tank reactor to fixed or fluidized bed reactors. When the oxidation of Cbz-ethanolamine was catalyzed by HLADH, the addition of semicarbazide was required in order to trap the Cbz-glycinal and prevent its oxidation to Cbz-glycine. In these conditions, the formation of semicarbazone-glycinal yielded 70%. Working in one-pot reactor for coupled oxidation and aldol addition resulted in the formation of aminopolyol compound. Apart from reaction yields, other factors such as enzyme requirement, ease of operation, enzyme stability and reuse were analyzed to compare both oxidation systems.
52
Acylpeptides as biosurfactants: Enzymatical synthesis and acylation of peptides by a cascade reaction
M. Andrea, C. Syldatka, J. Rudata a KIT Institute of Process Engineering in Life Sciences, Section II: Technial Biology, EnglerBunte-Ring 1, 76131 Karlsruhe, Germany E-mail: markus.andre@kit.edu Acylpeptides represent a group of biosurfactants of increasing interest for laundry and cosmetic applications. Because of its similarity to the skins surface structure, these peptides can be used as additives to increase the skin compatibility of otherwise irritating surfactants [1]. Our group already established the enzyme-catalysed synthesis of oligopeptides by cysteine proteases in a kinetically controlled approach according to Uyama et al. [2]. These oligopeptides consist of one amino acid species, resulting in the formation of homooligopeptides or of two different amino acids leading to heterooligopeptides. Depending on the kind of amino acids which are used in the synthesis the oligopeptides differ in chain length and water solubility. In a second step, biocatalysts will be used to couple these oligopeptides to fatty acids. We started screening for different enzymes able to carry out acylation of amino acid derivatives. Further we are testing the influence of the chain length of the fatty acids on the acylation efficiency.
Department of Organic and Inorganic Chemistry, University of Oviedo, Oviedo, Spain Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Graz, Austria E-mail: kedziorakinga@uniovi.es To gain access to valuable chiral intermediates in the most convenient way possible, a constant effort for developing new asymmetric methodologies that would meet the demanding criteria regarding green chemistry and atom economy is being made. Thus, chemoenzymatic cascade/tandem reactions that avoid the isolation of intermediates, perfectly fit into the requirements of the industrial sector. 1 In this area, the use of alcohol dehydrogenases (ADHs) represents an elegant approach to obtain enantiopure alcohols and if used with highly reactive dihalogenated compounds, the scope of potential intermediates can become significant. Encouraged by previous results obtained in our research group applied to the bioreduction of -halogenated ketones, 2 we have decided to extend our studies synthesizing and reducing ,-dihalogenated acetophenone derivatives by means of available ADHs (Figure 1).
________________ [1] Lang, S., Biological amphiphiles (microbial biosurfactants). Current Opinion in Colloid & Interface Science, 2002. 7(1-2): p. 12-20. [2] Uyama, H., et al., Protease-catalyzed regioselective polymerization and copolymerization of glutamic acid diethyl ester. Biomacromolecules, 2002. 3(2): p. 318-23.
Figure 1. Bioreduction of ,-dihalogenated ketones and possible chemical applications of the ,-dihaloalcohols obtained. Results obtained so far indicate not only excellent conversion and stereoselectivity for the bioreduction of bulky ketones but also remarkable diastereoselectivities in the production of -bromo--chloro alcohols. Furthermore, an oxidation study with the so-obtained ,dihalo alcohols is currently being developed to achieve the corresponding mandelic acid derivatives in a chemoenzymatic tandem process.
__________________ [1] J. H. Schrittwieser, J. Sattler, V. Resch, F. G. Mutti, W. Kroutil, Curr. Opin. Chem. Biol. 2011, 15, 249-256. [2] a) F. R. Bisogno, I. Lavandera, W. Kroutil, V. Gotor, J. Org. Chem. 2009, 74, 1730-1732; b) F. R. Bisogno, E. Garca-Urdiales, H. Valds, I. Lavandera, W. Kroutil, D. Surez, V. Gotor, Chem. Eur. J. 2010, 16, 11012-11019.
________________ [1] E. Kiljunen, L.T. Kanerva. Chloroperoxidase-catalysed oxidation of alcohols to aldehydes. Journal of Molecular Catalysis B: Enzymatic 9 (2000) 163-172. [2] L. Andersson, R. Wolfenden. A general method of -aminoaldehyde synthesis using alcohol dehydrogenase. Analytical Biochemistry 124 (1982) 150-157. [3]I. Ardao, T. Suau, J. Ruiz, S.D. Ros, G. lvaro, G. Caminal, M.D. Benaiges, G. Gonzlez, J. LpezSantn. DHAP-dependent aldolases in stereoselective synthesis: kinetic studies and immobilization. Journal of Biotechnology 131 (2007) S75.
October 2-6, 2011
84 53
One-pot reactions for the synthesis of non-natural carbohydrates: optimization by three different approaches.
Lara Babicha, Lieke van Hemertb, Aleksandra Burya, Louis Hartoga, Floris P. J. T. Rutjesb, Ron Wevera a Van t Hoff Institute for Molecular Sciences, University of Amsterdam, Science Park 904, 1090 GD Amsterdam, the Netherlands; b Radboud University Nijmegen, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, the Netherlands E-mail: l.babich@uva.nl Dihydroxyacetone phosphate (DHAP) is a valuable compound which is used in aldol reactions catalyzed by DHAP-aldolases resulting in a wide range of optically pure carbohydrates. DHAP can be synthesized from DHA with cheap PPi as phosphate donor using non-specific acid phosphatases (PhoN). DHAP can be coupled by aldolases to various aldehydes in a two-enzyme one-pot cascade reaction. The resulting phosphorylated sugar is then dephosphorylated by the phosphatase already present in the reaction, thus directing the thermodynamic equilibrium of the overall cascade (fig. 1) to the final product.[1] The advantage of this route is that many aldehydes can be used since the aldolases have a broad substrate tolerance. Moreover because all four DHAP-dependent aldolases are available the chirality of each chiral carbon center in the product can be steered with high stereoselectivity. With this method conversions up to 60% are easily obtained. The conversion has been increased to 100% by using three different approaches. By directed evolution of the acid phosphatase, the mutant PhoN-Se V78L gave 92% yield of aldol product.[2] High yields (>95%) have also been obtained in a fed batch and a continuous system with PhoN-Sf and aldolase immobilized on beads. This system is particularly advantageous when scalingup is required and reuse of valuable enzymes is essential. Quantitative yields were also obtained via a four-enzyme one-pot cascade reaction, in which DHAP is obtained via phosphorylation of glycerol and consequent oxidation by L-glycerol-3-phosphate oxidase (GPO, in combination with catalase). This reaction is cleaner and more efficient than the DHA cascade, because of the kinetic properties of the first phosphorylation step. The synthetic value of this catalytic cascade was demonstrated via a one-pot synthesis of the naturally occurring azasugar D-fagomine starting from glycerol and via the use of four different aldolases to obtain all the stereocomplementary products.

Rhamnulose and Fuculose Aldolases mediated synthesis of nitrocyclitols
Flora Camps-Bresab, Christine Gurard-Hlaineab, Carlos Fernandesab, Israel SnchezMorenoc, Eduardo Garca-Juncedac, Marielle Lemaireab* a Clermont Universit, Universit Blaise Pascal, Laboratoire SEESIB, BP 10448, F-63000 Clermont-Ferrand, France. b CNRS, UMR 6504, SEESIB, F-63177 Aubire, France. c Departamento de Qumica Bio-Orgnica, Instituto de Qumica Orgnica General. CSIC. Juan de la Cierva, 3. 28006 Madrid, Spain marielle.lemaire@univ-bpclermont.fr We recently developed a new methodology to access to nitrocyclitols and aminocyclitols via the fructose-1,6-bisphosphate aldolase (FBA). The key step was performed in one pot involving two enzymes and three reactions and where two carbon-carbon bonds were formed with high stereoselectivity. Four stereocenters were created and an enantiomerically pure compound was isolated for each aldehyde tested.
Oxidative biocatalysis 57
Pichia pastoris as whole-cell biocatalyst for (+)-nootkatone production
Tamara Wriessneggera, Peter Augustina, Anita Emmerstorfera, Monika Mllerb, Iwona Kaluznab, Peter Macherouxa,d, Helmut Schwaba,c, Harald Pichlera,c a ACIB - Austrian Center of Industrial Biotechnology, Graz, Austria; bDSM Innovative Synthesis B.V., Geleen, The Netherlands; cInstitute of Molecular Biotechnology, Graz University of Technology, Austria; dInstitute of Biochemistry, Graz University of Technology, Austria E-mail: tamara.wriessnegger@acib.at Membrane-attached cytochrome P450 monooxygenases (CYPs) are versatile and industrially important enzymes, playing major roles in drug metabolism as well as in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Functional expression of recombinant P450 enzymes is often complicated by the association of the enzymes with membranous structures, the need for co-expression of a P450-reductase and the availability of co-factors (NAD(P)H). The methylotrophic yeast P. pastoris, with its strong, inducible AOX1 promoter was used for heterologous expression of H. muticus premnaspirodiene oxygenase (HPO; CYP71D55) in combination with a cytochrome P450 reductase (CPR) from A. thaliana [1]. The plant enzyme HPO has been shown previously to convert the sesquiterpene (+)-valencene, which is a natural aroma compound of citrus fruits, into the highly sought-after grapefruit flavor (+)-nootkatone. Co-expression of HPO and CPR in P. pastoris was monitored by CO-difference spectra measurements and activity assays, respectively. Wholecell biotransformation of (+)-valencene resulted in the production of trans-nootkatol, which was subsequently oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Thus, the yeast P. pastoris represents a valuable whole-cell system for the stereo- and regioselective hydroxylation of (+)-valencene and therefore, the production of (+)-nootkatone.
85 58
Understanding laccases stability and glycosylation effect by integrating computational and experimental studies
Marco Foscatoa, Alexey Chernykhc,d, Valerio Ferrarioa, Ludmila Golovlevad, Loris Sinigoib, Diana Fattora, Cynthia Eberta, Lucia Gardossia Dipartimento di Scienze Chimiche e Farmaceutiche, Universit degli Studi di Trieste. Piazzale Europa 1, 34127 Trieste, Italy SPRIN S.p.A., Technologies for Sustainable Chemistry, via Flavia 23/1, 34148 Trieste, Italy c ICS-UNIDO, AREA Science Park, Trieste, Italy d G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms of RAS, Russia E-mail: marco.foscato@phd.units.it
a b
Further developments of this methodology will be presented with two aldolases Rhamnulose Aldolase (RhaA) and Fuculose Aldolase (FucA). They catalyze C-C bound formation with respectively R,S and R,R stereochemistries. Depending on the aldehyde used, the two enzymes have shown different behaviour. RhaA was generally less diastereoselective than FucA.
[1]Fructose-1,6-bisphosphate Aldolase Mediated Synthesis of Aminocyclitols, Analogues of Valiolamine and Their Evaluation as Glycosidase Inhibitors L. El Blidi, Z. Assaf, F. Camps-Bres, H. Veschambre, V. Thry, J. Bolte and M. Lemaire* ChemcatChem, 2009, 7, 463-471.
________________ [1] Takahashi, S., Yeo, Y.-S., Zhao, Y., OMaille, P. E., Greenhagen, B.T., Noel, J. P., Coates, R. M., Chappell, J.(2007) JBC 282, 31744-54
Figure 1. Scheme of the two-enzyme cascade and the four-enzyme cascade

_________________ [1] T. van Herk, A.F. Hartog, H.E. Schoemaker, R. Wever, J. Org. Chem. 2006, 71, 6244 [2] T. van Herk, A. F. Hartog, L. Babich, H. E. Schoemaker, R. Wever, ChemBioChem. 2009, 10, 2230 [3] J. C. L. van Hemert, L. Babich, et al. Submitted
The capability of laccases to form reactive radicals in lignin can be also used in targeted modification of wood fibers in order to improve the chemical or physical properties of the fiber itself. The main purpose of this study was to evaluate the possibility of using laccases from fungi Panus (Lentinus) tigrinus 8/18, Lentinus strigosus 1566 and Steccherinum ochraceum 1833 in biotechnological processes of lignin transformation.[1] Their activity and stability were investigated in the presence of organic co-solvents and under microwave irradiation, with the aim of exploring experimental conditions of potential interest for improving lignin degradation and modifications of further non-water soluble substrates. Laccase from S. ochraceum 1833 showed a remarkable stability towards temperature and denaturating organic solvents. Interestingly, laccase from Steccherinum ochraceum undergoes an increase of activity at 60C, even under microwave irradiation, whereas laccases from P. tigrinus and from L. strigosus loose totally their activity after 5 minutes of irradiation. At 80C laccase from Steccherinum ochraceum shows a gradual inactivation, and microwave radiation causes a pronounced temperature-indipendent effect that leads to the immediate loss of all the activity. The behavior of laccase from S. ochraceum 1833 suggested to further investigate conformational changes under experimental conditions considered. Tryptophan fluorescens and Circular Dicroism confirmed that laccase from S. ochraceum 1833 retains secondary and tertiary structure at 60C, while the incubation at 80C causes a severe denaturation. Interestingly, UV-Vis spectroscopy demonstrated that microwaves irradiation causes the release of one of the coordinated coppers, leading to complete loss of activity. Finally a computational study was approached in the attempt of correlating structural properties and laccase thermostability. The 3D-QSAR VolSurf based approach [2] demonstrated that there is no correlation between the superficial properties of the enzymes when only the protein is considered (apoproteins) despite such kind of correlations were already described for other different proteins. Indeed, the extended glycosylation of the laccases considered in the study seem to play a major role in determining superficial properties of the proteins, their conformational behavior and ultimately their stability. On that basis, a more refined 3D-QSAR model was developed which, by taking into account also the glycans, is able to correlate superficial properties and thermostability.
________________ [1] A. Chernykh, et al. J. Appl.Microbiol., 2008, 105, 2065. [2] P. Braiuca et al. Biotechnol. J., 2006, 24, 419-425.
55
In vitro metabolic pathway construction in Immobilized Microuidic Enzyme Reactor (IMER)
Amanatuzzakiah Abdul Halima and Frank Baganza a The Advanced Centre for Biochemical Engineering, University College London, Department of Biochemical Engineering, Torrington Place London, WC1E 7JE, United Kingdom. E-mail: a.halim@ucl.ac.uk The idea of de novo metabolic engineering through novel synthetic pathways offers new directions for multi-step enzymatic synthesis of complex molecules. This has been complemented by recent progress in performing enzymatic reactions on chips using immobilized microfluidic enzyme reactor (IMER)[1]. Microfluidics offers the advantage of cheaper, faster and controlled reactions while IMER further enhance the capability by enabling high efficiency, multi step synthesis of the desired molecule on a miniaturized chip format. This work is concerned with the construction of de novo designed enzyme pathways in microfluidic channels synthesizing chiral pharmaceutical intermediates. A particularly interesting compound for several pharmaceutical syntheses is a single diastereoisomer of 2-amino-1,3,4-butanetriol (ABT). This chiral amino alcohols can be synthesized from simple achiral substrate using two enzymes transketolase (TK) and transaminase (TAm) [2]. Here we describe the development of an IMER using His6-tagged TK and TAm enzyme immobilized onto Ni-NTA beads and packed into capillaries to enable multiple steps enzyme reactions. Single IMERs were cascaded in series, whereby the first enzyme, TK catalyzed a model reaction of hydroxypyruvate and glycoaldehyde into L-erythrulose, and the second unit of the IMER with immobilized TAm converted the L-erythrulose into 2-amino-1,3,4-butanetriol (ABT) using methylbenzylamine as amine donor. Results of the kinetic analysis of the two enzymes will be presented and the potential of the IMER system for as tool for in vitro pathway construction will be discussed. Key words: Immobilized microfluidic enzyme reactor (IMER), transketolase, transaminase, multistep enzymatic synthesis.
________________ [1] Matosevic et al (2010) Fundamentals and applications of immobilized microfluidic enzymatic reactors. J Chem Technol Biotechnol. 86. 325 334 [2] Ingram et al (2007) One-pot synthesis of amino-alcohols using a de-novo transketolase and -alanine:pyruvate transaminase pathway in Escherichia coli. Biotech Bioeng. 96. 559-569
56
Dynamic kinetic resolution of arylamines with lipase and Pd/BaSO4
YANG Lirong, FU Simin, XU Gang, WU Jianping Department of chemical and biological Engineering, Zhejiang University, Hangzhou 310027,China E-mail: lryang@zju.edu.cn Dynamic kinetic resolution (DKR) was recently proposed as a new method for the synthesis of enantiomerically pure compounds such as chiral amines and alcohols. The process combines the enzyme-catalyzed kinetic resolution(KR) with an in situ racemization step. While the critical drawback of KR is the limited maximum theoretical yield of 50%, DKR starts from racemic mixtures and in principle yields a single enantiomer product with a theoretical yield of 100%. A novel and efficient DKR process of arylamines was developed with Pd/BaSO4 as racemization catalyst. Firstly, the KR of 1-phenylethylamine catalyzed by lipase Novozym435 was established. Then, Pd/BaSO4 was prepared as racemization catalyst with excellent racemization capacity and applied to the DKR of 1-phenylethylamine successfully. To expand the application area, several substrate derivants with different substitution groups on its benzene ring were tested in this DKR system. It was found that 4-chlorophenyl valerate was the most suitable acyl donor, toluene was the most suitable medium for the KR reaction, and the addition of a small amount of NaHSO3 can reduce side reactions. The optimum ratio of amine and ester, amine concentration, and temperature were 1:0.8, 200 mmolL-1 and 40 oC, respectively. Under the optimal conditions, E value was greater than 200. A product enantiomeric excess(eep) of 99% was achieved with conversion of 50% after 6 h. Pd/BaSO4 was shown to have excellent racemization activity and good biocompatibility. During the process, the transesterification catalyzed by Pd/BaSO4 was found to be the main reason for the decreasing of eep. While acyl donors with complex structures were used, this transesterification was effectively suppressed. For example, excellent eep (>99%) at high conv. (>99%) can be achieved when 4-chlorophenyl valerate was used as acyl donor. Then the DKR of other arylamines was evaluated. The relationship of the substrate structure and the enantioselectivity was studied.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (No. 20936002), Key Project of Chinese National Programs for Fundamental Research and Development (Nos. 2009CB724706 and 2011CB710800), and Hi-Tech Research and Development Program of China (No. 2011AA02A209).
59
Automated evaluation of microscale linked process sequences for generation of scaleable bioprocess design data
J.Z. Baboo1, J.M. Ward2, G.J. Lye1, M. Micheletti1 Department of Biochemical Engineering1 & Institute of Structural and Molecular Biology2, University College London, Torrington Place, London, WC1E 7JE, UK j.baboo@ucl.ac.uk Oxidative bioconversions offer valuable opportunities in industrial pharmaceutical synthesis such as using Baeyer-Villiger monooxygenases for antibiotic synthesis1. However, a limiting factor is the identification of scaleable hydroxylation biocatalysts. Coupling high-throughput microscale techniques with automation enables the operation of linked process sequences for faster identification and characterisation of optimal conditions2. A fully automated microscale sequence involving fermentation, bioconversion, liquidliquid extraction for sample analysis and analytical techniques has been developed for the evaluation of whole cell Baeyer-Villiger monooxygenases. The automated approach has been shown to be robust and reproducible over multiple runs producing consistent results on different days. Rapid automated collection of quantitative kinetic data on a number of parameters has been applied to optimise biocatalyst expression and to identify new bioconversion substrates. Such studies have enabled the processing time to be halved and the biocatalysts specific activity to be more than doubled. Using the same methodology this automated platform is being applied to investigate novel cytochrome P450 biocatalysts for hydroxylation reactions3. Their vast reaction ability and essential role in both drug metabolism and bioremediation makes novel P450 biocatalysts an area of much interest. By using a matched oxygen transfer coefficient (kLa) approach both fermentation and bioconversion operations have been successfully scaled up to 75 L scale confirming the use of microscale approaches for process optimisation.
________________ [1] Strukul G (1998) Transition Metal Catalysis in the Baeyer-Villiger Oxidation of Ketones. Angewandte Chemie International Edition 37:1198-1209 [2] Lye GJ, Ayazi-Shamlou P, Baganz F, Dalby PA, Woodley JM (2003) Accelerated design of bioconversion processes using automated microscale processing techniques. Trends in Biotechnology 21:29-37 [3] Hussain HA & Ward JM (2002) Enhanced heterologous expression of two Streptomyces griseolus cytochrome P450s and Streptomyces coelicolor ferredoxin reductase as potentially efficient hydroxylation catalysts. Applied and Environmental Microbiology 69: 373-382
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Tryptophan oxidases involed in bis-indole antibiotics biosynthesis and application to tryptophan determination
a
Masafumi Kameyaa, Hiroyasu Onakaa, Yasuhisa Asanoa Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan E-mail: asano@pu-toyama.ac.jp Amino acids quantification has attracted more and more attention in recent years because it has become known as a useful biomarker to diagnose a variety of diseases. For rapid and convenient diagnoses, simple and high-throughput methods to determine amino acids are needed. While conventional HPLC methods are time-consuming to analyze multiple samples requiring bulky instruments, enzymatic assay methods enable simple, rapid, and economic quantification of amino acids. The aim of this study is to establish an enzymatic method to determine tryptophan by using tryptophan-oxidizing enzymes. Many L-amino acid oxidases have been reported to catalyze this reaction, such as those in snake venome. However, most of them also oxidize other amino acids besides tryptophan. In order to obtain tryptophan-specific oxidase, we focued on bis-indole antibiotics biosynthesis (Fig. 1). This metabolism has been known only among a limited number of bacteria, including staurosporine and violacein biosyntheses in Streptomyces sp. TP-A0274 [1] and Chromobacterium violaceum [2], respectively. In these unique metabolic pathways, tryptophan oxidation is catalyzed as the first reaction by StaO and VioA for staurosporine and violacein syntheses, respectively. In this study, StaO and VioA were heterologously expressed and purified from recombinant Escherichia coli. Enzymatic analysis revealed that these two enzymes show high affinity and high substrate specificity toward tryptophan unlike most of known L-amino acid oxidases, indicating that StaO and VioA are suited for tryptophan determination. Indeed, StaO and VioA assay systems estimated tryptophan concentration in human plasma samples as accurately as an instrumental analysis did. Therefore, it was clearly shown that these tryptophan oxidases offer an effective method to determine tryptophan in biosamples rapidly, inexpensively, and accurately.
Figure 1. Staurosporine and violacein biosynthetic pathways.

________________ [1] Onaka, H., Taniguchi, S., Igarashi, Y. & Furumai, T. (2002). J Antibiot 55, 1063-1071. [2] Hirano, S., Asamizu, S., Onaka, H., Shiro, Y. & Nagano, S. (2008). J Biol Chem 283, 6459-6466.
October 2-6, 2011
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Relationship between cell physiology and biocatalytic activity in E. coli expressing proline-4-hydroxylase
F. Falcionia, O. Fricka, L. Blanka, A. Schmida,b, and B. Bhlera a Laboratory of Chemical Biotechnology, TU Dortmund University, Dortmund, Germany. b Leibniz-Institut fr Analytische Wissenschaften (ISAS), Dortmund, Germany E-mail: francesco.falcioni@bci.tu-dortmund.de Microbial physiology and metabolism have rarely been investigated with respect to whole-cell biotransformations, although they constitute interesting engineering targets to improve the performance of oxygenase-based whole-cell biocatalysts. In this study, recombinant Escherichia coli containing proline-4-hydroxylase (P4H) from Dactylosporangium sp. RH1 [1], a non-heme iron and 2-oxoacid-dependent dioxygenase, was investigated as a model system (Figure 1). Initial resting cell activities achieved with E. coli BL21(DE3)pLysS carrying the P4H gene on the vector pET-HYP1, were in the range of 1-3 U/gCDW (1 U = 1 mol/min of product, CDW = cell dry weight). Stepwise optimization was achieved by improvement of the soluble P4H fraction via medium engineering, codon optimization and screening of alternative strains and vectors and growth temperatures. In particular, the addition of proline to a minimal medium significantly improved the production of active P4H. However, at 10-12 U/gCDW, a maximum of activity in resting cells was reached, which did not correlate with the levels of soluble enzyme and with the activity of permeabilized cells and cell extracts, indicating that whole-cell activities were limited either by substrate uptake or cell metabolism. Metabolic flux analysis (MFA) was used as a tool to investigate the effect of proline hydroxylation on metabolic network operation. Growing cells did not show the same behaviour as resting cells, and the metabolic network responded to both proline feed and catalytic activity. However, higher catalytic activities corresponded to lower proline uptake rates and TCA cycle activity, which negatively affect the overall catalytic efficiency. The catalytic capacity and metabolic flexibility of the host are discussed.
Directed evolution of Glucose oxidase for their application in biofuel-cells
Erik Zeithammel, Hemanshu Mundhada, Ulrich Schwanenberg Lehrstuhl fr Biotechnologie, RWTH Aachen University, Germany Glucose oxidase (GOx) is a flavoprotein which belongs to the family of oxidoreductases. GOx catalyses the oxidation of -D-glucose to D-glucono--lactone by reducing molecular oxygen to hydrogen peroxide. GOx is widely used for applications in biosensors, food and beverage industries and biofuel cells [1]. Biofuel cells convert blood sugars into electrical energy by using biocatalysts such as GOx. However the high Km value and less activity of GOx under physiological conditions (pH) [2] are key limitations for their applications in biofuel cells. Our research is focused on evolving of GOx to overcome the limitations of the enzyme for applications in biofuel-cells. We have developed a product based medium-throughput screening system (Glucose oxidase detection assay) for directed evolution of GOx [3]. The screening system allows the detection of D-glucono--lactone formation independent of oxygen consumption. The screening of two standard error-prone PCR mutant libraries employing Sacchaomyces cerevisiae as expression organism yielded a double mutant with improved thermal- and pH-stability as well as an improved kcat value (69.5/s WT; 137.7/s mutant) [4]. An ultrahigh throughput screening system using flow cytometry was developed to overcome limitations on high throughput screening. Thereby a mutant with 1.2 times decreased Km was identified [5]. For further improvement of GOx for lower Km values and a better performance under physiological conditions our established directed evolution platform are in use.
_____________ [1] Bankar S. B., Blue M. V., Singhal R. S. and Ananthanarayan L. (2009) Biotechnology. Advances 27, 489-501. [2] Kenausis G., Chen Q. and Heller A. (1997) Anal. Chem. 69, 1054-1060. [3] Zhu Z., Momeu C., Zakhartsev M. and Schwaneberg U. (2006) Biosensors and Bioelectronics 21, 2046-2051. [4] Zhu Z., Wang M., Gautam A., Nazor J., Momeu C., Prodanovic R. and Schwaneberg U. (2007) Biotechnol. J. 2, 241-248. [5] Prodanovic R., Ostafe R., Scacioc A. and Schwaneberg U. (2011) Combinatorial Chemistry & High Throughput Screening 14
OsO4Streptavidin - a tunable hybrid system for enantioselective olefin dihydroxylation
Valentin Khlera, Jingchen Maoa, Tillmann Heinischa,b, Anca Pordeaa, Alessia Sardoa, Yvonne Wilsona, Livia Knrra, Marc Creusa, Jean-Christophe Prosta, Tilman Schirmerb, Thomas R. Warda a Department of Chemistry, University of Basel, Basel, Switzerland;bBiozentrum, University of Basel, Basel, Switzerland E-mail: valentin.koehler@unibas.ch The asymmetric cis-selective OsO4-dependent dihydroxylation (AD) of olefins ranks among the most powerful laboratory methods for the synthesis of vicinal diols. Chiral ligands for the catalytic version are almost exclusively based on quinidine and quinine derivatives.[1] Encouraged by an early report from Kokubo[2] a range of proteins was investigated for their suitability as chiral modifiers. Streptavidin (SAV) was identified as a promising candidate.
a
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Robust enzyme preparations for the enzyme-catalysed stereoselective reduction
A. Scholza, M. B. Ansorge-Schumachera Technical University of Berlin, Institute of Chemistry, Department of Enzyme Technology (TC4), Str. des 17. Juni 124, D-10623 Berlin (Germany) E-mail: alexander.scholz@chem.tu-berlin.de Oxidoreductases are biocatalysts with tremendous potential in industrial processes due to their applicability under mild conditions, e. g., pH and temperature, obtaining remarkable chemo-, regio-, and stereoselectivity. The number of interesting monooxygenases, dehydrogenases, reductases, oxidases, and peroxidases is steadily increasing and the tailoring of a given biocatalyst has more and more become a standard technology [1]. For an industrial use of enzymes it is important to have high activity, stability and manageability. For that reason, biocatalysts are usually immobilized. However, for many industrial applications, the resulting preparations have only an insufficient long-term stability especially in terms of enzyme leaching and carrier integrity under process conditions. We recently reported that in non-aqueous reaction systems, both features can be considerably improved by the deposition of silicone on carrier bound enzyme preparations [2]. In this study, the deposition material was adapted in a way, that a transfer of the immobilization method to hydrophilic reaction systems can be achieved. The performance was investigated with an immobilized carbonyl reductase from Candida parapsilosis catalysing the asymmetric, cofactor-dependent reduction of acetophenone to chiral 1-phenylethanol as model system [Figure 1].
The application of OsO4SAV in the AD and the impact of single point mutations on the catalysis outcome will be presented. The interpretation of the effects of mutations in combination with X-ray structure analysis will be employed to discuss possible locations of the catalytically active osmium species in the host protein.
________________ [1]a) H. C. Kolb, M. S. VanNieuwenhze, K. B. Sharpless, Chem. Rev. 1994, 94, 2483- 2587; b) A. Baitsev, H. Adolfsson, Synthesis 2006, 11, 1725-1756. [2] T. Kokubo, T. Sugimoto, T. Uchida, S. Tanimoto, M. Okano, Chem. Commun. 1983, 769-770.
Figure 1. Reduction of acetophenone to 1-phenylethanol

________________ [1]Hollmann, F., I.W.C.E. Arends, and K. Buehler, Biocatalytic Redox Reactions for Organic Synthesis: Nonconventional Regeneration Methods. Chemcatchem, 2010. 2(7): p. 762-782. [2]Wiemann, L.O., et al., Composite Particles of Novozyme 435 and Silicone: Advancing Technical Applicability of Macroporous Enzyme Carriers. Chemcatchem, 2009. 1(4): p. 455-462.
Figure 1. Scheme of the hydroxylation of L-proline to trans 4-hydroxyproline by metabolically active recombinant E. coli
_______________ [1] T. Shibasaki, H. Mori, Biosci. Biotechnol. Biochem., 2000, 64, 746-75
63
Biosynthesis and Biotransformation of Santalene Derivatives from Indian Sandalwood, Santalum album
Pankaj P. Daramwara, Prabhakar Srivastavaa, Thulasiram H. V.*a,b aChemistry-Biology Unit, Organic Chemistry Division, National Chemical Laboratory, Pune, India. bInstitute of Genomics and Integrative Biology, Council of Scientific and Industrial Research (CSIR), Mall Road, New Delhi-110007, India. E-mail: pp.daramwar@ncl.res.in The endangered, Sandalwood or Chandan (Santalum album L.) belongs to the Santalaceae family, a medium-sized evergreen hemi-root parasitic tree, highly valued for its oil. The sesquiterpene alcohols, - and -santalol (1 and 3, Scheme 1), which are the major components (~90%) of essential oil from well matured tree (80 years old)[1], are responsible for most of the biological activity of the oil. -Santalol has particularly attracted increasing attention for its neuroleptic property[2] and chemopreventive effect[3] in in vitro and in vivo bioassay systems. These alcohols being isomers, are difficult to be separated by conventional separation techniques. Here we present the separation of santalols and santalenes using medium pressure liquid chromatography (MPLC) with silver nitrate coated silica gel as stationary phase. We also present our work on biotransformation and biosynthesis of Santalenes. Biotransformation of - and -Santalol (1 and 3) has been studied using a soil isolated versatile fungal strain which could able to hydroxylate at several positions on both - and -santalols. Santalenes are synthesized at the interface of the heartwood-sapwood of the sandalwood tree. Santalene synthase catalyzes the cyclization of Farnesyl diphosphate (FPP) to Santalenes which further undergo hydroxylation by monooxygenase system to form Santalols. In order to study the biosynthesis of Santalols, total RNA has been isolated from the interface of heartwood and sapwood of sandalwood tree and a cDNA library has been constructed. From the screening of cDNA library, we have isolated FPP synthase and santalene synthase for their biochemical characterization.
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Heterologous Expression of CYP71D55 in S. cerevisiae for (+)-Nootkatone Production
Anita Emmerstorfera, Tamara Wriessneggera, Peter Augustina, Monika Mllerc, Iwona Kaluznac, Peter Macherouxa,d, Helmut Schwaba,b, Harald Pichlera,b aACIB - Austrian Center of Industrial Biotechnology, Graz, Austria bInstitute of Molecular Biotechnology, Graz University of Technology, Austria cDSM Innovative Synthesis B.V., Geleen, The Netherlands dInstitute of Biochemistry, Graz University of Technology, Austria E-mail: emmersto@sbox.tugraz.at Membrane-attached cytochrome P450 monooxygenases (CYPs) play a central role in biosynthesis of important, natural compounds and are involved in drug metabolism. Therefore, these catalysts are very attractive for industrial application. However, a number of limitations have restricted their use in industrial processes including substrate specificity, the co-expression of a reductase, the need for a complex system of cofactor regeneration, low activity and poor stability. Our work focuses on functional expression of Hyoscyamus muticus premnaspirodiene oxygenase (HPO; CYP71D55) and cytochrome P450 reductase (CPR) from A. thaliana in Saccharomyces cerevisiae. As a eukaryote, S. cerevisiae shares the complex internal cell structure of plants and animals, but can easily be genetically modified and cultured. Besides P. pastoris, which has proven to be a potent host for functional expression of HPO and CPR (own unpublished data) S. cerevisiae was chosen as second expression system due to its well-known molecular and cellular biology. The enzyme of interest, HPO, is a monooxygenase derived from Egyptian henbane and, previously, has been shown to be an appealing biocatalyst for the generation of nootkatone, the most important and high-value flavor of grapefruits [1]. Coexpression of HPO and CPR from a 2-derived vector was optimized and enzyme activities were tested via whole-cell conversions. Formation of (+)-nootkatone from valencene was determined by GC-FID.
_____________________ [1] Takahashi, S., Yeo, Y.-S., Zhao, Y., OMaille, P. E., Greenhagen, B.T., Noel, J. P., Coates, R. M., Chappell, J.(2007) JBC 282, 31744-54
67
A Novel Biocatalyst for Oxidation of Secondary Alcohols
Inga Matijoyt, Milda Atrauskait, Aura Veteikyt, Rta Grukien, Edita Kleinait Vilnius University Institute of Biotechnology, Sector of Applied Biocatalysis, V.A. Graiino str. 8-255, LT-02241, Vilnius, Lithuania E-mail: inga.matijosyte@bti.vu.lt Although it has been more than two decades since the interest for biocatalysts and their application in industry has increased significantly, however, the enzymatic oxidation of secondary alcohols to desired ketones is still not an issue. At the present time only six oxidases are known which can perform oxidation of secondary alcohols [1]. However, they have very narrow substrate specificity: five of them are specific only for one desired substrate. Therefore, the aim of our work was to discover enzyme with broad substrate specificity for oxidation of secondary alcohols. Secondary alcohol oxidase (SAO) was discovered more than twenty years ago by investigating polymer (PVA) biodegradation [2], nevertheless, only a small number of enzyme properties have been established: a.a. sequence of SAO protein, gene sequence and the exact structure of catalytic center are not known. First of all, seven strains of Pseudomonas were identified and it was determined that three strains belong to P. aeruginosa species and four strains belong to P.putida species. The estimation of growth conditions for Pseudomonas species for the optimum expression of secondary alcohol oxidase (SAO) were determined by following cell population, protein concentration and SAO activity. The parameters such as pH of growth medium, temperature, inducer and its concentration for optimal expression of SAO were defined. The most suitable and optimal purification system for enzyme was settled which gave 99 % of purification yield (Figure 1). It was identified that SAO is a monomer and has a molecular mass of 52 kDA. The substrate specificity experiments indicated that our obtained SAO is specific for long chain alcohols.
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Medium chain-length alkane activation under mild conditions
M. Bordeauxa, A. Galarneaua, F. Fajulaa, J. Dronea aInstitut Charles Gerhardt de Montpellier UMR 5253, CNRS/ENSCM/ UM2/UM1, Montpellier, France E-mail: melanie.bordeaux@enscm.fr Alkane activation in compounds of higher added value is a promising scientific challenge. Due to the high energy required for the C-H bond cleavage, this reaction is difficult, especially in the case of terminal methyl positions. Chemical methods require the use of toxic reagents, important energy consumptions and are not selective. Therefore, we propose a sustainable alternative for aliphatic alkane oxidation by enzymatic means, inspired from nature. Cytochrome P450 monooxygenases can oxidize a broad variety of substrates. A member of this family, CYP153A13a from Alcanivorax borkumensis SK2 (A13), converts medium chain-length aliphatic alkanes (C6-C12) into terminal alcohols [1]. One limitation of A13 is its low catalytic rate, mainly due to a low electron transfer from the reduced cofactor (NADPH) to the cytochrome. To overcome this limitation, we fused A13 with the reductase domain from the self-sufficient cytochrome P450RhF of Rhodococcus sp. NCIMB 9784. The proximity between the reductase and A13 allows a higher efficiency in electron transfer from the cofactor to the catalytic center, and consequently enhances its activity [2].
Figure 1. Regioselective oxidation of alkanes (n = 4-10) into alcohols by the fusion protein A13-Red This artificial self-sufficient cytochrome P450 was highly chemoselective and harbored a regioselectivity of more than 99% for the C-terminal position [3] (Figure 1). This new room temperature alkane hydroxylation biocatalyst is an efficient green alternative for the selective activation of alkanes. Our results clearly exemplify the strength of biotechnologies, to answer the increasing request for cleaner and lower energy-consuming processes.
________________ [1] E. G. Funhoff, J. Salzmann, U. Bauer, B. Witholt, J. B. van Beilen, Enzyme Microb. Technol. 2007, 40, 806-812. [2] S. Li, L. M. Podust, D. H. Sherman, J. Am. Chem. Soc. 2007, 129, 12940-12941. [3] M. Bordeaux, A. Galarneau, F. Fajula, J. Drone, Angew. Chem. Int. Ed. 2011, 50, 2075-2079.
Scheme 1
______________ [1] J. Brand, p. Kimber, J. Streatfield, Sandalwood Research Newsletter 21 (2006) 1. [2] Hongratanaworakit, T.; Heuberger, E.; Buchbauer, G. Planta Med. 2004, 70, 37. [3] Kaur, M.; Agarwal, C.; Singh, R.P.; Guan, X.; Dwivedi C.; Agarwal, R.Carcinogenesis 2005, 26, 369380.
Figure 1. SDS PAGE gel electrophoresis of purification steps M marker, 1 supernatant after the growth of P.putida 4, 2 after Q-Sepharose column, 3 after Sepharose SP column.
________________ [1] W. Kroutil, et al., Advanced Synthesis & Catalysis, 346 (2004) 125-142 [2] F. Kawai and X.Hu, Applied Microbiology Biotechnology, 84 (2009) 227-237
October 2-6, 2011
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Regioselective oxidative alkene cleavage of dialkenes by trametes hirsuta
Caroline E. Paula, Aashrita Rajagopalanb, Ivn Lavanderaa, Vicente Gotor-Fernndeza, Wolfgang Kroutilb, Vicente Gotora a Department of Organic and Inorganic Chemistry, University of Oviedo, Calle Julian Clavera 8, 33006 Oviedo, Spain; bDepartment of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria E-mail: paulcaroline@uniovi.es The preparation of aldehydes and ketones acting as important intermediates in the synthesis of natural products and pharmaceuticals is of high value in organic synthetic chemistry, and more significantly the selective generation of these carbonyl groups directly from alkenes is of particular interest. Conventional methods for alkene cleavage mostly suffer from harsh and sometimes dangerous reaction conditions, showing in most cases a lack of chemo-, regio- and stereoselectivity.[1] As an alternative an enzymatic methodology may be suitable to achieve a regioselective oxidative alkene cleavage. The fungi Trametes hirsuta G FCC 041 was shown to perform oxidative alkene cleavage reactions over for a wide range of aryl alkene derivatives.[2] Here we show the first example of regioselective alkene cleavage, thus the differentiation between two terminal alkenes and between a terminal and a methylated alkene (Scheme 1).
Combination of a palladium-catalyzed Suzuki cross-coupling reaction with an asymmetric biotransformation towards a one-pot reaction in aqueous medium at room temperature
Sonja Borcherta, Edyta Burdaa, Bettina Blumenrdera, Werner Hummelb, Jrgen Schatza, Harald Grgera,c a Department of Chemistry and Pharmacy, University of Erlangen-Nrnberg, Henkestr. 42, 91054 Erlangen, Germany; bInstitute of Molecular Enzyme Technology at the HeinrichHeine University of Dsseldorf, Research Centre Jlich, Stetternicher Forst, 52426 Jlich, Germany; cDepartment of Chemistry, Bielefeld University, P.O. box: 10 01 31, 33501 Bielefeld, Germany E-mail: sonja.borchert@chemie.uni-erlangen.de Multi-step one-pot processes represent an attractive synthetic concept for the improvement of overall process efficiency by decreasing the required number of work up and purification steps. Although a range of successful combinations of chemo- and biocatalytic reactions towards chemoenzymatic one-pot processes in organic media were developed,[1] analogous processes one-pot processes running in aqueous medium are still rare. In this connection there has been a recent interest by various groups in the combination of a Suzuki crosscoupling reaction as an example for a palladium catalyzed synthesis with an enzymatic reduction in a one-pot process.[2]
Lipase-mediated reaction systems for the efficient epoxidation of monoterpenes
Dr. Lars O. Wiemanna and Prof. Dr. Volker Siebera,b a Fraunhofer Institute for Interfacial Engineering and Biotechnology, Project Group BioCat, Schulgasse 16, 94315 Straubing, Germany E-mail: lars.wiemann@igb.fraunhofer.de b Technische Universitt Mnchen, Chemistry of biogenic resources, Schulgasse 16, 94315 Straubing, Germany. The ongoing trend towards the greening of chemical production processes currently gives a boost to research activities trying to access novel biocatalytic or combined chemoenzymatic conversion routes for sustainable resources. In this context monoterpenes are technically interesting compounds as they accumulate in bulk amounts as extracts from wood pulp [pinene (1) and carene (2)] or from citrus plants [limonene (3)]. The value of these compounds can be increased via addition of further functionalities, e.g. by producing the corresponding epoxides as versatile synthetic intermediates. Bjrkling et al. (1992)1 showed that most lipases (EC 3.1.1.3) are able to form peroxyacids from carbonic acids in the presence of H2O2. Thus, hazardous peroxyacid can be produced in situ in organic solvents and directly be used for the epoxidation of organic substrates such as alkenes via Prileschajew reaction. Previous works have proven the general feasibility of this approach for the epoxidation of different monoterpenes as well.2,3
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Catalytic biofilms: Glycolic acid production by Pseudomonas diminuta
Bettina Rosche and Xuan Zhong Li School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney NSW 2052, Australia, b.rosche@unsw.edu.au Microbial biofilms have been proposed as robust, self-immobilized and self regenerating catalysts, and have attracted increasing attention over the past decade. One concern for their applicability is their potential variability. This investigation evaluates the production of glycolic acid from ethylene glycol by Pseudomonas diminuta biofilms. P. diminuta established an active biofilm in an aerated continuous trickle-bed reactor system with stainless steel structured packing. 20 cultures were grown from biofilm effluent cells and 14 of these cultures had a decreased specific productivity compared to all ten P. diminuta cultures that were not of a biofilm origin. Despite the variation of effluent cells, the glycolic acid productivity in the biofilm reactor remained steady at 1.6 gl-1h-1 over 700 h and excellent reproducibility between three independent operations was observed. This demonstrates that the variation of biofilm effluent cells would not necessarily be detrimental in an industrial process where consistency and reproducibility are important. In addition, process robustness was demonstrated as the reactor system quickly recovered from medium and air flow interruptions and feedstock contamination. The results illustrate the potential of biofilm as catalyst for the production of chemicals. Application of biofilms is recommended for whole-cell processes which require improved catalyst stability or catalyst retention for continuous operation.
Scheme 1. Regioselective oxidative alkene cleavage catalyzed by Trametes hirsuta. A series of dialkene substrates were specifically designed and synthesised to study the chemo- and regioselectivity of this process with Trametes hirsuta. For the synthesis of these substrates different methodologies such as Wittig olefination and Suzuki coupling were employed affording dialkene products in high yields. Once the substrates were prepared the next step was the optimisation of the biocatalytic reaction parameters such as the type of reaction vessel and reaction time. In several cases, the process exclusively afforded the desired benzaldehyde derivative with moderate to excellent conversion and chemoselectivity. Selected reactions were also easily performed on a preparative scale with >100 mg of substrate, obtaining leading to excellent results (>80% conversion, >99% chemoselectivity).
________________ [1] Hudlicky, M. Oxidation in Organic Chemistry, ACS Monograph 186, American Chemical Society, Washington, DC, 1990. [2] (a) Mang, H.; Gross, J.; Lara, M.; Goessler, C.; Schoemaker, H. E.; Guebitz, G. M.; Kroutil, W. Angew. Chem. Int. Ed. 2006, 45, 5201-5203; (b) Mang, H.; Gross, J.; Lara, M.; Goessler, C.; Schoemaker, H. E.; Guebitz, G. M.; Kroutil; W. Tetrahedron 2007, 63, 3350-3354. (c) Lara, M.; Mutti, F. G.; Glueck, S. M.; Kroutil, W. Eur. J. Org. Chem. 2008, 3668-3672
Scheme 1. Combination of a Suzuki cross-coupling reaction with an enzymatic reduction In continuation of our previous work[2a] we have now combined a Suzuki cross coupling reaction with a subsequent asymmetric enzymatic reduction in a one-pot process in aqueous reaction medium with both steps proceeding at room temperature (Scheme 1). For the Suzuki cross coupling reaction step, in this work we used a water-soluble palladium catalyst (4 mol%), which was prepared from PdCl2 and TPPTS. The chiral biaryl-containing alcohols were obtained with conversions up to >95% and excellent enantioselectivities of >99% ee. Figure 1. Pinene (1), 3-Carene (2) and Limonene (3) and corresponding epoxides In this work, we compared different lipase-based reaction systems for the epoxidation of cyclic and acyclic monoterpenes and report on methodologies to optimise the reaction conditions with respect to conversion, enzyme activity, selectivity and catalyst stability. Moreover, we investigated how to shift the product range towards the diepoxides e.g. for limonene (3) as shown in figure 1.
________________ [1] Bjrkling F. et al. Lipase catalyzed synthesis of peroxycarboxylic acids and lipase mediated oxidations. Tetrahedron. 1992, 48 (22), 4587-4592. [2] Rusch M. and Warwel S. Chemoenzymatic epoxidations of alkenes by dimethyl carbonate and hydrogen peroxide. Organic Letters. 1999, 1 (7), 1025-1026. [3] Skouridou, V. et al. A study on the process of lipase-catalyzed synthesis of pinene oxide in organic solvents. Biocatalysis and Biotransformation. 2003, 21 (6), 285-290.
_______________________ [1] a) O. Pamies, J.-E. Bckvall, Chem. Rev. 2003, 103, 3247; b) A. Bruggink, R. Schoevaart, T. Kieboom, Org. Process Res. Dev. 2003, 7, 622; c) H. Pellissier, Tetrahedron 2008, 64, 1563. [2] a) E. Burda, W. Hummel, H. Grger, Angew. Chem. 2008, 120, 9693; Angew. Chem. Int. Ed. 2008, 47, 9551; b) A. Prastaro, P. Ceci, E. Chiancone, A. Boffi, R. Cirilli, M. Colone, G. Fabrizi, A. Stringaro, S. Cacchi, Green Chem. 2009, 11, 1929; c) V. Gauchot, W. Kroutil, A. R.Schmitzer, Chem. Eur. J. 2010, 1748.
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Biocatalytic hydroxylation of n-butane with in situ-cofactor regeneration at low temperature and under normal pressure
Svenja Staudta, Christina A. Mllerb, Jan Marienhagenb, Christian Bingc, Stefan Buchholzd, Ulrich Schwanebergb, Harald Grgera,e a Department of Chemistry and Pharmacy, University of Erlangen-Nrnberg, Henkestrae 42, 91054 Erlangen, Germany; bInstitute of Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; cEvonik Oxeno GmbH, Paul-Baumann-Strae 1, 45772 Marl, Germany; dEvonik Degussa GmbH, Industrial Chemicals, Paul-BaumannStrae 1, 45772 Marl, Germany; eFaculty of Chemistry, Bielefeld University, Universittsstrae 25, 33615 Bielefeld, Germany E-mail: svenja.staudt@chemie.uni-erlangen.de The functionalization of C-H-bonds like those appearing in n-alkanes is one of the most challenging reactions in organic chemistry. We focused on the regioselective enzymatic hydroxylation of the liquid gas n-butane in aqueous media at low temperature and under normal pressure. Very recently Reetz et al. showed the hydroxylation of n-butane with a variant of the P450-monooxygenase in combination with high pressure (10 bar) and 25C.[1] In our research we used the 19A12-mutant of the P450-monooxygenase from Bacillus megaterium (P450 BM-3). This enzyme was able to hydroxylate n-butane exclusivly to 2-butanol by the consumption of NADPH as cofactor at normal pressure conditions. The expensive required cofactor was regenerated in situ by using the well-established D-glucose dehydrogenase / D-glucose system which was also applied by Reetz et al. (Scheme 1).[1,2]
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A yeast expression system for selectiondevelopment of whole cell hydroxylating catalysts
M.J. Masemea, N. van Rooyena,b, M. Labuschagnea, M.K.A. Mahlalaa, J. Albertyna, M.S. Smita,b a Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa; bSouth African DST-NRF Centre of Excellence in Catalysis, c*change E-mail: smitms@ufs.ac.za Hydroxylation reactions are very important for the introduction of additional functional groups into organic molecules. Hydroxylases are often monooxygenases that require cofactor regeneration. Application of hydroxylating enzymes in biocatalysis thus remains challenging and often requires the use of whole cells. Vanillyl alcohol oxidase (VAO) is a flavin containing oxidase that accomplishes hydroxylation without the requirement for any cofactor regeneration. VAO uses oxygen and water to hydroxylate p-alkyl phenols as well as p-allyl phenols such as eugenol. However, in our experience whole cell biotransformations still yield better results than cell free extracts. Our initial attempts to express VAO in E. coli gave only very low levels of active enzyme. Co-expression with the pRARE2 plasmid that encodes tRNAs that are scares in E. coli, significantly improved expression. Concentrated cell suspensions (4 gDCW L-1) of E. coli expressing VAO transformed 40 mM eugenol to coniferyl alcohol within 24 h. We also developed an integrative vector that allowed us to clone the VAO from Penicillium simplicissimum into seven different yeasts (Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, Kluyveromyces lactis, Arxula adeninivorans, Candida deformans and Hansenula polymorpha) for comparative whole cell biotransformation of eugenol to coniferyl alcohol. All the yeasts tested displayed VAO activity, with A. adeninivorans giving the most promising results. When a vector containing two copies of the VAO gene was transformed into A. adeninivorans, one transformant converted 80 % of 320 mM eugenol within 19 h to yield 200 mM coniferyl alcohol when a whole cell suspension (30 gDCW L-1) was used. A. adeninivorans is more prone to over oxidation of coniferyl alcohol than E. coli, and product recovery from A. adeninivorans was thus only around 60%. The results described here were obtained in analytical scale reactions under unoptimized conditions. We are currently investigating conditions for optimal biocatalyst production and whole cell biotransformations with E. coli and A. adeninivorans.
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MO14, a Baeyer-Villiger Monooxygenase from Rhodococcus jostii RHA1 Benjamin D. Summers*a Mike Lloydb and Gideon Grogana a York Structural Biology Laboratory, University of York, Department of Chemistry, Heslington, YO10 5DD, York, United Kingdom b Chirotech Technology Ltd., Dr Reddys Laboratories (EU) Ltd., Unit 410 Cambridge Science Park, Cambridge CB4 0PE UK * E-mail: bds500@ysbl.york.ac.uk The Baeyer-Villiger (BV) reaction is a significant oxygenation reaction in organic chemistry that uses peracids, such as m-CPBA, to convert aliphatic and alicyclic ketones to esters and lactones respectively. In Nature, equivalent reactions are catalysed by BaeyerVilliger Monooxygenases (BVMOs), flavin-containing enzymes that catalyse their oxidations using a flavin hydroperooxidate derived most usually from the reaction of reduced FAD with molecular oxygen. BVMOs have been the subject of considerable research as these enzyme catalysts catalyse BV reactions with excellent regio- and enantioselectivity not displayed by their abiotic counterparts.[1] We recently cloned and expressed a number of BVMOs from the organism Rhodococcus jostii RHA1 and demonstrated that these enzymes display regio- and enantio-selective properties that, for the most part are representative of sub-groups of enzymes that can be identified by amino-acid sequence.[2] One enzyme, encoded by the gene ro03437 and named MO14, proved distinctive in that it catalysed the regio- plus enantioselective kinetic resolution of the racemic model BVMO substrate bicycle[3.2.0]hept-2-en-6-one 1 to yield a single substrate enantiomer and the 2-oxa lactone 2 in high enantiomeric excess (Figure 1).
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A avoprotein monooxygenase displaying NADH-dependent Baeyer-Villigerase activity Chentel N. Jensen,*a Jared Cartwright,a Andrew Willetts,b Michael J. Allen,b Sohail Alib and Gideon Grogana a York Structural Biology Laboratory, University of York, Department of Chemistry, Heslington, YO10 5DD, York, United Kingdom b Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH * E-mail: cnj500@ysbl.york.ac.uk Flavoprotein monooxygenases, including Baeyer-Villiger monooxygenases (BVMOs) and flavin monooxygenases (FMOs) are a group of enzymes with potential for application in organic synthesis as green oxidation catalysts for reactions including Baeyer-Villiger (BV) oxygenations and heteroatom oxidations.[1] Many of these enzymes are dependent on the phosphorylated nicotinamide cofactor NADPH, which is used by the enzyme(s) to reduce enzyme-bound flavins, such as FAD, which then react with molecular oxygen to form a flavin hydroperoxide(ate) that is the active oxidant in catalysis. A screen of marine microorganisms using a colorimetric screen based on the oxidation of acetylindole[2] (Figure 1) yielded a strain of bacterium displaying BVMO activity. The sequence of the genome was determined and revealed flavoprotein monooxygenase genes that may be the source of BV activity in the organism. In this poster, the cloning and expression of the gene encoding one of these enzymes is presented. The relevant gene product was unusual in that it displays a nicotinamide cofactor preference for the cheaper cofactor NADH over NADPH in the oxidation of a range of substrates including fused cyclobutanones and prochiral sulfides. An analysis of biotransformations, kinetics and X-ray crystallographic studies of the enzyme (Figure 1) will be also be presented.
Scheme 1. Biocatalytic n-butane hydroxylation with enzymatic in situ-cofactor regeneration Low temperature appeared to be desirable due to the low boiling point of -0.5 C of the liquid gas n-butane. Thus our investigation also addressed the dependency of the reaction course shown in Scheme 1 on the reaction temperature which we varied from -5C to room temperature. So far the best result was obtained at 0C with a product concentration of 0.16 g/L of 2-butanol. Thus, interestingly the P450-monooxygenase showed a surprisingly high catalytic activity at low temperature.
______________________ [1] F. E. Zilly, J. P. Acevedo, W. Augustyniak, A. Deege, U. W. Husig, M. T. Reetz, Angew. Chem. 2011, 123, 2772-2776; Angew. Chem. Int. Ed. 2011, 50, 2720-2724; [2] S. Staudt, C. A. Mller, J. Marienhagen, C. Bing, S. Buchholz, U. Schwaneberg, H. Grger, manuscript submitted.
In this poster we will present further studies into this enzyme, which catalyses a unique stereoselective course for this biotransformation amongst BVMOs known to date. The pure enzyme has been subjected to kinetic studies to determine substrate binding and turnover constants and scale-up studies have been performed on the biotransformation of 1 to 2. In addition, the catalytic behaviour of the enzyme towards a library of prochiral sulfide substrates will be presented.
1. D.E. Torres Pazmio, H.M. Dudek and M.W. Fraaije (2010) Curr. Opin. Chem. Biol., 14, 138-144. 2. C. Szolkowy, L.D. Eltis, N.C. Bruce and G. Grogan (2009) Chembiochem, 10, 1208-1217. 1. W.J.H. van Berkel, N.M. Kamerbeek and M.W. Fraaije (2006) J. Biotechnol., 124, 670-689. 2. Kamerbeek, N. M. (2004) Ph.D. thesis, University of Groningen, NL.
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Cofactor regeneration in polymersome nanoreactors: enzymatically catalysed Baeyer-Villiger reactions Silvie A. Meeuwissena, Ana Rioz-Martnezb, Gonzalo de Gonzaloc, Marco W. Fraaijec, Vicente Gotorb, Jan C. M. van Hesta a Radboud University Nijmegen, Institute for Molecules and Materials, Department of Organic Chemistry, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands; bDepartamento de Qumica Orgnica e Inorgnica, Instituto Universitario de Biotecnologa de Asturias, Universidad de Oviedo, c/ Julin Clavera 8, 33006 Oviedo, Spain; cLaboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. ana.rioz@gmail.com Baeyer-Villiger monooxygenases (BVMOs) are flavoproteins able to selectively catalyze the Baeyer-Villiger reaction and the oxygenation of different heteroatoms.[1] To sustain their catalytic activity, BVMOs require an electron supplier, as for instance, natural nicotinamide cofactors [NAD(P)H]. As these coenzymes are too expensive and can inhibit the biocatalysts when used in stoichiometric amount, an efficient cofactor recycling system is required for BVMOs cell-free applications. Polymersomes, assemblies of amphiphilic block copolymers in aqueous medium, have drawn particular attention in the last years due to their resemblance to liposomes and cell membranes.[2] These polymeric vesicles present different properties depending on the nature of the copolymer used in their synthesis. Therefore, by using a block copolymer as for instance, polystyrene-b-poly(3-(isocyano-l-alanyl-amino-ethyl)-thiophene) (PS-b-PIAT), porous polymersomes can be obtained.[3] In this way, it is possible to employ them as a biocatalytic nanoreactors where a cofactor regeneration system is feasible. Herein, we report a novel cofactor regeneration system for BVMO-catalysed reactions with PS-b-PIAT polymersomes as scaffold for positional assembly of the essential enzymes (Figure 1.). Efficient coenzyme regeneration was performed when CRE2-PAMO or G6PDH were encapsulated in the lumen of the vesicles. Covalent immobilisation of PAMO onto G6PDH loaded vesicles was achieved, although a less efficient reaction was yielded.
Regioselective hydroxylation by CYP153 enzymes Daniel Schepsa, Sumire Honda Malcaa, Michael Breuerb, Heinz Wagnerb, Bettina M. Nestla and Bernhard Hauera a Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany b BASF SE, Fine Chemicals & Biocatalysis, 67056 Ludwigshafen, Germany E-mail: sumire.honda@itb.uni-stuttgart.de Cytochrome P450 monooxygenases are a very large and diverse superfamily of hemecontaining proteins found in all domains of life. The enzymes catalyze a variety of reactions including hydroxylation of C-H bonds, heteroatom oxygenation, heteroatom release (dealkylation), oxidative deaminations, dehalogenations, desaturations and epoxide formation. [1] They have long been the focus of biochemists because of their ability to introduce a single atom of oxygen from O2 into an organic substrate, resulting in the one-step synthesis of complex molecules. Most of these enzymes only work as part of a multiprotein complex with redox partners providing electrons from NAD(P)H to the heme domain. Selectively hydroxylated hydrocarbons are of great interest in the chemical industry, due to their role as intermediates for the synthesis of bulk and fine chemicals. CYP153 enzymes are such enzymes catalyzing the terminal hydroxylation of aliphatic, alicyclic and alkyl-substituted compounds with high regiospecificity under mild reaction conditions.[2] CYP153 enzymes were cloned and expressed in Escherichia coli. The activity of each P450 was reconstituted with artificial electron transfer partners and assayed in vitro towards C5C12 n-alkanes and C6-C12 primary alcohol substrates. [3] This work was performed within the scope of the Systems Biology in Pseudomonas for Industrial Biocatalysis project and financial support by the German Federal Ministry of Education and Research (BMBF) and BASF SE is gratefully acknowledged. ________________
[1] Isin E.M., Guengerich F.P. Biochim Biophys Acta, 2007, 1770, 314-329. [2] van Beilen J.B., Funhoff E.G., van Loon A., Just A., Kaysser L., Bouza M., Holtackers R., Rthlisberger M., Li Z., Witholt B. Appl Environ Microbiol, 2006, 72, 59-65. [3] Scheps D.*, Honda Malca S.*, Hoffmann H., Nestl B.M. and Hauer B. Org Biomol Chem, 2011, manuscript accepted (* first authors).
Microbiological production of 11alpha-hydroxyprogesterone from pregnenolone
Vyacheslav V. Kollerov, Andrey A. Shutov, Marina V. Donova Institute of Biochemistry & Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino, Prospect Nauki,5, Moscow region, Russia E-mail: svkollerov@rambler.ru 11-Hydroxy progesterone is well known as effective anti-inflammatory agent and high-value precursor in the synthesis of pharmaceutical steroids [1]. It can be obtained microbially from progesterone (pregn-4-ene-3,20-dione) with high yield using fungal strains of Rhizopus nigricans or Aspergillus ochraceus [2]. The production of 11-hydroxyprogesterone from pregnenolone (3-hydroxypregn-5-ene-20-one) in a single biotechnological operation is an alternative way to its synthesis via intermediate obtaining of progesterone. However, the information about microbial conversion of pregnenolone to 11-hydroxyprogesterone is rather limited. In this work, we screened 31 fungal strains of different taxonomy for the ability to carry out pregnenolone conversion. The target activity has been revealed for three strains of Aspergillus genus with its maximal level in Aspergillus niger VKM F-212. Four products were isolated and identified using mass spectrometry and 1H NMR analyses after 96hincubation of the strain with pregnenolone (0.5 g/l). 11-Hydroxyprogesterone was accumulated as a major product (52%), while progesterone, 6,11-dihydroxyprogesterone and 11,17-dihydroxyprogesterone were observed in minor amounts (totally, no more than 10%). Based on the time courses of products accumulation, the following route of pregnenolone transformation by A.niger VKM F-212 was proposed (Fig.1)
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Metabolic responses of escherichia coli to intracellular oxygenation of Cyclohexanone into -caprolactone
Ji-Yeong Yuna, Eun-Hee Dooa, Bruno Bhlerb, Daniel Kuhnb, Lars Blankb, Andreas Schmidb, Jin-Byung Parka a Department of Food Science & Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea; bLaboratory of Chemical Biotechnology, Dortmund University of Technology, D-44227 Dortmund, Germany E-mail: jbpark06@ewha.ac.kr Metabolic responses of the recombinant Escherichia coli to intracellular oxygenation of cyclohexanone to -caprolactone (Fig. 1) were investigated to evaluate the factors that influence the oxidative biotransformations involving toxic substrates and products. The wholecell biotransformation during continuous cultivation and analysis of central carbon and cofactor metabolism using 13C tracer based metabolic flux analysis showed that over 60% of NADPH regenerated in the PPP and TCA cycle was directed to generation of NADH (ultimately ATP equivalents) to meet the increased demands of cellular maintenance energy. Indeed, below 10% of NADPH regenerated could be exploited for oxygenation of cyclohexanone. These results indicated that the carbon and cofactor metabolism of the recombinant E. coli cells during oxygenation were significantly influenced by availability of ATP equivalents impacted by toxic effects of the reaction substrates and products. Therefore, the toxicity of reaction substrates and products, influencing cofactor distribution and availability, can be considered to be one of the critical factors influencing the efficiency of cofactor-dependent whole-cell biocatalysis.
Figure 1. The pathway of pregnenolone transformation by Aspergillus niger VKM F-212. Under the optimized conditions, the yield of 11-hydroxyprogesterone reached 65% at the 6-fold increased loading of pregnenolone.
________________________ 1. Matthew, C.; Peterson, M.D. (1995). Progestogens, Progesterone antagonists, progesterone, and androgens: synthesis, classification and uses. Clin Obstet Gynecol, 38, 823820 2. Fernandes, P., Cruz, A., Angelova, B., Pinheiro, H.M., Cabral, J.M.S., 2003. Microbial conversion of steroid compounds: recent developments. Enzyme Microb. Technol, 32, 688705
Figure 1. PAMO-oxidation of phenylacetone employing PS-b-PIAT polymersomes as cofactor regeneration reactors.

_______________ [1] G. de Gonzalo, M. D. Mihovilovic, M. W. Fraaije, ChemBioChem, 2010, 11, 2208-2231. [2] D. E. Discher, A. Eisenberg, Science, 2002, 297, 967-973. [3] H.-P. M. de Hoog, D. M. Vriezema, M. Nallani, S. Kuiper, J. J. L. M. Cornelissen, A. E. Rowan, R. J. M. Nolte, Soft Matter, 2008, 4, 1003-1010.
Figure 1. Oxygenation of cyclohexanone into -caprolactone by cyclohexanone monooxygenase expressed in E. coli
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Towards the evolution of 2-oxoglutarate dependent hydroxylases
Laila Roper* and Gideon Grogan York Structural Biology Laboratory, University of York, Department of Chemistry, Heslington, YO10 5DD, York, United Kingdom * E-mail: laila@ysbl.york.ac.uk 2-Oxoglutarate dependent oxygenases are a group of non-haem iron requiring enzymes catalysing oxidative reactions that require 2-oxoglutarate (2-OG) as a cosubstrate.[1] Proline hydroxylases belong to this family and catalyse the hydroxylation of proline at the C3 or C4 position to form hydroxyprolines. Reported enzymes have all been found to be highly regio- and enantiospecific. a) b)
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11-Hydroxylation of 6-uoro-16-methyl-deoxycorticosterone by filamentous fungi
Vyacheslav V. Kollerov, Victoria V. Fokina, Galina V. Sukhodolskaya, Andrey A. Shutov, and Marina V. Donova Institute of Biochemistry & Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Prospect Nauki, 5, Russian Federation E-mail: donova@ibpm.pushchino.ru More than 100 strains of filamentous fungi of 31 genera have been tested for the capability to transform 6-fluoro-16-methyl desoxycorticosterone 21-acetate (FM-DOCA, I). The target 11-hydroxylating activity had been firstly revealed for the representatives of Gongronella, Scopulariopsis and Epicoccum, and confirmed for Curvularia strains. The strains of Curvularia lunata VKM F-644 and Gongronella butleri VKM Ac-2033D expressed maximal activity. The major products and intermediates of FM-DOCA conversion by the two strains selected have been identified using TLC, HPLC, mass-spectrometry and 1H-NMR spectroscopy analyses as followings: 6-fluoro-16-methyl desoxycorticosterone (FM-DOC, II), 6-fluoro-16-methyl-11-hydroxy corticosterone (FM-C, III), 6-fluoro-16-methyl11-hydroxy corticosterone 21-acetate (11-OH-FM-DOCA, IV), 6-fluoro-16-methyl9-hydroxy corticosterone (9-OH-FM-DOC, V), and 6-fluoro-16-methyl-14-hydroxy corticosterone (14-OH-FM-DOC, VI) (Fig.1).
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Heterologous Expression and Characterisation of Galactose oxidase from Fusarium oxysporum in E. coli
Regina Paukner, Petra Whrer, Christian Leitner University of Natural Resources and Applied Life Sciences, 1190 Vienna, Austria regina.paukner@boku.ac.at Galactose oxidase is an enzyme with a unique active centre containing one copper atom and a tyrosyl radical covalently cross linked to a cysteine. It catalyses the oxidation of a variety of mono, di- and oligosaccharides at the OH group at position C6 to the corresponding aldehydes. Secondary hydroxyl groups or aldehydes are not oxidised. Interestingly, the enzyme has a high stereoselectivity regarding the C4 hydroxyl group leading to a high activity with galactose compared to no detectable activity with glucose. During this process oxygen is reduced to hydrogen peroxide. The so produced dialdehydes are interesting synthons in organic chemistry. During a screening we found that the enzyme is widespread among the Fusarium sp.. F. oxysporum was chosen as a novel producer of this enzyme. In this poster we will show the cloning of the galactose oxidase gene from F. oxysporum. Sequence identity was 77 % compared with the known sequence from F. graminearum. It was heterologously expressed in E. coli in good yields under control of the T7 promotor. By adding a C-terminal His-tag the enzyme could be purified via a one step affinity chromatography. The purified enzyme was characterised in detail.
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Exploring the ambiguity of P450 BM3: allylic oxidation vs. epoxidation
Katharina Neufeld, Jrg Pietruszka Institute of Bioorganic Chemistry, Heinrich Heine University Dsseldorf located in the Forschungszentrum Jlich, Stetternicher Forst Bldg. 15.8, 52426 Jlich, Germany E-mail: k.neufeld@fz-juelich.de Carbon-hydrogen activation of the allylic position is a hot topic in organic synthesis. Regularly transition metals are employed to achieve allylic functionalization.[1] One of natures alternatives is the utilization of monooxygenases. Due to their ability to catalyse the selective insertion of an oxygen atom from atmospheric dioxygen into non-activated carbon-hydrogen bonds of a variety of organic compounds, monooxygenases bear high potential for organic chemistry.[2] P450 BM3 is a well characterised, highly active and self-sufficient monooxygenase from Bacillus megaterium,[3] which was turned into a powerful biocatalyst concerning substrate spectra and hydroxylation patterns in engineering approaches. [4] In search of monooxygenases producing allylic hydroxylation products starting from olefins, a P450 BM3 triple mutant was observed to exhibit activity towards a terminal olefin producing the corresponding epoxide as the main product, whereas the wild-type enzyme was identified as an allylic alcohol forming catalyst. We will present our most recent efforts towards the enantioselective formation of allylic alcohols via monooxygenase mutants producing key-intermediates for active agent synthesis.
Figure I. The structures of FM-DOCA (I), major products and key intermediates by its bioconversion with Curvularia lunata VKM F-644 and Gongronella butleri VKM F-2033D
[1] Clifton, I. J. mcDonough, M. A. et al. and Schofield, C.J. (2006) J. Inorg. Biochem. 100 644-669 [2] Johnston, R. M., Chu, L. N., Liu, M., Goldberg, S.L, Goswami, A. and Patel, R. N. (2009) Enzym. Microb.Tech. 45 484-490
The different routes of FM-DOCA conversion by the two strains were observed. C.lunata pathway included FM-DOCA deacetylation (I-II) followed by hydroxylations at the positions 11, 9 and 14 to form the mixture of hydroxylated derivatives of FM-DOC (III, V, VI). The initial step of FM-DOCA transformation by mycelium of G.butleri was 11-hydroxylation to form 11-hydroxy derivative (IV) as a major product. The next step was deacetylation (IV - III). The compounds V and VI were formed in minor amounts. The enzyme accounting for 11-hydroxylation in G.butleri was found to be constitutive, and of membrane-binding localisation, in contrast to inducible 11-hydroxylase of C.lunata which located in microsomes. At the optimised conditions, the yield of target 6-fluoro-16-methyl-11-hydroxy corticosterone (III) from FM-DOCA (6 g/l) reached 72%. The results can be applied at the production of high-value precursors of pharmaceutical steroids.
Figure 1. Structure model of P450 BM3 (PDB: 1BVY) showing the heme- and reductase domain fused in a single polypeptide chain and potential oxy-functionalisation products.
________________ [1]G. Liu, Y. Wu, Top. Curr. Chem. 2010, 292, 195. [2]a) C. J. C. Whitehouse, S. G. Bell, H. G. Tufton, R. J. P. Kenny, L. C. I. Ogilvie, L. Wong, Chem. Commun. 2008, 966; b) H. M. Girvan, C. W. Levy, P. Williams, K. Fisher, M. R. Cheesman, S. E. J. Rigby, D. Leys, A. W. Munro, Biochem. J. 2010, 427, 455. [3]a) A. M. Sawayama, M. M. Y. Chen, P. Kulanthaivel, M. Kuo, H. Hemmerle, F. H. Arnold, Chem. Eur. J. 2009, 15, 11723; b) D. Appel, S. Lutz-Wahl, P. Fischer, U. Schwaneberg, R. D. Schmid, J. Biotech. 2001, 88, 167. [4]C. R. Otey, G. Bandara, J. Lalonde, K. Takahashi, F. H. Arnold, Biotech. Bioeng. 2006, 93, 494.
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Evaluating yeasts as hosts for heterologous expression of cytochrome P450 monooxygenases
C.W. Therona,b, M. Labuschagnea, J. Albertyna, R.K. Gudiminchia,b, M.S. Smita,b a Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa; bSouth African DST-NRF Centre of Excellence in Catalysis, c*change E-mail: theroncw@ufs.ac.za Cytochrome P450 monooxygenases catalyse reactions which are extremely difficult to achieve using traditional chemical methods. Therefore, biotransformations using P450s are of great interest.However, drawbacks to using these enzymes includes that they are often of a multi-component nature, as well as their requirement for co-factor recycling. These factors make their application as isolated enzymes very challenging. Therefore whole cell biocatalysis is considered, as enzymes in whole cells could have everything required for catalysis within their immediate environment. Since natural hosts are often unsafe and / or difficult to work with, hosts for heterologous expression are considered. Escherichia coli and Saccharomyces cerevisiae have been the traditional hosts of choice, but have numerous limitations. Yeasts are attractive alternative hosts, as they combine eukaryotic cellular machinery with prokaryotic ease of manipulation. In this study we compared S. cerevisiae to Kluyveromyces marxianus, Yarrowia lipolytica, and Arxula adeninivorans. The target genes were the eukaryotic CYP53B1, co-expressed with three different reductase (CPR) partners; as well as two self-sufficient P450s, the bacterial CYP102A1 and the eukaryotic CYP505A1. For a direct comparison between the yeasts, a common broad-range vector system was utilised, to facilitate comparison of the different yeasts with no yeast being favoured. The genes of interest were expressed under a translation elongation factor (TEF) promoter. All yeast cells were grown for 48h into their stationary phase of growth before assaying commenced. Using benzoic acid, the natural substrate for CYP53B1, we found heterologous enzyme activity in A. adeninivorans to be far superior to the other yeasts. Within 24h the poorer performing A. adeninivorans strain transformant produced ~28 mol gDCW-1 of p-hydroxybenzoic acid product compared to ~12 mol gDCW-1 produced by S. cerevisiae, which was the next best producer. One of the A. adeninivorans transformants, which probably contains two copies of the gene, produced ~52 mol gDCW-1. Furthermore, when co-expressing withthe different reductase partners, we found that the activity was further enhanced, particularly with the reductase from Ustilago maydis, where the production increased to ~190 mol gDCW-1. It is important to emphasise that these results were obtained under conditions that were not yet optimised, as the aim was to get an initial comparison of the expression hosts as a starting point. The most promising candidate will be used in future for optimisation studies. We have also demonstrated the expression of CYP505 and CYP102A1 in most of these yeasts, using hexylbenzoic acid as non-natural substrate. Again activity in A. adeninivorans was far superior to the others. Comparative expression of all these P450s in E. coli is currently also under investigation.
Potential application of bacterial vanadium-dependent haloperoxidases in biocatalytic antibiotic synthesis
M.A. van der Horsta, A.F. Hartoga, R. Wevera a University of Amsterdam, Van t Hoff Institute for Molecular Sciences, Science Park 904, PO Box 94157, 1090 GD Amsterdam, The Netherlands E-mail: m.a.vanderhorst@uva.nl Vanadium-dependent haloperoxidases catalyze the halogenation of organic molecules through oxygenation of a halide ion using hydrogen peroxide. This class of enzymes has been mainly found in eukaryotes [1], but recently a bacterial vanadium-dependent chloroperoxidase (VCPO) was identified in Streptomyces, which is involved in the biosynthesis of napyradiomycin antibiotics [2]. Since we want to explore the possibility to use these enzymes in stereoselective halogenations, a bioinformatic analysis was performed to study the presence of these enzymes in the bacterial kingdom. Because in bacteria functionally related genes are often clustered together in the genome, this yields more general insight in the biological function of these proteins. Furthermore, the discovery of more bacterial VCPOs can possibly lead to a new toolbox for biocatalytic antibiotic synthesis. We found several genes in sequenced bacterial genomes that show high sequence homology to the known VCPOs. From these, several candidates were found that likely encode a VCPO, based on a more thorough analysis of the conservation of active site residues. We are studying the putative VCPO from Rhodopseudomonas palustris and will present the first results here.
________________ [1] Wever, R. and Renirie, R. (2010) Structure and function of vanadium haloperoxidases in Peroxidases and Catalases: Biochemistry, Biophysics, Biotechnology and Physiology. H. B. Dunford (ed.). Wiley pp. 403 423 [2] Bernhardt, P., Okino, T., Winter, J.M., Miyanaga, A., Moore, B.S. (2011) A stereoselective VanadiumDependent Chloroperoxidase in Bacterial Antibiotic Biosynthesis. JACS 133, 4268-4270
Characterisation of a versatile bacterial laccase and its potential biotechnological application
Renate Reiss, Julian Ihssen & Linda Thny-Meyer Laboratory for Biomaterials, Empa, Swiss federal laboratories for Materials Science and Technology, Lerchenfeldstrasse, 5, CH-9014 St. Gallen, Switzerland renate.reiss@empa.ch Laccases (EC 1.10.3.2) are multi-copper oxidases that catalyse the one-electron oxidation of a broad range of compounds including substituted phenols, arylamines and aromatic thiols to the corresponding radicals. Laccases have been industrially applied for purposes ranging from detoxification of recalcitrant pollutants originating from the pulp and paper, textile and petrochemical industries as well as in the bioremediation of soil from herbicides and pesticides. Their catalytic ability has been demonstrated in the synthesis of pharmaceutical compounds such as steroid hormones and antibiotic derivatives. Owing to their wide substrate range, copper containing laccases are versatile biocatalysts, capable of oxidising natural and non-natural industrial compounds, with water as the sole by-product [1]. Developing bacterial laccases for biotechnological applications is advantageous in view that they can be produced in high yields and activity can be tailored using techniques such as directed evolution in cost-effective bacterial expression systems. We have cloned, expressed and purified a novel CotA-type laccase from Bacillus pumilus for biochemical characterisation [2]. Its catalytic activity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP) was investigated and the kinetic parameters, pH and temperature optimum were determined. Further, a comprehensive laccase substrate screen of natural and nonnatural compounds was recorded, revealing a wide substrate spectrum. A laccase oxidised substrate that forms a stable radical, which subsequently oxidises a secondary non-laccase substrate is referred to as a mediator. Preferably, mediators are good laccase substrates, stable in reduced and oxidised form and have no inhibitory effect upon enzyme activity. Once oxidised, mediators interact between the active site of the enzyme and the target molecule, thus broadening the substrate range. All substrates were tested for their ability to act as mediators in the decolourisation of various dyes, e.g. indigocarmine (IC). IC belongs to the group of carbonyl dyes and is widely used in the industrial production of denim. Dye effluents from the textile industry represent a major environmental pollutant. The decolourisation of this dye remains challenging and enzymatic degradation using laccase has great potential. The results of our substrate and mediator screen with B. pumilus laccase will be presented.
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The inuence of the cultivation conditions on the activity and specificity of the amino acid oxidases of the eucaryotic origin.
Magorzata Brzeziska-Rodaka, Anna Gnacha, Ewa ymaczyk-Dudaa, Magdalena Klimek- Ochaba, Pawe Kafarskia a Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocaw University of Technology, Wybrzee Wyspiaskiego 27, 50-370 Wrocaw, Poland E-mail: malgorzata.brzezinska-rodak@gmail.com D/L-Amino-acid oxidases catalyze the oxidative deamination of the amino acids with production of the corresponding 2-oxo acids and ammonia. Today, the flavoprotein D-amino acid oxidase (D-AO) is used at industrial scale as one of the two biocatalysts taking part in the enzymatic two-step conversion of cephalosporin C to 7-aminocephalosporanic acid [1]. The latter is a key compound for the production of many semisynthetic, cephalosporin - based -lactam drugs. Other important fields of application of the enantioselective D/L-A0 are the production of -keto acids [2] and pure L-amino acids [3]. This enzyme has applications as a part of the biosensors for L-amino acids [4,5] and catalysts in biotransformations [6]. That is why, the evaluation of the level of these enzymes production and the activity assignment as an implication of different gaining conditions is still under consideration. The activity of the amino acid oxidase toward suitable enantiomer of amino acids (D-alanine and L- lysine) in free-cells extract of the Rhodotorula gracilis strain was assayed. The influence of the external factors such as the composition of the cultivation media and the time of the incubation on the enzyme activity was examined. We applied a selective and sensitive peroxidase/o-dianisidine assay, detecting the formation of hydrogen peroxide. The best activity (27 Units/mg) was observed for R. gracilis DAAO cultured for 5 days in medium containing 0.1% D-alanine, comparing result for yeasts cultivated without any inductor, was: 0,91 Units/mg. Addition of 0.1% L-leucine increased the activity of LAAO only four times (0.88 Units/mg), while without the induction was 0,22 Units/mg.
Figure 1. Schematic representation of laccase-mediator redox cycle

________________ [1]a) S. Riva, Trends Biotechnol. 2006, 24, 219-226; b) S. Witayakran, A. J. Ragauskas, Adv. Synth. Catal. 2009, 351, 1187-1209. [2]R. Reiss, J. Ihssen, L. Thony-Meyer, BMC Biotechnol. 2011, 11, -.
This work was financed from Project Biotransformations for pharmaceutical and cosmetics industry No.POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund for the Innovative Economy. ________________ [1] D.H. Conlin, J. Baqal, K. Baker, Q. Shen, R. Wong, R. Nolles, C.W., Biotechnology and Bioengineering 46 (1995), 510-513. [2] S. Buto, L. Polegionlli, L. DAngiuro, M.S. Pilone, Biotechnology and Bioengineering 44 (1994), 12881294. [3] L. Fisher, R. Horner, F. Wagner, Annals of the New York Academy of Sciences 750 (1995), 415-420. [4] P. Sarkar, I.E. Tothill, S.J Setfords, A.P.F. Turner, Analyst (Lond.), 124 (6) (1999), 865870. [5] E. Akyilmaz, A. Erdogan, R. Ozturk, I. Yasa, Biosensors and Bioelectronics, 22(6) (2007), 10551060. [6] E. Takahashi, M. Furui, H. Seko, T. Shibatani, Applied Microbiology and Biotechnology, 47(2) (1997), 173179.
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Reengineering P450 BM-3 monooxygenase for industrial applications: production of bulk chemicals
Christina A. Mllera, Svenja Staudtb, Till Winklerc, Amol Shivangea, Harald Grgerb,d, Werner Hummelc, Ulrich Schwaneberga a Lehrstuhl fr Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; bDepartment of Chemistry and Pharmacy, University of ErlangenNrnberg, Henkestrae 42, 91054 Erlangen, Germany; cInstitut fr Enzymtechnologie der Heinrich-Heine-Universitt, Forschungs-zentrum Jlich, Stetternicher Forst, 52426 Jlich, Germany; dFaculty of Chemistry, Bielefeld University, Universittsstrae 25, 33615 Bielefeld, Germany E-mail: c.mueller@biotec.rwth-aachen.de Cytochrom P450 monooxygenases have the remarkable capability of using molecular oxygen as sole oxidizing agent for the functionalization of C-H bonds. A wide range of industrial relevant hydroxylation, epoxidation and other oxidative reactions are catalyzed by this enzyme family. In the last decade, a large variety of P450 monooxygenase mutants have been generated through directed evolution and rational design to overcome major limitations of P450 as a biocatalyst for industrial applications. Some of the limitations include poor activity towards non-natural substrates, low co-solvent stability and dependence on equimolar amounts of NAD(P)H as cofactor.[1] Here we present an attractive alternative to traditional chemo-catalytic processes for the one-pot direct oxidation of short alkanes. In our approach we couple the hydroxylation of n-heptane, using a P450 BM-3 monooxygenase variant, with the oxidation of the resulting alcohols by means of an alcohol dehydrogenase (Fig. 1).
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Cytochrome P450 Monooxygenase-Catalysed Hydroxylations
Sumire Honda Malca, Daniel Scheps, Bettina M. Nestl, Bernhard Hauer Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany E-mail: bettina.nestl@itb.uni-stuttgart.de Cytochrome P450 monooxygenases are very attractive biocatalysts as they are able to catalyse the monooxygenation of a large variety of hydrophobic chemical compounds, such as alkanes, xenobiotics, steroids and prostaglandins [1]. CYP153s are bacterial class I P450s that operate as three-component systems, comprised by the P450 itself and two additional redox protein partners. However, their cofactor requirements, their low activity and stability and their restricted electron supply are limiting the academic and industrial implementation of these biocatalysts [2].
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Biocatalytic C=C double bond cleavage by an enzyme from Trametes hirsuta: unexpected metal dependence
Aashrita Rajagopalana, Francesco G. Muttia, Markus Schobera, Wolfgang Kroutila a University of Graz, Heinrichstrasse 28, 8010 Graz, Austria; E-mail: aashrita.rajagopalan@uni-graz.at Alkene cleavage is a widely investigated oxidation reaction in organic chemistry.[1,2] Recently, the biocatalytic cleavage of C=C double bonds adjacent to an aromatic ring was found to be carried out by a cell-free extract of white-rot fungi Trametes hirsuta, with oxygen as the sole oxidant (Figure 1).[3,4] The reaction conditions were optimized to carry out the reaction with cell-free extract. Studies of the mechanism of the enzymatic reaction suggested a metal-dependence of the alkene cleavage activity.[5] The spectrophotometric study of the enzymatic reaction led to the unexpected finding that the enzyme was Mn(III) dependent. It was also observed that the loss of enzymatic activity during enzyme purification could be recovered by addition of Mn(III). This result enabled the purification of the enzyme. Degenerated primers were designed based on the short peptide sequences got from the MALDI-MS/MS and De-novo sequencing of the purified enzyme band from the 1D SDS PAGE. These could be used as primers for the PCR, with cDNA of T. hirsuta as the template. This work deals with the preliminary identification and amino acid sequence determination of an unknown enzyme with an Mn(III) dependence from a fungus whose genome is not yet sequenced.
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Mediated electron transfer with P450cin experimental data and prediction tools
S. Zengin ekia, D. Holtmanna, M. Fioronib K.M. Mangolda, U. Schwanebergb, J. Schradera a DECHEMA e.V., Karl- Winnacker-Institut, Theodor-Heuss-Allee 25, 60486 Frankfurt/ Main, Germany, bRWTH Aachen University, Department of Biotechnology, Worringerweg 1, 52074 Aachen, Germany E-mail: holtmann@dechema.de A key issue for catalytic applications of isolated P450s is the demand for a continuous electron supply to the heme group. In vivo, the electrons are supplied by NAD(P)H which is, however, too expensive for in vitro technical applications. Furthermore the redox partners of P450s are often unknown or not expressible in an active form. The substitution of NADPH as well as the redox partner by using electrochemical methods can help to overcome these problems. In the present study, different mediators and prosthetic groups (e.g. viologens, cobalt sepulchrate, phenosafranine, FAD, FMN) were tested with the model system P450cin, which catalyzes the stereoselective hydroxylation of 1,8-cineole at the 2 position. The product is a valuable intermediate in the synthesis of herbicides and fragrance compounds. Investigations were carried out in an electrochemical cell in which mediators were reduced on a metal electrode, which subsequently deliver the electrons to the heme of P450cin. Two different artificial electron transfer modes have been tested (Fig. 1). First, to mimic Figure 1: Schematic representation of the part of the natural electron transfer path we natural and mediator-driven electron transfer chain. used cindoxin which shuttles the electrons between the redox mediator and P450cin. Second, also cindoxin was omitted and a direct electron transfer from the redox mediator to P450cin was investigated. For both modes mediators have been identified which enable the electrochemically driven product formation. The experimental results were verified by enzyme-mediator docking studies. The combination of geometry-based molecular docking algorithm and calculation allowed the prediction of theoretical electron transfer rates. The predicted values correlate with the experimental data. With the help of docking and molecular dynamics studies we have identified further mediators for the electron transfer to P450cin and tested them successfully in the electrochemical cell. The combination of proteinmediator docking algorithms and quantum mechanics allows a prediction of suitable mediators for different P450, rational design of novel improved mediators and identification of important amino-acids for an effective electron transfer.
Figure 1. Hydroxylation of alkanes and primary alcohols under the catalysis of a cytochrome P450 monooxygenase/ferredoxin/ferredoxin reductase delivering electrons to the heme cofactor. In this project cytochrome P450 monooxygenases of the CYP153A family [3] from Mycobacterium marinum M. and Polaromonas sp. were selected as model enzymes for the selective terminal hydroxylation of medium-chain n-alkanes and primary alcohols. Alkanes are especially inert molecules, thus their selective transformation into high value intermediates continues to be a scientific challenge. Due to a negligible P450 oxidation activity in the application of CYP153A monooxygenases, putidaredoxin reductase (PdR or CamA) and putidaredoxin (Pdx or CamB) from Pseudomonas sp. showing high protein similarity to the natural redox partners, were used to reconstitute the activity of the CYP153A monoxygenases. Together with alkanes, various primary alcohols were hydroxylated by purified CYP153 monooxygenase, PdR and Pdx in in vitro reactions. Although, in experiments both enzyme systems yielded primary alcohols and ,-alkanediols, each one displayed a different oxidation pattern towards alkanes demonstrating -hydroxylation and ,-hydroxylation activity [4]. This project is financially supported by the Bundesministerium fr Bildung und Forschung (BMBF), Germany. ____________________
Fig. 1: Biocatalytic, coupled direct-oxidation of n-heptane to produce 2-heptanone The short-chain alkane-hydroxylating P450 BM-3 variant 19A12[2] was selected for reengineering to improve activity towards n-heptane. Eleven amino acid positions were selected based on literature reports for site saturation mutagenesis. Two substitutions (A74E, V184S) contributing to enhanced activity were identified. The substitutions are situated in the substrate binding pocket. The aliphatic amino acids (Ala/Val) were exchanged with sterically demanding amino acids (Glu/Ser), making the binding cavity more favorable to convert small substrates like n-heptane. The described approach was validated by coupling the monooxygenase reaction to a second oxidation using a (S)-specific alcohol dehydrogenase from Rhodococcus erythropolis. [3] Further development of the biocatalytic system has the potential to be advanced to an efficient process for the direct oxidation of bulk chemicals under mild conditions, using molecular oxygen as sole oxidizing agent and an enzyme-coupled regeneration of the costly cofactor NAD(P)H.
Figure 1: Biocatalytic alkene cleavage by T. hirsuta

_______________________ [1]F.E. Khn, R.W. Fischer, W.A. Hermann, T. Weskamp in Transition Metals for Organic Synthesis, Vol. 2 (Eds. M. Beller, C. Bolm), Wiley-VCH, Weinheim, 2004, pp. 427-436. [2] R. A. Berglund in Encyclopedia of Reagents for Organic Synthesis, Vol. 6 (Ed.: L. A. Paquette), Wiley, New York, 1995, pp. 3837-3843. [3] H. Mang, J. Gross, M. Lara, C. Goessler, H.E. Schoemaker, G. M. Gbitz, W. Kroutil, Angew. Chem., Int. Ed. 2006, 45, 5201-5203. [4] M. Lara, F.G. Mutti, S. M. Glueck, W. Kroutil. Eur. J. Org. Chem. 2008, 3668-3672. [5] M. Lara, F.G. Mutti, S.M. Glueck, W. Kroutil. J. Am. Chem. Soc. 2009, 131, 5368-5369.
________________ [1] V. B. Urlacher, S. Eiben, Trends Biotechnol. 2006, 24, 324-330. [2] R. Fasan, M. M. Chen, N. C. Crook, F. H. Arnold, Angew. Chem. Int. Ed. 2007, 46, 8414 8418. [3] W. Hummel, K. Abokitse, K. Drauz, C. Rollmann, H. Grger, Adv. Synth. Catal. 2003, 345, 153-159.
[1] R. Bernhardt, J. Biotechnol. 2006, 124, 128-145. [2] E. OReilly, V. Khler, S. L. Flitsch, N. J. Turner, Chem. Commun. 2011, 47, 2490-2501. [3] a) E. G. Funhoff, J. B. van Beilen, Biocatal. Biotrans. 2007, 25, 186-193; b) M. Bordeaux, A. Galarneau, F. Fajula, J. Drone, Angew. Chem. Int. Ed. 2011, 50, 2075-2079. [4] D. Scheps, S. Honda Malca, H. Hoffmann, B. M. Nestl, B. Hauer, manuscript submitted.
October 2-6, 2011
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Laccases: Potential biocatalysts for the synthesis of natural products
Katja Koschorreck, Vlada B. Urlacher Institute of Biochemistry II, Heinrich Heine University Dsseldorf, Universittsstr. 1, 40225 Dsseldorf, Germany E-mail: Katja.Koschorreck@uni-duesseldorf.de Laccases are multicopper oxidoreductases (E.C. 1.10.3.2) capable of oxidizing a vast variety of phenolic and non-phenolic compounds through the concomitant reduction of molecular oxygen to water. These enzymes are widely distributed among fungi, higher plants and bacteria. In nature they are involved in degradation of lignin, pigment production and pathogenesis. Due to their broad substrate spectrum and wide range of catalyzed reactions, which include cross-linking of monomers, degradation of polymers, and oxyfunctionalization of aromatic compounds, laccases are considered to be industrially relevant enzymes. Besides application in textile bleaching and the pulp and paper industry, laccases have gained in importance for the production of fine chemicals and pharmaceuticals. Their capability of homo- and heteromolecular coupling of low molecular weight phenolic molecules makes them of special interest for the synthesis of biologically active compounds like antibiotics, antioxidants and cytostatics. Several novel microbial laccases (T. versicolor, P. ostreatus, B. licheniformis, B. subtilis) have been heterologously expressed in Pichia pastoris and Escherichia coli and investigated regarding their C-C-coupling activity. Both, fungal and bacterial laccases demonstrate high activity upon dimerization of phenolic acids.[1-2] However, fungal laccases are the superior enzymes owing to their higher activity and selectivity in this reaction. Moreover, fungal laccases often possess a broader substrate spectrum compared to bacterial ones which might be attributed to their higher redox potential (0.4-0.8 V vs. 0.4-0.6 V). The use of small redox mediators in laccase-catalyzed reactions compensates to a certain extent for a lower redox potential and allows for the oxidation of bulky substrates with high redox potentials. So-called Laccase-Mediator-Systems (LMS) thus open new ways for the synthesis of sterically demanding compounds.
Laccase
Aerobic oxidations using alcohol dehydrogenases
Serena Gargiulo, Paul Knst, Isabel Arends, Frank Hollmann* Department of Biotechnology, Delft University of Technology, Julianalaan 136, Delft, The Netherlands E-mail: s.gargiulo@tudelft.nl Alcohol oxidations have great potential in synthetic organic chemistry. Enzymatic approaches display a variety of possible applications, including the conversion of diols to the corresponding lactones, the regio-selective oxidations of polyols and the resolution of racemic alcohols.[1] However, one issue that did not receive much attention is the regeneration of the oxidized nicotinamide cofactor NAD(P+). Currently the most popular approach is to recycle NAD(P)+ in situ by addition of an oxidized co-substrate. Even though established, this substrate-coupled approach has two major drawbacks: the poor thermodynamic driving force and the amount of waste produced due to the huge molar surplus of the co-substrate required.
Actinobacterial oxidases as novel cross-linking agents
Stephanie Burton, Zaida Palmer, Marilize Le Roes-Hill Biocatalysis and Technical Biology Research Group, Cape Peninsula University of Technology, PO Box 1906, Bellville, 7535, South Africa E-mail: burtons@cput.ac.za or Stephanie.burton@up.ac.za With new approaches in the fields of nanotechnology and biomaterials, new applications are emerging which involve the use of benign methods of achieving cross-linking reactions [1,2]. Over the past five years we have isolated some 1400 different strains, (mostly actinobacteria) from different environments, and screened them using customized discriminatory assay methods, specifically focusing on the detection of the oxidative enzymes, laccase, peroxidase, phenoxazinone synthase (PHS) and tyrosinase. These oxidative enzymes are well-known, especially those produced by fungi, but those from bacterial sources are not well understood. Using selection criteria focused on the suitability of these enzymes as industrial biocatalysts (viz., extracellular production, stability, activity at extremes of temperature and/or pH, stability in presence of organic solvents [3]), we have selected a sub set of some 9 enzymes with high activities. Each of the enzymes have been produced at small scale and shown to have some unusual properties which make them superior to their previously reported analogs (such as activity at different pH or with particular substrates). Each of the enzymes described above is capable of delivering controllable cross-linking (as opposed to chemical cross-linking which can be unpredictable and difficult to control). The different oxidases (tyrosinase, laccase, peroxidase and PHS) together provide a range of possible linkage pathways which are being exploited in our research programme. The results of this study and the potential for useful novel materials will be discussed.
________________ [1] Basnar, B., Xu, J., Li, D., Willner, I. (2007). Encoded and enzyme-activated nanolithography of gold and magnetic nanoparticles on silicon. Langmuir 23:2293-2296. [2] VTT publication 693, Selinheimo, E. (2008). Tyrosinase and laccase as novel crosslinking tools for food biopolymers. [3] Burton, S.G., Cowan, D.A., Woodley, J.M. (2002). The search for the ideal biocatalyst. Nature Biotechnology 20:37-44.
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Selective substrate oxyfunctionalizations using bacterial cytochrome P450 monooxygenases
Anett Schallmeya, Paula Braccoa, Dick B. Janssenb a Junior Professorship for Biocatalysis, Department of Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany; bBiochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands E-mail: a.schallmey@biotec.rwth-aachen.de Cytochrome P450 monooxygenases are very attractive enzymes for use in biocatalysis since they are able to directly introduce oxygen into various substrates using molecular oxygen from air. As an example, they catalyze the direct hydroxylation of non-activated carbon atoms a reaction that is very difficult to perform chemically [1]. We have studied a bacterial P450 monooxygenase from Nocardia farcinica for regio- and stereoselective hydroxylation of various steroids in order to produce hydroxylated products that are valuable intermediates for the synthesis of steroid hormones. Thus we could show in preparative biotransformations that the enzyme exhibits very high selectivity for hydroxylation in 16-position of most tested steroid substrates (Fig. 1). 16-hydroxylated steroids are important intermediates in the synthesis of highly active glucocorticoids [2]. For all tested substrates, application of resting cells for preparative-scale reactions outperformed, in terms of conversion and productivity, reactions using cell-free extract of P450-containing E. coli cells.
Figure 1: schematic representation of the approach used for the aerobic regeneration of NAD(P)+ . Alternatively, a system based on molecular oxygen as the final electron acceptor would provide a much higher driving force and would basically eliminate waste generation. We present two approaches developed in our group for the aerobic regeneration of NAD(P)+. The first method relies on the so called laccase-mediator system (LMS): a chemo-enzymatic system in which NAD(P)H reacts with an oxidized mediator to give NAD(P)+ and the reduced mediator, that is subsequently re-oxidized by laccase.[2,3] The enzymatic recycling of the mediator is paired to the reduction of molecular oxygen, yielding water. The second method we propose is based on the use of photo-excited flavins capable of shuttling reducing equivalents from NAD(P)H directly to molecular oxygen, yielding NAD(P)+. The rate of NAD(P)H oxidation is increased by two orders of magnitude upon illumination, thereby improving the performance of FMN as regeneration catalyst.[4] Both systems have been characterized and tested in situ, and their applicability to promote ADHs-catalysed oxidations on a preparative scale will be discussed.
________________ [1] W. Kroutil et al., Adv. Synth. Catal., 2004, 346, 125-142. [2] S.Riva Trends Biotechnol., 2006, 24, 219-226. [3] S. Aksu et al., Adv. Synth Catal, 2009, 351, 1211-1216. [4] S. Gargiulo et al., ChemCatChem, 2011, 3, 338-342.
R Figure 1. Laccase-catalyzed C-C coupling.
Mediator
Figure 1. Selective hydroxylation of testosterone at 16-position by the P450 monooxygenase from N. farcinica using FdR and Fdx as redox partners. Furthermore, we recently identified a moderately thermostable P450 monooxygenase, CYP154H1, from the thermophilic soil bacterium Thermobifida fusca [3]. The enzyme, exhibiting an apparent melting temperature of 67C, catalyzes the oxidation of mainly small arylaliphatic substrates like ethylbenzene, styrene and different arylaliphatic sulphides. Additionally, the P450 monooxygenase was found to exhibit a high regio- and stereoselectivity in the conversion of monoterpenes like -ionone and its derivatives. Currently, we are studying different thermostable redox partners of T. fusca in order to perform biocatalyses with CYP154H1 at elevated temperatures.
________________ [1] F. Hollmann, I. W. C. E. Arends, K. Bhler, A. Schallmey, B. Bhler, Green Chem., 2011, 13, 226-265 [2] M. Bureik & R. Bernhardt, 2007, in Modern Biooxidations, Wiley-VCH, 155-176 [3] A. Schallmey, G. den Besten, I. G. P. Teune, R. F. Kembaren, D. B. Janssen, Appl. Microbiol. Biotechnol., 2011, 89, 1475-1485
By applying a combined approach of random and site-directed mutagenesis the activity of a bacterial laccase towards phenolic compounds could be improved and the expression level significantly enhanced.[3] Thus, both fungal and bacterial laccases are quite promising enzymes for biotechnological applications.
________________ [1]Koschorreck K, Richter SM, Ene AB, Roduner E, Schmid RD, Urlacher VB (2008) Appl. Microbiol Biotechnol 79:217-224. [2]Koschorreck K, Richter SM, Swierczek A, Beifuss U, Schmid RD, Urlacher VB (2008) Arch Biochem Biophys 474:213-219. [3]Koschorreck K, Schmid RD, Urlacher VB (2009). BMC Biotechnol 9:12.
95
Phosphite-driven artificial self-sufficient cytochrome P450
a
96
Whole-cell biocatalysis for the controlled terminal functionalization of sp3 hybridized C-H bonds
Manfred Schrewe, Nadine Ladkau, Kerstin Lange, Eik Czarnotta, Mattijs K. Julsing, Andreas Schmid, Bruno Bhler Laboratory of Chemical Biotechnology, Faculty of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany E-mail: manfred.schrewe@bci.tu-dortmund.de The specific functionalization of sp3 C-H bonds remains a major challenge in organic chemistry. In contrast to traditional chemical means, biocatalysis offers multiple approaches to perform the desired reactions with outstanding stereo- and regioselectivity, high yields and turnover numbers [1, 2]. Here, the application of whole-cell biocatalysts was investigated for the formation of terminally (oxy)functionalized fatty acid methyl esters from renewable substrates (Fig. 1). Recombinant Escherichia coli carrying the alkane monooxygenase genes alkBGT from Pseudomonas putida GPo1 are able to catalyze three subsequent oxidation steps leading to terminal alcohols, aldehydes, and acids [3-5]. Detailed studies of the multistep reaction kinetics were the basis for the development of biotransformation strategies to selectively produce one product species. Two-liquid phase systems (2-LP) were used to control substrate availability and to ensure in situ product removal. Furthermore, the production of terminal amines was evaluated by the co-expression of an additional transaminase gene. The transamination reaction is coupled to oxygenase catalysis using the accumulating aldehyde as substrate. This study shows that, by making use of the reaction network and its kinetics, controlled product formation is possible. The constructed whole-cell catalysts are powerful tools even for complex multistep reactions allowing the production of specifically functionalized alkyl chains.
99
A recombinant -dioxygenase from rice to produce fatty aldehydes using E. coli
Fenja Khne, Markus Buchhaupt, Jens Schrader DECHEMA e.V. Karl-Winnacker-Institut, Biochemical Engineering, Theodor-HeussAllee 25, 60486, Frankfurt/Main, Germany buchhaupt@dechema.de Fatty aldehydes are an important group of fragrance and flavor compounds that are found in different fruits and flowers. A biotechnological synthesis of fatty aldehydes based on Escherichia coli cells expressing an -dioxygenase (DOX) from Oryza sativa (rice) is presented. -Dioxygenases are the initial enzymes of -oxidation in plants and oxidize long and medium-chain C( n ) fatty acids to 2-hydroperoxy fatty acids. The latter are converted to C( n - 1) fatty aldehydes by spontaneous decarboxylation. Successful expression of DOX in E. coli was proven by an in vitro luciferase assay. Using resting cells of this recombinant E. coli strain, conversion of different fatty acids to the respective fatty aldehydes shortened by one carbon atom was demonstrated. The application of Triton X 100 improves the conversion rate up to 1 g aldehyde per liter per hour. Easy reuse of the cells was demonstrated by performing a second biotransformation without any loss of biocatalytic activity.
Hiroshi Watanabea, Hidehiko Hirakawab, Teruyki Nagamunea,b Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan; bDepartmenet of Chemistry and Biotechnology, School of Engineering The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan E-mail: hirakawa@bio.t.u-tokyo.ac.jp
Cytochrom P450s (P450s) are potential biocatalysts because of their regio- and stereoselective monooxygenase activities. In many of bacterial P450s, their catalytic cycles require electrons from NAD(P)H through two kinds of proteins, ferredoxin reductases and ferredoxins. Therefore, there are two major charanges for their practical applications; i) requirement of high concentrations of ferredxin reductases and ferredoxins for efficient electron trasfer and ii) stoichiometric consumption of expensive NAD(P)H. Recently, we have demonstrated that Sulfolobus solfataricus PCNA-utilized heterotirimerization of Pseudomonasu putida P450 (P450cam), ptudaredoxin (PdX) and putidaredoxin reductase (PdR) dramatically enhanced its catalytic activity [1]. Subsequently, it is expected that cofactor regeneration in proximity to PdR could reduce the cofactor concentration. A phosphite dehydrogenase (PTDH) that catalyzes the oxidation from phosphite to phosphate with NAD+ is attractive for regeneration of NADH because PTDH activity is not inhibited by phosphate. In this study, we incorprated PTDH in the heterotrimer containing P450cam, PdX and PdR, and evaluated its cofactor regeneration (Figure 1). A triple fusion protein of PTDH-PCNA-PdX was successfully expressed in E. coli without any loss of function of PTDH. The fusion protein sponteneously formed a complex with two PCNA proteins fused with P450cam and PdR. PTDH fusion was effective to decrease the cofator concentration to keep the overall reaction rate compared with co-existing of PTDH.
Figure 1. Cofactor regeneration by PTDH fused with a PCNA-utilized heterotrimer of P450cam, PdX and PdR.
_______________ [1] H. Hirakawa and T. Nagamune, ChemBioChem, 11, 1517-1520 (2010).
Figure 1. Directed terminal functionalization of fatty acid methyl esters by recombinant whole-cells
________________ [1] W. A. Duetz et al., Curr. Opin. Biotechnol., 2001, 12, 419-25 [2] B. Buehler, and A. Schmid, J. Biotechnol., 2004, 113, 183-210 [3] S. W. May, and A. G. Katopodis, Enzyme Microb. Technol., 1986, 8, 17-21 [4] B. Witholt et al., Trends Biotechnol., 1990, 8, 46-52 [5] J. B. van Beilen et al.,. Enzyme Microb. Technol. 1994, 16, 904-911
October 2-6, 2011
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Immobilization of formate dehydrogenase from Candida boidinii on magnetic particles
Caterina G.C.M. Nettoa, Marcelo Nakamuraa, Leandro H. Andradeb , Henrique E. Tomaa a Supramolecular NanotechLab, Instituto de Quimica, Universidade de So Paulo, So PauloSP, Brazil b Laboratory of Fine Chemistry and Biocatalysis, Instituto de Quimica, Universidade de So Paulo, So PauloSP, Brazil E-mail: caterina@iq.usp.br Immobilization is widely used as a method for increasing the stability of enzymes and sometimes within immobilization, there is also an increase in enzyme activity and selectivity. [1] Among the supports and methods magnetic nano (or micro) particles are easily recovered by the simple use of an external magnetic field. In our study we performed the immobilization of FDH from Candida boidinii on different magnetite nanoparticles: MagNP-APTS, MagNP@SiO2-APTS and MagNP-Glioxyl Agarose in order to understand some parameters in immobilization processes and finding the best support among these three for the recycling of FDH. Formate dehydrogenase (FDH) is a NADH dependent enzyme which performs the conversion of CO2 to formate. [2] After optimization of the immobilization method we used these FDH-magnetic particles in activity studies for different pH and temperatures (figure 1).
Biotransformations in organic synthesis 101

Deep-eutectic-solvents: promising bio-based non-conventional media for biocatalysis
Zaira Maugeri, Pablo Domnguez de Mara Institute of Technical and Macromoelcular Chemistry (ITMC), RWTH Aachen, Worringerweg 1, 52074 Aachen, Germany E-mail: Dominguez@itmc.rwth-aachen.de Deep-eutectic-solvents (DES) are formed by complexion of quaternary ammonium salts (e.g. choline chloride) with hydrogen bond donors (HBD) such as amines, amides, alcohols, or carboxylic acids)[1]. The interaction of the HBD with the quaternary salt reduces the anioncation electrostatic force, thus decreasing the freezing point of the mixture. DES share many characteristics of conventional ionic liquids, yet being DES components often cheaper and biodegradable. Furthermore, DES syntheses do not require purification. Promising applications of DES have already been reported, e.g. in electrochemistry and electrodeposition[2], as (co-)solvent in organic[3] and inorganic syntheses[4], as well as in biocatalysis[5-7]. Herein novel choline-chloride-based DES are presented, by adding different HBD entirely derived from renewable resources (carboxylic acids and some saccharide-derived polyols). Interestingly, some of them (e.g. D-sorbitol) are chiral, and therefore chiral DES can be formed in a straightforward manner. Chiral solvents are usually expensive and their use is therefore limited. New DES were characterized by measuring melting point, pH, and water content. Addition of glycerol led to diminished viscosities in formed DES. Likewise, kinetic studies on water absorption were investigated in a closed system at specific humidity level. Finally, the solubilities in protic and aprotic organic solvents were assessed. Two phases were often formed with aprotic organic solvents like acetone, ethyl acetate or 2-methyltetrahydrofuran, whereas DES were soluble in water and in protic solvents as methanol. Overall, many new biocatalytic concepts can be envisaged in these netoreic, biobased, non-conventional media. Acknowledgement: Financial support from DFG training group 1166 BioNoCo (Biocatalysis in Non-conventional Media) is gratefully acknowledged.
________________ [1] Abott et al. J. Am. Chem. Soc., 2004, 126, 9142-9147. [2] Whitehead et al. J. Electrochem. Soc. 2010. 157, D328-D334. [3] Abott et al. Green Chem. 2005, 7, 705-707. [4] Abott et al. Chem. Eng. Data, 2006, 51, 1280-1282. [5] Lindberg et al. J. Biotechnol., 2010, 147, 169-171. [6] Gorke et al. Chem. Commun., 2008, 1235-1237. [7] Domnguez de Mara and Magueri, Curr. Op. Chem. Biol., 2011, 15, 220-225.

Recognition of remote chirality in lipase-catalyzed acylation application to the resolution of a sterically hindered alcohol
Ryohei Kobayashi, Hanghang Huang, Toshinori Higashi, Mitsuru Shoji, Takeshi Sugai Department of Pharmaceutical Science, Keio University, Tokyo 105-8512, Japan; E-mail: sugai-tk@pha.keio.ac.jp Stericol (1, Figure 1) is an important chiral auxiliary in asymmetric synthesis. Preparation of its enantiomerically pure form has been, however, difficult even by applying a variety of hydrolytic enzymes [1], because of very high steric hindrance around the secondary alcohol. In order to circumvent this problem, a new substrate, the corresponding hydroxyacetate 2a was provided. This has another hydroxy group, which would be susceptible to the enzyme-catalyzed reaction [2], and would avoid the steric interaction of the en- Figure 1. Both enantiomers of stericol zyme catalytic site and bulky isopropyl substituent (Scheme 1). Burkholderia cepacia lipase (Amano PS-IM) recognized its remote chiral center, and kinetic resolution proceeded with E=22.
97 105
Porous materials in Bioconversion of lipophilic compounds
Hamid Bou Saab, Anna Boulanger, Samuel Fouchard, Serge Neunlist Universit de Haute Alsace, Ecole Nationale Suprieure de Chimie de Mulhouse Laboratoire de Chimie Organique et Bioorganique 3 rue Alfred Werner, 68093 Mulhouse, France E-mail: serge.neunlist@uha.fr One of the major advantages of using biocatalysts in organic synthesis is that water constitute the reaction medium. However, water becomes a serious problem when bioconversion deals with lipophilic compounds, in particular those poorly soluble in water such as sterols [1]. Bioconversion of lipophilic compounds requires the use of huge amounts of chemical to increase their bioavailability to the biocatalysts in aqueous media. In fact, this process depends on the close contact between the hydrophobic substrate and the biocatalyst [2]. Green solvents used for bioconversion of lipophilic compounds are often expensive with limited applications, privileged for one pot biotransformation with enzymes and their key issues are still required [3]. In our laboratory, we study cell immobilization in three dimensional porous materials to increase the contact between microorganisms and lipophilic substrates. For the side chain cleavage of sterols as a model multistep microbial bioconversion, we showed, in a previous work, that the immobilization of Mycobacterium species on results in a 3 to 4-fold time increase of androstnones yield [4]. Here, we show that the dry fruits of Luffa cylindrica can be used in a 5 L jar fermentor with phytosterols as substrates (1 g/L) without addition of any chemicals or solubilizing agents. Products (0,057 g/L.j) were accumulated in the aqueous phase while substrate remains on the fibers of Luffa. A green semi-continous process is under investigation. The dry fruit of Luffa cylindrica has no influence on cells growth, it is natural, cheap, non toxic and mechanically strong.
Scheme 1. B. cepacia lipase-catalyzed enantioselective acylation of hydroxyacetate. To suppress the reactivity of hydroxy group, hopefully in (S)-isomer selectively, an electron-withdrawing cyano group was introduced to provide another substrate 3. This has secondary alcoholic hydroxy group instead of primary one in 2a and an additional chiral center. The diastereomeric pair was separated into two enantiomeric pairs [(2R,1R) + (2S,1S)-3, and (2R,1S) + (2S,1R)-3] and evaluation of reactivity and stereoselectivity in the treatment of each substrates with lipase PS-IM under acylating conditions is now under way (Scheme 2).
Figure 1. Formate to CO2 conversion at different pHs (A) and temperatures (B) for: MagNP-APTS-FDH (black line), MagNP-APTS-glioxyl agarose-FDH (red line), MagNP@SiO2-APTS-FDH (green line) and free FDH (blue line). When immobilized on MagNP-APTS and MagNP@SiO2-APTS, formate dehydrogenase gave higher conversions, but also, being more sensitive to temperature and pH changes. Values of the transition state entropy indicate that upon immobilization on MagNP-APTS and MagNP@SiO2-APTS there is a stiffening of FDH, while with MagNP-Glioxyl Agarose, there is more mobility, indicating that stiffening is a stabilization factor in immobilizations. ________________
[1] Yoshimoto M. ,Yamasaki R., Nakao M., Yamashita T.; Enzyme and Microbial Technology, 46 (2010), 588-593 [2] Schmidt T., Michalik C., Zavrel M., Spiess A., Marquardt W., Ansorge-Schumacher M.B., Biotechnology Progress, 26 (2010), 73-78
Scheme 2. Reaction with electron-defficient substrates.

________________ [1] J. Am. Chem. Soc., 2005, 127, 12228. [2] Tetrahedron: Asymmetry, 2007, 18, 994.
_______________ [1] J.M.S. Cabral, M.R. Aires-Barros, H. Pinheiro, D.M.F Prazeres, J Biotechnol, 1997, 59, 133. [2] B. Angelova, H. Schmauder, J Biotechnol, 1999, 67, 13. [3] M.P. Marques, F. Carvalho, C.C. de Carvalho, J.M. Cabral, P. Fernandes, P. Food Bioprod Process, 2010, 88, 12. [4] H. Bou Saab, S. Fouchard, A. Boulanger, P. Llopiz, S. Neunlist, Biocatal Biotransfor, 2010, 28, 387.
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Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from bacillus amyloliquefaciens and their stuctural analysis
Feng Tian, Lotthida Inthanavong, Fred Morin, Salwa Karboune Department of Food Science and Agricultural Chemistry, McGill University, 21111 Lakeshore Road, Ste. Anne de Bellevue, Qc, H9X 3V9, Canada. Email : salwa.karboune@mcgill.ca. Cumulative scientific evidences have demonstrated, in the last years, the potential benefits of non-digestible oligosaccharides (NDOs) to improve the intestinal health by selectively stimulating the growth of beneficial bacteria and to reduce the risk of colon cancer. The functional health attributes of NDOs were found to be dependent on their chemical structures, in particular their monosaccharide composition, their degree of polymerization as well as the position and the conformation of their glycosidic linkages. -(2-6)- and neo-fructo-oligosaccharides (FOSs) are of increasing interest because of their prebiotic effects that surpass those of -(2-1)-FOSs available for human consumption. Levansucrases (LSs), which are fructosyl-transferases belonging to the family 68 of glycoside hydrolases, catalyze the synthesis of -(2-6)-levan by transferring the fructosyl group of non-activated sucrose into the fructan chain. Interestingly, the formation of levan can be quantitatively replaced with the formation of homo- and hetero--(2-6)-FOSs in the presence of various acceptors. However, this attractive approach is still limited by the poor availability of LSs, their low stability and the lack of understanding of their mechanism. In the present study, LS from B. amyloliquefaciens was purified by fractionation with polyethylene glycols followed by size exclusion chromatography. The purified LS shows single band on SDS-PAGE with estimated molecule weight of 52 kDa. The kinetic parameters of LS reveal that the catalytic efficiency of the transfructosylation activity was higher as compared to that of its hydrolytic activity. This interesting characteristic of B. amyloliquefaciens LS of high transfructosylation to hydrolysis ratio makes it different from most of the previously described LS. Detailed studies of donor and acceptor specificities of LS were carried out. B amyloliquefaciens LS demonstrated a high acceptor specificity toward lactose and maltose. The novel FOSs were purified by ion exhange and Biogel P-2 gel permetaion chromatographies and their chemical structures were characterized by LCMS and 13C NMR. This study is the first to highlight the potential of B. amyloliquefaciens LS as a synthetic tool in the synthesis of FOSs.
________________ [1] Kilian S, Kritzinger S, Rycroft C, Gibson G, and Du Preez J, World J. Microbiol. Biotechnol., 18, 637-644 (2002) [2] Tian F, Inthanavong L, Karboune S, Biosci. Biotechnol. Biochem. Accepted. (2011)
103
Solvent free conditions in the efficient lipase-catalyzed kinetic resolution of primary amines
Mari Pivi, Pivi Perki, Liisa T. Kanerva Institute of Biomedicine/Department of Pharmacology, Drug Development and Therapeutics/Laboratory of Synthetic Drug Chemistry, University of Turku, FIN-20014 Turku, Finland E-mail: mari.paivio@utu.fi Lipases are widely used in the biocatalytic preparation of enantiopure amines due to their broad substrate specificity, easy accessibility and effortless handling. Lipase-catalyzed kinetic resolution of amines is broadly applied in industrial production of a variety of enantiopure amines.[1] Conventional solvent reactions are often limited by low concentrations of poor soluble aromatic primary amines in organic solvents. At the same time the enzyme loadings, enabling efficient reactions, have been relatively high rendering the lipase-catalyzed kinetic resolution methods less feasible. A simple solvent free kinetic resolution method utilizing Candida antarctica lipase B (CAL-B)-catalyzed N-acylation with equimolar amount of activated acyl donor under ambient conditions was developed. Elevated temperature and reduced pressure were not required to facilitate the fast kinetic resolution. Method optimization regarding reaction conditions was performed and the scope of the method was extended to 9 structurally different aromatic primary amines (Scheme 1). CAL-B immobilization and the recycling of the CAL-B preparations were also studied. The highly enantioselective kinetic resolution method provided enantiomers of the amines at 50 % conversion with enantiomeric excess 95 %.
106
Employing C-C hydrolase and transaminase in the preparation of non-natural amino acid
Elina Siirolaa, Francesco G. Muttia, Barbara Grischeka, Gideon Groganb, Wolfgang Kroutila a Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria; bDepartment of Chemistry, York Structural Biology Laboratory, University of York, YO10 5YW York, UK E-mail: elina.siirola@uni-graz.at Applying a sequence of stereoselective enzymatic reactions for the synthesis of diastereomers is an appealing option for the preparation of pharmaceutical intermediates. Here we report a chemoenzymatic concept for the preparation of a -amino acid methyl ester (1, Scheme 1) by employing two biocatalytic reactions whereby the first step is catalysed by a C-C hydrolase and the second by an -transaminase. For the stereoselective preparation of 3-substituted cyclohexylamines in general, organocatalytic cascade reaction[1] and a lipase catalysed resolution method[2] have been described. However, these methods have their limitations in stereoselectivity or yield. The -diketone hydrolase (6-oxo camphor hydrolase from Rhodococcus sp.)[3] catalysed the hydrolysis of a diketone like 2 to a corresponding keto-acid. The -transaminase catalysed amination of the corresponding methyl ester (3) led to a non-natural amino acid methyl ester (1), a building block in the synthesis of potential pharmaceuticals.[4] The CC-hydrolase defined the configuration of the first stereocenter (e.e. >99 %) and by choosing either a (S)- or (R)-selective transaminase, both diastereomers (1S/3R) and (1S/3S) could be obtained in good diastereomeric excess (Scheme 1).
107
A stereoselective inverting sec-alkylsulfatase for the deracemisation of sec-alcohols
Schober Ma, Knaus Tb, Macheroux Pb, Wagner Uc, Faber Ka a Department of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria; bInstitute of Biochemistry, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria; cInstitute of Molecular Biosciences, Structural Biology, University of Graz, Humboldtstrasse 50/III, A-8010 Graz, Austria Sulfatases catalyze the hydrolytic cleavage of the sulfate ester bond by liberating inorganic sulfate and the corresponding alcohol [1,2]. The secondary alkylsulfatase PISA1 from Pseudomonas DSM 6611 is the first molecularly characterized member of the class of -lactamase related alkyl sulfatases able to convert a broad range of secondary alkyl sulfates. PISA1 reacts under inversion of configuration at the carbon atom. Biotransformations in 18O labeled buffer using GC/MS for product analysis have shown that PISA1 triggers a nucleophilic attack of an activated water molecule at the chiral carbon atom. This mechanism does not have a counterpart in chemical catalysis, because base-catalysed hydrolysis of sulfate esters would generate the bad leaving group SO42- and thus is exceedingly difficult. Current structural studies indicate strong similarities between PISA1 and its only known relative, the primary alkylsulfatase SdsA1. Both share a conserved sulfate binding site and a closely related nucleophilic site. The only differences are located in the respective hydrophobic sites. SdsA1 was shown to have a high affinity for prim-sulfate esters, whereas PISA1 predominantly hydrolyzes sec-sulfate esters with excellent yields and enantioselectivities (E up to > 200). In combination with acid-catalyzed hydrolysis of the residual substrate that proceeds under strict retention of configuration [4], a two-step protocol leading to a single isomeric product from a racemate could be developed. This process was also performed on a preparative scale for 2-octyl sulfate with an E-Value >200 and an overall yield of 87%.
Scheme 1. Chemoenzymatic preparation of 1 by employing a C-C hydrolase and an -transaminase.
Scheme 1. CAL-B catalyzed kinetic resolution of aromatic primary amines.

______________ [1] Ditrich, K. Synthesis, 2008, 14, 2283.
________________ [1] J. Zhou, B. List, J. Am. Chem. Soc. 2007, 129, 74987499. [2] L. M. Levy, G. de Gonzalo, V. Gotor, Tetrahedron: Asymmetry, 2004, 15, 20512056. [3] a) G. Grogan, J. Graf, A. Jones, S. Parsons, N. J. Turner, S. L. Flitsch, Angew. Chem. Int. Ed. 2001, 40, 11111114. b) G. Grogan, G. A. Roberts, D. Bougioukou, N. J. Turner, S. L. Flitsch, J. Biol. Chem. 2001, 276, 1256512572. [4] Based on Reaxys survey, nine patents include 2-(3-aminocyclohexyl)acetic acid as a building block in the pharmaceutical synthesis.
Figure 1. Enantio-convergent process for the deracemisation of sec-alcohols with PISA1.

________________ [1] Gadler P and Faber K (2007) Eur. J. Org. Chem., 5527-5530 [2] Gadler P, Faber K (2007) Trends Biotechnol. 25, 83-88 [3] Hagelueken G, Adams TM, Wiehlmann L, Widow U, Kolmar H, Tuemmler B, Heinz DW, Schubert W-D, (2006) Proc. Natl. Acad. Sci. USA 103, 76317636 [4] Wallner SR, Nestl B, Faber K (2005) Tetrahedron 61, 1517-1521
October 2-6, 2011
98 108
Synthesis of n3-PUFAs ethyl esters: an efficient irreversible biocatalyzed solvent-free process
R. Morrone, N. DAntona, D. Lambusta, G. Nicolosi CNR Istituto di Chimica Biomolecolare, Via P. Gaifami 18 95126 Catania, Italy E-mail: raffaele.morrone@icb.cnr.it Nowadays biocatalysis is a methodology that has become a central part of the so-called Green Chemistry. The use of enzymes, the natural catalysts present in all living organisms and able to catalyze a wide range of chemical reactions, as matter of fact permits to change classical, and often not very eco-friendly, chemical processes into greener and sustainable ones. Undoubtedly, lipases represents the most commonly used enzymes in organic synthesis due to their high stability and to the abilities to accept a wide range of substrates and to operate efficiently in mild reaction conditions. This possibility to perform reactions in eco-friendly conditions is very important especially when using sensitive compounds such as the polyunsaturated fatty acids (PUFAs). The increasing interest toward PUFAs, and more specifically toward n3-PUFAs, is justified by their beneficial effects on human health [1], and is proven by their increasing consumption. Actually the n3-PUFAs used as dietary supplements are marketed as triglycerides or ethyl esters because in this chemical form are more resistant to oxidative processes [2] if compared to the free acids. Since the PUFAs represents natural substrates for lipases, these enzymes are optimal candidates to act as catalysts in the preparation of their corresponding ethyl esters. Hereby, these peculiarities of the lipases have been exploited for the preparation of the ethyl esters of the n3-PUFAs: -linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In our laboratories we have developed procedures to obtain irreversible biocatalyzed esterification reactions3,4 and here we show their application for the preparation of PUFA ethyl esters. Immobilized lipase B from Candida antarctica (Novozym 435) in presence of diethylcarbonate (DEC) or triethylorthoformate (TEOF) as alcohol donors (in equimolar ratio with respect to acidic substrates) and in solvent free conditions, was able to catalyze the synthesis of the ethyl esters of ALA, EPA and DHA with quantitative yields. The following figures show the esterification trend as time function.

An efficient chemoenzymatic dynamic kinetic resolution of N-heterocylic 1, 2-amino alcohols
Richard Lihammara, Renaud Milleta, Jan-E. Bckvalla a Department of Organic Chemistry, Arrhenius laboratory, Stockholm University, SE-106 91 Stockholm, Sweden. Enantio enriched N-heterocycles are highly attractive compounds for industrial and academic research.[1] Among them, chiral secondary alcohols such as 3-hydroxypiperidines or 3-hydroxypyrrolidines constitute an important class of molecules.[2] In this work both chemoenzymatic dynamic kinetic resolution (DKR) and kinetic resolution (KR) of nine N-substituted cyclic amino alcohols are described. To demonstrate the usefulness of the protocol, both removable N-protecting groups and stabile aliphatic substituents has been resolved. This range of substrates illustrates the viability of the protocol since the resolution can be performed both before and after a desired synthetic transformation. Various lipases have been studied as biocatalysts for the kinetic resolution. Dramatic changes in enzyme selectivity were observed depending on the technique used for immobilization. The DKR is efficient and all 3-acetoxypyrrolidines and piperidines were obtained in high yield and high enantiomeric excess after 24 to 36 hours.

Enzymatic deracemization of hydroxyaryl phosphine oxide
Sylwia Kaczmarczyk, Piotr Kiebasiski Department of Heteroorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 d, Poland E-mail: sylwiak@cbmm.lodz.pl We have recently reported on a successful synthesis of enantiomerically pure precursors 1 and 4 of bidentate and tridentate ligands (catalysts) via enzyme promoted kinetic resolution of 1 (eq. 1) or desymmetrization of 3 (eq. 2)[1]. Although the stereoselectivity of both reactions was very high, in the case of 3 we have encountered problems connected with its undesired cyclization to the spirophosphorane 5. To avoid this, we have decided to use another substrate, namely bis-(2-hydroxyphenyl)methylphosphine oxide 6, in which lack of a methylene group excludes such a cyclization. It should be mentioned that the analogous racemic 2-hydroxyphenyl(phenyl)methylphosphine oxide was earlier resolved into enantiomers via kinetic resolution with moderate stereoselectivity (E = 5) [2]. To obtain the desired monoacetate 8, two procedures of deracemization have been applied (eq. 3): enzymatic acetylation of the diol 6 (route a) and hydrolysis of its diacetyl derivative 7 (route b). We have found that the hydrolysis of 7 gives the product 8 with a much higher enantioselectivity than the acetylation of 6. The influence of the enzyme type and conditions on the outcome of the reaction, as well as application of the products as chiral catalysts in selected reactions of asymmetric synthesis, will be discussed.
99 113
Enzymatic synthesis of estolides using biodiesel from castor oil as raw material
Erika C. G. Aguieirasa, Cludia O. Velosob, Danielle O. Rosasc, Mnica A. P. da Silvaa, Marta A. P. Langoneb a Escola de Qumica, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, CEP 21949-909, Rio de Janeiro, RJ, Brazil, bPPGEQ, Universidade do Estado do Rio de Janeiro, Rua So Francisco Xavier, 524, PHLC, IQ, sl. 427, CEP 20559-900, Rio de Janeiro, RJ, Brazil, cPDAB/HPE, CENPES/PETROBRAS, Universidade Federal do Rio de Janeiro, Av. Horcio Macedo, 950, CEP 21941-915, Rio de Janeiro, RJ, Brazil E-mail: langone@uerj.br Estolides are polyesters based on vegetable oils that are formed when the carboxylic acid functionality of one fatty acid reacts at the site of unsaturation of another fatty acid [1] or by covalent ester bonds between hydroxyl moiety of one hydroxyl acid and the carboxyl moiety of another hydroxyl acid molecule [2]. These lubricants have been developed in order to overcome deficiencies associated with some characteristics of vegetable oils, which are known to have poor thermal oxidative stability, low hydrolytic stability and poor low temperature properties [1]. The estolides synthesis using lipases (triacylglycerol ester hydrolases E.C.3.1.1.3) have been investigated as an alternative to overcome the common problems that occur in the conventional route such as the low selectivity of acid catalyst, formation of undesired by-products and acid effluents, colouring and malodours of the products and corrosion of equipments [3]. The aim of this work was to study the synthesis of estolides from oleic acid or stearic acid and methyl ricinoleate (castor oil biodiesel), using commercial immobilized lipases (Novozym 435, Lipozyme RM IM and Lipozyme TL IM) in a solvent free medium. The effect of type of lipase, specificity of enzyme and water removal from the medium by free evaporation and molecular sieves on the conversion was evaluated. The reusability of immobilized lipase Novozym 435 was also investigated. The acidity index (acid value) was used to monitoring the progress of reactions. After 48 hours, the highest conversion was attained with Novozym 435 (33%), because it is a non-specific lipase. Novozym 435 showed no preference for the presence of unsaturation on the fatty acid. During 100 h of reaction, it was observed the same profile of reduction of acid value with time for both fatty acids studied and after 100 h it were obtained 49% and 50% of conversion in the reaction with oleic acid and stearic acid, respectively, with 6 wt. % of lipase at 80C. The use of molecular sieve increased the conversion in more than 10% after 48 h of reaction. Novozym 435 could be reused four times on the reactions between oleic acid and methyl ricinoleate with conversion of 15% after the fourth reaction at 80C.
_______________ [1] 1. Cermak SC, Isbell TA (2001) Synthesis of estolides from oleic and saturated fatty acids. J. Mol. Catal 78: 577565. [2] Bdalo-Santoyo A, Bastida-Rodriguez J, Mximo-Martin MF, Montiel-Morte MC, Murcia-Almagro MD, Ortega S (2009) Influence of the operating conditions on lipase-catalysed synthesis of ricinoleic acid estolides in solvent-free systems. Biochem. Eng. J 44: 214-219. [3] Bdalo-Santoyo A, Bastida-Rodriguez J, Mximo-Martin MF, Montiel-Morte MC, Murcia-Almagro MD (2008) Production of ricinoleic acid estolide with free and immobilized lipase from Candida rugosa. Biochem. Eng. J 39: 450-456.
Figure 1. General scheme for dynamic kinetic resolution of 1, 2 cyclic amino alcohols.
________________ [1] S. A. Lawrence in Amines: Synthesis, Properties and Applications; Cambridge University Press, Cambridge, 2006. [2] a) L. Pu; H.-B. Yu, Chem. Rev. 2001, 101, 757-824, and references cited therein. b) J. R. Liddell, Nat. Prod. Rep. 2002, 19, 773; c) H. M. Garraffo, J. Caceres, J. W. Daly, T. F. Spande, N. R. Andriamaharavo, M. J. Andriantsiferana, J. Nat. Prod. 1993, 56, 1016-1038; d) C. J. Moody, Chem. Commun. 2004, 13411351;
_______________ [1] H.E. Bays, A.P. Tighe, R. Sadovsky, M.H. Davidson, Expert Rev. Cardiovasc. Ther. 2008, 6, 391409. [2] T. J. Lin, S. W. Chen and A. C. Chang, Biochem. Engin. J. 2006, 29, 27-34. [3] R. Morrone, M. Piattelli, G. Nicolosi, Eur. J. Org. Chem. 2001, 4, 1441-1442. [4] R. Morrone, N. D'Antona, D. Lambusta, G. Nicolosi, J. Mol. Cat. B: Enzym. 2010, 65, 4951.
___________________
[1] S. Kaczmarczyk, M. Kwiatkowska, L. Madaliska, A. Barbachowska, M. Rachwalski, J. Baszczyk, L. Siero, P. Kiebasiski, Adv. Synth. Catal. 2011, in press. [2] A. Serreqi, and R. Kazlauskas, J. Org. Chem. 1994, 59, 7609-7615.
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Optimization of alcohol oxidation using galactose oxidase from Fusarium NRRL 2903 and application to one-pot cascade processes
Michael Fuchsa, Katharina Taubera, Johann Sattlera, Jan Pfefferb, Wolfgang Kroutila, Kurt Fabera a University of Graz, Heinrichstra 28, 8010 Graz, Austria; bEvonik Degussa GmbH, PaulBaumann-Strae 1, 45772 Marl, Germany E-mail: mihi_fuchs@yahoo.de Selective oxidation reactions are crucial organic transformations. Especially aldehydes are important targets due to their high reactivity and their versatility for further transformations. However, this reactivity intrinsically bears also the inherent disadvantage of limited shelf lifetime due to overoxidation and they often represent toxic and volatile intermediates, which complicates isolation and purification. Therefore, recent research has proposed to form the aldehyde precursor prior or even during the reaction and use it immediately without purification for the subsequent reaction step.[1] Galactose oxidase from Fusarium NRRL 2903 has proven to be a powerful enzyme for the selective oxidation of primary hydroxy functions to the corresponding aldehydes.[2] Here we describe the optimization of the oxidation reaction as well as the application of the latter reaction in high yielding onepot cascade processes, giving access to high-value organic intermediates.
O H R X
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Lipase-Catalyzed Kinetic Resolution of Aryltrimethylsilyl Chiral Alcohols
Leandro H. Andrade, Juliana C. Abreu, Dayvson J. Palmeira. Institute of Chemistry, University of So Paulo, Av. Prof. Lineu Prestes, 748, So Paulo SP, Brazil. ZIP code 05508-000. E-mail: dayvson@usp.br Aryltrimethylsilanes are recently attracting attention due to the possibility of using them to perform some interesting desilylative functionalizations.[1] Although silicon-containing organic compounds have a remarkable application in organic synthesis, just few publications have reported its use in enzyme-catalyzed transformations to date.[2] In this work, lipase-catalyzed kinetic resolution (KR) of aryltrimethylsilyl chiral alcohols via enantioselective transesterification was investigated. Initially, the compound 3 was chosen as substrate in a screening of lipases (13 lipase sources). Amano Lipase PS-C II led to complete KR of 3 (50% of conversion) with excellent enantiomeric excess for both remaining alcohol and acetylated product (ee > 99% in both cases). The amount of enzyme and time reaction were also studied. An optimal condition of 1 mg of Amano Lipase PS-C II and a shorter time reaction of 3 hours in the KR of 3, led to the same excellent results achieved previously. Therefore, these optimal conditions were applied in the KR of other aryltrimethylsilyl chiral alcohols (Scheme 1).
114
Optimized Kinetic Resolution of 1,3,6-tri-O-benzyl-myo-inositol by Novozym 435
Evelin Andrade Manoela, Karla Ceodaro Paisb, Aline Gomes Cunhac, Maria Alice Zarur Coelhod , Alessandro Bolis Costa Simase, Denise Maria Guimares Freiref a Master student Faperj/ UFRJ, Escola de Qumica, RJ, Brazil; bDoctoral student CNPQ/UFRJ, NPPN, RJ, Brazil, cDoctoral student- CNPQ/UFRJ, Instituto de Qumica, RJ, Brazil; dResearch scientist UFRJ/Escola de Qumica, RJ, Brazil; eResearch scientist UFRJ/NPPN, RJ, Brazil; fResearch scientist UFRJ/IQ, RJ, Brazil. E-mail: evelin@ufrj.br The phosphorylated myo-inositols play key roles in fundamental biological phenomena such as cellular signal transduction. Despite the high reputation of lipases [1] as catalysts for optical resolutions [2], most syntheses of chiral inositol derivatives still rely on derivatization. Following our previous results on this field, we recently established that DL-1,3,6-tri-O-benzyl-myo-inositol (DL-1, figure 1) could engage in successful kinetic resolution in hexanes (24h, 30oC) with vinyl acetate as acyl donor under catalysis by immobilized lipase from Candida antarctica (Novozym 435) [3]. Herein, we report on the optimization of this reaction. Thus, Factorial Design and Response surface methodology (RSM) were employed to evaluate the effects of parameters, such as temperature, enzyme concentration, concentration of DL-1 [4] and concentration of the acyl donor. Substrate concentration of myo-inositol and enzyme concentration proved to be the most important variables. High substrate concentration and high enzyme concentration showed a negative and positive effect, respectively. Based on the analysis of Statistic program results, the optimum conditions were determined, leading to high conversion (49,7%) and high enantiomeric excesses.
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Bioreduction of aldehydes aromatic with leaves of banana and corn plant.
Arturo Navarro-Ocaa,a Hctor Luna,b Liliana Hernndez-Vzquez.b Agustn Reyo.a a.Departamento de Alimentos y Biotecnologa, Facultad de Qumica, UNAM. Circuito Exterior Ciudad Universitaria, Mxico. DF b.Departamento de Sistemas Biolgicos, UAMXochimilco A. P. 23/181, Mxico, DF. MXICO. E mail: arturono@servidor.unam.mx Several alcohols are considered as a key starting materials for drug synthesis in the pharmaceutical and flavor industries. The biocatalytic methods for the preparation of alcohols is an important tool in organic chemistry. In a green context it is important to consider that there are several reports about the possibility of using parts of fresh plants as biocatalysts. For example, roots of D. carota have been used for reduction of ketones or aldehydes as alternative for traditional synthesis.[1] This methodology can also be and alternative to bakers yeast or cell culture.[2-4] In this work are described a general methodology for the efficient reduction of aromatic aldehydes to the corresponding alcohols with moderate to excellent chemical yield, using freshly cut leaves of banana and corn plant in aqueous medium and mild reaction conditions (Figure 1).
GalactoseO xidase, OH Pi Buffer R X pH =7 .0,4 barO 2
one-potc ascade processes
Scheme 1 Lipase catalyzed KR of aryltrimethylsilyl chiral alcohols.

C-C coupling Functional Group Interconversion
The results of this study are summarized in table 1.
X= CH, N
Scheme 1. Alcohol oxidation via Galactose Oxidase from Fusarium NRRL 2903
________________ [1] S. Imm, S. Baehn, L. Neubert, H. Neumann and M. Beller, Angew. Chem. Int. Ed., 2010, 49, 8126; B. Gnanaprakasam, J. Zhang, D. Milstein, Angew. Chem. Int. Ed. 2010, 49, 1468; V. P. Srivastava, R. Patel, L. D. S. Yadav, Adv. Synth. Catal. 2011, 353, 695. [2] B. Yuan, A. Page, C. P. Worrall, F. Escalettes, S. C. Willies, J. J. W. McDouall, N. J. Turner, J. Clayden, Angew. Chem. Int. Ed. 2010, 49, 7010; L. Sun, T. Butler, M. Alcalde, I. P. Petrounia, F. H. Arnold, ChemBioChem 2002, 3, 781.
Table 1 Results of lipase catalyzed KR of aryltrimethylsilyl chiral alcohols with Amano Lipase PS-C II. In summary, lipase catalyzed KR of ortho-TMS substituted chiral alcohols did not occur under the conditions evaluated. On the other hand, para-TMS substituted chiral alcohols showed better results than meta-TMS substituted analogues. The size of the alkyl chain attached to the chiral carbon, played an important role in the enantioselectivity of the KR. An increase in the size of the side chain caused a decrease on conversion, but excellent enantioselectivity.
[1] (a) Brenzovich, W. E.; Brazeau, J.-F.; Toste, F. D. Org. Lett. 2010, 12, 4728. (b) Ball, L. T.; Green, M.; Lloyd-Jones, G. C.; Russell, C. A. Org. Lett. 2010, 12, 4724. [2] (a) Fukui, T.; Kawamoto, T.; Tanaka, A. Tetrahedron: Asymm. 1994, 5, 73. (b) Li, N.; Zong, M.-H.; Peng, H.-S.; Wu, H.-C.; Liu, C. J. Mol. Catal. B: Enzym. 2003, 22, 7. (c) Zani, P. J. Mol. Catal. B: Enzym. 2001, 11, 279.W
Figure. 1 Kinetic resolution (enantioselective acetylation) of D-1 by Novozym 435

________________ [1] Gotor-Fernndez, V. et al. 2006. J. Mol. Cat. B: Enzym. 40, 111-120. [2] For leading refs. on the use of lipases (resolution) in the synthesis myo-inositols, see: Liu, Y. C-; Chen, C. S. Tetrahedron Lett. 1989, 30, 1617. Ling, L.; Ozaki, S. 1993, Tetrahedron Lett. 34, 25012504. Rudolf, M. T.; Schultz, C. 1996, Liebigs Ann. 533-537. Laumen, K.; Ghisalba, O. 1999, Biosci. Biotechnol. Biochem. 63, 13741377. [3] Manoel, E. A. et al., manuscript in preparation. [4] Simas, A.B.C. et al. 2009 Tetrahedron Lett. 50, 2744.
[1] F. Baldassare, G. Bertoni, C. Chiape, F. Marione J. Mol Cat B: Enzymatic 2000, 11, 55-58. [2] R. Bruni, G. Fantin, S. Maietti, A. Medeci, P, Pedrini, G. Sacchetti Tetrahedron Asymmetry, 2006, 17, 2287-2291. [3] J. C. C. Assuncao, L. L. Machado, T. L.G. Lemos, G. A. Cordell , F. J. Q. Monte, J. Mol Cat B: Enzymatic 2008, 52, 194-198. [4] L.L Machado, F.J.Q. Monte, M. C. F. Olivera, M. C. de Mattos, T. L. G. Lemos, V. Gotor-Fernandez, G de Gonzalo , V. Gotor J. Mol Cat B: Enzymatic, 2008, 54, 130-133.
Figure1. Reduction of aromatic aldehydes with leaves of banana of corn plant The leaves were thoroughly washed with a solution of 10 % (v/v) NaClO, followed with deionizer water. The leaves were cut into approximately 0.3 cm2 and added to a Erlenmeyer flask with of phosphate buffer (0.2 M pH 7.0), later the substrate dissolved in acetone was added. The reaction was allowed to proceed at room temperature in a rotatory shaker. The reaction products were obtained by simple filtration and extraction with EtOAc, the organic phase were then dried and concentrated in vacuum. The mixture were purified for CC and the isolated yield reported. The substrates were benzaldehyde, 4-methoxybenzaldehyde, piperonal. For reduction with leaves of banana, benzaldehyde was not reduced to phenylmethanol. The other aldehydes gave the alcohols with yield isolated of 30 to 85 % respectively. In these cases the yields were increased because the substituents in the ring. In the case of reduction with leaves of corn plant, benzaldehyde produce a 90 %, 4-metoxybenzaldyde 79 % and piperonal 98 % yield. ____________
October 2-6, 2011
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Visible light as driving force for biocatalysis
Frank Hollmann, Ekaterina Churakova, Serena, Gargiulo, Maria Mifsud Grau, Isabel W.C.E. Arends Department of Biotechnology, Delft University of Technology, Delft, The Netherlands. E-mail: f.hollmann@tudelft.nl Flavins are ubiquitous and versatile redox cofactors in cellular redox metabolism. Beyond that they also may play a central role in redox biocatalysis for organic chemistry. Especially their visible-light photoexcitability opens up some unexpected possibilities to promote biocatalytic redox reactions: (1) Photoexcited flavins are potent oxidants, which can be exploited for the regeneration of oxidized nicotinamide cofactors. Simply by visible-light illumination the hydridetransfer from NAD(P)H to e.g. FMN can be accelerated by at least two orders of magnitude. The resulting photocatalytic regeneration system for NAD(P)+ was successfully applied to promote alcohol dehydrogenase-catalyzed oxidation reactions.[1] (2) Photoexcited flavins can also oxidize simple sacrificial electron donors such as EDTA, formate, or ascorbic acid. The resulting reduced flavins (e.g. FMNH2) can deliver their reducing equivalents directly to redox enzymes such as monooxygenases or enoate reductases. Thereby, not only the costly nicotinamide cofactor can be circumvented but also higher chemoselectivity can be achieved in some cases.[2] (3) Finally, the high reactivity of reduced flavins with molecular oxygen can be exploited for in situ generation of H2O2 to promote peroxidase-catalyzed oxidation and oxyfunctionalization reactions. [3]

Development of a biocatalytic production process for (S)-2-hydroxy ketones
A. Baraibar, E. von Lieres, W. Wiechert, M. Pohl, D. Rother Institute of Bio- and Geoscience (IBG-1: Biotechnology), Forschungszentrum Jlich, D-52426 Jlich, Germany E-mail: a.baraibar@fz-juelich.de Enantiomerically pure 2-hydroxy ketones have several applications as building blocks in the pharmaceutical industry as well as chiral ligands and chiral auxiliaries. They can be found in antidepressants, anti-Alzheimer drugs, antifungals and in some antitumor agents [1]. Further they are valuable precursors for chiral 1,2-amino alcohols or 1,2-diols. The production of enantiomerically pure 2-hydroxy ketones is a challenging task, which can be mastered by the use of thiamine diphosphate (ThDP)-dependent enzymes. These enzymes are able to catalyze the carboligation of two aldehydes yielding different 2-hydroxy ketones with high enantiomeric purity and high regioselectivity. Whereas several (R)-selective enzymes are already known, only recently the lack of (S)-selective enzymes could be closed within our group. Thus, a complementary toolbox of enzymes is now available, which covers a broad range of mixed aromatic aliphatic 2-hydroxy ketones.

CMT - Center for Molecular Transformations
Dominik Behrendta, Jens Langankea, Stefan Olejnika, Anja Schillinga, Walter Leitnerb, Wolfgang Marquardtc, Ulrich Schwanebergd a CMT, RWTH Aachen, Germany; bITMC, RWTH Aachen, cAVT, RWTH Aachen, dBIOTEC, RWTH Aachen E-mail: cmt@cmt.rwth-aachen.de The Center for Molecular Transformations is a project house fostering research that spans over disciplines and scales. A new project house "Center for Molecular Transformations (CMT) was established at RWTH Aachen in 2010 to structurally support and promote the increasingly successful interdisciplinary research activities towards sustainable methods and processes for molecular transformations. The latter includes the quest for new value chains from alternative carbon resources (biomass, CO2) to fine and bulk chemicals. The ability to understand, predict, and control molecular transformations across all scales is the primary prerequisite to establish a sustainable chemical and energetic supply chain under the constraints of limited global resources and an increasing carbon footprint of human activities. The fundamental scientific challenges resulting from this aim can only be met at the interdisciplinary interface of chemistry, biology and (bio-)chemical engineering. These scientific challenges include the development of novel synthetic strategies for molecular transformations or the discovery and reengineering of efficient chemo- and biocatalysts by rational design and evolutionary algorithms.
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Engineering of N-acetyl-Amino Acid Racemase (NAAAR) for improved production of chiral amino acids
Scott Baxter,a Karen Holt-Tiffin,b Anna Fryszkowska,b Ian Fotheringham,c Dominic Campopianoa a The School of Chemistry, The University of Edinburgh, Joseph Black building, West Mains Road, Edinburgh, EH9 3JJ, UK, bChirotech Technology Ltd, 410 Science Park, Milton Road, Cambridge, CB4 0PE, UK cIngenza Ltd, Wallace Building, Roslin BioCentre, Midlothian, EH25 9PP, UK. E-mail: Dominic.Campopiano@ed.ac.uk Enzymatic, acylase-based production of optically active -amino acids via kinetic resolution of their N-acetyl- derivatives is a widely applicable and established industrial process. However, a flaw in this approach is the need for repeated chemo-catalytic racemisation steps of the non-desired enantiomer to achieve yields greater than 50%. A solution to this drawback would be an in situ racemisation step, affording a dynamic kinetic resolution (DKR) with a yield approaching 100% (Scheme 1). The racemisation step could be performed by an enzyme, such as N-acetyl-amino acid racemase (NAAAR, Scheme 1), however usually these biocatalysts display poor activity towards useful substrates, rendering it unsuitable for application on an industrial scale.
Examples for the aforementioned possibilities will be presented and discussed.

________________ [1] S. Gargiulo et al., ChemCatChem., 2011, 3(2), 338-342. [2] F. Hollmann et al., ChemCatChem, 2010, 2(7), 762782. [3] D. Perez et al., Chem. Commun., 2009, 6848 685
Figure 1. Carboligation of two aldehydes into a 2-hydroxy ketone catalyzed by an ApPDC variant. A variant of the Acetobacter pasteurianus pyruvate decarboxylase (ApPDC), catalyzing the formation of (S)-hydroxy ketones (such as phenylpropionyl carbinol) (Figure 1), opens the way of producing (S)-selective products which were unavailable by traditional organic chemical syntheses [2]. However, the stereoselectivity is currently not yet optimal with enantiomeric excess of 89% obtained in small scale batch experiments. This project focuses on the optimization of the reaction conditions for this new ApPDCE369G-variant via reaction engineering in order to achieve high enantiomeric excesses, high yields and high total turnover numbers of the process. The determination of the relevant parameters and their optimization using computational design of experiments (DOE) will be presented and should finally lead to an upscale of the process.
________________ [1] Hoyos, P.; Sinisterra, J.-V.; Molinari, F.; Alcntara, A. R.; and Domnguez de Mara, P.: Biocatalytic Strategies for the Asymmetric Synthesis of -Hydroxy Ketones Acc. Chem. Res. 43, 288299 (2010) [2] Rother, D.; Kolter, G.; Gerhards, T.; Berthold, C. L.; Gauchenova, E.; Knoll, M.; Pleiss, J.; Mller, M.; Schneider, G.; and Pohl, M. (2011). ChemCatChem, accepted for publication
Scheme1. Dynamic kinetic resolution of an -amino acid via coupled acylase- catalyzed hydrolysis and NAAAR-based in situ racemisation process. In an attempt to allow the industrial use of the coupled acylase/NAAAR process we have applied directed evolution to Amycolatopsis sp Ts-1-60 NAAAR enzyme [1]. We report the successful generation of NAAAR mutants with up to 7-fold improved activity towards a wide range of hydrophobic N-acyl-amino acids. Our results indicate that the coupled mutant NAAAR/acylase dynamic kinetic resolution of N-acetyl amino acids can be a cost effective process at scale for the industrial production of enantiomerically pure amino acids.
________________ [1] S. Tokuyama, and K. Hatano, Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60, Applied Microbiological Biotechnology, 1995, 42, 853
Figure 1: Research comprising scales To tackle this task CMT has established the Scientific Integration Team consisting of scientists from the involved disciplines. This team offers primarily scientific support based on its specific knowledge and administrative support for large scale projects.
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Sophorolipids: improvement of therapeutic potential through the design of the recovery process
Isabel A. Ribeiro, Vanessa H. Azevedo, Matilde F. Castro, Maria H.L. Ribeiro Research Institute for Medicines and Pharmaceutical Sciences (i-Med-UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal e-mail: mhribeiro@ff.ul.pt Sophorolipids are carbohydrate-based, amphiphilic biosurfactants with increased interest in pharmaceutical and environmental areas. They are biosurfactants, showing unique properties, namely biological activities, as antimicrobial and antiviral, growth inhibition and differentiation-inducing activities against human leukemia cells. Additionally, another important application is as gene delivery carriers with increased gene transfection in mammalian cells. The aim of this work is the production of the sophorolipids and its recovery from fermentation media, using new microstructured materials. The glycolipids were produced with Rhodotorula bogoriensis and the formation in the cell was followed by microscopy with image analysis. The subsequent extracellular release was evaluated by surface tension and high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD) analysis. Sophorolipids recovery from fermentation media was achieved using new adsorbents recently approved by Food and Drug Administration (FDA). The production of sophorolipids was carried out in the suitable fermentation media and adsorption performed in 24-well microtiter plates. Different amounts (50150 mg L1) of the microstructured materials were added to fermentation media previously centrifuged for biomass removal. The uptake of the sophorolipids to the different microstructure materials throughout the time was evaluated. The maximum sophorolipids adsorption from fermentation broths, at 25C and 40C, was evaluated. The adsorption data for the different sorbents were adjusted to isotherms models. The results presented in this work highlight the potential of sophorolipids selective adsorption to different neutral microstructured materials, a friendly process, as a suitable alternative to current processes of multiple extractions with organic solvents, leading to sophorolipids in a higher purity.
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Sensitive high-throughput screening for the detection of reducing sugars
Andrea Mellitzera, Karlheinz Flickerb, Roland Weisc, Christoph Reisingerd, Aleksandra Mitrovicb, Anton Gliedera,b a Institute of Molecular Biotechnology, Graz University of Technology, Graz, Austria; b ACIB GmbH, Graz, Austria; cVTU Technology GmbH, Grambach, Austria; dSd Chemie AG, Munich, Germany E-mail: aleksandra.mitrovic@acib.at The exploitation of renewable resources for the production of biofuels relies on efficient processes for the enzymatic hydrolysis of lignocellulosic materials. The development of enzymes and strains for these processes requires reliable and fast activity-based screening assays. Additionally, these assays are also required to operate on the microscale and on the high-throughput level. Herein, we report the development of a highly sensitive reducing-sugar assay in a 96-well microplate screening format. The assay is based on the formation of osazones from reducing sugars and para-hydroxybenzoic acid hydrazide. By using this sensitive assay, the enzyme loads and conversion times during lignocellulose hydrolysis can be reduced, thus allowing higher throughput. The assay is about five times more sensitive than the widely applied dinitrosalicylic acid based assay and can reliably detect reducing sugars down to 10 M. The assay-specific variation over one microplate was determined for three different lignocellulolytic enzymes and ranges from 2 to 8%. Furthermore, the assay was combined with a microscale cultivation procedure for the activity based screening of Pichia pastoris strains expressing functional Thermomyces lanuginosus xylanase A, Trichoderma reesei -mannanase, or T. reesei cellobiohydrolase 2.
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Biotransformation of silyl ether mediated by fungi
Leandro Helgueira Andrade, Dayvson Jos Palmeira and Patrcia Bulegon Brondani Instituto de Qumica, Universidade de So Paulo, Av. Prof. Lineu Prestes 748, SP 05508900, So Paulo, Brazil. E-mail: : leandroh@iq.usp.br In recent years, the chemistry of silicon containing compounds has emerged as a promising area in organic synthesis. Several types of organo-silanes are commonly used in organic synthesis.[1] Although organo-silanes have an well-established role in synthetic organic chemistry, there are a few studies involving these compounds and biocatalyzed reactions in enantioselective fashion.[2] In this work we have explored the biotransformations of silyl ether mediated by fungi. We performed reactions with substrates 1-3 with whole cells of Aspergillus terreus URM 3571, CCT 3320 and URM 3371 (Scheme 1).
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Enzymatic polar head modifications of phospholipids In the presence of ionic liquids
Paola DArrigoa,b, Lorenzo Ceriolia, Cinzia Chiappec, Andrea Melea,b, Walter Panzerid, Stefano Servia,b, Chiara Signa, Davide Tessaroa,b a Politecnico di Milano, Department of Chemistry, Materials, and Chemical Engineering, Piazza Leonardo da Vinci 32, 20133 Milano, Italy b The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universit degli Studi dellInsubria, via Mancinelli 7, 20131 Milano, Italy c Universit di Pisa, Dipartimento di Chimica e Chimica Industriale, Via Bonanno 33, 56126 Pisa, Italy d ICRM, CNR Via Mancinelli 7, 20131 Milano, Italy E-mail: stefano.servi@polimi.it Natural and synthetic phospholipids (PLs) have attracted considerable interest for the multiple scientific and practical applications which follow from their amphiphilic properties. PLs are the focus of biomedical research because of their possible use as pharmaceuticals, food additives, cosmetics, in liposome technology, in gene transfer therapy and in drug delivery systems. However, the investigations and applications rely upon the availability of a great number of purified PLs which can be obtained by semisynthesis from natural sources or from synthetic precursors [1]. The enzymatic modification of the polar heads of natural or synthetic PLs is perhaps the most commonly employed enzymatic modification in the preparation of modified PLs. Phospholipase D (PLD) (E.C. 3.1.4.4) catalyzes the in vivo hydrolysis of PLs to phosphatidic acid (PA). However, in the presence of an alcohol, PLD from Streptomyces PMF catalyzes the in vitro transphosphatidylation of the polar head [2]. This enzyme has been largely used for the transformation of the most abundant natural PL, phosphatidylcholine (PC), into many different PLs [3]. However, the presence of water causes the formation of PA as ubiquitous by-product, thus lowering reaction yields and forcing to further purification steps. Since the purification of this class of compounds is not straightforward, the enhancement of reaction selectivity opens the way to an easier preparation of the target compounds on a relatively large scale. The aim of this work is to investigate innovative reaction media such as ionic liquids for enzymatic PLs transformations in order to obtain a higher selectivity in the enzymatic transphosphatidylation reactions of natural PC catalyzed by PLD from Streptomyces PMF. The opportunity of using ionic liquids (ILs) in PLs chemistry had not been yet investigated. We have tested the stability and the activity of the PLD in various commercial ionic liquids. We illustrate the encouraging preliminary results of the use of ionic liquids as co-solvents in transesterification reactions with different alcohols. This reaction medium seems to be able to drive the reaction towards transphosphatidylation with high depression of competitive hydrolysis.
________________ [1] Eibl, H. Chem. Phys. Lipids 1980, 26, 405-429. [2] D'Arrigo, P.; de Ferra, L.; Piergianni, V.; Selva, A.; Servi, S; Strini, A. J. Chem Soc. Perkin Trans. I. 1996, 2651-2656. [3] D'Arrigo, P.; Servi, S. Trends Biotechnol. 1997, 15, 90-96.
Scheme 1 Biotransformation of silyl ether mediated by fungi. According to the reaction on progress, we observed the formation of racemic-4 after 24 h caused by buffer effect in compounds 1 and 2. However, the Aspergillus terreus has oxidoreductases wich were responsible for deracemization of the alcohol 4 (Table 1, entry 1-6). On the other hand, compound 3 was stable to aqueous solution and we observed a enantioselective biotransformation from silyl ether (3) to alcohol (4) (Table 1, entry 7 and 9).
Table 1 Optimized results of biotransformation of silyl ether into alcohol. In conclusion, we have showed a mild and enantioselective method for biotransformation of silyl ether 3 into alcohol, mediated by fungi. Unfortunately, compounds 1 and 2 were not stable in the reaction medium affording the racemic alcohol 4, wich suffered deracemization.
________________ [1] (a) Colvin, E. Silicon in Organic Synthesis; Butterworth London, 1981. (b) Weber, W. P. Silicon Reagents for Organic Synthesis; Springer-Verlag: New York, 1983. [2] (a) Fukui, T.; Kawamoto, T.; Tanaka, A. Tetrahedron: Asymm. 1994, 5, 73. (b) Li, N.; Zong, M.H.; Peng, H.-S.; Wu, H.-C.; Liu, C. J. Mol. Catal. B: Enzym. 2003, 22, 7. (c) Zani, P. J. Mol. Catal. B: Enzym. 2001, 11, 279.
October 2-6, 2011
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Kinetic study of 2-butanol O-acylation and sec-butylamine N-acylation catalyzed by Candida antarctica lipase B.
Le Joubioux F., Achour O., Bridiau N., Graber M. and Maugard T. UMR 6250 CNRS-ULR, LIENSS, LIttoral ENvironnement SocitS, Equipe Biotechnologie Environnementale, Dpartement Biotechnologie, UFR Sciences, Universit de La Rochelle, Btiment Marie Curie, Avenue Michel Crpeau, 17042 La Rochelle, France. E-mail: florian.le_joubioux@univ-lr.fr The aim of this work was to study the differential behavior shown by Candida antarctica lipase B during the O-acylation and N-acylation of monofunctional alcohols and monofunctional amines, starting from myristic acid as an acyl donor. To achieve this, 2-butanol and sec-butylamine were used as model acceptors (scheme 1). Yields, kinetics and enantioselectivity were studied for both reactions. Although a steady-state ordered ternary complex bi-bi mechanism was obtained for the O-acylation of 2-butanol, a ping-pong bi-bi mechanism was obtained for the N-acylation in case of low sec-butylamine concentrations. The values of apparent kinetics parameters were calculated: the enantiomeric ratios (E) were evaluated and confirmed the preference of Candida antarctica lipase B for the (R)-enantiomer, which was consistent with the literature [1]. The enantioselectivity was calculated for the alcohol (E 3.17) and for the amine (E 1.34). Concerning the O-acylation, the yields were found to be very similar for both enantiomers R and S. However, both initial rates and yields of the (R)-enantiomer N-acylation were higher than those of the (S)-enantiomer. In the last part of our study, the chemoselectivity of Candida antarctica lipase B was evaluated, showing that Candida antarctica lipase B was a chemoselective enzyme that preferentially catalyzed the O-acylation to the detriment of the N-acylation (C 92, for the selective acylation of (R)-enantiomers) [2]. These results provide new insights for the synthesis of products issued from the selective acylation of multifunctional substrates such as amino-alcohols.

Dehydration reactions catalyzed by coenzyme B12-dependent glycerol- and diol dehydratases
Christopher Geinitza, Dennis Wetzla, Bettina Nestla and Bernhard Hauera a Institute for Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, 70569 Stuttgart, Germany; E-mail: dennis.wetzl@itb.uni-stuttgart.de Coenzyme B12-dependent glycerol- (GDH) and diol dehydratases (DDH) are part of the anaerobic glycerol pathway of different enteric bacteria such as Citrobacter freundii and Klebsiella pneumoniae. GDH and DDH are known to catalyze the conversion of glycerol, 1,2-propanediol and 1,2-ethanediol to the corresponding aldehydes. These enzymatic dehydration reactions proceed by a radical mechanism involving coenzyme B12 as a cofactor. [1]1,3-propanediol (1,3-P) is an industrially interesting substitute for ethylene glycol in the production of polyesters, which is formed as a side product of the natural glycerol pathway. [2] In this project, we will present our results concerning the expression and characterization of glycerol- and the isofunctional diol dehydratases from Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca and Lactobacillus reuterii. The dehydratases were expressed in E. coli BL21(DE3) and the so-called Walker strains E. coli C41(DE3) and C43(DE3). A set of different short-chain aliphatic 1,2-diols was examined towards potential novel dehydration activities. The activities of the applied dehydratases were assayed and their substrate range was elucidated using HPLC methods.
________________ [1] R. Daniel, T. A. Bobik, G. Gottschalk, FEMS Microbiol. Rev. 1998, 22, 553566. [2] D.-I. Liao, G. Dotson, I. Turner, L. Reiss, M. Emptage, J. Inorg. Biochem. 2003, 93, 8491.

Biotransformations of cortisone
Stefania Costa, Paola Pedrini, Pier Paolo Giovannini Dipartimento di Biologia ed Evoluzione,Universit di Ferrara, C.so Ercole I dEste 32, 44121 Ferrara, Italy E-mail: pdp@unife.it Steroid compounds can be ranked among the most widely marketed products from the pharmaceutical industry.[1] Highly specific reactions are required to produce functionalized compounds with therapeutic use and commercial value. The complexity of steroid molecules renders the use of biocatalysts particularly interesting, due to the high regio- and stereo-selectivity of the reactions to be performed. In the field the biotrasformations of cortisone (17,21-dihydroxy-4-pregnene-3,11,20-trione) with various bacterial strains (collection of IEP GmbH) are reported.[2] The reactions are described in the Figure 1.
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Asymmetric Bioreduction of 'Activated' Esters
Gbor Tasndia, Dorina Clayb, Christoph K. Winklerb, Mlanie Hallb, Kurt Faberb a ACIB GmbH c/o bDepartment of Chemistry, Organic & Bioorganic Chemistry, University of Graz, Heinrichstrae 28, 8010-Graz, Austria E-mail: gabor.tasnadi@acib.at The asymmetric reduction of C=C bonds leads to the creation of (up to two) stereogenic centers. The biocatalytic variant, catalysed by NAD(P)H-dependent enoate reductases [EC 1.3.1.X] belonging to the old yellow enzyme (OYE) family, takes place in a strict transfashion, and is applicable to activated alkenes bearing an electron-withdrawing substituent [1]. In recent years, we successfully employed OYEs for the asymmetric reduction of structurally diverse activated alkenes [2]. However, it appears that simple ,-unsaturated esters (derivatives of acrylic and/or cinnamic acid) are not easily converted by enoate reductases, which is probably due to an insufficient degree of activation. Therefore, the addition of an additional electron-withdrawing group (e.g. allylic alcohol or -ether, halogen, thioether, acetal moiety), which alone is not sufficient to act as 'activator' may assist to construct socalled 'activated esters'.
Figure 1. Biotransformations of cortisone

________________ [1] P. Fernandes, A. Cruz, B. Angelova, H.M. Pinheiro and J. M. S. Cabral, Enz. Microb. Techn., 2003, 32, 688. [2] S. Costa, P.P. Giovannini, P. Pedrini, J. Mol. Catal. B: Enzym in press.
In order to elucidate the degree of activation required to render a successful substrate, a series of activated and non-activated ,-unsaturated carboxylic esters was subjected to the asymmetric bioreduction catalysed by OYEs. -Halogen substitution proved to be a suitable activating manner and allowed for the preparation of enantioenriched saturated -haloesters. Overall, the concept of 'activated esters' proved to be useful for the construction of monoester substrates for enoate reductases. Acknowledgements: This study was financially supported by BASF SE, the FFG and the FWF (P22722).
________________ [1] M. Hall, Y. Yanto, A. S. Bommarius The Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, Flickinger, M., Ed. Wiley: 2010; pp 2234; R. Stuermer, B. Hauer, M. Hall, K. Faber Curr. Opin. Chem. Biol. 2007, 11, 203. [2] M. Hall, C. Stueckler, W. Kroutil, P. Macheroux, K. Faber Angew. Chem. Int. Ed. 2007, 46, 3934; C. Stueckler, M. Hall, H. Ehammer, E. Pointner, W. Kroutil, P. Macheroux, K. Faber Org. Lett. 2007, 9, 5409; M. Hall, C. Stueckler, H. Ehammer, E. Pointner, G. Oberdorfer, K. Gruber, B. Hauer, R. Stuermer, W. Kroutil, P. Macheroux, K. Faber Adv. Synth. Catal. 2008, 350, 411; C. K. Winkler, C. Stueckler, N. J. Muller, D. Pressnitz, K. Faber Eur. J. Org. Chem. 2010, 6354; C. Stueckler, C. K. Winkler, M. Bonnekessel, K. Faber Adv. Synth. Catal. 2010, 352, 2663.
Scheme 1. Acylation of monofunctional alcohols and amines catalyzed by Candida antarctica lipase B in tert-amyl alcohol.
______________________ [1] V. Leonard, S. Lamare, M. Legoy, M. Graber, J. Mol. Catal. B: Enzym. 32 (2004) 5359 [2] F. Le Joubioux, O. Achour, N. Bridiau, M. Graber, T. Maugard. J. Mol. Catal. B: Enzym., 70 (2010), 108-113
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-glutamyltranspeptidase-catalyzed chemoenzymatic synthesis of avour enhancers from allium sp.
Speranza, G., Manitto, P., Morelli, C.F. Dipartimento di Chimica Organica e Industriale Universit degli Studi di Milano via Venezian, 21 20133 Milano - Italy E-mail: carlo.morelli@unimi.it The use of flavour enhancers in the food industry could be beneficial for several reasons: they ensure homogeneity of the final products, reduce costs for condiments and favor consumer's acceptance. On the other hand, the consumer's attention for convenient, minimally-processed, nutritious, healthy, yet tasty food prompts the food industry to an accurate choice of the ingredients. In this scenario, naturally occurring kokumi substances could play an important role. Kokumi is a japanese term that refers to mouthfulness, thickness and long-lasting savory sensations. Kokumi substances are represented mainly by -glutamyl derivatives of amino acids. They are nearly tasteless for themselves, but they elicit a strong taste sensation, expecially in conjunction with protein-rich food [1]. -glutamyl derivatives with kokumi properties are found in cheese, legumes, mushrooms, spices and vegetables such as garlic and onion. In vegetables of the genus Allium, kokumi substances were identified in -glutamyl derivatives of S-alkyl and S-alkenyl cysteines and their S-oxides [2, 3]. There is a number of difficulties connected with the supplying of these materials. Isolation from natural sources is laborious, and their content in vegetables varies with cultivation and storage. In addition, upon crushing the plant, they are enzymatically degraded to thiosulfinates, disulfides and other volatiles. The chemical synthesis is tedious and not economical, due to the need of protection/deprotection steps. With the aim of expanding our research field from umami chemoreception [4, 5] to kokumi sensations, we exploited the enzymatic synthesis at the laboratory scale of the -glutamyl derivatives of S-allyl cysteine, S-methyl cysteine and methionine, catalyzed by a commercially available mammalian GGT. This study is introductory to the use of a purified home-made bacterial GGT suited to food processing. At this preliminary stage, the reaction requires glutathione (-Lglutamyl-L-cysteinylglycine, GSH) as a -glutamyl donor and proceeds at low temperature in order to minimize side-products formation. The obtained compounds are flavour enhancers with kokumi properties found in garlic and other plants of the genus Allium.
127
Substrate Inhibition in -transaminase Catalyzed Reaction
a
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Use of transaminase enzymes for the synthesis of pharmaceutical intermediates
Alex Boura, Julio Martinez-Torresb, Ian Taylorc, Helen Hailesa, John Wardb Department of Chemistry, University College London, WC1H 0AJ London, UK; bInstitute of Structural and Molecular Biology, Division of Biosciences, University College London, WC1E, 6BT, UK; cChirotech Technology Ltd, 162 Science Park, Milton Road, Cambridge, CB4 0GH E-mail: uccaadb@live.ucl.ac.uk
a
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From simple molecules to more complex enantiopure imidates and phthalides using biotransformations
Vicente Gotor-Fernndez, Juan Mangas-Snchez, Eduardo Busto, Mara Lpez-Iglesias, Nicols Ros-Lombarda, Mara Rodrguez-Mata, Vicente Gotor a Department of Organic and Inorganic Chemistry, Instituto Universitario de Biotecnologa de Asturias. Universidad de Oviedo. Avenida Julian Clavera 8, 33006 Oviedo, Spain; E-mail: vicgotfer@uniovi.es The development of stereoselective synthetic strategies in heterocyclic chemistry has recieved extraordinary attention along the last century. In particular, chiral 3-substituted phthalides, also known as 1-(3H)-isobenzofuranones, are five-membered lactones which present themselves interesting pharmacological properties but also they can be employed as useful building blocks for the synthesis of biologically active compounds. Unfortunately very little attention has been paid to the design of robust chemoenzymatic synthesis for this class of oxygen-containing molecules.[1] As part of our ongoing project towards the preparation of optically active heterocyclic compounds,[2] ketobenzonitriles have been found as key precursors for the synthesis of interesting chiral imidates and phthalides (Figure 1). Oxidoreductases and hydrolases have been tested as possible enzymes in asymmetric processes,[3] focusing our attention in the development of a straightforward and general chemoenzymatic synthesis for a broad range of enantiopure phthalides. Further studies are currently in progress for the preparation of other related cyclic oxygenated and nitrogenated structures.
Naweed Al-Haquea, Pr Tufvessona, Rafiqul Ganib and John M. Woodleya Center for Process Engineering and Technology,bComputer Aided Process-Product Engineering Center, Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), DK-2800, Kgs. Lyngby, Denmark; E-mail: nah@kt.dtu.dk For many synthetic processes in the chemical and pharmaceutical industries, highly selective reactions are required. While biocatalysis frequently results in highly selective conversions [1], in many cases, the substrate and the product may inhibit or damage the biocatalyst or interfere with other components in the reaction medium above a critical concentration [2]. One solution is to introduce an auxiliary phase in the form of a solid resin [3]. A solid resin has several advantages over the alternative methods of supply such as organic solvents [4]. This technology uses the resin to act as a reservoir for the substrate and the product. The substrate, via mass transfer, will slowly diffuse into the solution. The slow release will maintain the aqueous concentration beneath an inhibitory level. The biocatalyst will convert the substrate to product which can subsequently be recovered by means of in-situ product removal (ISPR). When the resin is saturated, the product is eluted to give a high concentration solution. Such an approach will be investigated in this project. This therefore represents a novel way of intensifying bioprocesses, via the combination of controlled substrate supply, reaction and product removal in an integrated unit operation. In order to test this hypothesis, a -transaminase (-TAm) catalyzed reaction for the asymmetric synthesis of (S)-(-)-- methylbenzylamine (MBA) using isopropylamine (IPA) as amine donor and acetophenone (APH) as amine acceptor was selected. This reaction mechanism was selected since MBA is known to be a very important building block of several pharmaceutical products [5]. However, specific challenges have to be addressed in this system which includes high product and substrate inhibition, unfavourable equilibrium and low substrate solubility [6]. A broad range of polymeric adsorbents comprising various surface areas, pore volumes and functional groups were used in experiments to quantify the partition coefficient and overall capacity of the resin for the substrate and product. It is very critical to select the appropriate adsorbent as the rate of release of the substrate should complement the rate of reaction. The resin can also be loaded with a higher concentration of the substrate. With the selection of the appropriate resin, it was loaded with substrate and released into the reaction media. To make a comparison, the same reaction was carried out by adding the substrate directly to the reaction media. From the experimental findings, it can be stated that the presence of resins had a significant positive impact with respect to biocatalytic activity. The substrate inhibition could be alleviated and allow the reaction to go to completion.
________________ [1] Pollard D.J.; Woodley J.M. (2006). Trends Biotechnol., 25:66-73. [2] Tufvesson P.; Fu W.; Jensen J.S.; Woodley J.M. (2010). Food. Bioprod. Process.; 88:3-11. [3] Kim P-Y.; Pollard D. J.; Woodley J.M. (2007). Biotechnol. Prog., 23:74-82. [4] Straathof AJJ. (2003). Biotechnol. Prog., 19:755-762. [5] Tufvesson P.; Lima-ramos J.; Jensen SJ.; Al-Haque N.; Neto W.; Woodley JM. (2011). Biotech. Bioeng., 108: 1479 1493 [6] Seo J-H.; Kyung D.;Joo K.; Lee J.; Kim B-G. (2010). Biotech. Bioeng. 108: 253 263.
The past decade has seen a remarkable growth in interest in the potential use of microorganisms and enzymes for the synthesis of pharmaceutical intermediates. Chiral amines are highly sought after in the pharmaceutical and agrochemical industries as 70% of all pharmaceuticals are derivatives of these amines. Transaminases (TAms) are a class of metabolic enzymes that catalyze the transfer of an amino group from a donor (e.g. amino acid) to an acceptor ketone or aldehyde functionality. To date there are six classes of TAms based on their amino acid sequence similarity [1]. Each class therefore has unique substrate and co-substrate preferences depending on the enzyme active site. Conventional synthesis of chiral amines often involves the kinetic resolution of racemic mixtures using hydrolytic enzymes, however, only a 50% maximum yield can be achieved [2]. The use of TAms in the asymmetric synthesis of chiral amines provides a strategy that may yield up to100% of the product with less waste and at lower cost. We are interested in the identification of novel TAms for use in organic synthesis. The strategy employed for the selection of novel -TAms (and their subsequent cloning and over-expression), begins with the generation of a phylogenetic tree derived from the BlastP search of the amino acid sequence of a known -TAm [1]. Enzyme activity is determined using an HPLC-based screening assay with the substrate pair -methylbenzylamine (MBA) and pyruvate. The reaction yields alanine and acetophenone (AP). The enantioselectivity of the enzyme is also determined, as it is known that some classes of TAms prefer the amine group of one enantiomer over the other [1]. This is achieved via chiral HPLC analysis of the isolated amine product alongside the commercially available amine (derivatised if need be) [3]. Our TAm library of over 100 enzymes will be screened thoroughly to match different classes of enzymes with their preferred functional groups (i.e. aliphatic, aromatic, cyclic, carboxylic acids ketones and aldehydes). Our unique approach to the discovery of novel enzymes coupled with a thorough substrate screen will contribute to our understanding of the substrate preferences of TAms while providing a cheap and succinct route for the biotransformation of ketones and aldehydes to amines.
1. Ward, J.M and Wohlgemuth, R., Curr. Org. Chem., 14 (2010) 1-13 2. Kaulmann, U., Smithies, K., Smith, M.E.B., Hailes, H.C., Ward, J.M., Enzyme Microb. Technol., 41 (2007) 628. 3. Truppo, D.M., Rozzell, J.D., Moore, J., Turner, N.J., Org. Biomol. Chem., 7 (2009) 395-398
Figure 1. General struture of 3-alkyl-imidates and 3-alkyl-phthalides

________________ [1] (a) T. Izumi, O. Itou, K. Kodera, J. Chem. Tech. Biotechnol. 1996, 67, 89-95. (b) T. Kitayama, Tetrahedron: Asymmetry 1997, 8, 3765-3774. [2] J. Mangas-Snchez, E. Busto, V. Gotor-Fernndez, V. Gotor, Org. Lett. 2010, 12, 3498-3501. [3] (a) V. Gotor, I. Alfonso, E. Garca-Urdiales, Asymmetric Organic Synthesis with Enzymes; WileyVCH, Weinheim (Germany) 2008. (b) W.-D. Fessner, T. Anthonsen, Modern Biocatalysis. Stereoselective and Environmentally Friendly Reactions. Wiley-VCH, Weinheim (Germany) 2009. (c) E. Busto, V. Gotor-Fernndez, V. Gotor, Chem. Rev. 2011, DOI: 10.1021/cr100287w.
________________ [1] A. Dunkel, J. Kster, T. Hofmann, J. Agric. Food Chem. 2007, 55, 67126719. [2] Y. Ueda, M. Sakaguchi, K. Hirayama, R. Miyajima, A. Kimizuka, Agric. Biol. Chem. 1990, 54, 163169. [3] Y. Ueda, T. Tsubuku, R. Miyajima, Biosci. Biotechnol. Biochem. 1994, 58, 108110. [4] P. Cairoli, S. Pieraccini, M. Sironi, C. F. Morelli, G. Speranza, P. Manitto, J. Agric. Food Chem. 2008, 56, 10431050. [5] C. F. Morelli, P. Manitto, G. Speranza, Flav. Fragr. J., in press. Available as early view at wileyonlineibrary.com DOI 10.1002/ffj.2053.
October 2-6, 2011
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Enzymatic transesterification of egg-yolk phosphatidylcholine with castor oil
Witold Gadkowski, Anna Chojnacka, Czesaw Wawrzeczyk Department of Chemistry, Wroclaw University of Environmental and Life Sciences, 50375 Wroclaw, Poland E-mail: glado@poczta.fm The introduction of hydroxy acids into the natural phospholipids is the subject of increasing research interest. Hydroxylated phospholipids have useful surface properties, higher moisture retention and increased water dispersibility therefore they can find the application in food industry as the emulsifying agents or in bakery as staling retardants [1]. They are also used in cosmetic and pharmaceutical industry as the source of biologically active hydroxyacids. The most common hydroxy fatty acid is ricinoleic acid which occurs in castor seeds (Ricinus communis). Few chemical and enzymatic methods have been developed so far to produce phospholipids containing ricinoleic acid [2-4]. Enzymatic approach have advantages: mild reaction conditions and regio-and stereospecificity of the biocatalysts. In this communication we report the results of direct incorporation of ricinoleic acid to the lecithin isolated from egg yolk using direct transesterification with castor oil as the natural acyl donor. We applied two enzyme preparations: lipase from Candida antarctica (Novozym 435) and lipase from Thermomyces lanuginosa (Lipozym TL IM). The effect of various conditions (enzyme dose, temperature, castor oil:lecithin molar ratio, water activity - aw) on the incorporation of ricinoleic acid was studied. The highest content of ricinoleic acid in modified lecithin (31%) was achieved in following conditions: temperature 50oC, enzyme dose 30% (w/w of total substrates), molar ratio castor oil:lecithin 5:1, aw=0.22. Detailed results of the enzymatic reactions will be presented. Acknowledgements: This work was co-financed by the European Union within the European Regional Development Fund (Project no. POIG.01.03.01-00-133/08)
__________________ [1] Schmidt J.C., Orthoefer. Lecithin in baking applications. In: Szuhaj B.F. List G.R., eds, Lecithins, Champaign, IL: American Oil Chemists Society, 203-211, (1985). [2] Borsotti G., Guglielmetti G., Spera S., Battistel E. Tetrahedron 58, 10219-10227, (2001). [3] Vijeeta, T., Reddy, J.R.C., Rao, B.V.S.K., Karuna, M.S.L., Prasad, R.B.N. Biotechnol. Lett. 26, 10771080, (2004). [4] Das S., Bhattacharyya D.K. J. Am. Oil Chem. Soc. 83, 1015-1020, (2006).

Obtaining of -Hydroxyesters Through Dynamic Processes Catalysed by Alcohol Dehydrogenases
Ivn Lavanderaa, Anbal Cuetosa, Ana Rioz-Martneza, Fabricio R. Bisognoa, Barbara Mautnerb, Cristina Rodrgueza, Gonzalo de Gonzaloa, Wolfgang Kroutilb, Vicente Gotora a Organic and Inorganic Department, Instituto de Biotecnologa de Asturias, University of Oviedo, C/ Julin Clavera 8, 33006 Oviedo, Spain; bDepartment of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria E-mail: lavanderaivan@uniovi.es In the last few years, the employment and development of dynamic protocols in order to obtain enantio- or diastereomerically pure compounds starting from easily available racemic starting material has largely increased. In this sense, more and more processes involving enzymatic and homogeneous catalysis in dynamic conditions are designed to achieve successful transformations to synthesise optically pure high-added value derivatives.[1] One of the most typical examples of a dynamic process is the reduction of -substituted -ketoesters (Scheme 1) to obtain the corresponding alcohols with high diastereomeric excesses (des).[2] This process has been successfully achieved using both metal- and enzyme catalysis due to high acidity of the -hydrogen that ensures a fast substrate racemisation even at neutral pHs (Scheme 1).

Rhamnulose-1-Phosphate aldolase from Thermotoga maritima as a new biocatalyst for the synthesis of nitrocyclitols
Isabel Oroz-Guineaa, Flora Camps-Bresbc, Christine Gurard-Hlainebc, Israel Snchez-Morenoa, Eduardo Garca-Juncedaa* and Marielle Lemairebc* a Departamento de Qumica Bio-Orgnica, Instituto de Qumica Orgnica General, CSIC. 28006 Madrid, Spain. b Laboratoire SEESIB, Clermont Universit, Universit Blaise Pascal, 24 avenue des Landais, F-63177 Aubire. c CNRS, UMR 6504, 24 avenue des Landais, F-63177 Aubire. E-mail: isabel.oroz@iqog.csic.es Recently, Prof. Marielle Lemaires group has developed a new methodology to access to nitrocyclitols and aminocyclitols analogs of valiolamine, validamine and valienamine which have a high potential activity as molecular chaperons for glycosidases1. The key step in this chemo-enzymatic synthesis is the aldol reaction between dihydroxyacetone phosphate (DHAP) and an aldehyde catalyzed by a DHAP-dependent aldolase. A highly stereoselective intramolecular cyclization (Henry reaction) takes place during the aldolase-catalyzed reaction and is then followed by a phosphatase-catalyzed hydrolysis step. Enantiomerically pure nitrocyclitols with four new stereocenters for each aldehyde tested were obtained in one pot. We are involved now in extending this strategy using the rhamnulose-1-phosphate aldolase (Rham-1PA) from Thermotoga maritima. This enzyme has been previously cloned and characterized in our group and, as a hyperthermophilic enzyme, offers intriguing possibilities for practical catalysis like high stability under extreme (temperature, presence of organic co-solvents, etc.) and storage conditions. In this communication we describe the synthetic application of rhamnulose-1-phosphate aldolase from Thermotoga maritima as a new biocatalyst for the synthesis of nitrocyclitols according to the strategy described above. Acknowledgements: We thank the Spanish Ministerio de Ciencia e Innovacin (Grant CTQ2010-15418) and Comunidad de Madrid (Grant S2009/PPQ-1752) for financial support. I. Oroz-Guinea is a JAEPredoc fellow from CSIC.
________________ L. El Blidi, Z. Assaf, F. Camps-Bres, H. Veschambre, V. Thry, J. Bolte and M. Lemaire* ChemcatChem, 2009, 7, 463-471.
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Via enzyme toolboxes to chiral diols
Justyna Kuliga, Robert Simonb, Wolfgang Kroutilb, Frank Hollmannc, Wolfgang Wiecherta, Martina Pohla, Drte Rothera aInstitute of Bio- and Geosciences 1: Biotechnology, Forschungszentrum Jlich GmbH, 52425 Jlich, Germany; bDepartment of Chemistry, Karl-Franzens-Universitt Graz, Heinrichstrasse 28, 8100 Graz, Austria; cDepartment of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands E-mail: j.kulig@fz-juelich.de Chiral diols find versatile application as synthons for chemical catalysts, agrochemicals and pharmaceuticals.[1,2] The concept of combining two toolboxes of enzymes opens the access to diversely substituted enantiocomplementary vicinal diols (Fig 1).
Figure 1. Combining ThDP-dependent enzymes and oxidoreductases toolboxes First, the carboligation of cheap aldehydes by thiamine diphosphate (ThDP)-dependent enzymes yielding 2-hydroxy ketones with high chemo- and stereoselectivity takes place. [3] Subsequently the 2-hydroxy ketones are further reduced via NAD(P)H-dependent oxidoreductases (Fig 2).
Scheme 1. Bioreduction of -substituted -ketoesters affording a mixture of diastereomeric alcohols employing ADHs. Concerning the bioreduction of these and related derivatives, the first examples were shown employing whole cells such as bakers yeast, and although in some cases excellent conversions and enantiomeric excesses (ees) were achieved, the presence of several active enzymes also depleted the selectivity in many cases.[2] More recently, the development of these dynamic protocols using purified or overexpressed alcohol dehydrogenases (ADHs),[3] has overcome this problem although in hand with efficient techniques to recycle the expensive cofactor needed in these processes. Herein we would like to show the use of several purified and overexpressed ADHs applied to the bioreduction of several -substituted -ketoesters in order to obtain the corresponding alcohols with excellent enantiomeric and diastereomeric excesses. Obviously, depending on the enzyme and the substrate structure, different results were observed and they will be discussed to gain a deeper insight into the different possibilities that these biocatalysts can provide.
________________ [1] (a) H. Pellissier, Tetrahedron 2011, 67, 3769-3802. (b) J. Steinreiber, K. Faber, H. Griengl, Chem. Eur. J. 2008, 14, 8060-8072. [2] (a) H. Stecher, K. Faber, Synthesis 1997, 1-16. (b) R. S. Ward, Tetrahedron: Asymmetry 1995, 6, 1475-1490. [3] S. M. A. de Wildeman, T. Sonke, H. E. Schoemaker, O. May, Acc. Chem. Res. 2007, 40, 1260-1266
Figure 2. Stereoselective 2-step enzymatic synthesis of chiral 1,2-diols The main focus is the stereoselective reduction of especially bulky-bulky substrates.[4,5] Promising reactions are optimised via reaction engineering and an appropriate process for the enzymatic 2-step synthesis with cofactor regeneration will be evaluated.
________________ [1] Daussmann, T., Hennemann, H. G., Rosen, T. C. and Dnkelmann, P., Chem. Ing. Techn. 78, 249255, 2006. [2] Lavandera, I., Holler, B., Kern, A., Ellmer, U., Glieder, A., de Wildeman, S. and Kroutil, W., Tetrahedron-Asymmetry 19, 1954-1958, 2008. [3] Pohl M., Gocke D., Mller M. Handbook of Green Chemistry, Green Catalysis, Vol. 3 Biocatalysis Weinheim: Wiley-VCH, 75-114, 2008. [4] Lavandera, I., Kern, A., Ferreira-Silva, B., Glieder, A., de Wildeman, S. and Kroutil, W., J. Org. Chem. 73, 6003-5, 2008 [5] Lavandera, I., Kern, A., Schaffenberger, M., Gross, J., Glieder, A., de Wildeman, S. and Kroutil, W., ChemSusChem 1, 4
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Chemistry and synthetic biology in symbiosis for the synthesis of benzylisoquinoline alkaloids
Thomas Pesnot,1 Markus C. Gershater,2 John M. Ward2 and Helen C. Hailes1 1 Department of Chemistry, University College London, 20 Gordon Street, London 2 Structural and Molecular Biology, University College London, Gower Street, London t.pesnot@ucl.ac.uk In nature, benzylisoquinoline alkaloids (BIAs) are produced by plants as a defence mechanism against herbivores or pathogens. Many BIA natural products were also identified as biologically active and are now prescribed for the treatment of a wide array of diseases including HIV, cancer and microbial infections as well as for the treatment of pain.[1] Thus, BIAs provide excellent scaffolds for the development of new compounds with enhanced biological activities. The chemical synthesis of BIAs is however hindered by many challenges including their optical activity, high polarity and multiple functionalities.[2] In order to address these issues and readily synthesise libraries of novel BIAs, we investigated the concerted use of synthetic biology and chemical synthesis.
135
Enzymatic Carboxylation of Aromatic Compounds
a
138
A chemoenzymatic process for the synthesis of acyclic nucleoside analogues involving the use of rabbit muscle aldolase (RAMA)
Martn A. Palazzoloa, Adolfo M. Iribarrena,b, Elizabeth S. Lewkowicza a Laboratorio de Biocatlisis y Biotransformaciones, Departamento de Ciencia y Tecnologa, Universidad Nacional de Quilmes, Roque Senz Pea 352, Bernal (CP 1876), Argentina; bLaboratorio de Qumica de cidos Nucleicos, INGEBI-CONICET, Vuelta de Obligado 2490, Ciudad Autnoma de Buenos Aires (CP 1428), Argentina E-mail: mpalazzolo@unq.edu.ar For more than 40 years, nucleoside analogues have shown to be a valuable source of antiviral and antitumor agents [1]. In particular, acyclic nucleosides received great interest since the discovery of acyclovir in 1978- due to their extraordinary chemical and biological stability. Unlike natural nucleosides, the high flexibility of the C-C linkages enables the acyclic analogues to adopt conformations that favor the formation of complexes with the enzymes involved in their metabolism [2]. Often, the absolute configuration of the stereogenic centers plays an important role in determining both their biological activity and selectivity [3]. Aldolases are enzymes that catalyse reversible, stereospecific C-C bond condensation reactions. In general, they possess strong dependence on the donor compound but show low specificity towards the acceptor substrate [4]. Owing to these facts, they are a very interesting tool for the synthesis of a variety of chiral compounds. In this work, we describe a chemoenzymatic process for the preparation of thymidine and adenosine acyclic analogues (Figure 1). This strategy involved the suitable N-alkylation of the nucleosidic bases to generate the acceptors for the subsequent aldol condensation reaction biocatalysed by rabbit muscle aldolase (RAMA). The expected acyclic nucleoside analogues were obtained in moderate to high yields.
139
Operating windows for transaminase processes using thermodynamic and biocatalyst constraints
Pr Tufvesson, Joana Lima-Ramos, Jacob S. Jensen and John M Woodley Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), DK-2800 Lyngby, Denmark E-mail: pt@kt.dtu.dk Transaminase catalysed reactions have been demonstrated as an attractive alternative to conventional resolution strategies for chiral amines, both in the laboratory and to a much more limited extent in industrial processes [1]. Indeed, one challenge that needs to be overcome when developing such process for industrial application is how to achieve high yields in the face of an unfavorable reaction equilibrium. Several different solutions have been suggested to overcome this, such as evaporation of a volatile co-product or the in-situ enzymatic degradation of the co-product, e.g. pyruvate. This work describes a methodology to analyse the feasibility of a process by combining a set of physical and biological constraints to create an operating window. For instance the equilibrium constant of the reaction determines the concentration of the product and co-product in solution that must be maintained in order to meet a target reaction yield [2]. Additionally, the performance of the biocatalyst (including inhibition) gives another set of constraints. The methodology is demonstrated for the development of a biocatalytic transamination reaction and shows how it can be used to guide the selection of technologies and configurations, as well as identification and establishment of development targets.
Christiane Wuenscha, Silvia M. Gluecka and Kurt Faberb Austrian Centre of Industrial Biotechnology, c/o bDepartment of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria christiane.wuensch@acib.at
Due to the increasing abundance of CO2 as a major greenhouse gas, the development of CO2-fixation reactions for the production of chemicals is one of the main challenges in synthetic organic chemistry [1]. The Kolbe-Schmitt reaction [2], which is one of the major industrial processes for the production of aromatic carboxylic acids, requires harsh reaction conditions (high pressure and temperature). In contrast, the use of (de)carboxylases represents a green alternative method for the regioselective carboxylation of aromatic compounds. To test the viability of this novel enzymatic process various aromatic substrates were subjected to several benzoic acid (de)carboxylases in bicarbonate buffer. Among the seven tested enzymes the decarboxylases from Aspergillus oryzae [3], Rhizobium sp. [4] and Trichosporon moniliiforme [5] were found to be highly active. Depending on the enzyme and substrate, respectively, the carboxylated product was formed in up to 42% conversion.
Figure: Enzymatic and biomimetic syntheses of norcoclaurine Biosynthetically, the first committed step to BIAs is catalysed by norcoclaurine synthase (NCS) which couples dopamine with 4-hydroxyphenylacetaldehyde (4-HPAA) to yield (S)-norcoclaurine (Figure).[3] During our studies, we carried out expressions of native NCSs in BL21 E. Coli and measured their respective enantioselectivities. The substrate tolerance of selected NCSs was screened using our newly developed fluorescence-based assay. The assay highlighted substrate features which are essential for NCS activity and helped in understanding its catalytic mechanism. Hits from the assay were scaled up and enantiomeric excesses measured. Biomimetic reactions conditions were also developed and utilised for the rapid synthesis of naturally occurring as well as non-natural BIAs.[4] In combining chemical synthesis with synthetic biology, a wide variety of BIAs pharmaceutical candidates were produced.
________________ 1.Facchini, P. J. Annu. Rev. Plant. Physiol. Plant Mol. Biol. 2001, 52, 29-66 2.Chrzanowska, M. and Rozwadowska, M. D. Chem. Rev. 2004, 104, 3341-3370 3.Samanani, N. and Facchini, P.J. J. Biol. Chem. 2002, 277, 33878-33883 4.Pesnot, T.; Gershater, M. C.; Ward, J. M. and Hailes, H. C. Chem. Commun. 2011, 47, 3242-3244
Figure 1. Biocatalytic carboxylation of various aromatic substrates. Acknowledgemets: Financial support by the FGG is gratefully acknowledged.
________________ [1] S. M. Glueck, S. Gms, W. M. F. Fabian and K. Faber, Chem. Soc. Rev., 39, 313 (2010) [2] A. S. Lindsey and H. Jeskey, Chem. Rev., 57, 583 (1957) [3] R. Santha, N. A. Rao and C. S. Vaidyanathan, Biochim. Biophys. Acta, 1293, 191 (1996) [4] M. Yoshida, N. Fukuhara and T. Oikawa, J. Bacteriol., 186, 6855 (2004) [5] K. Kirimura, H. Gunji, K. Kino, R. Wakayama, T. Hattori and Y.Ishii, Biochem. Biophys. Res. Commun., 394, 279 (2010)
Figure 1. Chemoenzymatic synthesis of acyclic nucleoside analogues.

[1] De Clercq, E. Annu. Rev. Pharmacol. Toxicol. (2011), 51, 1-24 [2] DAlonzo, D.; Guaragna, A.; Palumbo, G. Chem. Biodiversity (2011), 8, 373-413 [3] Paju, A. et al. Nucleosides, Nucleotides Nucleic Acids (2010), 29, 707720 [4] Claps, P.; Fessner, W-D.; Sprenger, G. A.; Samland, A. K. Curr. Opin. Chem. Biol. (2010), 14, 154167 ________________ [1] Savile, Janey, Mundorff, Moore, Tam, Jarvis, Colbeck, Krebber, Fleitz, Brands, Devine, Huisman and Hughes, Science, 2010, 329, 305-309 [2] Tufvesson, Lima-Ramos, Jensen, Al-Haque, Neto and Woodley, Biotech. Bioeng., 2011, 108, 14791493
October 2-6, 2011
106 140
Acyl-CoA synthetase (ACS) can catalyze the amide bond formation reaction between fatty acid and amino acid
Dai-ichiro Kato, Takahiro Kamon, Eriko Nishimura, Masahiro Takeo, Seiji Negoro Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo, 2167 Shosha, Himeji, Hyogo 671-2201, Japan E-mail: kato@eng.u-hyogo.ac.jp Acyl-CoA synthetase (ACS) is an enzyme that catalyzes the thioesterification reaction between the fatty acid and coenzyme A (CoASH) using ATP as cofactor. Although the natural substrates of this enzyme are the n-fatty acids in vivo, ACS can also accept some artificial carboxylic acids such as 2-arylpropanoic acid (profen compound) and 2-aryloxypropanoic acid. Formerly we have reported that an ACS from Brevibacterium ketoglutamicum KU1073, which belongs to a family of medium-chain acyl-CoA synthetase (MACS), can differentiate the absolute configuration of 2-(4-chlorophenoxy)propanoic acid and catalyze the S-enantioselective thioesterification reaction [1]. Although information on the specificity toward carboxylic acid substrates was stored by our recent studies, we have little information about the substrate acceptance in thiol compounds. Thus for this presentation, we have examined the specificity for thiol compounds of MACS. Using the octanoic acid as carboxylic acid substrate, thiol compounds such as pantetheine, N-acetylcysteamine and cystein were subjected to the reaction on behalf of CoASH. From this experiment, we confirmed that all these compounds were acceptable for MACS, though the specific activities fell in. Interestingly enough, we discovered that the product between octanoic acid and cystein was amide compound, N-octanoylcystein, which was not the thioester compound (Figure 1). This result indicated that MACS can catalyze the two types of reactions, thioesterification and amidation, depending on the structure of thiol compound. The Km value for cystein of amidation reaction with octanoic acid was about 2 mM. The same amidation activity has already reported by Abe et al. in an acetyl-CoA synthetase, but the affinity and the reaction rate were quite low [2]. MACS, however, displays the good affinity for cystein and the catalytic rate was also high. Moreover, the substrate specificity toward amino acid was broad and methionine, lysine, and glutamic acid were acceptable for this biotransformation system. Thus we will present the enzymatic properties of MACS catalyzed amidation reaction between the fatty acid and amino acid because this enzymatic system should provide us a new technique for the production of N-acylamino acid derivatives.

Enzymatic 2-step synthesis of chiral amino alcohols combining ThDP-dependent lyases and -transaminases
T. Sehla, H.C. Hailesb, J. M. Wardc, W. Wiecherta, M. Pohla, D. Rothera a Institute of Bio- and Geosciences 1: Biotechnology, Forschungszentrum Jlich GmbH, Ger. b Department of Chemistry, University College London, UK c Department of Biochemistry and Molecular Biology, University College London, UK t.sehl@fz-juelich.de, do.rother@fz-juelich.de, m.pohl@fz-juelich.de. Chiral 2-amino alcohols are versatile synthons used as chiral auxiliaries and for the synthesis of biologically active compounds. A well known example is L-ephedrine, which is prepared via the chemical reductive amination of phenylacetylcarbinol. Here we describe a novel two-step synthesis of 2-amino alcohols such as ephedrine and analogues, catalysed by the combination of thiamine diphosphate (ThDP)-dependent lyases and -transaminases.

Optimisation of the chemo-enzymatic dynamic kinetic resolution for the synthesis of benzoin and related chiral hydroxy ketones
R. Niegutha, M. B. Ansorge-Schumachera a Technical University of Berlin, Institute of Chemistry, Department of Enzyme Technology (TC4), Str. des 17. Juni 124, D-10623 Berlin (Germany) E-mail: rene.nieguth@chem.tu-berlin.de The demand for enantiomerically pure compounds has distinctly increased in recent years, especially in the fine chemical and pharmaceutical industries. An example for an important class of such compounds are -hydroxy ketones. In principal, these can be obtained by several different strategies, whereas the enzymatic kinetic resolution (KR) of racemic mixtures can be regarded as particularly useful.[1] However, an important drawback of kinetic resolutions (KR) is the intrinsic limitation of the maximum theoretical yield to 50%. This can be overcome by coupling the KR with a racemisation reaction to a dynamic kinetic resolution (DKR) allowing theoretical yields of 100%. It has been reported that the DKR of bulky hydroxy ketones such as benzoins (1,2-diaryl2-hydroxyethanone) can be effectively performed on lab-scale by Lipase TL (from Pseudomonas stutzeri) combined to the Shvos catalyst [Figure 1].[2] In this study, we aim at an optimisation of this approach with regard to industrial application emphasising operational stability, reusability and, in particular, catalytic performance of the biocatalyst.
107 145
Kinetic characterization of the NADPH-dependent 7- and 7-hydroxysteroid dehydrogenases from Clostridium absonum
Giulia M. Bertolesia , Daniela Montia, Erica E. Ferrandia, Sergio Rivaa a ICRM-CNR, via Mario Bianco 9, 20131 Milano, Italy; E-mail: daniela.monti@icrm.cnr.it NADPHdependent 7- and 7-hydroxysteroid dehydrogenases (7-HSDH and 7-HSDH) from Clostridium absonum, a perfringens-like Clostridium, catalyze the oxidation-reduction at position 7 of primary bile salts and may be suitable as biocatalyst for the synthesis of bile acids derivatives [1]. After purification by affinity chromatography, the enzymes were submitted to kinetic analysis for determination of Michaelis constants (Km), specificity constants (kcat/Km) and substrate inhibition in the presence of different substrates. Specifically, the tested derivatives showed different oxidation levels at positions 3, 7 and 12 (Figure 1), making them suitable as substrates in regio- and stereoselective oxidation or reduction reactions catalyzed by 7-HSDHs. 7-HSDH showed a higher specificity for the oxidative reactions substrates, cholic acid and 12-ketochenodeoxycholic acid being practically equivalent both in terms of Km and kcat/Km values. 7-HSDH showed a higher specificity for ursocholic and ursodeoxycholic acid in oxidation reactions, whereas 7-ketolitocholic acid was the preferred substrate in reduction reactions. Both enzymes showed a very strong substrate inhibition with all tested substrates. The highest KS/Km values were observed with chenodeoxycholic acid as a substrate in the case of 7-HSDH, whereas ursodeoxycholic acid was the most effective inhibitor of 7-HSDH activity.
Fig. 1: Novel two-step synthesis of 2-amino alcohols catalysed by ThDP-dependent lyases and transaminases A toolbox of ThDP-dependent enzymes was assembled to access a broad range of enantiocomplementary 2 hydroxy ketones. Preparative routes toward several chiral 2-hydroxy ketones by carboligation of cheap aldehydes have already been established using batch, repetitive batch and semicontinuous methods [2]. In a follow-up reaction the reductive amination of chiral 2-hydroxy ketones (R and R are different), catalysed by -transaminases will yield chiral 2 amino alcohols. Therefore a second toolbox of -transaminases from different organisms [3] was made available. We established a novel screening assay for -transaminase activity based on the consumption of 2 hydroxy ketones in 96-well plates. With this the substrate ranges of the wildtype enzymes was fingerprinted. Promising reactions were scaled-up and analyzed in batch reactions in order to built up a platform of enantiocomplementary and diversely substitute 2 amino alcohols. Products like (1R,2S)-Norephedrine and derivatives thereof are enzymatically accessible with high enantioselectivities (ee > 99 %).
_____________________________ [1] Mller M., Gocke D., Pohl M. (2009). FEBS Journal 276 (11): 2894-904 [2] Roger P.L., Shin H. S. (1995). Appl. Microbiol Biotechnology 44: 7-14 [3] Kaulmann U., Smithies K., Smith E.B., Hailes H.C., Ward J.M. (2007). Enzyme and Microbial Technology 41 (5): 628-637
Figure 1. DKR of -hydroxy ketones catalysed by Lipase TL (from Pseudomonas stutzeri) and Shvos catalyst as example for a suitable racemisation catalyst In a first step, a suitable carrier for adsorption of the enzyme was identified. Best results were obtained with the strongly hydrophobic Accurel MP1001, which significantly increased the specific activity in the desired transesterification reaction. In a second step, silicone was deposited on the carrier as this is known to enhance both leaching and mechanical stability[3]. For the optimisation of the racemisation step, transition-metal-mediated racemisation via hydrogen transfer as well as alternative heterogeneous racemisation catalysts were applied.
________________ [1] B. Martin-Matute, M. Edin, K. Bogar, F.B. Kaynak, J. Bckvall, J. AM. CHEM. SOC. 2005, 127, 8817-8825 [2] P. Hoyos, M. Fernandez, J.V. Sinisterra, A.R. Alcantara, J. Org. Chem. 2006, 71, 7632-7637 [3] L.O. Wiemann, R. Nieguth, M. Eckstein, M. Naumann, O. Thum, M.B. Ansorge-Schumacher, ChemCatChem 2009, 1, 455-462.
Figure 1. MACS can catalyze two types of reactions depending on the substrate structure
________________ [1] D.Kato, H.Yoshida, M.Takeo, S.Negoro, H.Ohta, Biosci. Biotechnol. Biochem., 2010, 74, 2405-2412. [2] T.Abe, Y.Hashimoto, H.Hosaka, K.Tomita-Yokotani, M.Kobayashi, J. Biol. Chem., 2008, 283, 11312-11321
Figure 1. Bile acids tested for kinetic characterization of 7-HSDHs.

____________________ [1] Monti D. et al., Adv. Synth. Catal. (2009), 351,1303-1311.
142
Chemoenzymatic synthesis of green tea aroma compounds
Valentina Venturi, Pier Paolo Giovannini, Silvia Maietti, Gianni Sacchetti, Paola Pedrini Dipartimento di Biologia ed Evoluzione, Universit di Ferrara, C.so Ercole I dEste 32, 44121 Ferrara, Italy E-mail: valentina.venturi@unife.it Many volatile compounds identified in complex biological matrixes are chiral molecules. The chirality evaluation is of great importance in drugs and pheromone chemistry, but chirality also plays a very important role in flavour and fragrance chemistry. Analyses of these products are performed to determine their composition, to control their quality and to identify adulterations or product counterfeiting. In this respect, proper characterizations of the enantiomeric composition can provide information on the genuineness, geographic origin, biogenesis and quality of the product. 3-hydroxy-3-methyl-2,4-nonanedione 1, 1-methyl-2-oxopropyl-hexanoate 2 and 1-methyl-2-oxopropyl-acetate 3 are volatile compounds contributing to the characteristic flavour of a green tea brew.[1] Here we report the stereoselective chemo-enzymatic synthesis of compound 1 with acetylacetoin synthase (AAS)[2] and its use to determine the stereochemistry of the natural product by chiral GC-MS analysis. Furthermore, on the basis of known rearrangements of -hydroxy--diketones[3], we propose an alternative mechanism for the formation of 3 and 4 based on thermal and/or basic degradation of 1.
143
Enzymatic Synthesis of Amoxicillin in the Presence of Ionic Liquids at Controlled Water Activity
S.C. Pereiraa, R. Bussamarab, J. Dupontb, R.L.C. Giordanoa, R.C. Giordanoa Department of Chemical Engineering, Federal University of So Carlos, Via Washington Luiz, km 235, 13565-905, So Carlos, SP, Brazil; bBiotechnology Center, Federal University of Rio Grande do Sul, Avenue Bento Gonalves, 9500, 91501-970, Porto Alegre, RS, Brazil; E-mail: roberto@ufscar.br
a
146
Kinetically controlled transesterification of glycerine catalyzed by chemically glycosylated CAL-B.
Oscar Romeroa, Melissa L.E. Gutarraa,b, Olga Abianc,d, Denise M.G. Freireb, Jose M. Guisana, Jose M. Palomoa. a Departamento de Biocatlisis. Instituto de Catlisis (CSIC), Madrid, Spain; bInstituto de Qumica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; cInstituto IIS Aragn. Unidad de Investigacin Traslacional, Zaragoza, Spain; dInstituto de Biocomputacin y Fsica de Sistemas Complejos (BIFI), Universidad de Zaragoza, Spain. oscar.romero@icp.csic.es The exponential current increase in biodiesel production also promotes a dramatic increase in the production of very low-cost glycerol. In this way, the use of this by-product as raw material for the production of commodities and fine chemicals is becoming a very exciting field of research [1]. Therefore, we study the kinetically controlled transesterification of diferents methyl and ethyl esters with glycerol catalysed by a chemically glycosylated Candida antarctica lipase (fraction B) (Cal-B). The selective modification of proteins has been a longstanding challenge of modern chemical biology with many applications, ranging from therapeutic use [2] to improve biocatalysts properties [3]. For that reason, different tailor-made oligosaccharides were investigated for a specific surface glycosylation of Cal-B previously immobilized on octyl-Sepharose by interfacial activation. The chemical modification was performed in the N-terminal aminoacid enzyme residue by using low oxidized aldehyde-dextrans via a reductive amination. SDS-PAGE indicated that polymer/enzyme conjugates were obtained in all cases. Circular dichroism experiments revealed interesting conformational changes in secondary and tertiary structures of the protein after modification. Additionally, the formed immobilized chemically glycosylated lipase biocatalysts were more stable, active and selective toward different substrates than unmodified Cal-B. The synthesis of glyceryl esters was carried out at significant water concentrations (10% v /v), neutral pH and room temperature obtaining high synthetic yields [4]. The Dext1500Cal-B biocatalyst exhibited the highest synthetic activity in the transesterification reaction between methylbutyrate and glycerol with 80% yield of monoglyceryl ester at 100% conversion (Figure 1) compared to 57% yield obtained with unmodified Cal-B or others polymer-lipase conjugates.
147
Selective Biotransformation of Phenylacetonitrile Derivatives by Brazilian Marine Fungi
Julieta R. de Oliveira (PG)a, Mirna H. R. Seleghim (PQ)b, Andr L. M. Portoa aInstituto de Qumica de So Carlos, Universidade de So Paulo, Av. Trabalhador Socarlense, 400, 13560-970, So Carlos, So Paulo, Brazil bDepartamento de Ecologia e Biologia Evolutiva, Universidade Federal de So Carlos, 235, 13565-905, So Carlos, So Paulo, Brazil julietarangel@iqsc.usp.br; almporto@iqsc.usp.br Nitriles are important intermediates in organic synthesis as well as in the chemical industry for the production of amides, carboxylic acids, and other compounds.[1,2] There is a great interest in the utilization of biocatalysts for the conversion of nitriles to their corresponding carboxylic acids and/or amides. Enzymatic hydrolysis of nitriles can proceed by nitrilases, that directly hydrolyse nitriles to the corresponding carboxylic acids, or nitrile hydratases which convert nitriles to the amides as an intermediate which under the influence of amidades can be converted into the acids.1 Studies in the literature shown that the biotransformation of nitriles by bacteria is common, and are more rare by filamentous fungi.[3] Initially, the screenings of marine fungi for biotransformation of phenylacetonitrile (1) were based on the adaptation of 12 fungal strains (genus Aspergillus, Penicillium, Cladosporium, Bionectria). The microorganisms were grown in Petri dishes containing mineral medium supplemented with glucose and phenylacetonitrile (1), as carbon and nitrogen sources, respectively. These experiments showed that the addition of phenylacetonitrile (1) promoted the induction of the cell colonies growing in agar up to 8 days. The control experiments were conducted using 12 strains, inoculating the marine fungi in the mineral medium containing inorganic salts, glucose and without the addition of phenylacetonitrile (1). In these experiments the marine fungi did not grown on the agar colonies. In order to investigate the biotransformation of phenylacetonitrile (1) by marine fungi, the experiments in liquid culture medium were investigated. All the marine fungi catalyzed the biotransformation of phenylacetonitrile (1) to 2-hidroxyphenylacetic acid (5), when these fungi were induced in mineral liquid medium. The marine fungi Aspergillus sydowii Ce19 was selected for catalyzed the biotransformation of 4-fluorophenylacetonitrile (2), o-methylbenzylcyanide (3) and m-methylbenzylcyanide (4), producing the corresponding carboxylic acids 6-8 in excellent isolated yields (> 60%). The screening of marine fungi adapted in mineral solid and liquid medium supplemented with glucose and phenylacetonitrile (1), promoted specific biotransformation of phenylacetonitrile derivatives in excellent yields.
Penicillin G acylase (PGA) from Escherichia coli (EC 3.5.1.11) is widely used for the hydrolysis of penicillin G to produce 6-aminopenicillanic acid (6-APA), which is a key intermediate in the synthesis of semi-synthetic penicillins [1], including amoxicillin, which has a broad spectrum of action against bacterial infections. Industrially, -lactam semisynthetic antibiotics are produced through chemical processes, which require drastic reaction conditions, protection/deprotection of side groups, use of organochloride solvents, and generation of non-recyclable waste. The enzymatic synthesis is a more attractive alternative from the point of view of environmental impacts, since it occurs in aqueous solution at room temperature and pH close to neutrality. However, a major obstacle to its industrial implementation is the limited yield due to side hydrolytic reactions of the precursor substrate, which could be partially avoided by reducing the activity of water (aw) in the reaction medium. One attempt to reduce the hydrolytic reactions in order to turn economically viable the enzymatic synthesis of -lactam antibiotics relies on the use of organic co-solvents. However, organic solvents are volatile, often toxic and may be harmful to the environment as well. Ionic liquids have emerged as an alternative to conventional organic media. They are salts with low melting point which form liquids entirely ionized. They are potential solvents for a large number of chemical and biochemical transformations, due to their high thermal stability, negligible vapor pressure, ability to dissolve a large number of compounds (organic, organometallic and inorganic), ability to dissolve gases, non-flammability, easy recycling and immiscibility in many organic solvents, allowing their use in multiphase systems [2]. Within this context, this work assesses the catalytic activity and selectivity of different enzymes (PGAs from E. coli) in the reaction of synthesis of amoxicillin using different ionic liquids, all based on dialkylimidazolium cations. The activity of water is measured with the aid of a hygrometer (AquaLab Series 4 TEV Decagon Devices). The tests used PGA in soluble form, and immobilized on different supports: Sepabeads resin activated with epoxy groups [3], commercial PGA supported on acrylic resin activated with epoxy or amino groups (Sprin Technologies, Italy) and IPA-750 biocatalyst (Hunan Flag Biotechnology, China).
________________ [1] Giordano RC, Ribeiro MPA, Giordano RLC (2006) Kinetics of -lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization. Biotechnol Adv 24:27-41. [2] Sheldon RA (2005) Green solvents for sustainable organic synthesis: state of the art. Green Chem 7:267-278. [3] Mateo C, Graz V, Pessela BCC, Montes T, Palomo JM, Torres R, Lpez-Gallego F, FernndezLafuente R, Guisn JM (2007) Advances in the design of new epoxy supports for enzyme immobilizationstabilization. Biochem Soc Trans 35:1593-1601.
Figure 1
________________ [1] C:H. Zhou, et. al. Chem. Soc. Rev. 37 (2009) 527549. [2] A. Diaz-Rodriguez, B.G. Davis. Curr Opin Chem Biol 15 (2010) 19 [3] C. Godoy, et. al. Process Biochem 45 (2010) 534-541. [4] A: Acosta, et.al. Bioresource Technol. 102 (2011) 507512.
_____________ [1] R. Naef, A. Jaquier, A. Velluz, B. Maurer. J. Agric. Food Chem. 2006, 54, 9201-9205 [2] P.P. Giovannini, V. Venturi, P.Pedrini, G. Fantin, A. Medici. J. Mol. Catal. B: Enzymatic, 2010, 64, 113-117. [3] M. B. Rubin, S. Inbar. J. Org. Chem. 1988, 53, 3355-3358
[1] Banerjee, A; Sharma, R; Banerjee, U. C. Appl. Microbiol. Biotechnol., 2002, 33-34. [2] Rustler, S; Stolz, A. Appl. Microbiol. Biotechnol., 2007, 899-908. [3] Kaplan, O. Et Al.; J. Ind. Microb. Biotechnol., 2006, 891-896.
October 2-6, 2011
108 148
Regioselective enzymatic acylation of aureolic acids to obtain novel analogues with improved activity
Javier Gonzlez-Sabna, Luz E. Neza, Alfredo F. Braab, Carmen Mndezb, Jos A. Salasb, Vicente Gotorc, Francisco Morsa a Entrechem S.L., Edificio Cientfico Tecnolgico, Campus El Cristo, 33006 Oviedo, Spain; bDepartamento de Biologa Funcional e Instituto Universitario de Oncologa del Principado de Asturias (I.U.O.P.A.), Universidad de Oviedo, 33006 Oviedo, Spain c Departamento de Qumica Orgnica e Inorgnica, Facultad de Qumica, Universidad de Oviedo, 33006, Oviedo, Spain E-mail: jgsabin@entrechem.com The aureolic acids, mithramycin, chromomycin A3, olivomycin, UCH9 and durhamycin A, constitute a family of polyglycosidic aromatic polyketides bearing a tryciclic core.[1] They are all antineoplastic antibiotics against Gram-positive bacteria and also stop the proliferation of tumor cells. Mithramycin (1), was approved as an anticancer drug in 1970 and used originally for the treatment of several types of cancer. Nowadays and due to ist severe toxicity, its clinical use is limited to the control of hypercalcaemia in patients with malignant diseases.[2] In recent years there are several reports of polyhydroxylated natural products where the atachment of an acyl moiety leads to analogues with improved activity, stability and pharmacokinetic properties as well as lower toxicity. Herein we report our findings in the lipase-catalyzed acylation of 1 and two derivatives, the genetically engineered mithramycin SK and mithramycin SDK.[3] Depending on the substrate, lipase and acyl donor considered the acylation took place in the aglycone moiety or in the sugar B-unit, a plethora of acylated derivatives being efficiently obtained. Most of the novel acylated derivatives showed antitumor activity at sub-micromolar level concentration, in some cases significantly improving the activity of the parent drugs.

Lipase catalyzed Michael addition reactions
a

A recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius highly enantioselective on diaryl diketone benzil
Angela Pennacchio, Mos Rossi, and Carlo A. Raia a Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, I-80131 Naples, Italy E-mail: a.pennacchio@ibp.cnr.it Alcohol dehydrogenases (ADHs) that catalyze ketone reductions are reliable source of high enantiomeric excess chiral alcohols matching and often exceeding the ability of chemical catalysts to perform the same reaction [1]. Our aim was to identify and characterize novel ADHs for the selective bioreduction of sterically demanding prochiral ketones, preferably NAD(H) dependent and endowed with good operational stability. We have focused our attention on the genome of thermophilic microorganisms containing genes coding putative ADHs belonging to the short-chain dehydrogenase/reductases (SDRs) superfamily. The gene encoding a novel dehydrogenases/reductase that belongs to the SDRs superfamily has been identified in the Sulfolobus acidocaldarius genome [2]. The saadh gene has been heterologously overexpressed in E. coli and the protein, named SaADH2, has been purified to homogeneity and characterized. The enzyme has remarkable thermophilicity and thermal stability, and shows good tolerance to common organic solvents. SaADH2 has a strict requirement for NAD(H) as the coenzyme, displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and -ketoesters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The potential applicability of SaADH2 was demonstrated by way of an efficient in situ NADH-recycling system involving a second thermophilic NAD(H)-dependent ADH. The novel archaeal enzyme catalyses the reduction of benzil to (R)-benzoin with both excellent conversion (98%) and optical purity (98%) in analytical scale at 50C and after 24 h reaction.
________________ [1] a) Kroutil W, Mang H, Edegger K, Faber K (2004) Curr Opin Chem Biol 8, 120-126; b) Goldberg K, Schroer K, Ltz S, Liese A (2007) Appl Microbiol Biotechnol 76, 237-248. [2] Chen L, Brgger K, Skovgaard M, Redder P et al. (2005) J Bacteriol 187, 4992499.
109 153
Asymmetric Reduction of alfa-Keto Esters with Thermus thermophilus NADH-Dependent Carbonyl Reductase using Glucose Dehydrogenase and Alcohol Dehydrogenase for Cofactor Regeneration
Angela Pennacchio,a Assunta Giordano,b Mos Rossi,a and Carlo A. Raiaa a Istituto di Biochimica delle Proteine, CNR, Via P. Castellino 111, I-80131 Naples, Italy b Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, I-80078 Pozzuoli, Italy E-mail: ca.raia@ibp.cnr.it Optically active hydroxy esters provide very versatile building blocks widely used as chiral intermediates for the synthesis of pharmaceuticals and other fine chemicals. Among them, methyl (R)-mandelate 1a and methyl (R)-o-chloromandelate 2a are valuable synthons used in organic synthesis (Scheme). O-protected 1a is used as an intermediate for the synthesis of pharmaceuticals [1] and 2a is an intermediate for the anti-thrombotic agent, (S)-clopidogrel, commercialized under the brand name Plavix (clopidogrel sulphate) [2]. The enantioselective bioreduction of methyl benzoylformate 1 and methyl o-chlorobenzoylformate 2 was investigated using a NADH-dependent carbonyl reductase from Thermus thermophilus (TtADH) and, separately, Thermoplasma acidophilum glucose dehydrogenase (TaGDH) and Bacillus stearothermophilus alcohol dehydrogenase (BsADH) for NADH regeneration. The optimal reaction time and substrate concentration in the absence and presence of organic solvents were determined. The enantiofacial selectivity of TtADH was shown to be inversely correlated to the hydrophobicity of the short-chained linear alcohols employed as co-substrates of the bacillar ADH.
Jaime Escalante-Garcaa, Edmundo Castillob, Jos Domingo Riveraa Centro de Investigaciones Qumicas. Universidad Autnoma del Estado de Morelos. Av. Universidad 1001, Cuernavaca, Mor. MEXICO, 62210. bInstituto de Biotecnologa, Universidad Nacional Autnoma de Mxico. Av. Universidad 2001, Cuernavaca, Mor. MEXICO, 62210. aghomes315@hotmail.com In the last years, the synthesis of enantiopure -amino acids has generated interest due to their potential as building blocks in the obtention of bioactive compounds.[1,2] Most of the chemo-enzymatic paths for obtaining enantiopure -amino acids are based on the kinetic resolution of racemic -amino acids or some of their derivates.[3] As an alternative the development of a strategy allowing the synthesis and resolution of these compounds in one-pot reactions would be interesting. Recently we reported a solvent engineering strategy for controlling the chemoselectivity of an aza-Michael type addition reaction.[4] Accordingly, in non polar solvents the Michael addition reaction was favored, while in polar solvents amide formation was preferred. In order to synthesize enantiopure -amino acids the aza-Michael type addition of benzylamine (2) to ,-unsaturated esters (1) catalyzed by Candida antarctica Lipase B is reported. It was found that excepting Eq. 3, in all studied systems aza-Michael addition products were obtained (Eq. 1, 2, 4 ,5). In some cases a second aza-Michael addition product was observed (Eq. 2, 5b). In agreement to our previous report, in non polar solvents such as n-hexane the aza-Michael products were preferentially formed. In contrast, in reactions carried out in 2-methyl-2-butanol the amide products were the main products obtained. This results confirmed that the chemoselectivity of this kind of process is strong dependent on the polarity of reaction media.
Scheme 1. Enzymatic acylation of the aureolic acid mithramycin (1).

________________ [1] Lomb, F., Menndez, N., Salas, J. A., Mndez, C., Appl. Microbiol. Biotechnol. 2006, 73, 1-14. [2] a) Remers, W. A., The Chemistry of Antitumor Antibiotics, Wiley-Interscience, New York, 1979, 1, 133-175; b) Skarbek, J. D., Speedie, M. K., Antitumor Compounds of Natural Origin, CRC Press, Boca Raton, Florida, 1981, 1, 191-235. [1] Remsing, L. L., Gonzlez, A. M., Nur-e-Alam, M., Fernndez-Lozano, M., Braa, A. F., Rix, U., Oliveira, M. A., Mndez, C., Salas, J. A., Rohr, J., J. Am. Chem. Soc. 2003, 125, 5745-5753.
[1]. a) Wei, M; Grger, H. Letters 2009, 8, 1251-1254; b) Flores. P.; Escalante, J.; Castillo, E. Tetraedron: Asymmetry 2005, 16, 629-634; c) Juaristi, E.; Escalante, J.; Lamatsch, B.; Seebach, S. J. Org. Chem. 1992, 57, 2396-2398. [2] E. Juaristi, E.; Soloshonok, V. Enantioselective Synthesis of -aminoacids; 2nd Ed. Wiley-VCH: New York, 2005. [3]. Liljeblad, A.; Kanerva, L. Tetrahedron, 2006, 62, 5831-585. [4]. Priego, J.; Ortiz, C.; Carrillo, M.; Lpez-Mungua, A.; Escalante, J.; Castillo, E. Tetraedron 2009, 65, 536-539.
TaGDH and BsADH regeneration modes gave methyl (R)-o-chloromandelate with good isolated yield and 92-95% ee.
________________ [1] Y. Kobayashi, Y. Takemoto, Y. Ito, S. Terashima, Tetrahedron Letters 1990, 31, 3031; C. Pousset, M. Haddad, M. Larchevque, Tetrahedron 2001, 57, 7163. [2] A. Bousquet, A. Musolino, PCT. Int. Appl., WO9918110 A1, Chem. Abs., 1999, 130, 296510e.
150
Asymmetric Synthesis of - and -Amino Acid Derivatives using EneReductases
Mlanie Halla, C. K. Winklera, C. Stuecklera, Kurt Fabera a Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria E-mail: melanie.hall@uni-graz.at The enzyme-catalyzed production of enantiopure -amino acid derivatives currently relies on kinetic resolutions based on the cleavage of amide- or ester bonds using proteases or lipases/esterases, while enzymatic asymmetric synthetic approaches are rare [1]. Various ene-reductases from the Old Yellow Enzyme family [2] were found to catalyze the reduction of ,-dehydroamino acid derivatives. Whereas N-acylamino substituents were tolerated in the -position, -analogs were generally unreactive. Optically enriched N-acyl derivatives of alanine and aspartic acid esters were obtained in high enantiopurity (97% and up to >99% ee, resp.). For aspartic acid derivatives, the stereochemical outcome of the bioreduction using OYE3 could be switched by variation of the size of the N-acyl protective group to furnish the corresponding (S)- or (R)-amino acid derivatives. This was explained by a change in the substrate binding as proven by 2H-labelling experiments [3]. The bioreduction to (R)-amino acid thus comprises a route equivalent to the production of -amino acid derivatives and therefore opens new perspectives for the enzymatic asymmetric synthesis of -amino acids.
151
Nicotinamide-Independent Asymmetric Bioreduction of C=C Bonds via Disproportionation of Enones Catalyzed by Ene-Reductases
Christoph K. Winkler,b Gbor Tasndi,a Dorina Clay,b Mlanie Hall,b Kurt Faberb a ACIB GmbH c/o bDepartment of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria; E-mail: christoph.winkler@edu.uni-graz.at According to early reports, ene-reductases from the old yellow enzyme (OYE) family do not only catalyze the trans-specific asymmetric reduction of activated C=C bonds [1], but are also able to form equimolar amounts of cyclohexanone and phenol from two molecules of cyclohex-2-enone in a disproportionation (dismutation) reaction [2].
154
Organic phase biotransformations using a lipase-immobilized silica monolith bioreactor
Koei Kawakami, Ryo Takahashi, Yasuhiro Oda, Masaki Ueno Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan E-mail: kawakami@chem-eng.kyushu-u.ac.jp A Burkholderia cepacia lipase (BCL)-immobilized butyl-substituted silica monolith was successfully applied to the following two biotransformation systems: 1) Kinetic resolution of racemates in the transesterification of (R, S)-1-phenylethanol with vinyl acetate, and 2) Biodiesel production through methanolysis of crude Jatropha oil. In both the systems, the enzymatic activities of BCL were highly enhanced by immobilization into the hydrophobic silicates. Kinetic resolution of racemates in the transesterification of (R, S)-1-phenylethanol with vinyl acetate The effect of water content was first investigated in a batch reactor. The optimal water activity was 0.11 giving the highest activity and high enantioselectivity of 240. The continuous kinetic resolution was successfully performed in a lipase-immobilized silica monolith micro-bioreactor with the inside diameter of 0.25-1.6 mm. The steady state conversion of 40% and the enantiomeric excess more than 98% were maintained over the time period of 100h (Fig.1). Biodiesel production through methanolysis of Jatropha oil The oil used contained 18% free fatty acid, which caused a favorable miscibility between the oil and methanol. The biodiesel yield of 90% was reached in 12h of the batch reaction under the condition of a stoichiometric mixture of methanol and oil (3:1) and water content of 0.6% (w/w) at the temperature of 40C. The continuous production of biodiesel was also performed using a lipase-immobilized silica monolith bioreactor. The steady state yield of 95% was attained at the low flow rate of 0.6 ml h-1 (2.79 g-lipase h ml-1) and 80% of the initial yield was retained even after the continuous operation for almost 50 days (Fig.2).
155
Stereochemical preference in Candida parapsilosis ATCC 7330 mediated Deracemization: (E) vs. (Z)-Alkyl-2-hydroxy-4-arylbut-3-enoate
Thangavel Saravanana, Anju Chadhaa,b a Laboratory of Bioorganic Chemistry, Department of Biotechnology and bNational Center for Catalysis Research, Indian Institute of Technology Madras, Chennai 600 036, India. E-mail: tsaravananiitm@gmail.com; anjuc@iitm.ac.in Biocatalytic deracemization is an efficient method to synthesize biologically important optically pure secondary alcohols.[1] Among the strategies for deracemization, onepot stereoinversion i.e. oxidation followed by reduction, using microbial whole cells is a good alternative to the usual enzymatic resolution, as it allows for transformation of racemates into a single enantiomerically pure product with 100% yield and cofactor regeneration.[2] Earlier reports from our lab showed that Candida parapsilosis ATCC 7330 mediated deracemization of racemic (E)-alkyl 2-hydroxy-4-arylbut-3-enoates gave optically pure S enantiomers with high ee (up to 99%) and isolated yields (up to 85%) via stereoinversion.3 The present study addresses the important question of stereoselectivity in C. parapsilosis ATCC 7330 mediated deracemization with respect to cis/trans configuration in (Z) vs. (E) alkyl 2-hydroxy-4-arylbut-3-enoates. Initial results show that the substrate, (Z)-ethyl-2-hydroxy-4-phenylbut-3-enoate did not get deracemized to the optically pure product even after 12h (ee = 0%) unlike the (E) isomer reported earlier. [3] Interestingly, alkyl 2-hydroxy-4-arylbut-3-ynoate intermediates which are precursors for (Z) alkyl 2-hydroxy-4-arylbut-3-enoates were deracemized in excellent enantiomeric excess (up to >99%) and good isolated yields (up to 78%) by C. parapsilosis ATCC 7330. [4] Results of the studies carried out for deracemization of cis and trans alkyl-2-hydroxy4-arylbut-3-enoates will be presented.
Figure 1. Ene-Reductase Catalysed Disproportionation of Cyclohex-2-enone. Based on this side-reaction, we recently developed a coupled-substrate-protocol for the C=C-bond bioreduction via direct hydrogen-transfer from a sacrificial hydrogen donor, which neither requires a second recycling enzyme nor a nicotinamide cofactor [3]. Although this study demonstrated the proof-of-principle, it was impeded by incomplete conversions, which were attributed to enzyme inhibition caused by the phenolic by-product, which forms a charge-transfer complex with the flavin cofactor [2].
Figure 2. Nicotinamide-Independent C=C Bond Reduction. In order to drive the bioreduction to completion, we developed strategies to overcome coproduct inhibition by scavenging the inhibiting phenol as well as engineering of the corresponding co-substrate. Acknowledgements: Financial support by BASF SE, the FFG and the FWF (P22722) is gratefully acknowledged.
Figure 1. Two distinct substrate binding modes leading to opposite enantiomers with OYE3. BASF and the Austrian Science Fund (FWF) [P22722] are thanked for financial support.
________________ [1] J. Rehdorf, M. D. Mihovilovic, U. T. Bornscheuer, Angew. Chem. Int. Ed., 2010, 49, 4506; M. A. Swiderska, J. D. Stewart, Org. Lett., 2008, 8, 6131; A. Liljeblad, L. T. Kanerva, Tetrahedron 2006, 62, 5831. [2] M. Hall, Y. Yanto, A. S. Bommarius, in The Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, John Wiley & Sons, 2010, 2234. [3] C. Stueckler, C. K. Winkler, M. Hall, B. Hauer, M. Bonnekessel, K. Zangger, K. Faber, Adv. Synth. Catal., 2011, 353, 1169.
________________ [1] M. Hall, Y. Yanto, A. S. Bommarius, in: The Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, Flickinger, M., Ed. Wiley: 2010; pp 2234; R. Stuermer, B. Hauer, M. Hall, K. Faber Curr. Opin. Chem. Biol. 2007, 11, 203. [2] A. D. N. Vaz, S. Chakraborty, V. Massey Biochemistry. 1995, 34, 4246; J. Buckman, S. M. Miller Biochemistry, 1998, 37, 14326; P. A. Karplus, K. M. Fox, V. Massey FASEB J., 1995, 9, 1518. A. S. Abramovitz, V. Massey J. Biol. Chem., 1976, 251, 5327. [3] C. Stueckler, T. C. Reiter, N. Baudendistel, K. Faber Tetrahedron, 2010, 66, 663.
Fig.1 Operational stability of the lipase-immobilized silica monolith micro-bioreactor in the continuous transesterification of (R, S)-1-phenylethanol with vinyl acetate. The substrate solution containing 20 mM (R, S)-1-phenylethanol and 0.4 M vinyl acetate in isooctane was continuously fed at a fixed flow rate of 7.2 mlh-1 to the micro-bioreactor (1.6 mm x 15 cm) loaded with 81.3 mg silica monolith containing 26.1 mg lipase.
Fig. 2 Operational stability of the lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel. The substrate solution comprised of methanol and oil at 3:1 molar ratio and 0.6% (w/w) water was fed continuously at a constant flow rate of 0.06 mlh-1 to the bioreactor (10 mm x 10 cm) loaded with 1.87 g silica monolith containing 0.63 g lipase.
_______________ [1] Straathof, A. J. J.; Panke, S.; Schmid, A. Curr. Opin. Biotechnol 2002, 13, 548-556. [2] Voss, C. V.; Gruber, C. C.; Kroutil, W. Angew. Chem. Int. Ed. Engl. 2008, 47, 741- 725. [3] Baskar, B.; Pandian, N. G.; Priya, K.; Chadha, A. Tetrahedron, 2005, 61, 12296-12306. [4] Saravanan, T; Chadha, A. Tetrahedron: Asymmetry, 2010, 21, 2973-2980.
October 2-6, 2011
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Stereoselective chemoenzymatic synthesis of cyclic -aminophosphonates through lipase-catalyzed processes
Mara Rodrguez-Mataa, Vicente Gotor-Fernndeza, Alicia Arizpeb, Francisco J. Sayagob, Carlos Cativielab,*, Vicente Gotora,* a Departamento de Qumica Orgnica e Inorgnica, Instituto Universitario de Biotecnologa de Asturias, Universidad de Oviedo, C/ Julin Clavera, 8, 33006, Oviedo, Spain; b Departamento de Qumica Orgnica, Instituto de Ciencia de Materiales de Aragn, Universidad de Zaragoza-CSIC, C/ Pedro Cerbuna, 12, 50009, Zaragoza, Spain. E-mail: rodriguezmmaria@uniovi.es -Aminophosphonic acids and their corresponding -aminophosphonates are the phosphorous analogues of both natural and unnatural -aminocarboxylic acids. Nowadays they are receiving much interest in organic and medicinal chemistry because of their presence in many peptides, which posses remarkable biological profiles such as enzyme inhibitors, antitumor or antibacterial agents.[1] In addition, the incorporation of cyclic amino acids of medium ring size into key positions in peptide chains plays an important synthetic role to conformationally constrained peptidomimetics, a tool in modern drug discovery. In view of their different biological and chemical applications, development of suitable stereoselective synthetic methodologies for -aminophosphonates in optically pure form constitutes a topic of great interest.[1] In this context, the combination of chemical and enzymatic methods allows the selective modification of polyfunctional compounds under mild reaction conditions.[2] Recently, we have focused our efforts towards the asymmetric synthesis of cyclic -aminophosphonic acid derivatives with different ring sizes through a chemoenzymatic approach (Scheme 1). To achieve this objective, their preparation was carried out in a five-step route starting from the corresponding lactams. Once the racemic compounds were obtained, their enzymatic kinetic resolutions were attempted employing commercially available lipases and different carbonates. This path can be considered as an alternative method for their previously and unique recently described synthesis.[3]

Lipase-catalyzed transesterification of krill oil and 3,4-dihydroxyphenyl acetic acid in solvent-free medium using response surface methodology
Sarya Aziza, Pierrre Dutilleulb, Richard St-Louisc, Marya Aziza, Selim Kermashaa a Department of Food Science and Agricultural Chemistry, McGill University, 21,111 Lakeshore, Ste-Anne de Bellevue, Qc, Canada H9X 3V9; bDepartment of Plant Science, , McGill University, 21,111 Lakeshore, Ste-Anne de Bellevue, Qc, Canada H9X 3V9; cInstitut des Sciences de la Mer de Rimouski, UQAR, 310 alle des Ursulines, Rimouski, Qubec, Canada G5L 3A1. E-mail: selim.kermasha@mcgill.ca The enzymatic transesterification in solvent-free medium of krill oil with selected phenolic acids (PAs), using two immobilized lipases, was investigated. The use of Novozym 435 with 3,4-dihydroxyphenylacetic acid resulted in the highest bioconversion yield (BY). The central composite rotatable design was used to evaluate the effects of the concentrations of PA and lipase as well as the agitation speed (AS) on the BY of phenolic lipids. The initial results indicated that the stationary point was a saddle point. To overcome this saddle point, sequential experiments have been carried out at fixed PA concentrations, with lipase concentration and AS as the two independent variables. For the models with PA, fixed at 10 and 20 mM, the results revealed that the lipase concentration had a quadratic significant effect on the BY, whereas a linear significant effect was only obtained at PA concentration fixed at 20 mM. Only for the model with PA, fixed at 10 mM, the AS had a significant quadratic effect on the BY. At fixed PA concentration of 20 mM, the response surface model predicts a BY of 75%, using lipase concentration (62 mg/mL) and AS (154 rpm). The subsequent verification experiment under these conditions confirmed the validity of the prediction. Keywords: Krill oil, Phenolic lipids, Lipase, Response surface methodology, Solvent-free media, Bioconversion yield.

Immobilization of E.coli containing a recombinant -aminotransferase for production of chiral amines
Gustav Rehn1, Carl Grey1, Watson Neto1, Cecilia Branneby2, Ulf Sjstrand2, Patrick Adlercreutz1 1 Department of Biotechnology, Center for Chemistry and Chemical engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden 2 Cambrex Karlskoga AB, Bjrkborns Industriomrde, SE-691 85 Karlskoga, Sweden Email: gustav.rehn@biotek.lu.se Aminotransferases (transaminases) can be used to produce chiral amines, which are valuable products and intermediates in the pharmaceutical industry. In order to achieve good process economy, immobilization to facilitate reuse of the biocatalyst may be necessary. Purified -transaminases have previously been immobilized covalently on different support materials [1] and by encapsulation in a sol-gel matrix [2], whereas whole-cells expressing transaminases have been immobilized by entrapment in Ca-alginate beads [3, 4]. The objective of a present study is to characterize and compare several alternative methods for immobilization of E.coli cells containing a recombinant -transaminase to be used for production of chiral amines. Cells were immobilized using principally different techniques including gel entrapment in Ca-alginate, carrageenan or polyacrylamide and surface immobilization on hydrous titanium oxide. The stability of the -transaminase was much improved by adding the cofactor pyridoxal5-phosphate (PLP), as previously reported for the -transaminase from V.fluvialis [5]. However, the concentration of PLP was drastically reduced in the presence of some substrates, such as substituted tetralons. The disappearance of PLP may cause a decrease of the stability of the biocatalyst. This effect is further investigated in an ongoing study.
__________________________ [1] Yi, S.S., et al., Covalent immobilization of w-transaminase from Vibrio fluvialis JS17 on chitosan beads. Process Biochemistry, 2007. 42(5): p. 895-898. [2] Koszelewski, D., et al., Immobilization of -transaminases by encapsulation in a sol-gel/celite matrix. Journal of Molecular Catalysis B: Enzymatic, 2009. 63(1-2): p. 39-44. [3] Martin, A.R., et al., Characterization of free and immobilized (S)-aminotransferase for acetophenone production. Applied Microbiology and Biotechnology, 2007. 76(4): p. 843-851. [4] Shin, J.S., B.G. Kim, and D.H. Shin, Kinetic resolution of chiral amines using packed-bed reactor. Enzyme and Microbial Technology, 2001. 29(4-5): p. 232-239. [5] Shin, J.S., et al., Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17. Applied Microbiology and Biotechnology, 2003. 61(5): p. 463471.
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Immobilization of Oleic Acid Hydratase
Aida Hiseni, Rosario Franco Berriel, Linda G. Otten, Isabel W.C. E. Arends Delft University of Technology, Department of Biotechnology, Biocatalysis and Organic Chemistry Group, Julianalaan 136, 2628 BL Delft, The Netherlands E-mail: I.W.C.E.Arends@TUDelft.nl Hydroxy fatty acids have specific physical and chemical properties i.e. high viscosity and reactivity, which make them suitable for the production of a number of products, including resins, nylons, plastics, waxes, cosmetics and coatings. They can be synthesized by hydrolyzing the unsaturated moiety of the corresponding unsaturated fatty acid. Oleic acid is an unsaturated fatty acid (C18:1), and is one of the cheapest and most abundant biological raw material. The hydration of oleic acid to produce 10-hydroxystearic acid has been studied for many years, but only recently Bevers et al. [1] were able to recombinantly express and characterize oleate hydratase (OHase) from Elizabethkingia meningoseptica (formerly known as Pseudomonas sp. 3266). This hydratase represents a new type of hydro-lyases, which is able to hydrate an isolated, non-activated carbon-carbon double bond (Fig. 1). From an industrial point of view it would be thus interesting to investigate the possibility of making this enzyme suitable for industrial processes in order to make high value products out of two cheap liquid phases (eg. oleic acid + water). Therefore, we have immobilized OHase as Cross-Linked Enzyme Aggregates [2] and characterized its properties such as thermal stability, pH activity and recycling of the biocatalyst. Subsequently the obtained results were compared with those obtained for OHase in cell-free extracts and whole cells.
Scheme 1. Synthetic route to chiral -aminophosphonates via lipase-catalyzed alkoxycarbonylation reactions.

________________ [1] M. Ordez, H. Rojas-Cabrera, C. Cativiela, Tetrahedron 2009, 65, 17-49. [2] (a) W.-D. Fessner, T. Anthonsen, Modern Biocatalysis. Stereoselective and Environmentally Friendly Reactions; Wiley-VCH: Weinheim, Germany, 2009; (b) Hudlicky, T.; Reed, J. W. Chem. Soc. Rev. 2009, 38, 3117-3132; (c) V. Gotor-Fernndez, V. Gotor, Curr. Opin. Drug Discov. Dev. 2009, 12, 784-797. [3] F. Wuggenig, A. Schweifer, K. Mereiter, F. Hammerschmidt, Eur. J. Org. Chem. 2011, 1870-1879.
Figure 1. Reaction catalyzed by OHase: the conversion of oleic acid into 10-hydroxystearic acid.
________________ [1] L. E. Bevers, M. W. H. Pinkse, P. Verhaert and W. R. Hagen. Oleate Hydratase Catalyzes the Hydration of a Nonactivated Carbon-Carbon Bond. Journal of Bacteriology. (15) 191 (2009), 5010-5012. [2] R. A. Sheldon. Cross-linked enzyme aggregates (CLEA (R) s): stable and recyclable biocatalysts. Biochemical Society Transactions. 35 (2007), 1583-1587.
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Exploiting regio-selectivity of lipase catalysis for obtaining high yields of six-membered cyclic carbonates from diols and dimethylcarbonate
Sang-Hyun Pyo and Rajni Hatti-Kaul Department of Biotechnology, Lund University, Box 124, SE-221 00 Lund, Sweden E-mail: Sang-Hyun.Pyo@biotek.lu.se Cyclic carbonates are potential monomers for isocyanate free polyurethanes and polycarbonates. Six-membered cyclic carbonates were prepared by reaction between diols with dimethylcarbonate (DMC) mediated by Novozym435, and thermal cyclization in a solvent-free medium. The highest yield of cyclic carbonate obtained was 99.3% in the reaction with 3-methyl-1,3-butanediol having a primary- and a tertiary alcohol groups, while reactions with 1,3-propanediol having two primary alcohols and 1,3-butanediol having a primary and secondary alcohols resulted in corresponding cyclic carbonate yields of 43.2% and 85.5%, respectively. Novozym435 and DMC were recovered and reused in the biocatalytic reaction with high reproducibility. This process provides a novel and more environmentally friendly approach for synthesis of cyclic carbonates without using toxic organic solvents, phosgene and isocyanate.
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Immobilized Solanum tuberosum epoxyde hydrolase. Application for the resolution and the "deracemization" of racemic epoxides in a repeatedbatch reactor.
Vaidya Ba., Archelas A.a, Mateo C.b, Fernandez-Lafuente R.b, Guisan J.M.b a ISM2-UMR6263 Biosciences, case 432, Facult Sciences et Techniques de St Jrome, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France. bDepartamento de Biocatalisis, Instituto de Catalisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain E-mail: a.archelas@univ-cezanne.fr Epoxide Hydrolases (EHs) are very efficient biocatalysts that are able to catalyse enantioand regioselective hydrolysis of epoxides.[1] This versatility makes them very useful to prepare enantiopure epoxides an/or their corresponding vicinal diols that are highly valuable chiral synthons used in numerous syntheses of biologically actives molecules. These last years biocatalylic processes with high efficiencies (high substrate concentration, high TON and TOF) implying Epoxide Hydrolases (EH) have been described in the literature but generally these processes did not preserve the enzyme activity.[2] A solution to this inconvenience can be the enzyme stabilization by immobilization allowing an effective recycling of the biocatalyst. In this context, we have recently carried out the covalent immobilization of the recombinant Solanum tuberosum EH (StEH) using a Glyoxyl-agarose support allowing us to obtain an immobilizate exhibiting excellent thermal and chemical stability.[3] These results clearly paved the way for the efficient use of this novel biocatalyst for carrying out either the biocatalyzed hydrolytic resolution of an epoxide or the enantioconvergent preparation of the corresponding diol by running a repeated-batch reactor. To illustrate the synthetic potentialities of such a reactor two preparative processes will be presented:
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Endo-1,4--galactanase from Emericella nidulans catalyzes hydrolysis of potato galactan and rhamnogalacturonan I to potential prebiotics
Malwina Michalaka, Lise V. Thomassena, Henna Roytiob, Arthur C. Ouwehandb, Anne S. Meyera, Jrn D. Mikkelsena a Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark b Health and Nutrition, Danisco Finland, Sokeritehtaantie 20, 02460 Kantvik, Finland E-mail: mmi@kt.dtu.dk Prebiotics are non-digestable food ingredients that beneficially affect the host by selective stimulation of the growth of a limited number of bacteria in the colon. Potato pulp is a side-stream from industrial potato starch manufacturing. Potato pulp is particularly rich in pectin, notably galactan branched rhamnogalacturonan I polysaccharides, which are highly bifidogenic when solubilized[1]. Galactan is also known for its prebiotic activity[2]. The objective of this study was to use these potato polysaccharides for targeted enzyme catalysis to provide new opportunities for designing functional food with significant health benefits. A novel enzyme, endo-1,4--galactanase from Emericella nidulans was produced by fermentation in a recombinant Pichia pastoris strain. The enzyme was purified by affinity column chromatography and the optimal biocatalysis of designed prebiotic oligosaccharides was determined by a statistical experimental design. The E. nidulans enzyme was very active on both potato galactan and the more complex structure of rhamnogalacturonan I. The enzyme generated a broad spectrum of hydrolysis products, i.e. oligo- and polysaccharides, which were further fractionated by membrane filtration. Prebiotic properties of each fraction were evaluated by growth of beneficial gut microbes and pathogenic bacteria. The galactan derived products with a molecular weight less than 3 kDa and rhamnogalacturonan I with molecular weight less than10 kDa promoted growth of probiotic strains of Bifidobacterium longum and Lactobacillus acidophilus and suppressed the propagation of Clostridium perfringens. Growth of B. longum was almost 4 times higher on galactan and rhamnogalacturonan I derived products than on fructooligosaccharides. In contrast, the growth rate of C. perfringens on these samples was lower than that on fructooligosaccharides.
________________ [1] Thomassen, L.V., Vigsns, L.K., Licht, T.R., Mikkelsen, J.D., Meyer, A.S., 2011. Appl. Microbiol. Biotechnol. 90, 873- 884 [2] Rastall, B., Gibson, G., 2006 Prebiotics. J. Wiley & Sons, Inc
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A Chemoenzymatic Synthesis of Deoxy Sugar Esters Involving Stereoselective Acetylation of Hemiacetals Catalyzed by CALB
Ly Villoa, Malle Kreena, Marina Kudryashovaa, Andrus Metsalaa, Sven Tampa, lo Lillea, Tnis Pehkb, Omar Parvea a Department of Chemistry, Tallinn University of Technology, Akadeemia tee 15, 12618 Tallinn, Estonia; bDepartment of Chemical Physics, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618 Tallinn, Estonia e-mail: lee@chemnet.ee The objective of the work was to extend the chemoenzymatic strategy of the synthesis of stereochemically pure pyranose deoxy sugar (DOS) esters [1] also to the synthesis of furanose DOS derivatives [2] (Scheme 1). Carboxylic acids as deoxycholic acid (DCA), -methoxyphenylacetic acid (MPA), N-Boc-L-phenylalanine (BOC-Phe) and N-Boc-Ltyrosine (BOC-Tyr) were used. Racemic 2-bromo-4-ethanoyloxy-butanal (1) was used as DOS precursor [3] to generate furanose DOS esters of DCA, MPA, BOC-Phe and BOCTyr. For the latter two carboxylic acids also 2-bromo-5-ethanoyloxy-pentanal (2) was used to afford corresponding pyranose DOS esters. In all cases the lipase-catalyzed stereoselective acetylation of furanose or pyranose hemiacetal moiety as a key step afforded one desired stereochemically pure acetylated hemiacetal DOS ester of trans-(2R) geometry in high de.
Resolution of 100 g of styrene oxide at high substrate concentration (50 to 100 g/L) Figure 1. Possible synthesis pathway of carbonates from diols and dimethylcarbonates.
Scheme 1. The strategy for the synthesis of deoxy sugar esters

_______________ [1] Villo, L.; Danilas, K.; Metsala, A.; Kreen, M.; Vallikivi, I.; Vija, S.; Pehk, T.; Saso, L.; Parve, O. J.Org. Chem. 2007, 72, 5813-5816. [2] Villo, L.; Kreen, M.; Kudryashova, M.; Metsala, A.; Tamp, S.; Lille, .; Pehk, T.; Parve, O. J. Mol. Cat.B: Enzym., 2011, 68, 44-51. [3] Villo, L.; Metsala, A.; Parve, O.; Pehk, T. Tetrahedron Lett. 2002, 43, 3203-3207.
Acknowledgements: The work was supported by the Swedish Foundation for Strategic Environmental Research (Mistra) and Perstorp AB.
"Deracemisation" of 14g of meta-chlorostyrene oxide at 20g/L

[1] W. J. Choi Appl. Microbiol. Biotechnol. 2009, 84, 237-239. [2] J. Deregnecourt, A. Archelas, F. Barbirato, J.M. Paris, R. Furstoss Adv. Synth. Catal. 2007, 349, 14051417 [3] C. Mateo, R. Fernandez-Lafuente, A. Archelas, J.M. Guisan, R. Furstoss Tetrahedron: Asymmetry 2007, 18, 1233-1238.
October 2-6, 2011
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Enantiomerically enriched forms of aromatic halohydrins by whole-cell biocatalyst-mediated asymmetric reduction
Shohei Taketomi, Masayoshi Asano, Toshinori Higashi, Mitsuru Shoji, Takeshi Sugai Department of Pharmaceutical Science, Keio University, Tokyo 105-8512, Japan; E-mail: sugai-tk@pha.keio.ac.jp To elaborate chemo-enzymatic routes to enantiomerically enriched forms of aromatic halohydrins (1), microorganisms were screened which reduce -chloroacetophenones 2a and 2b, with high enantioselectivity. Among them, cultured whole-cell biocatalyst of the Scheme 1. W. californica-catalyzed yeast Williopsis californica (JCM 3600) reduction of 2. was found, and the reduction proceeded in a highly si-enantiofacially selective manner by applying glycerol to enhance co-factor regeneration to give 1a in 71% yield with 96.0% ee and 1b in 80% yield with 98.4% ee, respectively. For the comparison of halogen atoms with different size, electronegativity, and leaving ability (Cl, Br) attached to -position of carbonyl group, dual substrate screening was attempted by incubating simultaneously two substrates 2b and 2c, which were derived from commercially available and inexpensive 3. Reduction of -chloroketone 2b was much Table 1. Dual substrate screening faster than that of 2c. Diverse enantiofacial with 2b + 2c. selectivities were shown among tested microorganisms (Table 1). To provide two free hydroxy groups which are involved in 2a, and are also requisite to MOM-protected 2b, Candida kefyr (JCM 21874) was found by the screening of microorganisms with diacetate 2d as the substrate. This hydrolysis proceeded very smoothly (95%) under mild conditions, without any damage on -chloroketones. The hyrolysis was compatible with that by applying isolated Scheme 2. C. kefyr-catalyzed deprotection under mild conditions. enzyme preparations, such as C. antarctica and B. cepacia lipases.

Effect of saponins from Agave sisalana and Ziziphus joazeiro on digestive enzymes
Bernardo Dias Ribeiro, Raisa de Souza Santos, Yang de Almeida Vegele Sousa, Maria Alice Zarur Coelho School of Chemistry, Biochemical Engineering Department, Federal University of Rio de Janeiro, Brazil. E-mail: dias.bernardo@gmail.com Saponins are triterpenic or steroidal glycosides largely distributed in nature and have important physicochemical (foaming, emulsification, solubilization, sweetening, bitterness) and biological (hemolytic, antimicrobial, molluscacide, insecticide) properties, which are being explored in many applications in food, cosmetic and pharmaceutical industries. The use of saponins in food has been limited by the possibility of inhibition of enzymes from digestive tract, causing a poor nutrient absorption; however this fact is being reconsidered due to health benefits of saponins such as hypocholesterolemic and anticancer properties[1,2]. The aim of this work is evaluating effects of saponins from sisal mucilaginous waste (Agave sisalana) and from ju bark (Ziziphus joazeiro) on activity of some digestive enzymes as lipase, protease and amylase from porcine pancreas. The saponins were obtained by methanolic (50% v/v) extraction of raw materials previously defatted, with partition to butanol and washing with aqueous NaOH solution (1% w/v), and finally, lyophilized. Thus, the saponins content was 0.92% in sisal mucilaginous waste and 3.88% in jua bark. All enzyme activities were determined spectrophotometrically. Lipase activity used p-nitrophenyl laurate (NPL) as substrate in concentrations ranging from 0.1 to 1.0 mM, while protease activity utilized azocasein (0.025 0.25% w/v). For amylase activity starch was used as substrate (0.1 to 1.0% w/v) and reducing sugars formed were measured with 3,5-dinitrosalicylic acid (DNS). Saponins concentration employed were 0.001, 0.005 and 0.01% w/v. Some results were shown in Figures A and B, related to ju and sisal saponins, respectively. These profiles varied with substrate concentration, enzyme and saponin types, for example, with 1.0 mM NPL in the presence of 0.01% ju saponin, lipase activity was inhibited in 50%, while in the presence of sisal saponin this activity was increased in 356%. Then, application of ju and sisal saponins in food industry is limited due to partial inhibition of lipase and amylase activities.

Kinetic resolution of 3-hydroxycyclohexanone using different lipases
Sanjib Kumar Karmee, Remco van Oosten, Ulf Hanefeld Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, The Netherlands. E-mail: S.K.Karmee@tudelft.nl 25-hydroxy-19-norvitamin D3 analogs are known for their antiproliferative activities on prostate cells. Along this line, optically enriched 3-hydroxycyclohexanone is used as a precursor for the synthesis of 25-hydroxy-19-norvitamin D3 analogs. However, the synthesis of optically enriched 3-hydroxycyclohexanone is laborious and requires multistep chemical synthesis. For instance, staring from (-)-quinic acid, Arai et al. have reported a seven step synthesis of (S)-3-hydroxycyclohexanone [1]. The main objective of this project is to develop a concise method for the synthesis of optically enriched 3-hydroxycyclohexanone.
113 169
Effects of stabilizing additives on the activity of alpha-chymotrypsin in organic solvent
a
Gloria Parinia,b, Gabriella Rodab, Gabriel L. Barlettac, Francesco Secundoa Istituto di Chimica del Riconoscimento Molecolare, CNR,v. Mario Bianco 9, Milan, Italy; b Dipartimento di Scienze Farmaceutiche "Pietro Pratesi", Universit degli Studi di Milano, Via Mangiagalli 25, 20133 Milan, Italy; cDepartment of Chemistry, University of Puerto Rico at Humacao,CUH Station, Humacao, Puerto Rico. E-mail: francesco.secundo@icrm.cnr.it The preparation of the biocatalyst is often a crucial step for successful biocatalytic applications at industrial level, especially if enzymes are used in organic solvents, where they have lower catalytic activity than in water. In particular, to improve the catalytic activity of enzymes in organic solvents, the use of lyoprotectants, that is, additives that preserve the enzyme functionality after the dehydration or lyophilization process, allows the preparation of hydrolase formulations that show an activity comparable to that of the same enzymes in water. In this study we aim to achieve information on the ability of ectoin, hydroxyectoin, glycoin (these three molecules are known as extremolytes),[1] glutaraldehyde and methoxypoly(ethylene glycol) (MeOPEG) to act as stabilizing additives of alpha-chymotrypsin (-CT), during the enzyme preparation process (dissolution and dehydration or lyophilization). In fact, although MeOPEG is known to increase the enzyme activity of some hydrolases in organic solvents,[2] little is known about the usefulness of extremolytes as lyoprotectants. Furthermore, the effect of glutaraldehyde, commonly employed for enzyme cross-linking reactions during the preparation of enzyme formulations such as CLEA or CLECs or for enzyme immobilization on amino functionalised supports, is also taken into account. Interestingly, the extremolytes increased the activity of -CT. However, the highest increase of activity was obtained with glutaraldehyde (up to 300-fold). Because of the different structures and chemical functionalities, the additives might lead to an improvement of enzyme activity in organic solvents by different mechanisms, whose possible rationale will be suggested and discussed.
Figure 1. PPL catalysed resolution of 3-hydroxycyclohexanone.
Table 1. Kinetic resolution of 3-hydroxycyclohexanone using different lipases In this work, the optically enriched 3-hydroxycyclohexanone was synthesized via a two step method involving chemical and biocatalysisbiocatalytic steps. In the first step, rac3-hydroxycyclohexanone was obtained in 40% isolated yield by the oxidation of cyclohexane-1,3-diol. Then the obtained rac-3-hydroxycyclohexanone was subjected to the lipase catalyzed resolution using vinyl acetate as a solvent and acylating agent. Among the lipases screened, Porcine pancreatic lipase (PPL)-II gave the optically enriched 3-oxocyclohexyl acetate in 95% ee (Table 1). Furthermore, the absolute stereochemistry of the obtained optically enriched 3-oxocyclohexyl acetate was determined and found to be R. The isolated (R)-3-hydroxy-cyclohexanone was used for the stereo selectivity determination of Michael hydratase alcohol dehydrogenase (MhyADH) [2].
_______________ 1. M. A. Arai, R. Tsutsumi, H. Hara, T. C. Chen, T. Sakaki, N. Urushino, K. Inouye and A. Kittaka, Heterocycles, 2005, 66, 469-479. 2. J. Jin, P. C. Oskam, S. K. Karmee, A. J. J. Straathof and U. Hanefeld, Chem. Commun., 2010, 46, 8588-8590.
_______________ [1] Gl-stndag, ; Mazza, G; Critical Reviews in Food Science and Nutrition, 47 (3), 231, 2007. [2] Sparg, S. G.; Light, M. E.; Van Staden, J.; Journal of Ethnopharmacology, 94, 219, 2004.
________________ [1] G. Lentzen, T. 2006. Schwarz Extremolytes: natural compounds from extremophiles for versatile applications. Appl Microbiol Biotechnol 72:623634. [2] F. Secundo, G.L. Barletta, G. Mazzola. 2008. Role of methoxypolyethylene glycol on the hydration, activity, conformation and dynamic properties of a lipase in a dry film. Biotechnol Bioeng101:255-262.
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Electroenzymatic synthesis with in situ electrochemical reduction of NAD analogs
Hye Jung Leea, Chan Beum Parkb, Keehoon Wona* a Department of Chemical and Biochemical Engineering, Dongguk University-Seoul, Seoul 100-715, Republic of Korea; bDepartment of Material Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea E-mail: keehoon@dongguk.edu Redox enzymes are very promising in organic synthesis in that they catalyze reactions involving oxygen insertion, hydrogen extraction, or electron transfer. Nicotinamide adenine dinucleotide, reduced form (NADH) is an essential coenzyme for oxidoreductasecatalyzed useful reactions such as reduction, hydroxylations, and epoxidations. However, it is too expensive to be supplied in stoichiometric quantities. Therefore, efficient regeneration of this reduced coenzyme is essential for successful applications of redox enzymes to industrial synthetic processes. Although an electrochemical regeneration method is attractive due to no requirement of impurities such as secondary enzymes and co-substrates, the performance is not still satisfactory compared with enzymatic methods. In this work, we introduced NAD analogs in order to improve the efficacy of electrochemical cofactor regeneration. Four commercially available NAD analogs with the substituted pyridine ring were examined: thionicotinamide adenine dinucleotide (thio-NAD), 3-acetylpyridine adenine dinucleotide (APAD), 3-pyridinealdehyde adenine dinucleotide (PAAD), and nicotinic acid adenine dinucleotide (NAAD). It was unveiled that APAD and PAAD analogs were electrochemically reduced more efficiently while their reduced products showed coenzyme activity comparable to original NADH (Figure 1). Moreover, stability of APADH and PAADH was revealed to be much higher than NADH. Finally, indirect electrochemical reduction of the NAD analogs were coupled to synthesis reactions catalyzed by dehydrogenases.
167
Biocatalytic Chiral Amine Synthesis
Rachel Heatha, Kirk J. Malonea, and Nicholas J. Turnera School of Chemistry, The University of Manchester, Manchester Interdisciplinary Biocentre, 131 Princess Street, Manchester, M1 7DN, UK E-mail: kirk.malone@manchester.ac.uk
a
170
Screening, immobilization and utilization of whole cell biocatalysts to mediate the ethanolysis of babassu oil
Silva G.S.a, Freitas L.a, Oliveira P.C.a, De Castro H.F.a a Engineering School of Lorena, University of So Paulo PO Box 116, 12602-810, Lorena, SPBrazil E-mail: grazielle@debiq.eel.usp.br The screening, biomass growth of lipase-producing fungus isolated from different sources and available at culture collection URM (University Recife Mycologia-Brazil), as well as, the immobilization and utilization of the whole cells for the transesterification of babassu oil were investigated. Rhizopus oryzae (URM 3231, 4692), Mucor circinelloides (URM 4140, 4182) and Penicillium citrinum (URM 4216) were considered better intracellular lipase producers than those from Mucor hiemalis (URM 4144) and Mucor piriformis (URM 4145). All fungi strains produced maximum amount of intracellular lipase by batch cultivation after three days of incubation in medium containing olive oil at 30C and pH 6.5. Whole cells containing high lipase activities were immobilized on different biomass support particles (BSPs) and excepting Penicillium citrinum URM 4216 all the others biomass exhibited high lipolytic activity (20-50 U.g-1) when immobilized in situ using polyurethane foam particles. Transesterification activities of the immobilized whole cells were evaluated in the ethanolysis reaction with babassu oil and higher performances were attained by M. circinelloides URM 4182 and Rhizopus oryzae URM 4692 strains. Both immobilized whole cells were able to form ethyl esters from all fatty acids present in the babassu oil and the highest ethyl ester concentrations were in regard to laurate followed by mirystate and oleate esters. In agreement with these results, among the screened strains, lipase from M. circinelloides URM 4182 showed the highest activity towards the transesterification of babassu oil with ethanol attained 83.22 3.68% ester yield in lees than 96 h reaction. The purified product (biodiesel) was straw yellow in color and essentially odorless, exbiting viscosity (6.63 cSt) close to the value specified by ASTM D6751 to be used as biofuel. Results are favorable compared with data already reported in the literature and demonstrated that M. circinelloides URM 4182 whole cells is a cheaper biocatalyst that can be also used in the biodiesel synthesis.
Acknowledgments: FAPESP, CAPES, CNPq.
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Natural Product Synthesis by Squalene-Hopene Cyclases (SHCs)
Miriam Seitza, Stephan Hammera, Janosch Klebensbergera, Bettina Nestla, Michael Breuerb, Bernhard Hauera a Institute of Technical Biochemistry, Universitaet Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany, bBASF SE, Fine Chemicals and Biocatalysis Research, D-67056 Ludwigshafen, Germany E-mail: miriam.seitz@itb.uni-stuttgart.de Considering the membrane fraction of cells, one difference between bacteria and eukaryotes is the absence of sterols as membrane constituents. In contrast to eukaryotes, it is considered that some eubacteria produce pentacyclic triterpenes of the hopanoid class as structural and functional equivalents of sterols [1-2]. Hopanoids are synthesised by squalene-hopene cyclases (SHC; EC 5.4.99.17), which catalyse the cyclisation of triterpenes via cationic intermediates in one of the most complex and powerful one-step reactions known in biochemistry. Most of our understanding about the biochemical and molecular mechanism of this reaction has been obtained by the characterisation of a SHC from Alicyclobacillus acidocaldarius (AaSHC; GI: 1435434). In our study, we characterised a novel SHC from the gram-negative, alcohol producing bacterium Zymomonas mobilis (ZmSHC1; GI: 56552444) and compared its activity and substrate spectrum with another, previously described squalene-hopene cyclase (ZmSHC2; GI: 6466213) from the same organism [1, 3]. In order to do this, we optimised a protocol for the expression of these enzymes in Escherichia coli and the conditions for the enzymatic reaction. Subsequently, we determined the enzymatic activity of ZmSHC1 with a variety of substrates including citronellal, homofarnesol and squalene. Despite the differences in chain length (C10-C30) and the presence of C=C double bounds or functional groups like aldehydes at the position where protonation needs to occur for the initiation of the reaction, conversion could be found for all of these substrates. Beside the conversion of squalene to hopene, the cyclisation of homofarnesol to ambroxan and citronellal to isopulegol is of particular interest, as these compounds are commonly used in the manufacturing of fragrance and flavour concentrates or could provide a bio-catalytic access for the production of menthol, respectively. Furthermore, our results revealed significantly higher conversion rates of ZmSHC1 in comparison to previously described rates of the squalene-hopene cyclase AaSHC from A. acidocaldarius. [4] From these results, we conclude that the squalene-hopene cyclase ZmSHC1 from Z. mobilis has a high bio-catalytic potential for a large variety of industrial applications.
________________ [1] Reipen, I., Poralla, K., Sahm, H., Sprenger, G., Microbiology, 1995, 141: 155-161. [2] Abe, I., Rohmer, M., Prestwich, G., Chem. Rev., 1993, 93: 2189-2206. [3] Wendt, K., Schulz, G., Corey, E., Liu, D., Angew. Chem., 2000, 112: 2930-2952. [4] Neumann, S., Simon, H., Biol. Chem. Hoppe-Seyler, 1986, 367: 723-729.
Amines are one of the most important families of compounds for the chemical manufacturing industries, produced in bulk for agrochemicals, pharmaceuticals and speciality chemical applications. The traditional manufacture of amines often involves the use of precious metal reagents and wasteful protection strategies. Biocatalytic routes offer great potential for improving the large scale production of amines with reduced cost and waste production. We have targeted three groups of enzymes to develop into the next generation of core biocatalytic technologies for chiral amine production (Figure 1). These are transaminases (C=O to C-N), ammonia lyases (C=C to C-N) and monoamine oxidases (deracemisation of racemic amines). Each enzyme class is being tailored to industrial amine synthesis through a combination of high throughput screening, directed evolution and the development of larger scale bioprocesses using enhanced biocatalysts. This research is part of a flagship EU project entitled BIONEXGEN. A consortium of 17 leading European academic and industrial partners are researching key industrial bioprocesses, the goal being to develop the next generation of biocatalysts to be used for eco-efficient manufacturing processes in the chemical industry. [http://bionexgen-fp7.eu]
________________ [1] S. H. Lee, K. Won, H.-K. Song and C. B. Park, Small, 2009, 5, 2162. [2] F. Hollmann and A. Schmid, Biocatal. Biotransform., 2004, 22, 63.
Figure 1. Electrochemical Reduction of the NAD Analogs in the Presence of [Cp*Rh(bpy)H2O]2+ and Coenzyme Activity of Their Reduced Products.
Figure 1. Biocatalytic synthesis of chiral amines
October 2-6, 2011
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Innovative enzymatic (R,S)-urbiprofen resolution in continuous ow reactor with lyophilized cells of Aspergillus oryzae
Diego Romano,a* Lucia Tamborini,b Arianna Bertolani,a,b Maria Celeste Iannuzzi,b Federica Mastronardi, Paola Conti,b Francesco Molinaria a Department of Food Science, Technology and Microbiology (DISTAM), University of Milan, via Celoria 2, 20133 Milan, Italy; bDepartment of Pharmaceutical Sciences Pietro Pratesi (DISFARM), University of Milan, via Mangiagalli 25, 20133,Milan, Italy. *e-mail: diego.romano@unimi.it In our previous work dry mycelia of Aspergillus oryzae, displaying high enantioselectivity towards (R)-flurbiprofen, have been efficiently used in pure organic solvent for the resolution of (R,S)-flurbiprofen through esterification.[1] The use of the lyophilized mycelia facilitated the separation process so that in one step the two enantiomers of flurbiprofen, which are both valuable for pharmaceutical applications,[2] were easily separated. Reaction was performed and optimized in usual batch system. New possibilities of process-improvement using the advantages - in terms of processing flexibility, easy scaling factors, heat and mass transfer, reduced substrate and product inhibition phenomena[3] - of a flow chemical approach have been investigated. A column reactor, packed with lyophilized cells of Aspergillus oryzae, was continuously flushed with a solution of (R,S)-flurbiprofen and ethanol. After optimization of the reaction parameters (solvent, temperature, reactor configuration, flow rate) we were able, using the same amount of cells, to increase reaction rate of about 12 times (2 h compared to 24 h in the batch reaction) maintaining the same degree of molar conversion and enantiomeric purity of product. Moreover the cells could be repetitively used for several days, thus allowing for an easy scale up of the process. Future development and optimization of the continuous process for the potential industrial application will also be discussed.

Penicillin acylase-catalyzed stereoselective acylation of amines in aqueous medium using non-activated acyl donor
Dorel T. Gurandaa,b, Gennady A. Ushakovb,c,Vytas K. vedasa,b a Faculty of Bioengineering and Bioinformatics and bBelozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Vorobjev hills 1-73, Moscow 119991, Russia E-mail: dorel@belozersky.msu.ru Until recently, the possibility of enzymatic acylation of amines had been demonstrated mainly in non-aqueous water-free organic media with the use of lipases as catalysts of the acyl transfer from the activated acyl donors to the amines [13]. Even though the use of lipases has been fairly successful, biocatalysis in non-aqueous media has a number of complications, the most obvious being low enzymatic activity [4], as well as the necessity to control the use of ecologically toxic organic solvents, which constitute the bulk of pharmaceutical industrial waste [5]. Recently it was demonstrated also that effective and stereoselective acylation of amines can take place in 100% aqueous solution using penicillin acylase-catalyzed acyl transfer [6,7]. This was made possible due to the unique catalytic properties of the penicillin acylase from Alcaligenes faecalis [8] which possess high catalytic activity and stability at alkaline conditions (pH ~ 10) optimal for the acylation of basic primary amines. Moreover at sufficient optimization the enzymatic acylation of amines in aqueous media could be more effective than was previously thought. The possibility of performing an effective and enantioselective acylation of amines in an aqueous medium catalyzed by penicillin acylase from Alcaligenes faecalis without using activated acyl donors was demonstrated for the first time as the example of direct condensation of phenylacetic acid and racemic 1-phenylethylamine. Direct condensation of the acid and the amine took place at mild reaction conditions with a high initial rate (3.3 mole/(lh)), degree of conversion (80% acylation of active amine enantiomer), and enantioselectivity (enantiomeric excess of the product was more than 95%). The suggested approach has remarkable advantages compared to enzymatic reactions in organic media and is of practical value for the biocatalytic preparation of enantiomerically pure compounds at mild conditions using readily available reagents.
1. Jaeger K.-E., Liebeton K., Zonta A., Schimossek K., Reetz M. T., Appl. Microb. Technol. 1996, 46, 99105. 2. Takayama S., Lee S.T.; Hung, S.C., Wong C.H., Chem. Commun. 1999, 127128. 3. Iglesias L.E., Rebolledo F., Gotor V., Tetrahedron: Asymmetry, 2000, 11, 10471050. 4. Klibanov A.M., Trends Biotechnol. 1997, 15, 97101. 5. Carey J.S., Laffan D., Thomson C., Williams M.T., Org. Biomol. Chem. 2006, 4, 2337-2347. 6. Guranda D.T., Van Langen L.M., Van Rantwijk F., Sheldon R.A., vedas V.K., Tetrahedron: Asymmetry, 2001, 12, 1645-1650. 7. Guranda D.T., Khimiuk A.I., Van Langen L.M., Van Rantwijk F., Sheldon R.A., vedas V.K., Tetrahedron: Asymmetry, 2004, 15, 2901-2906. 8. vedas V., Guranda D., Van Langen L., Van Rantwijk F., Sheldon R., FEBS Lett. 1997, 417 (3), 414-418. This work was supported by the Russian Foundation for Basic Research (grant 10-08-01243).

Efficient Synthesis of (S)--Bromohydrins via Biocatalytic Asymmetric Reduction of -Bromoacetophenones
Jie Ren, Benqing Yu, Qiaqing Wu, Dunming Zhub National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Park, Tianjin 300308, China E-mail: zhu_dm@tib.cas.cn Optically pure halohydrins are useful building blocks for the synthesis of optically active molecules of pharmaceutical and agrochemical interest, such as adrenergic receptor agonists, bronchodilators, anti-depressants and HIV-1 protease inhibitor[1]. Both chemical and biological approaches have been developed to obtain enantiomerically pure halohydrins, such as asymmetric reduction of -haloacetophenones by organocatalytic, transition metal-based catalytic and biocatalytic methods. Because the bromo group can be more conveniently substituted by amino group or other nucleophilic groups, bromohydrins are more active than chlorohydrins and should be preferable precursor for the synthesis of most adrenergic receptor agonists and other active compounds. However, only a few literatures reported the asymmetric reduction of -bromoacetophenone and their derivatives with moderate enantiomeric excess and even low yield via whole cell bioreduction. The reduction product of -bromoacetophenone usually contained by-products such as -chlorohydrin, phenylethandiol, acetophenone and phenylethanol, because the bromo group was substituted by hydroxyl or chloride ion in the culture medium, or lost to form acetophenone which was further reduced to phenylethanol. Therefore, the reduction of -bromoacetophenones affording high yield of -bromohydrins with high optical purity still presents a significant challenge in synthetic organic chemistry. Recently, we have reported that a carbonyl reductase from Candida magnolia catalyzed the reduction of various ketones including - and -ketoesters and aliphtic and aromatic ketones.[2] More importantly, this enzyme reduced -chloroacetophenone to optically pure (S)--chlorohydrin. As such, we applied this carbonyl reductase in the reduction of various -bromoacetophenone analogues. To our delight, this enzyme catalyzes the reduction of various -bromoacetophenones to give (S)-bromohydrins with isolated yields being 74-92%. Most of the reduction products are essentially optically pure, with the only exceptions for meta-methoxy and para-methoxy substituted -bromoacetophenones, which have enantiomeric excess of 97% and 93%, respectively. This demonstrated that use of suitable isolated carbonyl reductase would be a promising approach for the efficient synthesis of enantiomerically pure -bromohydrins via reduction of -bromoacetophenones.
______________ [1] R. N. Patel, A. Banerjee, C. G. McNamee, D. B. Brzozowski, L. J. Szarka, Tetrahedron: Asymmetry 1997, 8, 2547-2552. [2] D. Zhu, Y. Yang, L. Hua, Journal of Organic Chemistry, 2006, 71, 4202-4205.
115 177
Batch process for lipase catalyzed monoacylglycerol synthesis
Ivaldo Itabaiana Junior a*, Felipe K. Sutili a, Ivana R. C. Leal (PQ)b, Leandro Soter de Mariz e Mirandaa e Rodrigo Octvio Mendona Alves de Souzaa a Grupo de Biocatlise e Sntese Orgnica, Instituto de Qumica, Universidade Federal do Rio de Janeiro, CEP 22941-90, Brazil. bFaculdade de Farmcia, Universidade Federal do Rio de Janeiro, CEP22941-909,Brazil. E-mail: ivaldo@bossgroup.com.br Monoacylglycerols(MAG) like monostearin, are well known as biodegradable and biocompatible surfactants, widely used in food, pharmaceutical and industrial applications. They are commonly produced based on alkaline-catalyzed chemical glycerolysis of natural oil and fats at high temperatures (220-250C)[2], responsible for the formation of dark-colored and burned-tasting products. In this work we have optimized by response surface methodology (RSM), the lipase catalyzed esterification reactions between 1-2,isopropylideneglicerol (solketal) and both stearic acid and a industrial waste from palm fatty acids (stearic, oleic and palmitic) as a potential substitute to the traditional chemical glycerolysis, since lipases as biocatalysts demand milder reaction conditions and minimize the energy costs. First we evaluated the performance of 4 free lipases and 4 immobilized lipases in the esterification of solketal (150mM) and stearic acid (75mM) in n-heptane at 60 C and 250 rpm. The percentages of conversion were analyzed by a modification of the Lowry-Tinsley method[1]. As a result, the immobilized lipases of T. Lanuginosus (TL IM) and R. miehei (RM IM) showed conversion rates of 92 and 94% respectively after 4h of reaction. Aiming an industrial applicability, we proposed a new batch of reactions by applying a 1:1 ratio of reagents (75mM each) with the selected enzymes, whose could be observed 81% of conversion for lipase TL IM and 82% for the enzyme RM IM. In order to determine the variables that most interferes on the reaction, a two level factorial design was adopted. First, we carried out the fractional factorial designs 24-1 for each biocatalyst, with 22 experiments, being eleven trials for each enzyme. Then, a central composite rotatable design (CCRD) was employed to lipase RM IM, that presented 95% of conversion with 0.1% of biocatalyst. The response surface (figure 1a) for this reaction in function of temperature and concentration of the substrate is showed below. A scale-up of 20g of this product, using 5% of biocatalyst and a solvent-free reaction was performed, achieving 80% of conversion. The acetals present in the final product were cleaved according to Hess et al, 1995. Using a waste from palm fatty acids as starting inputs for the production of monoacylglycerols by esterification with solketal (75mM of each one in n-heptane) and aiming to find the best reaction conditions and biocatalyst, the same designs of experiments was applied with the lipases studied before. As a result, RM IM and TL IM had demonstrated similar behavior in esterification reactions, with conditions that offered 96 and 95% of conversion, respectively. The response surface for these reactions are showed also in figure 01.
[1] Spizzo, P. et al., Tetrahedron 2007, 63, 1100511010. [2] Grosch, S. et al., Biochem. Pharmacol. 2005, 69, 831839. [3] Baxendale, I. R. Ley, S. V. in: Seeberger, P. H. and Blume, T. Editors, New Avenues to Efficient Chemical SynthesisEmerging Technologies Vol. 3, Springer, Berlin, Heidelberg (2007), pp. 151185.
Figure 01. Response surface for the esterification of solketal catalyzed RM IM (A), and the esterification of solketal and waste from palm fatty acids catalyzed by TL IM (B) and RM IM (C).
_____________________________________________ [1] Machado, et al. J. Mol. Cat. B: Enzymatic. 2010 [2] R.R. Lowry, I.J. Tinsley, J. Am. Oil Chem. Soc. 53 (1976) 470-472
174
Carboligation reactions with benzaldehyde lyase immobilized on surface modified magnetic nanoparticles
Bilsen Tural1,2*, lke imek1, Blent elebi2, brahim Yalnkl2, Ayhan S. Demir1 1 Department of Chemistry, Faculty of Arts and Sciences, Middle East Technical University, Ankara 06531, Turkey 2Department of Chemistry, Faculty of Education, Dicle University, 21280, Diyarbakir, Turkey E-mail: bilsentural@gmail.com The value of coupling biological molecules such as enzymes to solid materials has long been recognized. To date, protein immobilization onto such surfaces often involves covalent coupling, encapsulation, or non-specific adsorption techniques. In this work we demonstrate two different techniques for immobilization of benzaldehyde lyase (BAL, EC 4.1.2.38), a thiamine pyrophosphate (TPP) dependent enzyme, isolated from fluorescens Biovar I. In the first part we designed a heterocatalyst system which consists of a carboligating enzyme benzaldehyde lyase and silica coated maghemite particles. The supporting material for immobilization is Co2+-NTA functionalized -Fe2O3 particles[1,2]. Than it was possible to immobilize the histidine tagged recombinant enzyme starting from crude cell extract. In the second part BAL is efficiently immobilized to surface-modified magnetic particles with covalent binding via epoxy ring opening process. Fe3O4@SiO2 coreshell magnetic nanoparticles were synthesized using a solgel method[3,4]. They particles are characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transforms infrared spectroscopy (FTIR) methods. The immobilization processes are used for purification and carboligation steps. The efficiently immobilized BAL on two different types magnetic solid supports are used as a heterogeneous catalyst for some representative BAL-mediated carboligation reactions (homo and cross coupling reactions). The high yield, high selectivity and reusability obtained from the carboligation reactions that were performed with this simple and convenient heterogeneous biocatalysts were comparable to that of free enzyme-catalyzed reactions.
References [1] Sopaci B, imek , Tural B, Volkan M, Demir AS., Org. Biomol. Chem, 2009;7:1658-1664. [2] J. S. Kim, A. Valencia, R. Liu and W. Lin, Bioconjugate Chem., 2007, 18, 333341; [3] Z. Ma, Y. Guan, H. Liu, J Magn Magn Mater, 2006, 301, 469. [4] X.Q. Liu, J.M. Xing, Y.P. Guan, G.B. Shan, et al, Colloids Surf. A, 2004, 238, 127.
175
Microbial production of phase I and phase II metabolites of propranolol
a
178
Enhancement of the selectivity of Rhizopus oryzae lipase in desymmetrization reactions.
Zaida Cabreraa, Jos M. Palomob. a Pontificia Universidad Catolica de Valparaiso,General cruz 34, Valparaiso, Chile; bInstituto de Catalisis y Petroleoquimica (CSIC), C/ Marie Curie 2, Madrid, Espaa. E-mail: zaida.cabrera@ucv.cl Rhizopus oryzae lipase (ROL) is an enzyme widely used in transesterificaction reactions. Its application in desymmetrization reactions, however, has been very little explored. In this work, we have evaluated the potential of an immobilized preparation of ROL to catalyze the synthesis of a chiral compound, (R)-3-phenylglutaric acid (building block of important chiral drugs) [1], by the asymmetric hydrolysis of dimethyl 3-phenylglutarate (DMFG), prochiral compound. ROL was immobilized on Lewatit-105 resin activated with aldehyde groups in the presence of dithiothreitol (DTT) at pH 7 and 25 C [2], by single point covalent attachment (Figure 1). The immobilization yield was >60% (in terms of activity).
179
Development and characterisation of a two-step microuidic reactor for the biocatalytic asymmetric synthesis of chiral amino diol
Homam Al-Bahrania, Brian OSullivana, James Lawrencea, Helen C. Hailesb, Frank Baganza, Roland Wohlgemuthc, Nicolas Szitaa a Department of Biochemical Engineering, University College London, Torrington Place - London - WC1E 7JE, United Kingdom. bDepartment of Chemistry, University College London, 20 Gordon Street - London - WC1H 0AJ, United Kingdom. cSigma-Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland. E-mail: ucbehal@ucl.ac.uk The idea of de novo metabolic engineering [1, 2] through novel synthetic pathways has offered future directions for multi-step enzymatic synthesis of complex molecules and has been complemented by recent progress in performing enzymatic reactions on-chip using microfluidic reactors [3]. These reactors have made a significant impact in the field of analytical studies [4], yet relatively few studies have explored the biocatalytic synthesis of chiral compounds [5, 6]. In this work a microfluidic reactor utilising clarified lysates for continuous small-scale synthesis is reported. We focus on two model reactions, namely the transketolase-catalysed conversion of hydroxypyruvate (HPA) and glycolaldehyde (GA) to produce L-erythrulose (ERY), followed by the conversion of ERY to aminoerythrulose (EAT) in the presence of methybenzylamine (MBA) by -transaminase. High conversion was obtained for the first model reaction system, providing a constant stream of ERY for the second enzyme step, thus allowing the EAT to be continually synthesised also. The rate of conversion in the microfluidic reactor was also compared with batch reactions. This study investigates the potential of microfluidic enzyme reactors for the continuous synthesis of commercially important compounds.
Cyrille Marvalina, Xavier Morgea, Cecile Olivariusa, Robert Azeradab Bertin Pharma, 10 bis avenue Ampre, Parc d'Activits du Pas du Lac, 78180 Montignyle-Bretonneux, France ; bLaboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universit Paris Descartes, 45 rue des Sts Pres, 75006 Paris, France E-mail: robert.azerad@parisdescartes.fr Purpose: To obtain regio- and stereospecific hydroxylated and glucuronidated metabolites of (R,S)-propranolol (a classical -adrenoreceptor antagonist and typical substrate of CYP2D6) by microbial transformation, and to prepare multimilligram amounts of authentic standards for kinetic, metabolic and pharmacological studies.
Methods: Adequate fungal strains were selected from an exhaustive screening to convert (R,S)-propranolol to its 4- or 5-(R,S)-hydroxy derivatives. In addition, a bacterial strain was shown to convert (R,S)-propranolol and its 4- or 5-(R,S)-hydroxyderivatives into the corresponding O--D-glucuronides, regio- and stereoselectively. The selected strains were incubated with substrates (0.1-1 g/L) in liquid cultures at +28C and 200 rpm orbital shaking during 48-168 hours. The metabolites were either extracted with organic solvents or adsorbed on XAD-16 and purified using reverse phase chromatographic methods. All metabolites were characterised by MS, 1H- and 13C-NMR and purity (>98%) was controlled by HPLC-UV/MS. When necessary, chiral chromatography was employed to determine the enantioselectivity of the reactions. Results: Strain M50002 transformed (R,S)-propranolol into 4-hydroxy-(R,S)-propranolol (20% total yield in 72 h) corresponding to the main animal and human metabolite. Strain M50036 transformed (R,S)-propranolol into 5-hydroxy-(R,S)-propranolol (70% total yield in 72 h). The transformations were regio- but not enantioselective. Hydroxy derivatives were obtained in multimilligram amounts by scaling up the incubations. When (R,S)-propranolol was incubated with strain 52104, the 10-O--D-glucuronide diastereomers were slowly formed in a 1:3 (S/R) ratio. Separation on semipreparative reverse phase HPLC allowed to obtain in multimilligram amounts pure diasteromeric glucuronides (>98% purity), identified by coelution with the products of separate incubations with optically pure R- or S-propranolols. Similarly, 4- or 5-(R,S)-hydroxypropranolols were quantitatively glucuronidated by strain M52104 (100% yield in 48 h) to give the corresponding 4- or 5-O--D-glucuronides (>98% purity), as a 1:1 mixture of unseparated diastereomers in each case. Conclusion: Propranolol is commonly used as a typical substrate for hydroxylation by CYP2D6 and O-glucuronidation by various UGTs. Owing to the poor commercial availability of most of these derivatives, particularly pure glucuronide conjugates, the easier preparation of such compounds in good yields, compared to chemical synthesis, in a one step microbial reaction, presents an outstanding interest by affording useful analytical standards.
Figure 1: Immobilization mechanism on Lewatit 105-aldehyde with DDT at pH 7 In all cases, asymmetric hydrolysis of DMFG was carried out at pH 7 and 25 C. The effect of the presence of different solvents as additives on the enzyme selectivity was tested. Very interestingly, the process exhibited significant enantioselectivity, producing mainly the R-monomethyl ester. The enantioselectivity value in the absence of solvent (phosphate buffer) was obtained with an enantiomeric excess (ee) of 62%. The presence of 20% solvent significantly improved the enantioselectivity of the immobilized preparation reaching an ee of 72%, 80% and 82% in the presence of dimethyl sulfoxide, diglyme and dioxane, respectively. The immobilized enzyme stability was studied in the presence of 40% solvent, interestingly, more than 90% of its activity was retained for at least 24 hours in all the solvents tested thus allowing the reuse of the catalyst for several production cycles. In conclusion, ROL is an enzyme that has an interesting potential for catalyzing selective desymmetrization reactions with a high stability in the presence of solvents with the possible reuse of the biocatalyst for several batches. Acknowledgements: Work supported by Grant 11090321 from Fondecyt, Chile.
________________ [1] Fryszkowska, A., et al. Tetrahedron: Asymmetry 2005; 16:2475-2485. [2] Bolivar, J.M., et al. Enzyme and Microbial Technology 2009 45:477483.
Figure 1. Dual-enzyme reaction scheme showing the TK-catalysed production of L-erythrulose (ERY) followed by TAm-catalysed production of aminoerythrulose (EAT) from ERY and methybenzylamine (MBA).
________________ [1] Meyer, A. et al. (2007), Curr. Opin. in Microbiol., 10, 246-253 [2] Tyo, K. E. et al. (2007), Trends in Biotech., Vol 25 (3), pp. 132-137 [3] Krenkov, J. et al. (2004), Electrophoresis, 25, 35503563 [4] Urban et al. Biotechnol. Adv. 2006, 24, 42 [5] Koch et al. Biotechnol. Bioeng. 2008, 99, 1028 [6] Thomsen et al. Eng. Life Sci. 2008, 8, 40
October 2-6, 2011
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Enantioselective synthesis of (R)-1- phenyl ethyl acetate by immobilized StcI recombinant esterase of Aspergillus nidulans
Mondragn Mara Elenaa, Esquivel Ricardoa, Navarro Arturoa, Farrs Ameliaa a Universidad Nacional Autnoma de Mxico, Chemistry Faculty. Food and Biotecnology Department. Av. Universidad 3000, 04510, Mxico City, Mxico. E-mail: carpem72@yahoo.com Carboxylic ester hydrolases (EC 3.1.1.x) are a diverse group with high regio- and stereospecificity, which makes them attractive biocatalysts for the production of optically pure compounds in fine-chemicals synthesis. Enantiomers have different biological properties and pure compounds are vital in the pharmaceutical and agrochemical industries. Only a few microbial carboxylesterases has been used for the synthesis of optically pure compounds and they show moderate enantioselectivity. In this study, we analyzed if immobilized recombinant StcI esterase from A. nidulans could showed enantioselective synthesis of a compound of industrial interest. We optimized the kinetic resolution of this compound and it was compared with a commercial enzyme (Novozym 435). StcI esterase was expressed in P. pastoris [1]. After immobilization in Accurel MP 1000, solvent stability was evaluated after 72 h incubation in hexane, diisopropyl eter, t-butanol and acetone; at different temperatures (25, 37 y 50C). (R,S)-1-phenylethanol was chemically synthesized and their structure was corroborated by NMR. For synthesis reactions, 1-phenylethanol and vinyl acetate (each 200 mM) were disolved in 3 mL of hexane (molecular sieves 4 at different Aw), then 40mg de immobilized StcI 15 mg de Novozym 435 were added and molecular sieves 4 (10%). It was incubated at 37 C, 200 rpm and kinetic was followed during 120 h. Products were evaluated by TLC, GC and chiral column separation. After this, the enantiomeric excess (%ee), enantioselectivity (E) and conversion percentage (%C) were determined, as shown in the table. After StcI immobilization we found that 94.11% of protein was adsorbed; however esterase activity was compromised (1.6 fold). The highest activity of immobilized enzyme was observed at 37C in hexane and synthesis reactions were done in these conditions. Chromatograms showed that generated (StcI) and commercial (Novozym 435) biocatalysts have selectivity for (R)-enantiomer. Low Aw and long time reaction favored E value. Kinetic resolutions with E>20 are useful for synthesis, and then obtained E values for both tested enzymes is useful (31.831 for StcI and 67.47 for Novozym 435). Best condition reactions were 120 h in hexane and Aw of 0.066. Results are resumed in the following table.

Biocatalyzed synthesis of chiral phosphinates with two stereogenic centers
Kinga Kozyraa, Magdalena Klimek-Ochaba, Magorzata Brzeziska-Rodaka and Ewa ymaczyk-Dudaa a Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocaw University of Technology, Wybrzee Wyspiaskiego 27, 50-370 Wrocaw, Poland E-mail: kinga.basalyga@gmail.com Biotransformations are biocatalytic conversions of non-natural substrates to structurally diverse products, they belong to the standard means of green chemistry [1, 2]. Phosphonates are compounds with direct, stable bond between phosphorus and carbon atoms. They can be considered as antibacterials, herbicides or neuromodulators [3]. In the most cases, compounds exerting biological activity are chiral molecules with defined absolute configuration. This rule also concerns the molecules with P-C bond. Microbial bioconversions of racemic chemically synthesized starting materials - into optically pure derivatives of organophosphorus compounds, are still not fully explored fields of biotechnology. Thus whole cells of Saccharomyces cerevisiae strain were applied in quite unique manner, it means for the stereospecific oxidation of particular enantiomers of ethyl 1-hydroxy-1-(3,4dimethoxyphenyl)methane(P-phenyl)phosphinate resulting in the unstable keto phosphinates, which immediately split under water conditions. It implies in the isolation of the enantiomerically enriched mixture of products - opposite unreacted enantiomers. It resulted in obtaining pure stereoisomers from each pair of enantiomers. The configuration of the products was determined using (S)-(+)-MTPA-Cl and 1H NMR technique. To enforce the yeasts cells to oxidation of the particular isomer of the phosphinate, the biotransformation conditions were desired to mimic the physiological cofactor regeneration system. Thus, the substrate - hydroxy phosphinate was an exogenous source of proton for oxidized cofactors and the additive was an acceptor, so was subsequently reduced, enriching the oxidized forms in the dehydrogenases coenzymes pool.

Dynamic Kinetic Resolution of unsymmetrical benzoins via lipase-metal combo catalysis: a rational strategy based on the enzyme recognition pattern.
Pilar Hoyosa, Andy Maraiteb, Jos Daniel Carballeirac, Alvaro Corts-Cabrerad, Marion B. Ansorge-Schumacherb, Jos V. Sinisterraa,e, Andrs R. Alcntaraa a Department of Organic and Pharmaceutical Chemistry, Pharmacy Faculty, Complutense University of Madrid, Madrid, Spain;bInst. of Chemistry, Dept. of Enzyme Technology, Technical University of Berlin, Germany;cDepartment of Molecular Biology, University of Cantabria, Santander, Spain.dUnidad de Bioinformtica, Centro de Biologa Molecular Severo OchoaCSIC-UAM, Madrid, Spain; eIndustrial Biotransformations Unit, Scientific Park of Madrid, Tres Cantos, Madrid, Spain. E-mail: andresr@farm.ucm.es Enantiomerically pure benzoins (1,2-diaryl-2-hydroxyethanone structures) are important building blocks in organic and pharmaceutical chemistry due to their wide versatility. These compounds are involved in the synthesis of many natural products, drugs and heterocycles[1]. Different chemical strategies have been developed for the synthesis of this kind of compounds, mainly focused on symmetrical benzoins[1]. However, only scarce examples can be found in literature describing the synthesis of chiral unsymmetrical benzoins, containing two different aromatic moieties at both sides of the acyloin core. In this communication, we describe the synthesis of optically pure unsymmetrical benzoins esters through a chemoenzymatic DKR process, attending to the lipase (Lipase TL, from Pseudomonas stutzeri) substrate recognition pattern, which has been previously defined based upon a homology modelling [2]. In this way, due to the interaction of the carbonyl group with different residues of the active site, the lipase would accommodate the benzoyl moiety in the medium-size pocket, in an "Anti-Kazlauskas fashion; thus, the increase of the dimensions difference of the substituents at both sides of the hydroxyl group, by the introduction of a bulky substituent on the aryl moiety next to the alcohol function, definitively enhances the lipase preference towards these last substrates, allowing for the first time the synthesis of chiral unsymmetrical benzoin esters in high yields and enantiomeric purity
117 185
Aspartate ammonia-lyase (AspB) approaches. Application to the biosynthesis of L-aspartate
A. Max Crdenas-Fernndez, Marta Jimnez, Carmen Lpez, Gregorio lvaro, Josep Lpez-Santn Department of Chemical Engineering, Applied Biocatalysis Unit associated to IQAC (UAB-CSIC). School of Engineering. Universitat Autnoma de Barcelona. 08193-Bellaterra (Cerdanyola del Valls), Catalunya, Spain E-mail: josep.lopez@uab.es L-Aspartate ammonia lyase or aspartase (AspB) from the bacterium Bacillus sp. YM55-1 catalyzes the reversible elimination of ammonia from L-aspartate to yield fumarate. AspB is a homotetramer with a molecular weight of 200 KDa; it is a thermostable enzyme, stereoselective and with a high activity and specificity for its natural substrate [1]. L-aspartate (L-asp) is a non-essential amino acid and it is produced by using immobilized wild or recombinant E. coli cells; moreover, L-asp is one of the principal ingredients to manufacture the artificial sweetener aspartame [2]. The aims of this work are to immobilize AspB by different methods and to use the immobilized enzymatic derivatives for L-aspartate synthesis. The recombinant AspB expressed in E. Coli (supplied by C-lecta, Leipzig - Germany) was immobilized by multipoint covalent attachment method on agarose-amine support (MANA) and on the hydrophobic support Eupergit C; and by entrapment in the commercial polymer Lentikat (polyvinylalcohol). All the immobilization processes were carried out in mild conditions and low enzymatic charge. The immobilization yields were 98%, 90% and75%; and retained activities 60%, 28% and 23% for the AspB immobilized on MANA, Eupergit C and Lentikat, respectively. The time of the immobilization process was shorter in MANA and Lentikat (3 h) than Eupergit C (18 h). In order to choose the appropriate immobilized derivative, its performance on L-aspartate synthesis will be also considered. The synthesis of L-asp with the soluble AspB and the three immobilized derivatives were carried out in presence of fumarate 0.5 M and NH4Cl 1 M in buffer HEPES 0.1 M pH 7.5 at 37C. We obtained a high conversion rate and productivity after 24 h with the soluble and immobilized enzymes. In conclusion, we have established and optimized methods to immobilize AspB in three different supports which lack of diffusional limitations; moreover, these immobilized enzymatic derivatives can be used to synthesize high concentrations of the amino acid Laspartate.
________________ [1]. Kawata Y, Tamura K, Yano S, Mizobata T, Nagai J, Nuboyoshi E. Purificatio and characterization of thermostable Aspartase from Bacillus sp. YM55-1. 366 (1) (1999) 40-46 [2]. Viola. R.E. L-aspartase: new tricks from an old enzyme. Advances in Enzymology. 74 (2000) 295-341.
Acknowledgements. PAPITT IN2148092. Programa 127 de la Facultad de Qumica

_______________________ [1] Pea-Montes C, Lange S, Castro-Ochoa D, Ruiz-Noria K, Cruz-Garca F, Schmid R, Navarro-Ocaa A, Farrs A. (2009). J. Mol. Catal. B: Enzym. 61, 225-234.
Figure 1. Ethyl 1-hydroxy-1-(3,4-dimethoxyphenyl)methane(P-phenyl)phosphinate

________________ [1] Kiebasiski, P.; Omelaczuk, J.; Mikoajczyk, M. Tetrahedron Asymm. 1998, 9. [2] Sobera, M.; Wieczorek, P.; Lejczak, B.; Kafarski, P. Toxicol. Environ. Chem. 1997, 61. [3] Kafarski, P.; Lejczak, B. Curr. Med. Chem. Anti-Cancer Agents. 2001, 1, 301. ______________ [1] Hoyos, P. et al. Acc. Chem. Res. 2010, 43, 288-299. [2] Hoyos, P. et al. Manuscript in preparation.
182
Mutated Variant of Candida antarctica lipase B in (S)-selective dynamic kinetic resolution of secondary alcohols
Karin Engstrma, Richard Lihammara, Ylva Wikmarka, Michaela Vallinb, Per-Olof Syrnb, Karl Hultb, Jan-Erling Bckvalla a Department of Organic Chemistry, Stockholm University, Arrhenius Laboratory, SE106 91 Stockholm, SWEDEN; bDepartment of Biochemistry, School of Biotechnology, Royal Institute of Technology (KTH), Albanova University Center,SE-106 91 Stockholm, SWEDEN. E-mail: karin@organ.su.se A variant of Candida antarctica lipase B (CalB) with a mutation in one of the residues limiting the size of the stereoselectivity pocket, Trp104, was published in 2005.[1] This CalB variant, where Trp104 was changed into an alanine (CalB W104A) has an enlarged stereospecificity pocket. A recent study of CalB W104A showed that its stereospecificity pocket can accommodate 1-phenylalkanols with an alkyl chain of up to eight carbon atoms and the (S)-enantiomer was preferred.[2] We have combined the kinetic resolution of CalB W104A with a racemization catalyst to obtain a dynamic kinetic resolution. 1-Phenylalkanols of different size was efficiently acylated at 60 C to give the product in 90-97% enantiomeric excess.3 This is the first (S)-selective DKR performed with an enzyme that can be used at elevated temperatures.
183
Lipolytic fungi as catalysts for desymmetrization of hydroxyphosphinate derivatives
Magdalena Klimek-Ochaba, Ewa ymaczyk-Dudaa, Magorzata Brzeziska-Rodaka, Paulina Majewskaa, Barbara Lejczaka, Pawe Kafarskia a Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wybrzee Wyspiaskiego 27, 50-370 Wrocaw, Poland E-mail: magdalena.klimek-ochab@pwr.wroc.pl Biocatalytic reactions as way for resolving racemic mixtures have gained increasing value. Chemoselectivity, regioselectivity and especially stereoselectivity of biocatalysts are particularly attractive features of biotransformations, because they allow obtaining enantiomerically pure compounds. Organophosphonates are compounds of special interest because of the large scope of their biological activities [1]. Phosphonic and phosphinic acids derivatives constitute a group of both biogenic and synthetic compounds with the stable, covalent carbon to phosphorus bond. The desymmetrization of chiral phosphonate derivatives through lipase-catalyzed reaction is extensively studied [2, 3] but focused mainly on the purified enzymes. From economical point of view the use of whole-cell biocatalysts instead of isolated enzymes is further more reasonable. Thus, several fungal strains from genus Penicillium sp. Fusarium sp. Pichia sp. were screened for their ability to resolve the racemic mixture of the substrate (2-butyryloxy-2-[ethoxy-P(phenyl)]phosphinoacetic acid) under the kinetically controlled conditions. The important feature of substrate used, is the presence of two stereogenic centers in the molecule. So, in optically pure form, this compound is very attractive for further application as chiral building block in chemoenzymatic synthesis. Biocatalysts growth conditions such as availability of carbon and nitrogen source, the presence of activators and inhibitors, surfactants, oxygen tension were studied as strongly influenced both lipase production and lipase mediated hydrolysis of hydroxyphosphinic substrate. The mixture of the products was consisted of unreacted substrate and particular optical isomer of the hydrolyzed product and was analyzed by means of NMR with the addition of quinine as a chiral discriminator.
This work was financed from Project Biotransformations for pharmaceutical and cosmetics industry No.POIG.01.03.01-00-158/09 part-financed by the European Union within the European Regional Development Fund for the Innovative Economy. ______________ Reference [1] Kafarski P., Lejczak B., 2001, Curr Med Chem.-Anti-Cancer Agents, 1, 301-312 [2] Majewska P., Lejczak B., Kafarski P., 2010, Phosp Sulf Silic, 185(9), 1915-1920 [3] Majewska P., Kafarski P., Lejczak B., 2006, Tetrahedron Asymm, 17(20) 2870-2875
186
Controlling regioselectivity in the enzymatic synthesis of acyl- and glucosyl-derivatives of resveratrol
P. Torresa, A. Povedab, J. Jimnez-Barberoc, J. L. Parrad, F. Comellesd, A.O. Ballesterosa and F.J. Ploua. a Instituto de Catlisis y Petroleoqumica, CSIC, 28049 Madrid, Spain; bServicio Interdepartamental de Investigacin, UAM, 28049 Madrid, Spain; cCentro de Investigaciones Biolgicas, CSIC, 28040 Madrid, Spain; dInstituto de Qumica Avanzada de Catalua, CSIC, 08034 Barcelona, Spain E-mail: pamela@icp.csic.es Resveratrol (trans-3,5,4-trihydroxystilbene) is one of the most promising polyphenolic antioxidants in the pharmacological field, which is highlighted by around 1,100 published articles in 2010. This phytochemical compound, found in grapes and wine, is biosynthesized in response to pathogenic attack or stress conditions. It possesses a variety of biological activities: anti-inflammatory, cardio- and neuroprotective, immunomodulator, SIRT-1 activator, etc. Thus it is a suitable target to develop more potent preventive drugs against aging. Polyphenols exhibit poor absorption, resulting in very low concentrations in the body. The modification of physicochemical properties such as solubility and partition coefficient by acylation or glycosylation of the phenolic groups seems to exert a positive influence on the entry of polyphenols into enterocytes and, in consequence, on their absorption. After that, polyphenols are conjugated with glucuronic acid or sulphate in the liver. Acylation or glycosylation may also exert other benefits for the application of resveratrol, including better formulation, protection from oxidation by masking phenolic groups, and thermal resistance. In the present work, we report the enzymatic synthesis of various -glucosyl resveratrol derivatives by a transglucosylation reaction catalyzed by cyclodextrin glucanotransferases (CGTases, EC 2.4.1.19). In addition, resveratrol was regioselectively transacylated with vinyl esters in the presence of several immobilized lipases. The compounds, most of them novel, were characterized using MS and NMR. Their antioxidant and surfactant properties were evaluated. The glucosides showed a remarkable surfactant activity which could be useful to include them in different delivery systems and administration forms. Our results suggest that the regioselectivity of the process can be controlled by an adequate selection of the biocatalyst. Such modifications yielded derivatives bearing different acyl- or glucosyl- groups in different positions, which may beneficially affect their bioavailability and/or pharmacological properties.
________________ [1] Regioselective lipase-catalyzed synthesis of 3-O-acyl-derivatives of resveratrol and study of their antioxidant properties. P. Torres, A. Poveda, J. Jimnez-Barbero, A. Ballesteros and F.J. Plou. Journal of Agricultural and Food Chemistry, 58, 807-813 (2010) [2] Enzymatic synthesis of -glucosides of resveratrol with surfactant activity. P. Torres, A. Poveda, J. Jimnez-Barbero, J. L. Parra, F. Comelles, A.O.Ballesteros and F.J. Plou. Advanced Synthesis and Catalysis, accepted (2011).
187
Unusual reactions catalysed by OYEs: bio-nef-reaction, oxazete-formation and C=C-bond isomerisation
a
Durchschein K.a, Fabian W. M. F.a, Zangger K.a, Macheroux P.b, Faber K.a Department of Chemistry, Organic & Bioorganic Chemistry, Karl-Franzens-University, Heinrichstr. 28/II, 8010 Graz, Austria; b Department/Institute of Biochemistry, University of Technology, Petersgasse 12/2, 8010 Graz, Austria E-mail: durchsck@stud.uni-graz.at Flavoproteins from the OYE-family are well-known for their ability to catalyse the asymmetric reduction of activated C=C-bonds[1]. Surprisingly, we discovered three novel reactions, which demonstrate the broad catalytic promiscuity of these flavoproteins: (A) The bioreduction of nitroalkene (E/Z)-1 using PETN-, morphinone-reductase and OPR3 did not stop at the (expected) nitroalkane 2a, but proceeded further via the nitrosoalkane, which tautomerised to oxime 2b. The latter was further reduced to the imine, which (spontaneously) hydrolysed to aldehyde 2c. Overall, this pathway constitutes a biocatalytic equivalent to the Nef-reaction[2]. (B) On the other hand, reduction of (E/Z)-1 using XenA gave nitrosoalkene 3a, which (spontaneously) cyclised to rac-3b. Retro-[2+2]-cycloaddition at elevanted temperatures gave ketone 3c and HCN.
In addition to the published DKR of phenylalkanols, the substrate scope is currently being increased to include functionalized R-groups, that can be used in further transformations subsequent to the DKR.
________________ [1] a) Magnusson, A. O.; Takwa, M.; Hamberg, A.; Hult, K. Angew. Chem. Int. Ed. 2005, 44, 45824585. b) Magnusson, A. O.; Rotticci-Mulder, J. C.; Santagostino, A.; Hult, K.ChemBioChem 2005, 6, 1051-1056. [2] Vallin, M.; Syrn, P.-O.; Hult, K. ChemBioChem 2010, 11, 411-416. [3] Engstrm, K.; Vallin, M.; Syrn, P.-O.; Hult, K.; Bckvall, J.-E. Org. Biomol. Chem. 2011, 9, 81-82.
(C) Bioreduction of ,\-unsaturated lactones 4a and 4c by NCR, EBP1 and YcnD gave the expected saturated lactone 4b with high stereoselectivity. In contrast, OYE1, OYE2, KYE and XenA catalysed the isomerisation of 4a to furnish 4c as the major product. Alkene isomerisation catalysed by flavoproteins is a rare phenomenon, whose mechanism is so far unclear[3].
This study was performed within the DK Molecular Enzymology and financial support by the FWF (project W9) is gratefully acknowledged.
_____________________
[1] Stuermer R., Hauer B., Hall M., Faber K. (2007) Curr. Opinion Chem. Biol. 11, 203. [2] Durchschein K., Ferreira-da Silva B., Wallner S., Macheroux P., Kroutil W., Glueck S. M., Faber K. (2010) Green Chem. 12, 616. [3] Thibodeaux C. J., Chang W., Liu H. (2010) J. Am. Chem. Soc. 132, 9994.
October 2-6, 2011
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Effect of reaction conditions and enzyme source on the selectivity of lactulose synthesis
Cecilia Guerreroa, Carlos Veraa, Francisco Ploub, Andrs Illanesa. Pontificia Universidad Catlica de Valparaso, Av. Brasil 2950, Valparaso, Chile. b Institute of Catalysis and Petroleum Chemistry, CSIC, 28049 Madrid, Spain E-mail: aillanes@ucv.cl
a

Enzymatic diastereo- and enantioselective synthesis of -alkyl-,dihydroxyketones
Pier Paolo Giovannini, Paola Pedrini, Valentina Venturi, Alessandro Medici Dipartimento di Biologia ed Evoluzione,Universit di Ferrara, C.so Ercole I dEste 32, 44121 Ferrara, Italy E-mail: gvnppl@unife.it Enantiopure tertiary alcohols are very valuable building blocks for the synthesis of many different natural products and pharmaceuticals. Recently we reported the syntheses of chiral and prochiral -alkyl--hydroxy--diketones catalyzed by acetylacetoin synthase (AAS) from Bacillus stearothermophilus.[1] This approach, together with the recently proposed use of YerE,2 represents the first example of an asymmetric aldehyde-ketone carboligation reaction catalyzed by a ThDP-dependent enzyme affording chiral tertiary alcohols. In this communication a fully enzymatic strategy for the preparation of optically pure -alkyl-,-dihydroxyketones is reported. A set of -alkyl--hydroxy--diketones 2a-g and 3b-d, obtained by acetylacetoin synthase (AAS) catalyzed homo- and cross-coupling of -diketones 1a-f, have been regio- and enatioselectively reduced with diacetyl(acetoin) reductase (DR) to -alkyl-,-dihydroxy ketones 4a-e and 4g. AAS and DR have been obtained from the same bacterial source and used in a crude form (Scheme).

Inuence of Ionic Liquids on Enzymatic Synthesis of -Galactosidases: a Surface Plasmon Resonance and Molecular Modeling Analysis.
Manuel Sandovala, Carlos Bayna, Sergio Navarretea, lvaro Corts-Cabrerab, Pedro Lozanoc, Jos Berenguerb, Jos V. Sinisterraa,d, Mara J. Hernaiza,d* a Department of Pharmaceutical and Organic Chemistry, Complutense University of Madrid, Campus de Moncloa, 28040, Madrid, Spain; bCenter of Molecular Biology Severo Ochoa. Faculty of Biology. CSIC-Autonoma University, Madrid; cDepartment of Biochemistry and Molecular Biology. Faculty of Chemistry. University of Murcia. Campus de Espinardo. E-30.100. Murcia. Spain; dMolecular Interactions Unit and Biotransformations Unit, Science Park of Madrid. Campus de Moncloa, 28040, Madrid. Spain E-mail: mjhernai@farm.ucm.es The use of enzymes for catalysis in the chemical industry is of great interest as an environmentally friendly alternative. [1,2] The combination of biocatalysts with the appropriate solvents is key to the successful construction of a bioprocess. As many enzymes can catalyze reactions in organic solvents, there is much interest in the use of ionic liquids (ILs) as (co)solvents to create reaction media for such biocatalytic processes. ILs have emerged as attractive green recyclable alternative to environmentally harmful organic solvents. [1,2] The use of enzymes in ILs has proven to have many advantages, such as, high conversion rates, high enantioselectivity, and better enzyme stability. Several authors have explained this behaviour in terms of enzyme-ILs molecular interactions but to our knowledge nobody has tried to quantify them until now. In this communication we have studied for the first time the influence of ILs as co-solvents in the enzymatic synthesis catalyzed by two pure -galactosidases (TTP0042 from Thermus thermophilus and -gal-3 from Bacillus circulans. TTP0042 leads to high yields of (Gal-[14]GlcNac) in one pot process, while for -gal-3 change in the classical regioselectivity from (13) toward (16) linkages is observed. This behaviour is believed to be caused by the interactions between the enzyme and ILs that were studied by molecular modelling and quantified using surface plasmon resonance (SPR) (Figure 1). [3]
119 193
Improved biotransformation rates for hydrophobic substrates using the outer membrane protein AlkL in E. coli
Mattijs K. Julsing, Manfred Schrewe, Sjef Cornelissen, Andreas Schmid, Bruno Bhler Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund, Germany E-mail: mattijs.julsing@bci.tu-dortmund.de Whole cell biocatalysis is often hindered by a limited mass transfer of hydrophobic substrates over the outer membrane [1,2]. General approaches, such as solvent treatment and the addition of surfactants, have shown to improve the substrate availability of Gram-negative bacteria [3]. However, these strategies often lead to permeabilization of the cells, leakage of compounds out of the cell or even lysis. The development of a more selective method to increase substrate availability of hydrophobic compounds is of high interest for the development of productive processes. In this study, the use of a membrane protein was investigated in order to increase substrate availability. For the -oxyfunctionalization of methyl dodecanoate by recombinant E. coli expressing the alkane monooxygenase system AlkBGT of P. putida GPo1 low activities have been observed, which were caused by a limitation in mass transfer. Coexpression of alkL, a gene encoding for an outer membrane protein, resulted in a 67-fold increased maximal activity for the -oxyfunctionalization of methyl dodecanoate. In a two-liquid phase system with resting E. coli cells and methyl dodecanoate as organic phase a product formation rate of 87 U gcdw L-1 was achieved. The relation between the expression levels of alkL, the activity of the whole cell biocatalyst, and its stability was further investigated. Next to that, the coexpression of alkL has shown to improve the performance of the biotransformation of limonene by recombinant E. coli containing a cytochrome P450 enzyme as well. Therefore, the presence of AlkL in the outer membrane of E. coli may provide a general tool to enhance whole-cell oxyfunctionalization rates for hydrophobic substrates.
________________ [1] Doig SD et al, Enz Microb Technol (2003) 32:347-355 [2] Fontanille P and Larroche C, Appl Microbiol Biotechnol (2003) 60:534-540 [3] Chen RR, Applied Microbiology Biotechnology (2007) 74:739-738
Lactulose (4-O--D-galactopyranosyl-D-fructose) is a lactose-derived non-digestible oligosaccharide of well documented prebiotic action. It is now produced by isomerization of lactose using chemical catalysts. However, it can be synthesized by a kinetically controlled reaction of transglycosylation from lactose using fructose as galactosyl acceptor and-galactosidase as catalyst. Both fructose and lactose can act as acceptors so actually a mixture of lactulose and galacto-oligosaccharides (GOS) is produced. The hypothesis behind this work is that, depending on the enzyme source and reaction conditions, different product compositions (lactulose-GOS ratios) can be obtained at will, which is important in terms of the prebiotic effect of the mixture. Commercial -galactosidases form Aspergillus oryzae (Enzeco Fungal Lactase), Kluyveromyces lactis (Lactozym 3000 L) and Bacilus circulans (Lactoles L3) were tested. Syntheses were conducted at 40 C, pH 4.5, 6.5 and 8.5, 4/1, 2/1, 1/1, 1/2, 1/4, 1/6 and 1/8 lactose/fructose molar ratios, 40, 50 and 60 % w/w total sugars initial concentrations, and 200, 300, 400 and 600 IU/g lactose enzyme/substrate ratios. Results show that selectivity of lactulose synthesis is only affected by the enzyme source and the molar ratio of substrates. Only the enzyme from A. oryzae was able to synthesize both lactulose and GOS with significant yields and was therefore selected to determine the effect of the operational variables on the selectivity of lactulose synthesis. Varying lactose/fructose ratio an ample range of variation in lactulose/GOS ratios was obtained. At 2/1 lactose/fructose molar ratio (Figure 1a) synthesis of GOS prevailed over lactulose synthesis, while at 1/4 y 1/8 ratios GOS synthesis was reduced favoring lactulose synthesis (Figure 1b and 1c). Therefore different product compositions can be obtained at will by manipulating the initial lactose to fructose ratio, which is important since it has been claimed that mixtures of GOS and lactulose have a higher prebiotic index that the compounds alone. Acknowledgements: Work supported by Grant 110050 from Fondecyt, Chile.
Scheme The absolute configuration of enantiopure 3R,4S-dihydroxy-3-methyl-pentan-2-one 4a was determined by NOE spectroscopy on the corresponding 2,2-dimethyl-1,3-dioxolane derivatives and confirmed by its conversion to (+)-citreodiol, the antipode of the natural product. On the analogy of 4a, NOE experiments on the dioxolane derivatives, allowed to determine the configuration of the -alkyl-,-dihydroxy ketones 4b-e and 4g and accordingly of the starting chiral -methyl--hydroxy--diketones 2b-e and 2g.
________________ [1] P. P. Giovannini, P. Pedrini, V. Venturi, G. Fantin and A. Medici, J. Mol. Catal. B: Enzym., 2010, 64, 113. [2] P. Lehwald, M. Richter, C. Rhr, H. Liu and M. Mller, Angew. Chem. Int. Ed., 2010, 49, 2389
Figure 1. Effect of lactose to fructose molar ratio on product distribution at different lactose conversions (fraction of initial lactose converted into products) in the synthesis of lactulose from lactose and fructose with A. oryzae -galactosidase at 40 C, pH 4.5, 50 % w/w total sugars initial concentration, 200 IU/ g lactose, and lactose to fructose molar ratios: 2/1 (a); 1/4(b); 1/8 (c) . : lactulose; (): total GOS.
Figure 1.General procedure for the study of IL-enzyme interactions.

________________ [1] M.J. Herniz, A.R. Alcntara, J.I. Garca, J.V. Sinisterra, Chem. Eur. J., 2010, 16, 9422 9437. [2] M. Prez, J.V. Sinisterra, M.J. Herniz, Curr. Org. Chem., 2010, 14, 2366-2383. [3] M. Sandoval et al., Angew. Chem. Int. Ed., Submitted, 2011
190
One-pot enzymatic resolution of sec-alcohols
a
191
Lipase mediated synthesis of six-membered cyclic carbonates a green route to isocyanate free polycarbonate and polyurethane
Sang-Hyun Pyo, Amin Mollaahmad and Rajni Hatti-Kaul Department of Biotechnology, Lund University, Box 124, SE-221 00 Lund, Sweden E-mail: Rajni.Hatti-Kaul@biotek.lu.se Cyclic carbonates are potential monomers for an isocyanate free route to production of aliphatic polycarbonates and polyurethanes. These polymers are currently produced using toxic phosgene and/or isocyanate. Production of cyclic carbonates from polyols and dialkylcarbonate using different catalysts has been reported, however with low yields. We have investigated lipase catalysed transesterification of trimethylolpropane (TMP) and dimethylcarbonate (DMC) for the production of six-membered cyclic carbonate [1]. The products formed were TMP-esters and cyclic carbonates (Figure 1), the levels of which depended on the reaction conditions used, e.g. solvent system, temperature, TMP:DMC ratio. The reaction conditions were optimized to obtain maximum yield of TMP-monoester and the corresponding cyclic carbonate in a solvent-free system. The monoester was then converted to the cyclic carbonate in a subsequent thermal treatment step with a high overall yield. Different immobilized lipase preparations were compared with respect to catalytic activity, operational stability and cost for cyclic carbonate production.
194
The effects of organic co-solvents on the efficiency and regioselectivity of N-acetyl-lactosamine synthesis, using Bacillus circulans -galactosidase in hydro-organic media
Nicolas Bridiau, Neyssne Issaoui, Thierry Maugard UMR 6250 CNRS-ULR, LIENSS, Equipe Biotechnologie Environnementale, Universit de La Rochelle, Avenue Michel Crpeau, 17042 La Rochelle cedex 1, France. E-mail: nicolas.bridiau@univ-lr.fr N-acetyl-lactosamine (LacNAc) -D-Galp-(1-4)-D-GlcpNAc is one of the most representative core structures in oligosaccharide components of glycoproteins and glycolipids. It plays a central role in many cellular recognition phenomena and can be involved in various diseases such as infections, autoimmune and inflammatory pathologies or cancer. In particular it is known to form the common core structure of polylactosamine chains as well as the sialyl Lewis x antigen, which have been identified as tumor-associated carbohydrate antigens that are tissue-specifically expressed on carcinoma cells, and have been established as functional ligands for galectins and selectins, respectively. Sialyl Lewis x antigen is known to be involved in the transendothelial migration of some tumor cells utilizing the selectin-mediated adhesion mechanisms and is considered for these reasons as a target to elaborate compounds that could inhibit tumor cell/vascular endothelial cell interactions and would potentially have an antimetastatic activity [1]. The enzymatic synthesis of complex oligosaccharides via the use of glycosidases in non-conventional media represents an important axe of research in our laboratory. We thus studied the enzymatic synthesis of LacNAc 3b by the transgalactosylation of Nacetyl-D-glucosamine 1, catalysed by Bacillus circulans -galactosidase (Lactoles L3; EC 3.2.1.23, glycoside hydrolase family 42), starting from o-nitrophenyl--Dgalactopyranoside 2 as a galactosyl donor, in both aqueous solutions and aqueous organic solvent mixtures (Figure 1). This enzyme is mainly regioselective towards the formation of a (1-4) linkage. Nevertheless, it has been shown that its regioselectivity is still susceptible to favour the formation of a (1-6) linkage, depending on the experimental conditions used, notably the kinetic conditions [2]. Variability of glycosidase regioselectivity is a major drawback for their use in organic synthesis. The control of glycosidase regioselectivity represents for this reason a great challenge. Our work consisted in this context to examine the effects of six organic co-solvents on the regioselectivity and the yield of the synthesis [3].
195
Analysis and purification of -acyl sucrose regio-isomers by RP-HPLC with evaporaive light scattering and charged aerosol detection
Lars Haastrup Pedersena, Aleksander Liea, Sinthuwat Ritthithamb a Aalborg University, Sohngaardsholmsvej 49 DK-9000 Aalborg, Denmarky; bSilpakorn University, Nakhon Pathom 93000, Thailand E-mail: lhp@bio.aau.dk A milligram scale purification procedure obtaining seven different O-decanoyl sucrose regio-isomers, was developed using LC followed by reversed phase (RP) HPLC with ELSD [1]. The purificaion was perfomed in two steps beginning with separation of the crude reaction mixture by solid phase extraction (SPE-C18) followed by preparative C18 RP-HPLC for separation of the mono-ester regio-isomers. In the latter step improved separation was obtained by an initial stepdown in the acetonitrile concentration from 40% (v/v) to 35% (v/v) after sample injection. With the optimized gradient four dominating regio-isomers (2O-, 3-O-, 6-O- and 3-O-decanoyl sucrose) were purified in yields (w/w) of 14.2%, 34.6%, 34,5% and 43.6% respectively. While the sucrose monoester 6-O- and 6-O- regio-isomers were efficiently synthesised in lipase catalysed reactions (C. antarctica) in tertiary alcohols [2], the corresponding 2-O-, 3-O and 3-O regio-isomers were synthesised in a protease (B. pseudofirmus AL89) catalysed process using a reaction mixture of DMF and DMSO. The formation of the 2-O-regio-isomer proved to be controlled kinetically [3]. This selectivity showed to be relatively unique compared to other alkaline proteases [4]. In the purified preparations of the 2-O- and 3-O-decanoyl sucrose respectively, acyl migration was observed as a result of the final drying process with lyophilisation resulting in the highest purities (w/w) of 96% and 100% respectively. Regarding detection of O-acyl sucrose, the sensitivity of charged aerosol detection is compared to evaporative light scattering detection. The two step chromatography procedure reported here can be used to provide mg quantities of O-acyl sucrose regio-isomers in purity of 96% to 100% for structural and functional studies. Presently we are working on further scale up of the enzyme catalysed syntheses and down stream processing.
________________ 1)Ritthitham, S.; Wimmer, R.; Stensballe, A. and Pedersen, L.H. (2009) Analysis and purification of Odecanoyl sucrose regio-isomers by reversed phase high pressure liquid chromatography with evaporative light scattering detection. Journal of Chromatography A,1216: 49634967 2)Ritthitham, S.; Wimmer, R.and Pedersen, L.H. (2011) Polar co-solvents in tertiary alcohols effect initial reaction rates and regio-isomeric ratio ranging from 1.2 to 2.2 in a lipase catalysed synthesis of 6-O- and 6-O-stearoyl sucrose. Process Biochemistry 46: 931935 3)Ritthitham, S.; Wimmer, R.; Stensballe, A. and Pedersen, L.H. (2009) Selectivity and stability of alkaline protease AL-89 in hydrophilic solvents. Journal of Molecular Catalysis B: Enzymatic 59: 266-273 4)Pedersen, L.H.; Ritthitham, S. and Kristensen, M. (2009) Activity and stability of proteases in hydrophilic solvents in Modern Biocatalysis (eds. Fessner, W and Anthonsen , T) Wiley VCH, ISBN 978-352732071-4 pp 55-66
Carlos M. Monteiroa, Nuno M. T. Lourenob, Carlos A. M. Afonsoa iMed.UL, Faculdade de Farmcia da Universidade de Lisboa, Av. Prof.Gama Pinto, 1649-003 Lisboa, Portugal b IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Tcnico, Av. Rovisco Pais,1049-001 Lisbon, Portugal E-mail: nmtl@ist.utl.pt Over the last years we have been pursuing the development of attractive, competitive and more environmentally friendly processes for the enzymatic- resolution of secondary alcohols. In this line, our effort have been made on the development of new strategies for the one-pot resolution-separation of free sec-alcohols by the use of new acylating agents.[1] The resolution-separation is based on the selective reaction of one alcohol enantiomer with acylating agent, where one enantiomer stays in medium, leaving the other enantiomer free to be removed. The anchored enantiomer can be isolated by a second enzymatic reversible reaction (Figure 1). With this methodology is possible to obtain both free enantiomers using only the biocatalyst and a sustainable acylating agent. The great advantage of this approach is the possibility to circumvent the limitations of the common existing technology, specifically the use of chromatography separations, the use of organic solvents and post-chemical transformations for the isolation of free enantiomers. This methodology come out to be quite simple, robust and reliable allowing the reuse of the medium and enzyme. Herein is present the progresses achieved on the different strategies for the enzymatic one-pot resolution-separation of several sec-alcohols.
Figure 1.
Acknowledgments: We thank Fundao para a Cincia e Tecnologia (POCI 2010), FEDER (SFRH/ BD/48395/2008 and SFRH/BPD/41175/2007, PTDC/EQU-EQU/104552/2008) and ACS Green Chemistry Institute (Ref. GCI-PRF#49150-GCI for financial support. ________________ [1] Loureno, N.M.T., Afonso, C. A. M. Angew. Chem. Int. Ed., 2007, 46, 8178; Monteiro, C.A.; Loureno N. M. T.; Afonso C.A.M., Tetrahedron: Asymmetry, 2010, 952-956; Loureno N.M.T, Monteiro C.M., Afonso C.A.M, Eur. J. Org. Chem., 2010, 69386943.
Figure 1. Lipase mediated reaction between TMP and dialkylcarbonate

Acknowledgements: Financial support from the Swedish Foundation for Strategic Environmental Research (Mistra) and Perstorp AB is gratefully acknowledged. ________________ [1] S-H. Pyo, P. Persson, S. Lundmark and R. Hatti-Kaul (2011) Solvent-free lipase-mediated synthesis of six-membered cyclic carbonates from trimethylolpropane and dialkylcarbonates. Green Chemistry DOI:10.1039/C0GC00783H _______________ [1] Bay S and Freire T, Rev Francoph Lab., 2006; 381:39-46. [2] Vetere A and Paoletti S, Biochem Biophys Res Commun., 1996; 219:6-13. [3] Bridiau N, Issaoui N and Maugard T, Biotechnol. Prog., 2010; 26, 1278-1289.
Figure 1. Enzymatic synthesis of LacNAc 3b using Bacillus circulans -galactosidase.
October 2-6, 2011
120 196
Substrate mimetics for papain: dipeptide synthesis & computational rationale
Roseri J. A. C. de Beera, Sander B. Nabuursb, Timo Nuijensc, Peter J. L. M. Quaedfliegc, and Floris P. J. T. Rutjesa a Institute for Molecules and Materials, Radboud University Nijmegen, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands b Center for Molecular and Biomolecular Informatics, NCMLS, Radboud University Nijmegen Medical Centre, P.O. Box 9101 6500 HB Nijmegen , The Netherlands c DSM Innovative Synthesis BV, P.O. Box 18 NL-6160 MD Geleen, The Netherlands E-mail: r.debeer@science.ru.nl The importance of peptides in the fields of health care and nutrition probably renders the amide bond the most synthesised one of all chemical bonds. However, to date no universal enzymatic method is available to synthesise these peptidic amide bonds. Enzymatic hydrolysis of the peptide product during the coupling reaction and the limited recognition of many proteinogenic and non-proteinogenic amino acids, restrict such a universal application. In the substrate mimetics strategy the latter problem is overcome by incorporation of the recognition part in the ester leaving group, instead of in the side chain of the amino acid (Figure 1) [1].

Substrate imprinting as a way to enhance lipase enantioselectivity
Graber M.a, Balloumi A.a,Chaput L a and Marton Z.a a Universit de La Rochelle, UMR 6250 LIENSs CNRS-ULR, Avenue Michel Crpeau, 17042 La Rochelle, France b Laboratoire de Biotechnologie, Biocatalyse et Biorgulation, UMR 6204 du CNRS, Facult des Sciences et des Techniques, 2, rue de la Houssinire, 44322 Nantes Cedex 3, France E-mail: marianne.graber@univ-lr.fr Lipase enantioselectivity is strongly influenced by substrate structure. In the case of lipasecatalyzed resolution of racemic alcohols via transesterification, it has been shown that structures of both acyl donor (first substrate) and alcohol to be resolved (second substrate) have an influence on enantioselectivity. As in the second tetrahedral intermediate, the acyl part and chiral alcohol moieties are brought close in space in the active site, it is normal for acyl structure to have an effect on the selectivity of the reaction. However, there is no obvious reason, why the alkyl part of the acyl donor, which is the leaving group during first reaction step, should influence the enantiospecificity. Nevertheless different enantioselectivities have been obtained when varying the alkyl part alone of acyl donor, even if the reaction proceeds via the same acyl-enzyme. From these studies, it appears that the acyl donor bearing the leaving group structurally matching with the substrate which has to resolved, enhances enzyme activity and selectivity [1,2]. The explanation could be that, in the first tetrahedral intermediate, the enzyme would lightly mould its active site structure around first substrate.(=imprinting phenomenon) This change could be maintained in the acyl-enzyme intermediate and therefore it could display a greater enantioselectivity towards second substrate. We have investigated the ability of acyl donor, whose leaving group mimics second substrate which has to be resolved, to enhance Candida antarctica lipase B enantioselectivity. The experiments have been performed in continuous solid/gas reactor, both in presence of the imprinting compound in gaseous stream and with enzyme co-lyophilized with this imprinting compound. In some cases, in particular with chiral esters, improved enantioselectivity was obtained.

Magnetic cross-linked enzyme aggregates (cleas): pushing the limits of enzyme recovery and recycling
Betti Kondor, Menno Sorgedrager & Roger A. Sheldon CLEA Technologies BV, Delftechpark 34, 2628XH Delft, the Netherlands E-mail: r.sheldon@clea.nl An effective method for enzyme immobilization, thereby increasing operational stability and facilitating recovery and recycling, is often a conditio sine qua non for commercial viability of a biotransformation. Immobilization methods can conveniently be divided into three types: binding to a support (carrier), encapsulation, for example in a polymeric gel, or via cross-linking techniques. A distinct disadvantage of carrier-bound enzymes is the dilution of catalyst productivity resulting from the introduction of a large proportion of non-catalytic mass, often 90 to >99% of the total mass. Cross-linking of enzyme molecules does not suffer from this disadvantage. Cross-linked enzyme aggregates (CLEAs) are obtained by precipitating the enzyme from aqueous solution and cross-linking the resulting physical aggregates of enzyme molecules with e.g. glutaraldehyde. CLEAs have many advantages in the context of industrial applications [1]. The method is inexpensive and applicable to a broad range of enzymes [2-4], including crude preparations, affording industrial biocatalysts with low or no allergenicity and enhanced stability towards denaturation by heat, organic solvents and autolysis. Furthermore, they are stable towards leaching in aqueous media. and readily recycled via simple filtration or centrifugation. The technique can also be applied to the preparation of combi CLEAs containing two or more enzymes for use in enzymatic cascade processes. In a more recent extension of the CLEA technology, we have developed magnetic CLEAs consisting of a three-dimensional network of cross-linked aggregated enzyme particles and magnetic nanoparticles. We have shown that there is no change in structure or loss of activity as a result of the introduction of the magnetic particles and the magnetic strength of the mCLEA can be fine tuned for a particular application. The technique can, in principle, be applied to any enzyme. An important additional benefit of mCLEAs in the context of commercial applications is that separation is easily achieved by magnetic decantation or in a magnetically stabilized fluidized bed. In this lecture we shall present examples of mCLEAs prepared from, inter alia, lipases, glucose oxidase and catalase. We envisage potential applications in the pharmaceutical and food and feed industries and in diagnostic devices.
________________ [1] For recent reviews see R. A. Sheldon, Org. Proc. Res. Dev. (2011), 15(1), 213-223; R. A. Sheldon, Biochem. Soc. Trans. (2007), 35 (6) 1583. [2] D. I.Perez, F. van Rantwijk and R. A. Sheldon, Advan. Synth. Catal. (2009), 351, 2133. [3] S. van Pelt, S. Quignard, D. Kubac, D. Y. Sorokin, F. van Rantwijk, and R. A. Sheldon, Green Chem. (2008) 10, 395. [4] F. Cabirol, P. Loo Tan, B. Tay, S. Cheng, U. Hanefeld and R. A. Sheldon, Advan. Synth. Catal. (2008), 350, 2329.
121 201
Engineering of highly active and easily recyclable nanobiocatalysts via reversible occulation: efficient enantioselective reduction of ketone with cofactor recycling
Thao Phuong Nguyen Ngo, Wen Wang, Wei Zhang, and Zhi Li* Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576 E-mail: g0800030@nus.edu.sg Enzymes are widely used for chemical and pharmaceutical production. However, the practical application of isolated enzymes is hampered by the unsatisfied stability and high cost. These limitations can be overcome by using enzyme immobilization technology which helps to enhance enzyme stability and enable the recycling of enzymes, thus significantly decreasing the production cost. Compared to micrometric supports, nanoparticles appear to be promising due to high catalyst loading and less mass transfer limitation. Nevertheless, the main difficulty is to recycle these nanobiocatalysts. Even when magnetic nanoparticles (MNPs) are used, the separation is still very difficult. Here we develop a general and facile approach for fabrication of highly active and easily recyclable nanobiocatalysts via reversible flocculation. As an example, alcohol dehydrogenase was chosen to demonstrate this concept with regeneration of the costly co-factor for practical biotransformation. This enzyme was covalently immobilized on stable MNPs and then flocculated in phosphate buffer (7mM, pH8) to form flocs of nanobiocatalysts (FNB). The FNB was dissociated by shaking and showed the same high activity and >99% enantioselectivity as the free enzyme. The catalyst was highly stable in wide range of pH and reaction temperature and easily separated by magnet in a few seconds. The FNB remained 80% of its original activity after 14 times of cycling and reuse in the bioreduction of 7-methoxy-2-tetralone, producing totally 125 mM valuable product with high TTN of co-factor recycling. This system is expected to find applications in many important catalytic processes.
Figure 1. Principle of the substrate mimetics strategy. The guanidinophenyl ester (OGp), a known substrate mimetic for trypsin and chymotrypsin [2], is now applied for the first time in combination with papain. Furthermore, efforts have been undertaken to find simpler and more cost-efficient substrate mimetics, which are not prone to spontaneous hydrolysis and can be used on industrial scale. A small set of new potential mimetics has been designed, synthesised and evaluated for dipeptide formation. The scope of the reaction, the influence of the amino acid and the nucleophile have been investigated. The experimental results have also been rationalised by computational studies.
________________ [1] Schellenberger, V., Jakubke, H.-D., Zapevalova, N. P. and Mitin, Y. V., Biotechnol. Bioeng. 38 (1991) 104. [2] Bordusa, F., Curr. Protein Pept. Sci. 3 (2002) 159.
_________________
[1] Gonzalez-Sabin J., Gotor V., Rebolledo F. (2004) Tetrahedron Asymmetry 15(3) 481-488. [2] Lee D., Choi Y. K., Kim M.-J. (2000) Organic Letters 2(16) 2553-2555.
Figure 1. Reduction of ketone with cofactor recycling catalyzed by the flocs of nanobiocatalysts (FNB)
198
Microbial transformation of xanthohumol as a method of increasing the antioxidant properties
Tomasz Tronina, Ewa Huszcza, Agnieszka Bartmaska, Jarosaw Poposki Department of Chemistry ,Wrocaw University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocaw, E-mail: tomasz.tronina@gmail.com Microbial transformation of xanthohumol (1), isolated from agro-residue (spent hops), by Aspergillus ochraceus was investigated. A new aurone, (Z)-2-(2-hydroxyisopropyl)dihydrofurano-[2,3-6,7]-3,4-dihydroxy-4-methoxyaurone (2), was obtained as a main transformation product. Three minor metabolites were identified as 2-(2-hydroxyisopropyl)- dihydrofurano-[2,3:3,4]-2,4-dihydroxy-6-methoxychalcone (3), (2S,2S)-2-(2-hydroxyisopropyl)-dihydrofurano-[2,3:7,8]-4-hydroxy-5-methoxyflavanone (4a) and (2S,2R)-2-(2-hydroxyisopropyl)-dihydrofurano-[2,3:7,8]-4hydroxy-5-methoxyflavanone (4b). Their structures were elucidated on the basis of spectroscopic evidences. The antioxidant properties of xanthohumol (1) and its metabolites were investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. The major biotransformation product, was 8.6-fold stronger antioxidant than xanthohumol (1) and 2.3-fold then ascorbic acid.
199
Production and application of an His-tagged dehydrogenase
Daniela Quaglia*, Francesca Paradisi Center for Synthesis and Chemical Biology, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland E-mail: daniela.quaglia@ucd.ie
202
Employing -transaminases in different reaction media
Francesco G. Muttia, Christine S. Fuchsa,b, Desiree Pressnitza, Johann H. Sattlera, Wolfgang Kroutila a Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria; bACIB GmbH, Department of Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria. E-mail: francesco.mutti@uni-graz.at -Transaminases are pyridoxal 5-phosphate (PLP)-dependent enzymes, which employ the cofactor PLP to transfer ammonia and electrons from an amino donor to a ketone acceptor. These enzymes received recently a tremendous attention, leading to the launch of an industrial process.[1] The advantage of using -transaminases compared to chemocatalysis stems from the milder reaction condition (i.e. atmospheric pressure, ambient temperature), broad substrate specificity, high enantioselectivity and the absence of heavy metals. However, the equilibrium for the formal asymmetric reductive amination lies significantly on the side of the ketone (Keq. 10-4 10-5), thus an excess of alanine is used (5 eq.) and simultaneously the co-product pyruvate is removed employing either LDH or AlaDH (Fig. 1a).[2] Continuing our studies, various ketones were converted to chiral amines with excellent conversion and enantioselectivity (>99% ee), using R or S selective -transaminases of known amino acid sequence. Some -transaminases demonstrated to tolerate organic solvents as sole reaction medium. For instance, employing a wild type -transaminase from Arthobacter sp. the reductive amination of methoxy-acetone was carried out in ethyl-acetate affording the corresponding amine in quantitative conversion and optically pure form. Only 2 equivalents of donor were used without the need to shift the equilibrium (Fig. 1b). Hence, the amination in nonaqueous media enables: i) a more favourable equilibrium, ii) higher substrate solubility, iii) the use of alternative amino donors, iv) an easier work-up and recycle of the enzyme.
O
203
Biocatalytic synthesis of -hydroxy--keto esters and ,-dihydroxy esters
Anna Bariotaki, Dimitris Kalaitzakis, Ioulia Smonou, Michael Orfanopoulos Department of Chemistry, University of Crete, Division of Organic Chemistry, Heraklion 71003, Crete, Greece orfanop@chemistry.uoc.gr Asymmetric reduction of ketones is particularly important, since chiral alcohols are present in a wide spectrum of pharmaceutical and other high value compounds, or can be used as chiral building blocks in the synthesis of larger more complicated compounds. Optically active ,- dihydroxy esters represent important classes of chiral intermediates that have been used in the synthesis of many natural products and pharmaceuticals.[1,2] Being interested in the application of biocatalytic methods for the synthesis of optically active compounds with biological activity, our group has developed a method for the preparation of optically active keto alcohols, diols and hydroxy esters with high chemical yield and high optical purity.[3,4] NAD(P)H-dependent ketoreductases (KREDs) were used as chemo-, stereo-, and regioselective catalysts, which can reduce a wide range of ketones and have been proved very efficient catalysts. In this contribution, we will present our new results for the efficient synthesis of optically pure -hydroxy--keto esters and ,-dihydroxy esters by the use of ketoreductases. The biocatalytic reduction of tert-butyl 3,5-dioxo-hexanoate led to the formation of all possible single stereoisomers in each case.
Figure 1. EE-HLADH One of the main reasons for working with a polyhistidine-tagged enzyme is the access to the IMAC technology (Immobilized Metal Chelating Chromatography). Nevertheless, this is not the only advantage. In our work we use the His-tag technology for immobilizing Horse Liver Alcohol Dehydrogenase (HLADH) onto a solid support.[1] For an enzyme such as HLADH, which is already used in biocatalysis, the obtainment of an immobilized form is of paramaount importance. In fact, this allows for the recovery of the enzyme from the reaction mixture and it can help to improve its stability in organic solvents, which are needed for solubilizing the majority of the substrates of interest.[2,3] Figure1. Metabolites formed by Aspergillus ochraceus AM456 (2, 3, 4a and 4b) from xanthohumol (1). Acknowledgement This work is a part of research supported by Polish Ministry of Science and Higher Education, project N N312 149438. This poster will illustrate our work focused on improving the production and purification conditions of the His-tagged HLADH with the further aim of immobilizing the enzyme onto derivatized insoluble polymeric beads (resins kindly provided to us by Resindion S.R.L).
________________ [1] J.M. Guisan, Immobilization of enzymes and cells, 2006, 11, 117-128. [2] D.Giacomini, P. Galletti, A. Quintavalla, Chem Comm, 2007, 4038-4040. [3] D.Giacomini, P. Galletti, E. Emer, G. Gucciardo, A. Quintavalla, M.Pori, Org and Biom. Chem., 2010, 8, 4117-4123.
a)
-Transaminase, PLP1 mM Pi buff er,p H7 ,1 00mM,3 0C, 24 h with/without DMSO 15%, v:v R Alanine( 5e q.) AlaDH Pyruvate NH4 +
NH2 R * R LDH Lactate
50 mM
H 2O
LDH-System NAD + NADH
AlaDH-System NAD+ NADH
b)
O O
-Transaminase-PLP EtOAc, RT,2 4h
NH2 O conv.> 99% ee >99%
______________
References [1] Quirk, J.; Thornton, M.; Kirkpatrick, P. Nat. Rev. Drug Discovery 2003, 2, 769. [2] Kumar, R. N.; Meshram H. M. Tetrahedron Lett. 2011, 52, 1003 1007. [3] Kalaitzakis. D, Rozzell, J. D.; Kambourakis, S.; Smonou I. Org. Lett 2005, 7, 4799. [4] Kalaitzakis, D.; Rozzell, J. D.; Smonou, I.; Kambourakis S. Adv. Synth. Catal. 2006, 348, 1958.
Amino-donor( 2e q.)
Co-product
________________ [1] a) C. K. Sevile et al., Science 2010, 329, 305-309; b) F. G. Mutti et al., ChemCatChem 2011, 3, 109-111. [2] a) D. Koszelewski et al., Angew. Chem. Int. Ed. 2008, 47, 9337-9340; b) D. Koszelewski et al., ChemCatChem. 2010, 2, 73-77.
Figure 1. Strategies for formal asymmetric reductive amination using -transaminases
October 2-6, 2011
122 204
Production of galacto-oligosaccharides by the -galactosidase from Kluyveromyces lactis: comparative analysis of permeabilized cells versus soluble enzyme
B. Rodriguez-Colinasa, M.A. de Abreub, L. Fernandez-Arrojoa, R. de Beera, A. Povedac, J. Jimenez-Barberod, D. Haltriche, A.O. Ballesterosa, M. Fernandez-Lobatob and F.J. Ploua a Instituto de Catlisis y Petroleoqumica, CSIC, 28049 Madrid, Spain; bCentro de Biologa Molecular Severo Ochoa (UAM-CSIC), 28049 Madrid, Spain.; cServicio Interdepartamental de Investigacin, Universidad Autnoma de Madrid, 28049 Madrid, Spain; d Centro de Investigaciones Biolgicas, CSIC, 28040 Madrid, Spain; eFood Biotechnology Laboratory, BOKU University of Natural Resources and Applied Life Sciences, Vienna, Austria E-mail: fplou@icp.csic.es Under appropriate conditions, -galactosidases catalyze transgalactosylation reactions in which lactose (as well as the glucose and galactose produced in the normal hydrolytic reaction) serve as galactosyl acceptors yielding a series of di, tri- and tetrasaccharides (and eventually of higher polymerization degree) called galacto-oligosaccharides (GOS). In this work, the transgalactosylation activity of Kluyveromyces lactis cells was studied in detail. Cells were permeabilized with ethanol and further lyophilized to facilitate the transit of substrates and products. The resulting biocatalyst was assayed for the synthesis of GOS and compared with two soluble -galactosidases from K. lactis (Lactozym 3000 L HP G and Maxilact LGX 5000). Using 400 g/l lactose, the maximum GOS yield measured by HPAEC-PAD analysis was 177 g/l (44% w/w of total carbohydrates). The major products synthesized were the disaccharides 6-galactobiose [Gal-(16)-Gal] and allolactose [Gal-(16)-Glc], as well as the trisaccharide 6-galactosyl-lactose [Gal-(16)-Gal-(14)-Glc], which was characterized by MS and 2D-NMR. Structural characterization of another synthesized product was carried out, and found to be the disaccharide Gal-(13)-Glc. GOS yield obtained with soluble -galactosidases was slightly lower (160 g/l for Lactozym 3000 L HP G and 154 g/l for Maxilact LGX 5000); however, the typical profile with a maximum GOS concentration followed by partial hydrolysis of the newly formed oligosaccharides was not observed with the soluble enzymes. Results were correlated with the higher stability of -galactosidase when permeabilized whole-cells were used.

Immobilization of Bacillus circulans -galactosidase in glyoxyl-agarose at different protein loadings for galacto-oligosaccharides synthesis
Paulina Urrutiaa, Nicole Faura, Erick Arayaa, Lorena Wilsona, Andrs Illanesa a Pontificia Universidad Catlica de Valparaso, Av.Brasil 2147 Valparaso, Chile. E-mail: aillanes@ucv.cl Galacto-oligosaccharides (GOS) are non-digestible lactose-derived oligosaccharides, recognised as prebiotics, which can be produced from lactose with -galactosidases from different microorganisms. Enzymes are inherently labile catalysts being immobilization an attractive alternative for the production of robust biocatalysts for industrial processes. Besides operational advantages associated with the use of an insoluble catalyst, the development of stable and high loaded biocatalysts may have a strong impact on processing costs. However, high protein loading may affect immobilized enzyme kinetic, as a consequence of the strong impact of mass transfer limitations, affecting biocatalyst performance. The purpose of this work is the evaluation of the effect of the enzyme load of Bacillus circulans -galactosidase immobilized in glyoxyl-agarose, in the production of a biocatalyst appropriate for industrial GOS production. Biocatalysts stability, reactivation, lactose conversion yield, and specific productivity of GOS were evaluated. Three different protein-loaded biocatalysts were produced, corresponding to 10, 44 and 75% of the maximum capacity of protein loading in glyoxyl-agarose. Protein immobilization yields were 89.7, 86.6 and 83.1%, whilst enzyme immobilization yields expressed in terms of activity of synthesis were 79.4, 51.5 and 46.1% respectively. One international unit of -galactosidase activity of synthesis (IU) was defined as the amount of enzyme producing 1 mol of transgalactosylated galactose per minute from 50% w/w lactose solution in 0.1 M citrate-phosphate buffer pH 6 at 60C. Specific activity of synthesis for each biocatalyst was 615, 1793 and 2844 IU/g, respectively. Stability at 60C was evaluated showing that biocatalyst thermal stability decreased with protein loading (half life of each biocatalyst was 633, 134 and 90 min, respectively). These results may be explained by a decrease of multipoint covalent interaction between enzyme and support. However, it should be highlighted that thermal stability was done under non reactive conditions and therefore does not predict biocatalysts behaviour under reactive conditions. Biocatalysts reactivation was assessed for catalysts inactivated down to 25% residual activity at 60C. Each derivate was reactivated by two strategies: direct incubation in aqueous media at 30C and incubation in the same conditions after unfolding with 8 M guanidine. In all cases, better results were obtained in the second strategy and the level of recovered activity was inversely correlated with protein loading, highest reactivation (more than 60% of initial activity) being obtained with the less protein loaded biocatalyst. GOS production with each biocatalyst was evaluated, lactose conversion yield into GOS not varying with protein loadings, which is in agreement with the yield reported for the free enzyme (40% conversion yield of GOS at 60% lactose conversion), suggesting that diffusion limitations were not significant even for the high protein loaded biocatalysts. Specific productivity of GOS were 2.4, 6.5 and 11.3 gGOS/(g.h) respectively with the biocatalysts at the three different protein loadings. It can be concluded that protein loading in B. circulans -galactosidase immobilized in glyoxyl-agarose, does not alter GOS production profile, offering the advantage of higher specific productivities. When enzyme reactivation is considered, use of a lower protein loaded biocatalyst may be advantageous.
Acknowledgements: Work funded by Grants 1100050 and 1100323 from Fondecyt, Chile. Support from Conicyt, Chile, for a PhD fellowship to Ms. Urrutia is acknowledged.

Transesterification reaction of boron-containing carboxylic esters mediated by lipase
Leandro H. Andrade and Joel S. Reis Institute of Chemistry, University of So Paulo, Av. Prof. Lineu Prestes, 748, So Paulo, SP, Brazil. CEP: 05508-000 E-mail: jsreis@usp.br The chemistry of boron-containing compounds is an area of growing interest, mainly due to the wide application of these compounds in the formation of new carbon-carbon bonds. [1] Both chiral and achiral boron compounds are useful building blocks in organic synthesis and can be prepared by several synthetic methodologies. On the other hand, enzymemediated processes are considered elegant and useful methodologies for anantiosselective transformations. The use of lipases in organic synthesis is well established and has become an interesting area for organic chemists.[2] Therefore, enzymatic routes to achieve boron compounds enantiomerically enriched are very rare. To our best knowledge, there is only one report in the literature, published by our group,[3] containing that purpose. Herein, we present the results about the transesterification reaction of boron-containing carboxylic esters catalyzed by lipase (Table 1).
Bpin CO2R1 1 (0.05 mmol) R2OH, 24 h CAL-B (10 mg) "Condit ions" Bpin
123
CO2R1
Bpin
12
CO2R2
Bpin =
O O
Table 1 Transesterification reaction of boron-containing carboxylic esters mediated by lipase. Several reaction conditions were tested at 30 C, such as the use of different alcohols (MeOH, EtOH, BnOH, n-BuOH, OctOH), however, we have no observed the desired reaction (entries 1-3). On the other hand, by using PTSA as additive a good conversion was observed, but the achievement of racemates have revealed a non-enzymatic process. (entry 4). When the reaction was carried out at 70 C, after 24 hours, we have detected an enzymatic transesterification reaction leading the formation of optically pure product. By employing more concentrated media, higher conversion was obtained and the enantiomeric purity of the product was preserved (entries 5-6).In summary, we have shown an enzymatic route to achieve optically active boron-containig carboxylic esters.
________________ 1 For reviews, see: (a) Martin, R.; Buchwald, S. L. Acc. Chem. Rev. 2008, 41, 1461. (b) Molander, G. A.; Ellis, N. Acc. Chem. Rev. 2007, 40, 275. (c) Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 24. 2 Ghanem, A. Tetrahedron, 2007, 63, 1721. 3 Andrade, L. H.; Barcellos, T. Org. Lett. 2009, 11, 3052.
206
Penicillium citrinum immobilized on chitosan efficiently catalyse the reduction of ketones
Lenilson C. Rocha, Paulo S. C. Filho, Andr L. M. Porto Universidade de So Paulo, Instituto de Qumica de So Carlos, Av. Trabalhador Socarlense, 400, 13560-970, So Carlos, So Paulo, Brazil E-mail: almporto@iqsc.usp.br Chitosan is a N-deacetylated derivative of chitin - copolymer built of -(1,4)-2-amino-2deoxy--D-glucose and -(1,4)-2-acetamido-2-deoxy--D-glucose units [1]. Biocatalytic processes can take advantage of the habitat-related properties of marine enzymes, such as salt tolerance, hyperthermostability, barophilicity, cold adaptivity, and so on, while also taking into consideration substrate specificity and affinity [2]. The purpose of the present study is to evaluate the potential use of Brazilian marine fungus Penicillium citrinum F53 immobilized on chitosan for the stereoselective reduction of -chloroacetophenone (1) and p-methoxyacetophenone (2). The strain of P. citrinum was cultivated in liquid culture medium (1 L) agitated at 130 r.p.m., for 96 h at 30 C [3]. The mycelium of the strain was harvested by Buchner filtration. The fungal mycelium was immobilized in a 250 mL Erlenmeyer flask containing fresh mycelia (5.0 g) and chitosan (5.0 g) in 100 mL of 0.1 M phosphate buffer (Na2HPO4/KH2PO4; pH 7). The mixture was incubated at 32C for 24 h on an orbital shaker (130 r.p.m.). Afterwards, the immobilized material was filtered and used in the biocatalytic reduction of ketones 1-2. The control reactions were conducted using fungal mycelium without immobilization. The marine fungus P.citrinum immobilized on chitosan catalyzed the bioreduction of ketones 1-2 to alcohols 1a-2a (Figure 1). Interestingly, the p-methoxyacetophenone 2 was not reduced by using free whole cells of marine fungi. In this study, the cells of P. citrinum immobilized on chitosan favored the activity of the biocatalyst. The hydrolysis of the chitosan by the fungus enzymes can liberate oligosaccharides, which should have also favored the reduction reaction. These hypotheses are under investigation in our group.
207
Asymmetric reduction of acetophenone by conidial fungi from the Brazilian semi-arid region
Serly Santiago Machadoa, Enesio Rodriguez Nascimento Netoa, Elisa Teshimaa, Osmar Calderon Sanchezb, Heiddy Marquez Alvareza, Ivan Sergio Cols Gonzlesa, Luis Fernando Pascholati Gusmoa, Anglica Maria Lucchesea a Universidade Estadual de Feira de Santana, Av Transnordestina SN, Feira de Santana, Bahia, Brazil; bUniversidad de la Habana. Zapata y G. CP 10400. Ciudad Habana. Cuba E-mail: angelica.lucchese@gmail.com Enantioselective syntheses of chiral alcohols are processes with a great industrial interest due to their potential use in the production of pharmaceuticals and other high value compounds1. In comparison with conventional chemical routes for the production of enantiomeric pure chiral alcohols, biocatalysis has many advantages such as higher chemo-, regio- and stereoselectivity and the use of mild reactional conditions[1,2]. Whole-cell biological systems as fungi, yeast and bacteria can be used as catalysts to this conversion, and studies seeking new sources of ketoreductases have been carried out2. In this work a screening for the identification of microorganisms with reductase activity was performed with 56 conidial fungi, isolated from dead plant material in Brazilian semi-arid region. The bioreduction of acetophenone to 1-phenylethanol with conversion rates up to 55% was achieved with 34 fungi. It was found that seven fungi (Cladosporium sp, Periconia sp, Idriella sp, Stachybotrys sp1, Myrothecium sp, Stachybotrys sp2, Periconia hispidula) were the most effective biocatalysts for this enantioselective bioreduction, with enantiomeric excesses (ee) higher than 86% and moderate conversion rates (30-55%). The Prelogs rule was followed in six bioreduction procedures yielding the S enantiomer preferentially (85 to 99% ee). One fungus (Cladosporium sp) catalysed the bioreduction to the anti-Prelog R product but with lower stereoselection (86% ee). Various parameters such as agitation, co-solvent and time of incubation were also evaluated to optimize the process using these seven fungi as biocatalysts. Conversions up to 100% with >99% enantiomeric excesses (ee) were obtained with ethanol as co-solvent and without agitation. Although there was a regular decrease in conversion speed with the increase of incubation time (up to 10 days), enantioselectivity of 1-phenylethanol remained the same (>99%) throughout the seven reactions. The best conversion rates were obtained with Cladosporium sp (86%) in 10 days and Periconia sp (91%) in 6 days.
________________ [1] Carvalho, C.C.C.R. Biotechnology Advances, 2011, 29, 75. [2] Matsuda, T.; Yamanaka, R.; Nakamura, K. Tetrahedron: Asymmetry, 2009, 20, 513
Figure 1. Bioreduction of ketones 1-2 by P. citrinum immobilized on chitosan

________________ [1] Chen K.-S.; Lee, C.-H.; Lin, H.-R.; Lin, F.-H.; Chen, T.-M. Mater. Sci. Eng. C 2005, 25, 472478. [2] Trincone, A. (Editor) in: Marine Enzymes for Biocatalysis - Sources, Biocatalytic Characteristics and Bioprocesses of Marine Enzymes, ISBN 9781907568800. [3]Rocha, L.C.; Ferreira, H.V.; Pimenta, E.F.; Berlinck, R.G.S.; Seleghim, M.H.R.; Javorati, D.C.D.; Sette, L.D.; Bonugli, R.C.; Porto, A.L.M. Biotechnol. Lett. 2009, 31, 1559-1563.
October 2-6, 2011
124 209
New Fructose-6-Phosphate Aldolase from E. coli (fsa B): characterization, kinetic study and comparison with FSA ( fsa A)
Christine Gurard-Hlainea,b, Israel Sanchez Morenoa,b, Agnes Pinetc,d,e, Jean-Louis Petitc,d,e, Vronique de Berardinisc,d,e and Marielle Lemairea,b* a Clermont Universit, Universit Blaise Pascal, Laboratoire SEESIB, BP 10448, F-63000 Clermont-Ferrand, France ;bCNRS, UMR 6504, SEESIB, F-63177 Aubire, France. c CEA, IG, Genoscope, Evry, France; dCNRS, UMR8030, Evry, France ; eUniversite dEvry Val dEssone, Evry, France. E-mail:christine.helaine@univ-bpclermont.fr D-fructose-6-phosphate aldolase from Escherichia coli (FSA A, gene fsa A), discovered in 2001[1], has attracted a great deal of attention due to its tremendous catalytic potential in asymmetric synthesis of polyhydroxylated compounds. This enzyme was found to catalyze the S,R (syn) stereoselective reversible aldol addition of dihydroxyacetone (DHA), hydroxyacetone (HA), 1-hydroxy-2-butanone (HB) and more surprisingly glycolaldehyde (GA) on various aldehydes to afford carbohydrates and analogues.[2]
Discovery and design of new biocatalysts 210

Immobilization of recombinant lipase (Pf2001) from Pyrococcus furiosus by hydrophobic interaction and covalent bond
Roberta V. Brancoa, Jose M. Palomob, Jos M. Guisnb, Denise M. G. Freirea and Rodrigo V. Almeidaa. a Instituto de Qumica, Universidade Federal do Rio de Janeiro, Av. Athos da Silveira Ramos, 149, CEP 21949-909, Rio de Janeiro, RJ, Brazil, bDepartamento de Biocatlisis, Instituto de Catlisis-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain E-mail: volcan@iq.ufrj.br In this work the recombinant thermostable lipase (Pf200160) from the hyperthermophilic archaeon Pyrococcus furiosus was immobilized on supports with different interaction types: hydrophobic (butyl, phenyl, octyl-agarose and octadecyl Sepabeads), one-point covalent (glyoxyl agarose - DTT) and multipoint covalent (glyoxyl agarose). The effects of the temperature (70C), organic solvent and amination on stability were analyzed. The enzyme immobilized in hydrophobics supports showed successful immobilization. The best results of activity among the immobilized biocatalysts via hydrophobic adsorption were found for the enzyme immobilized on octyl agarose when it was used different temperatures and substrates. Furthermore the immobilized enzyme on octyl agarose showed hyperactivation (199%) and immobilization efficiency (60%). In addition, the same biocatalyst showed 76% residual activity after 24 hours of incubation at medium containing sodium phosphate buffer with ethanol 50%. This immobilization process was a simple purification method of this P. furiosus enzyme, suggesting a selective adsorption on hydrophobic supports. The enzyme immobilized by multipoint covalent attachment showed best stability at 70C (around 80% of residual stability) and worst stability in medium containing sodium phosphate buffer with ethanol. The immobilized lipase on octyl agarose showed decrease of activity and stability when the concentrations of 1-ethyl-3-(dimethylamino-propyl) carbodiimide used to amination were increased.

Methyltransferase NovO a versatile catalyst for enzymatic Friedel-Crafts alkylation
Martin Tengga,b, Harald Stechera, Peter Remlera, Helmut Schwaba,b, Mandana GruberKhadjawia a ACIB GmbH, Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria; bInstitute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria E-mail: martin.tengg@acib.at The alkylation of aromatic compounds, known as Friedel-Crafts alkylation is a reaction of great importance in organic chemistry. For the first time this reaction was achieved by the use of an enzyme.[1] Transfer of methyl- and other alkyl groups, namely allyl, crotyl, propynyl, butynyl, and benzyl to coumarin- and naphthalene containing substrates was accomplished by the methyltransferase NovO (Fig.1). The characterization of NovO revealed a type I methyltransferase, which performs its action as a dimer in solution and contains a conserved S-adenosyl-L-methionine (SAM) binding site and a catalytic base. Determination of kinetic parameters verified the evolutionary design of the enzyme towards its natural substrate configuration, but also showed the adaptability of this biocatalyst for an alkylation of unusual substrates. The quantum chemical advantages of reacting groups (cofactor) that consist of carbon-carbon double bonds were accurately described.
125 214
Improving the thermostability and catalytic activity of D-tagatose 3-epimerase, for the production of the rare sugar D-psicose
a
Andreas Bossharta, Nina Wagnera, Matthias Bechtolda, Sven Pankea ETH Zrich, Department of Biosystems Science and Engineering (D-BSSE) Mattenstrasse 2, 64058 Basel, Switzerland E-mail: andreas.bosshart@bsse.ethz.ch Uncommon monosaccharides (rare sugars) such as D-psicose have lately attracted considerable interest as chiral building blocks, low-calorie sweeteners or active pharmaceutical ingredients [1]. However, such compounds are typically difficult to make by organic synthesis due to elaborate protection group chemistry [2] However, of the hexose stereoisomers can be directly produced via enzymatically-catalyzed epimerization and isomerizations reactions from cheap starting substrates such as D-glucose. D-fructose, D-sorbose D-galactose. To overcome the intrinsic yield limitation imposed by the unfavourable thermodynamic equilibrium of such biotransformations an in-situ product removal approach is required which has been successfully implemented in our lab for the production of D-psicose [3]. Arguably, D-tagatose epimerases (DTEs) constitute the central enzymes for the generic production of rare sugars as they give access to the C3-epimers of the available cheap starting substrates. However, in order to apply DTEs on industrial scale, high activity and operational stability are required. DTEs from A. tumefaciens (DTEtu) and P.chicorii (DTEci) were investigated which both showed insufficient stability and moderate catalytic activity. In order to improve stability and activity of DTEci a sequential approach of (i) rational protein stability engineering and (ii) subsequent activity engineering based on growth-dependent directed evolution was applied. (i) We applied iterative saturation mutagenesis based on B-factors developed by Reetz et al [4]. The 10 most flexible residues of DTEci were identified and randomized by saturation mutagenesis. Preliminary results of the first rounds provided two improved variants with moderately improved thermostability. (ii) In order to enhance the catalytic activity of DTEci toward the efficient production of Dpsicose, a powerful selection system coupling enzyme activity of DTE with a viability phenotype of the host E. coli is established (see Figure 1).
A second E. coli FSA named FSA B was also found in 2001 by Sprengers group. This enzyme displaying a lowered activity towards fructose-6-phosphate cleavage than FSA A has not been fully studied. In the light of more recent results showing that fructose6-phosphate was not the best substrate even for FSA A[3], FSA B regained some interest. This work presents the characterization and biochemical studies of FSA B (clonage, overexpression, purification, oligomeric form, thermostability) and a comparison of its catalytic properties with FSA A (kinetic constants with DHA, HA, glycolaldehyde, Dglycraldhyde-3-phosphate, fructose-6-phosphate, fructose and sorbose).
________________ [1] M. Schrmann, G. A. Sprenger, J. Biol. Chem. 2001, 276, 11055. [2] A. L. Concia, C. Lozano, J. A. Castillo, T. Parella, J. Joglar and P. Claps, Chem. Eur. J. 2009, 15, 3808 [3] X. Garrabou, J.A. Castillo, C. Gurard-Hlaine, T. Parella, J. Joglar, M. Lemaire, P. Claps, Angew. Chem. Int. Ed., 2009, 5521
Figure 1. Alkyltransfer catalyzed by NovO. R1 = methyl, allyl, crotyl, propynyl, butynyl, benzyl.
_______________ [1] H. Stecher, M. Tengg, B. J. Ueberbacher, P. Remler, H. Schwab, H. Griengl, M. Gruber-Khadjawi, Angew. Chem. Int. Ed. 2009, 48, 9546-9548.
Figure 1: Selection system for improved conversion of D-psicose to D-fructose (host: E. coli W3110 E.O)
Abbrev: AlsABC = allose transporter; LRI = L-rhamnose isomerase; DTE = D-tagatose-3-epimerase; DFK = manno(fructo)kinase
________________ [1] Takata, M.K. et al. Journal of Bioscience and Bioengineering 100, 511-516 (2005). [2] Fessner, W. D., Aldolases: Enzymes for making and breacking C-C bonds. In Asymmetric Organic Synthesis with Enzymes Gotor, V.; Alfonso, I.; Garcia-Urdiales, E., Eds. Wiley-VCH: Weinheim, 2008; p 275. [3] Wagner, N. et al. Org Process Res. Dev. (submitted) [4] Reetz, M. T., J. D Carballeira, et al. (2006). Angewandte Chemie-International Edition 45(46): 77457751.
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Large-scale exploration of enzyme biodiversity to discover novel biocatalysts
De Berardinis Va,b,c, Petit JLa, Mariage Aa, Perret Aa,b,c, Tricot Sa,b,c, Zaparucha Aa,b,c, Marliere Pd, Salanoubat Ma,b,c , Weissenbach Ja,b,c a CEA, DSV, IG, Genoscope, 2 rue Gaston Crmieux, 91057 Evry, France; bCNRSUMR8030, 2 rue Gaston Crmieux, 91057 Evry, France; cUniversit dEvry Val dEssonne, boulevard Franois Mitterrand, 91057 Evry, France. dIsthmus, 91000 Evry. vberard@genoscope.cns.fr Some years ago, Genoscope, the French Sequencing Centre, had decided to diversify its activities to include large-scale functional genomics studies and to develop a high throughput platform (HTP) for cloning genes and biochemical screenings of enzymes from prokaryote genomes and metagenomes. This HTP is used in two different projects: (1) a systematic enzymatic screening of large enzyme families to discover new biocatalysts for synthetic chemistry and metabolic engineering and (2) a systematic functional annotation focused on genes with unknown predicted function of a bacterial ORfeome to explore its metabolism. Enzymes could have a large spectrum of activities (example: dehydrogenases) and its not rare that a protein which has been assigned to an enzymatic reaction could also perform another chemical reaction, albeit marginally. Exploiting enzyme catalytic promiscuity might lead to new and efficient catalysts with, as yet, unprecedented activity for reactions where no enzyme alternatives exist today. Today, novelty in terms of biocatalysts is one of the most challenging problems. Moreover, a large majority of the known enzymatic reactions were discovered in cultivated bacteria but now we are faced with a new situation in which sequences from metagenomes (consortium of bacteria) which are orphans of organisms technically become available. These metagenome sequences provide a reservoir of genes and represents an interesting source of novel proteins that will indubitably extend our knowledge of enzymatic reactions and lead to discover novel biocatalysts to fulfill the needs of synthetic biology or industrial processes and to propose alternatives to chemical synthesis.
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Recombinant cellulases from extremophilic microorganisms active in ionic liquids
Michele Girfoglioa,b, Thomas Schmidtb, Julia Beckerb, Philip Engelc, Antje Spiessc, Rainer Fischerb,d, Ulrich Commandeurb and Raffaele Cannioa,e a Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy b Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany c AVT Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany d Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6 52074 Aachen, Germany e Institute for Microelectronics and Microsystems, CNR, Via P. Castellino 111, 80131, Naples, Italy E-mail: thomas.schmidt@molbiotech.rwth-aachen.de Plant-derived lignocellulose is the most abundant renewable resource on earth and is used as raw material to obtain glucose for second generation biofuel production processes. Pretreatment and conversion of lignocellulolytic biomass for biofuel production usually takes place under extreme conditions, which are not ideal for biocatalysts, lowering the turn-over rates and lifetime of those enzymes. Therefore, enzymes are commonly used after the physicochemical pretreatment process. Here, we describe the activity of recombinant cellulolytic enzymes at high concentrations (80%) of ionic liquids (ILs), a class of compounds used for the dissolution and extraction of cellulose from lignocellulosic biomass. A hyperthermo- and acidophilic endoglucanase from Sulfolobus solfataricus as well as a halophilic endoglucanase from Halomonas sp66-4 show to be stable and active at those concentrations of ILs allowing the dissolution and enzyme-catalyzed breakdown of cellulose in one step. The combination of tolerance to ILs combined with a superior cellulose hydrolyzing performance of these biocatalysts represents a breakthrough in the development of a sustainable and economical lignocellulose conversion process.
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Escherichia coli: still unexhausted source for new biocatalysts
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Alkalophilic laccases by in vitro evolution
P. Torres, I. Ghazi, A.O. Ballesteros, F.J. Plou and M. Alcalde. Instituto de Catlisis y Petroleoqumica, CSIC, 28049 Madrid, Spain; E-mail: malcalde@icp.csic.es Fungal laccases (EC 1.10.3.2) are remarkable oxidases with a broad substrate specificity, which permits their use in several industrial sectors, from bioremediation to novel green processes. Fungal laccases are typically highly stable at pH above 7.0 although their activity is almost negligible close to neutrality. Indeed, their optimal pH falls in the range 3.0-5.0. Several applications demand fungal laccases that are active at neutral or alkaline pH (e.g. lignocellulose treatment, construction of biosensors and biofuel cells, dyestuffs processing, etc.). In former research, our laboratory was involved in the engineering of the laccase from Myceliophthora thermophila (MtL) by directed evolution in order to: i) attain functional expression in Saccharomyces cerevisiae [1] , and ii) enhance the tolerance towards organic co-solvents[2]. During these evolutionary routes (15 cycles), the pH activity profile of MtL was noticeable shifted, showing a change in the maximum activity from pH 3.0 to pH 5.0. Using as point of departure the evolved MtL highly active at pH 5.0, in this work five rounds of directed evolution were performed to engineer a fungal alkalophilic laccase. Over 10,000 clones were explored to detect shifts in the pH activity profile. To screen for alkalophilic mutants, the ratio between the activity at pH 8.0 and the activity at pH 5.0 was employed as a discriminatory factor. The generation of diversity combined error-prone PCR with different in vitro and in vivo DNA recombination methods (IvAM, StEP) as well as combinatorial saturation mutagenesis.
________________ [1] Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution. T. Bulter, M. Alcalde, V. Sieber, P. Meinhold, C. Schlachtbauer and F. Arnold. Applied and Environmental Microbiology, 69, 987-995 (2003). [2] In vitro evolution of a fungal laccase in high concentrations of organic solvents. M. Zumarraga, T. Bulter, S. Shleev, J. Polaina, A. Martinez-Arias, F.J. Plou, A. Ballesteros and M. Alcalde. Chemistry and Biology, 14, 1052-1064 (2007).
Andr Picka, Broder Rhmanna, Jochen Schmida, Volker Siebera Chair of Chemistry of Biogenic Resources TU Munich, D-94315 Straubing, Germany E-Mail: a.pick@tum.de
Escherichia coli represents one of the best-studied model organisms. It is the workhorse in the laboratory concerning genetics and molecular biology and proved of value as a production organism in many biotechnology applications. Already more than 10 years ago the complete genome was sequenced revealing 4288 genes of which almost 80% are thought to be known by function [1]. Nevertheless E.coli can still contribute as a crucial source for identification of new biocatalysts in the field of industrial biotechnology. Conversions of renewable resources constitute a promising area of application for biocatalysts concerning the complex structure of the substrates. With this focus investigation for an aldehyde reductase catalyzing the selective reduction of complex substrates with primary carbonyl function was done. As a promising candidate the well-known zinc-containing dehydrogenase YqhD from E.coli was first tested for activity against our substrate [2]. The enzyme showed no activity but further research led to two zinc-containing dehydrogenases of E.coli with improved properties compared to YqhD. Also both enzymes show a better performance like YqhD only one of the two dehydrogenases had the required activity for the investigated substrate. In matters of metabolic engineering approaches the use of E.colis own enzymes bearing advantages concerning over-expression and compatibility. Furthermore the uses in cell-free systems with an appropriate co-factor recycling are possible applications for the newly identified alcohol dehydrogenases.
________________ [1.] Blattner, F.R., et al., The complete genome sequence of Escherichia coli K-12. Science, 1997. 277(5331): p. 1453-62. [2.] Jarboe, L.R., YqhD: a broad-substrate range aldehyde reductase with various applications in production of biorenewable fuels and chemicals. Appl Microbiol Biotechnol. 89(2): p. 249-57.
Figure: a 0,5% w/v -cellulose solution in [emim]Ac was incubated with S. solfataricus endoglucanase (0.18 g/L). Samples were withdrawn at several time points and the IL was removed; 60 L of reaction samples with and without enzyme (lane C, incubation of 16 h) were spotted on silica plates and developed; b Sample of -cellulose solution in [emim]Ac was incubated with Sso1354 for 48 hours and afterwards analysed by SEC
October 2-6, 2011
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A microbial carbamate hydrolase for the deprotection of N-benzyloxycarbonyl derivatives
Michle Maurs, Francine Acher, Robert Azerad Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Universit Paris Descartes, 45 rue des Sts Pres, 75006 Paris E-mail : robert.azerad@parisdescartes.fr The benzyloxycarbonyl group (Cbz) is widely used to protect amino groups in organic synthesis reactions. Deprotection is usually achieved by catalytic hydrogenation, with some drawbacks associated with the presence of double bonds, nitro groups or sulphur heteroatoms, or a particular resistance to hydrogenation. In order to develop an enzymatic hydrolytic deprotection of N-Cbz derivatives, mainly N--Cbz-amino acids and oligopeptides, but eventually other N-Cbz-protected derivatives, we have screened microorganisms for their ability to hydrolyze Cbz-L-glutamic acid and identified some promising strains. We describe in this poster the comparative cleavage of Cbz-L-Glu and various Cbz-amino acids with a crude lyophilized preparation of one of these strains, its ability to react with other benzyloxycarbamate derivatives and to carry out the deprotection and optical resolution of these compounds. An optimisation of the experimental conditions for efficient deprotection (pH, temperature, additives,) has been also carried out in order to apply for preparative resolutions of synthetic non-natural N-Cbz-DL-amino acids.

A single amino acid directs Candida rugosa lipases enantioselectivity towards 2-halogeno substituted aryl acetic acid esters
Rungtiwa Piamtongkama,d, Sophie Duquesnea-c, Sophie Barbea-c, Isabelle Andra-c, Warawut Chulalaksananukule* and Alain Martya-c a Universit de Toulouse; INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; bCNRS, UMR5504, F-31400 Toulouse, France; cINRA, UMR792 Ingnierie des Systmes Biologiques et des Procds, F-31400 Toulouse, France; dProgram in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phyathai Road, Patumwan, Bangkok, 10330, Thailand; eDepartment of Botany, Faculty of Science, Chulalongkorn University, 254 Phyathai Road, Patumwan, Bangkok, 10330, Thailand E-mail: duquesne@insa-toulouse.fr Enzymatic resolution of racemate is a reaction of pharmaceutical interest that often involves few amino acids located in the active site of the enzyme. Candida rugosa produces 5 highly homologous lipase isoforms, whose enantioselectivity was often pointed out [1]. Here, we expressed 3 of these lipases, namely Lip1, Lip3 and Lip4, in the optimised expression host Yarrowia lipolytica[2]. This newly developed expression system enabled the production of extracellular active enzymes. Their capacity to resolve racemates of 2-bromophenylacetic octyl esters, an important class of pharmaceutical intermediates[3], was then studied. We showed that the three lipases exhibited a high enantioselectivity, Lip4 preferring the R-enantiomer (E-value=15), while Lip1 and Lip3 showed an S-enantioselectivity > 200. The involvement of position 296, which is one of the very few amino acids located in the binding site that differs between the 3 isoforms was then studied. Variants of Lip1 and Lip4 in which position 296 was replaced by G, A, L or F were constructed by site-directed mutagenesis and assayed for their enantioselectivity. We showed that the amino acid in position 296 controls both the enantioselectivity and enantiopreference of C. rugosa lipases: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. Preliminary modelling study of the tetrahedral intermediates formed by the wild type lipases with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers provided some insight regarding the determinants responsible for lipase enantiodiscrimination. This work was published in Biotechnology and Bioengineering in 2011[4].

Enzyme catalyzed laurolactam synthesis in aqueous solution
Nadine Ladkau, Inna Hermann, Bruno Bhler, and Andreas Schmid Laboratory of Chemical Biotechnology, Faculty of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany nadine.ladkau@bci.tu-dortmund.de Lactam formation from -aminocarboxylic acids is thermodynamically unfavored in aqueous solution and therefore difficult to achieve. Recently, laurolactam hydrolysis activity of several soil bacteria was reported [1,2] and the enzymes were identified as -laurolactam hydolases. In the present study, the -laurolactam hydrolases from Acidovorax sp. T31 and Cupriavidus sp. U124 were investigated regarding their potential to convert 12-aminododecanoic acid to laurolactam (Figure 1). The genes were expressed in Escherichia coli BL21 (DE3) and the activity of the -laurolactam hydrolases was investigated in vivo and in vitro. For this purpose, the concepts of equilibrium controlled and kinetically controlled peptide syntheses [3,4] were applied to synthesize laurolactam.
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Evaluation of genetically encoded FRET-biosensors as tools for quantitative metabolome analysis
Roland Moussaa, Anna Baierla, Wolfgang Wiecherta, Martina Pohla a Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jlich GmbH, 52425 Jlich, Germany; E-mail: r.moussa@fz-juelich.de Fluorescent labels have shifted the boundaries in cell biology. In recent years, a huge set of genetically-encoded fluorescence biosensors have been developed, which allow to detect signaling intermediates and metabolites in real time with subcellular spatial resolution [1]. Numerous of these biosensors are based on fluorescent proteins such as the green fluorescent protein (GFP) and its color variants. Several function via Frster Resonance Energy Transfer (FRET) [2]. They represent the basis for numerous non-invasive cell-based assays to monitor signal transduction in living cells. The FRETbased biosensors used in this work are composed of a periplasmic binding protein (PBP) and two fluorescent proteins, which are fused to the C-and N-termini, respectively [3]. Upon binding of a metabolite, PBPs transform their domain movement into increased FRET between the two terminal fluorescent proteins (Fig. 1a). As the FRET-signal changes significantly in the range of the Kd of the sensor, such sensors principally allow also quantitative metabolite analyses (Fig. 1b). In order to allow quantification, the influences of environmental factors like pH, temperature or halide ions on the signal intensity must be known. As GFP and many of its variants were shown to be highly sensitive toward various environmental parameters [4, 6] a broad range of less sensitive variants has been generated [5, 6]. In order to evaluate the FRET-based biosensors [Fig. 1b] for quantitative metabolite determination in vivo, we have thoroughly studied the effect of pH, ionic strength and temperature on the signal intensity and Kd of a glucose and a maltose sensor. The results demonstrate that all investigated sensors show different, but significant degrees of influence by the environmental parameters, indicating to be careful interpreting measurements with such techniques.
Figure 1. Condensation reaction catalyzed by a -laurolactam hydrolase to produce laurolactam. A cyclization of 12-aminododecanoic acid to laurolactam was not observed in wholecell biotransformations or crude extract assays, as already expected from thermodynamic considerations. However, the utilization of 12-aminododecanoic acid methyl ester, as activated form of 12-aminododecanoic acid, and the increase of the reaction pH to pH 10, resulted in cyclization of the methyl ester to laurolactam. Lactam formation was observed with maximum rates of 7.7 and 11.0 U gCDW-1 in biotransformations using recombinant E. coli harboring the -laurolactam hydrolases from Acidovorax sp. T31 and Cupriavidus sp. U124, respectively. Both recombinant strains showed similar maximum laurolactam yields of 17.1% and 14.5%. Furthermore, at pH 10 the application of whole-cell biocatalysts enabled ten-fold higher specific laurolactam formation rates in comparison to free enzymes. Although the hydrolase catalyzed cyclization of -aminocarboxylic acids to the corresponding lactams is unfavored in aqueous solution, laurolactam formation was achieved by selection of an appropriate substrate and reaction pH. _______________ [1] Y. Asano et al. (2008) Biosci. Biotechnol. Biochem. 72, 2141-2150 [2] Y. Fukuta et al. (2009) Biosci. Biotechnol. Biochem. 73, 980-986 [3] H.-D. Jakubke et al. (1985) Angew. Chem. Int. Ed. Engl. 24, 85-93 [4] D. D. Petkov (1982) J. Theor. Biol. 98, 419-425
Figure 1. Enantiopreference of C. rugosa Lip1 for (S)-2-Br-phenylacetic acid octyl ester _______________ [1] F. Manetti et al., Biochimica Biophysica Acta 2000, 1543(1),146 [2] F. Bordes et al., J. of Microbiological Methods 2007, 70, 493 [3] D. Guieysse et al., Tetrahedron: Asymmetry 2001, 12(17), 2473 [4] R. Piamtongkam et al., Biotechnology and Bioengineering 2011 Mar 9. doi:10.1002
________________ [1]Okumoto S., (2010). Anal Biotech, 21(1):45-54, 0958-1669. [2]Tsien, RY., (1998).Annu Rev Biochem 67: 509-544. [3]Frommer WB, et al., (2002). Proc Natl Acad Sci USA 99(1):98469851. [4]Laptenok SP, et al., (2010). PLoS ONE 5(11): e13862. [5]Zhang, J., R. E. Campbell, et al. (2002). Nat Rev Mol Cell Biol 3(12): 906-918. [6]Tsien RY, et al., (2001). J Biol Chem 276(31):29188-94.
Figure 1: a) Substrate-induced conformational change of a periplasmatic binding protein. b) Enhanced FRET-signal through increasing metabolite concentration, resulting in a Kd value.
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Characterisation of a novel bacterial p-hydroxybenzoate-1-hydroxylase from the metagenome
Sebastian Hofzumahausa, Sebastian Salentina, Ite G.P. Teuneb, Dick B. Janssenb, Anett Schallmeya a Junior Professorship for Biocatalysis, Institute of Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany; bBiochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands E-mail: s.hofzumahaus@biotec.rwth-aachen.de Para-hydroxybenzoate-1-hydroxylases (PHB1H) catalyze the conversion of various p-hydroxybenzoic acids into the corresponding 1,4-benzenediols (Figure 1) [1]. These enzymes, which were so far only identified in yeast and fungi, are involved in the degradation of aromatic acids derived from lignin and tannin [2]. By screening a metagenomic library derived from microorganisms originating from vanilla pods we were able to isolate the first bacterial p-hydroxybenzoate-1-hydroxylase [3]. The metagenomic clone exhibiting the respective hydroxylase activity was identified using an agar plate-based activity assay. The insert of the metagenomic clone of roughly 5 kb was sequenced revealing >80% sequence identity on nucleotide level to a genomic DNA fragment of Pantoea sp. At-9b, a g-proteobacterium. The insert contained several open reading frames, one coding for a putative FAD-dependent monooxygenase, which exhibited the respective p-hydroxybenzoate-1-hydroxylase activity.
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Ribavarin: A complementary universal base to P for Sequence Saturation Mutagenesis Method (SeSaM)
Anna Joelle Ruffa, Jan Marienhagenb, Hemanshu Mundhadaa, Ulrich Schwaneberga Chair of Biotechnology, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany; bIBG-1: Biotechnologie, Forschungszentrum Juelich, 52425 Juelich, Germany. E-mail: aj.ruff@biotec.rwth-aachen.de
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Research of new biocatalysts from prokaryotes: the nitrilases and their application in organic synthesis
Bordier. F.2,3, Vergne Vaxelaire. C.1,2,3, Mariage. A. 1, Pellouin. V. 1, Debard A .1, Besnard M.1, Petit. J.L. 1, De Berardinis. V.1,2,3, Zaparucha. A.2,3 1 CEA, DSV, IG, Genoscope, 2 rue Gaston Crmieux, 91057 Evry, France; 2CNRSUMR8030, 2 rue Gaston Crmieux, 91057 Evry, France; 3Universit dEvry Val dEssonne, boulevard Franois Mitterrand, 91057 Evry, France. E-mail: fbordier@genoscope.cns.fr Enzymes are enjoying increasing popularity in the chemical industry as environmentally friendly, economical and high selective catalysts. The search for new specific enzymatic activities allows enlargement of the biocatalyst applications in organic synthesis. Our multidisciplinary approach aims at identifying new enzymatic transformations performed by prokaryotic organisms, based on genomic analysis. After selection of candidate enzymes and activity screening on a pool of appropriate substrates, the selected enzymes are tested for the biocatalytic transformation of valuable synthons. One of our ongoing projects concerns the discovery of new nitrilases. Nitrilases, / hydrolases belonging to the nitrilase superfamily [1], have been widely acknowledged as valuable alternatives to chemical synthesis as they have proved to transform a variety of nitriles under mild conditions and often with stereospecificity [2]. This transformation requires normally harsh conditions by chemical hydrolysis and so may undergo to the formation of many by-products (Figure 1).
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Production of novel biocatalysts by directed evolution for the production of enantiomerically pure L-amino acids
Asma Alhaj Tamar, Paul C. Engel UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland E.mail: Asma.alhaj@ucd.ie The increasing demand for greener chemistry in industry has led to the application of synthetic routes other than the traditional chemical methods. Biocatalysis has gained tremendous popularity in enantiopure synthesis of non-natural amino acids that are used as precursors or components of drug molecules. Protein engineering combined with highthroughput screening or selection systems have led to the development of novel enzymes that are also more tolerant, compared to the native enzymes, to industrial process conditions. A number of engineered phenylalanine dehydrogenases[1] have proved to be successful in the production of enantiopure non-natural L-amino acids[2,3,4] and to be tolerant to different organic solvents[4,5]. The current work further investigates the application of the engineered PheDHs for enantiopure synthesis of novel amino acids under different reaction conditions to determine their potential for application at industrial level.
________________ References: [1] Seah S.Y.K., Britton K.L., Baker P.J., Rice D.W., Asano Y. and Engel P.C. (1995) Alteration in relative activities of phenylalanine dehydrogenase towards different substrates by site-directed mutagenesis. FEBS Lett. 370, 93-96. [2] Busca P., Paradisi F., Moynihan E., Maguire A.R. and Engel P.C. (2004) Enantioselective synthesis of non-natural amino acids using phenylalanine dehydrogenases modified by site-directed mutagenesis. Org Biomol Chem. 2, 2684-2691. [3] Paradisi F., Collins S., Maguire A.R. and Engel P.C. (2006) Phenylalanine dehydrogenase mutants: Efficient biocatalysts for synthesis of non-natural phenylalanine derivatives. J Biotechnol. 128, 408-411. [4] Chen S., Engel P.C. (2007) An engineered mutant, L307V of phenylalanine dehydrogenase from Bacillus sphaericus: High activity and stability in organic-aqueous solvent mixture and utility for synthesis of non-natural L-amino acids. Enzyme and Microbial Tech. 40, 1407-1411. [5] Cainelli G, Engel P.C., Galletti P., Giacomini D., Gualandi A. and Paradisi F.(2005) Engineered phenylalanine dehydrogenase in organic solvents: homogeneous and biphasic enzymatic reactions. Org Biomol Chem. 3, 4316 4320.
Methods to generate random mutant libraries in directed evolution are limited in diversity generation. Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method, overcomes most limitations of an error-prone PCR (epPCR) based mutagenesis methods [1]. SeSaM targets each nucleotide equally avoiding mutagenic hot spots. The control over mutational bias is achieved through universal bases and is independent from employed DNA-polymerases. In order to explore protein sequence space efficiencies, the bias of an epPCR was complemented by a transversion enriched protocol, in which the universal PBase was used [2]. Recently, the method was further advanced generating through consecutive mutations (36 %) a protein sequence space unobtainable by epPCR [3]. To further increase the amino acid sequence space that can be covered by the SeSaM method, we investigated the inclusion of ribavirin as complementary universal base to the employed P base. The nucleoside ribavirin (dRTP) is a well-known antiviral drug for hepatitis C. The base functions as purine analog and pairs thus with T and C (ratio of 1:1) [4]. Incipient experiments validated dRTP as universal base for the SeSaM method. Sequencing results showed an equal incorporation of T and C in the complementary DNA strand to Rbase. After optimisation of the different steps of the SeSaM method, several SeSaM libraries with the universal R-base were successfully generated with a transversion preference. Concluding, the ribavirin base can be used as complementary universal base in the SeSaM method. The combination of the 2 validated universal bases P and R allows for the mutated positions to cover nearly to a full extent the theoretical protein sequence space. The latter further advanced the powerful SeSaM to modulate mutational biases according to control targeted properties and employed protein folds. Acknowledgement: EU funded 7th framework project Oxygreen (212281)
________________ [1] Wong, TS., Tee, KL., Hauer, B., Schwaneberg, U., Nucleic Acids Res., 2004, 32(3): e26. [2] Wong, TS., Roccatano, D., Loakes, D., Tee, KL., Schenk, A., Hauer, B., Schwaneberg, U., Biotechnol. J., 2008, 3(1): p. 74-82. [3] Mundhada, H., Marienhagen, J., Scacioc, A., Schenk, A., Roccatano, D., Schwaneberg, U., Chembiochem, 2011, accepted. [4] Crotty, S., Maag, D., Arnold, JJ., Zhong, W., Lau, JY., Hong, Z., Andino, R., Cameron, CE., Nat Med., 2000, 6(12): p. 1375-9.
Figure 1: Conversion of p-hydroxybenzoate derivatives by the p-hydroxybenzoate-1-hydroxylase isolated from the metagenome. In order to investigate the substrate spectrum of the bacterial p-hydroxybenzoate-1-hydroxylase, the PHB1H gene was cloned and heterologously expressed in E.coli. The purified enzyme was used in biocatalysis of several p-hydroxybenzoate derivatives. Thus it was found that benzoates without hydroxyl group in para-position were not converted. Furthermore it could be shown that the enzyme is strictly NADH-dependent. The kinetic parameters of the PHB1H towards all identified substrates were also determined revealing the highest activity for vanillic acid.
________________ [1] van Berkel et al. (1994) FEMS Microbiol. Lett., 121, 207-216 [2] Eppink et al. (1997) J. Bacteriol., 179, 6680-6687 [3] Gabor (2004) Doctoral thesis, Groningen, Netherlands
Figure 1. This presentation describes the screening of substrates for the nitrilase activity of about 160 putative enzymes. Applications in the biocatalytic transformation of substrates of interest will be presented.
________________ [1] [1] a) Singh, R. ; Sharma, R. ; Tewari, N. ; Geetanjali ; S.Rawat, D. Chemistry & Biodiversity 2006, 3, 1279-1287 ; b) OReilly, C. ; Turner, P.D. J. Appl. Microbiol. 2003, 95, 1161-1174 ; c) Pace, H.C. ; Brenner, C. Genome Biology 2001, 2(1) :reviews0001.1-0001.9. [2] See for example : Ankati, H. ; Zhu, D. ; Yang, Y. ; Biehl, E.R. ; Hua, L. J. Org. Chem. 2009, 74, 1658-1662.
October 2-6, 2011
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Ralstonia eutropha H16 as source for new enzymes and as cell factory for biotechnological application
Zalina Magomedova, Petra Kfinger, Daniel Schwendenwein, Steffen Gruber, Helmut Schwab Institute of Molecular Biotechnology, Graz University of Technology E-mail: zalina.magomedova@tugraz.at Ralstonia eutropha is a Gram-negative, strictly respiratory facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole sources of energy and carbon in the absence of organic substrates. It has attracted great interest for biotechnology for its ability to degrade a large list of chloroaromatic compounds and chemically related pollutants. Furthermore it has already been applied for the production of biodegradable polymer polyhydroxyalkanoates on an industrial scale [1]. R. eutropha serves as a model organism for the mechanisms involved in the control of autotrophic carbon dioxide fixation, hydrogen oxidation and denitrification. In addition this bacterium is able to metabolize heavy metals [2]. In our project we are going to use Ralstonia eutropha as an organism for establishing specialized cell factories by genetic engineering. Homologous and heterologous protein expression as well as carboxylation and oxidoreductase reactions will be performed. For this purpose new genetic tools will be developed. The Calvin-Benson-Bessham reductive pentose phosphate cycle is used as the metabolic pathway of CO2 assimilation. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) is the key enzyme of the cycle. One approach is to identify additional carboxylases which catalyze reactions fixing CO2. In this line we will also overexpress carbonic anhydrases which provide bicarbonate as substrate for the carboxylases. Recent work of Steinbchel et al. [3] provided detailed knowledge about the enzymes involved in the conversion of C3 and C4 compounds. In the first attempt we will study various carboxylases of the C3/C4 carbon metabolism in view of their use in biotechnological applications.
[1] Steinbchel, A., Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example; Macromol Bioscience 1, 2001: 124 [2] Mergeay, M. et al. Alcaligenes eutrophus CH34 is a facultative chemolitotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 162, 1985: 328334 [3] N. Bruland, I. Vo, C. Brmer and A. Steinbchel, Unravelling the C3/C4 carbon metabolism in Ralstonia eutropha H16; Journal of Applied Microbiology 2010 Jul; 109(1):79-90

Cloning, expression, purification and characterization of a novel L-Arabinose isomerase from Lactobacillus reuteri
Petra Wuehrera, Dietmar Haltricha and Clemens Karl Peterbauera a Department of Food Sciences and Technology, BOKU University of Natural Resources and Life Sciences, Vienna, Austria E-mail: petra.wuehrer@boku.ac.at L-Arabinose isomerase (L-AI, EC 5.3.1.4) catalyzes the conversion of L-arabinose into L-ribulose in vivo, as well as the isomerization of D-galactose into D-tagatose in vitro [1]. D-tagatose can be used as a low-caloric sweetener with sweetness comparable to sucrose. D-tagatose has numerous health benefits, including anti-plaque, prebiotic and anti-biofilm properties, exhibits no glycemic effect and flavour-enhancement. Furthermore it can be used as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. D-tagatose can be produced from D-galactose by calcium-catalyzed chemical isomerization, but this process is followed by complex downstream operations and yields various by-products. For these reasons, production of D-tagatose using biocatalysts has been studied intensively in recent years [2]. We present here the cloning of the arabinose isomerase gene (araA) from Lactobacillus reuteri DSMZ17509. The gene was expressed heterologously in E. coli under control of the T7 promoter. The coding region was fused with a C-terminal His-tag for efficient purification by IMAC (Immobilized Metal Affinity Chromatography), and the purified enzyme was characterized in detail. Furthermore we produced the enzyme in L. plantarum, an organism with GRAS-status (Generally Recognized As Safe), using a novel host-vector system without antibiotic resistance markers and foreign, non-lactobacillal DNA sequences [3].
________________ [1] Rhimi, M., Ilhammami, R., Bajic, G., Boudebbouze, S., Maguin, E., Haser, R., Aghajari, N. (2010) Bioresour. Technol. 101, 9171-9177. [2] Oh, D.-K. (2007) Appl. Microbiol. Biotechnol. 76, 1-8. [3] Nguyen, T.-T., Mathiesen, G., Fredriksen, L., Kittl, R., Nguyen, T.-H., Eijsink, V.-G., Haltrich, D., Peterbauer, C.-K. (2011) J. Agric. Food Chem. 59 (10), 5617-24.

Towards the Directed Evolution of an L-Aspartate Oxidase
Charlotte Leese Franck Escalettes and Gideon Grogan a York Structural Biology Laboratory, University of York, Department of Chemistry, Heslington, YO10 5DD, York, UK b Ingenza Ltd, Wallace Building, Roslin BioCentre, Roslin, Midlothian, EH25 9PP UK * E-mail: charlotte@ysbl.york.ac.uk
*a b a
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Functional expression of CYP119 from a hyperthermophile sulfolobus acidocaldarius in thermus thermophilus
Ji-Won Song, Jang-Won Yun, Jin-Byung Park Dept. of Food Science & Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea E-mail: jbpark06@ewha.ac.kr The functional expression of the genes of thermophiles and hyperthermophiles in mesophiles can be difficult to achieve because molecular environment exposed to newly synthesized polypeptides can be different and thus folding processes would not be properly proceeded to. In this study, functional expression of CYP119 the cytochrome P450 oxygenase originated from hyperthermophilic archaeon Sulfolobus acidocaldarius was investigated in a mesophile Escherichia coli and in a thermophilic eubacterium Thermus thermophilus HB27. According to the spectroscopic study of the enzymes produced in the bacteria, expression level of CYP119 was much higher in the recombinant E. coli cells (1.67 M) than in the T. thermophiles (0.69 M). However, the specific activity of the enzymes produced in the T. thermophilus with respect to oxidation of Amplex Red was 0.17 min-1, which is ca. 7.1-fold greater compared to that of the enzymes produced in the E. coli. Therefore, it was assumed that the functional expression of the genes of hyperthermophiles can be obtained more efficiently in hyperthermophiles.
Amino acid oxidases (AAOs) are flavoenzymes that catalyse the enantioselective oxidation of amino acids to imino acids, which spontaneously hydrolyse to form keto acids. AAOs can be used to deracemise their chiral substrates when used in an oxidation-reduction cycle with a chemical reductant. These processes are useful for making enantiomerically pure chiral intermediates for the agrochemical and pharmaceutical industries. However natural AAOs have limited substrate specificity.[1] The L-aspartate oxidase from Pseudomonas putida shows activity against L-aspartate and L-asparagine. Thirteen active site residues in the known structure of a homologous E. coli L-aspartate oxidase[2] are conserved in the P. putida oxidase (Figure 1). These thirteen residues were split into five sequentially close groups, which were subjected to site-directed saturation mutagenesis. A Library of 600 transformants targeting residues H351 and Y352 was created and screened for activity against L-homoserine, L-alanine, and wild-type substrates L-aspartate and L-asparagine.
Figure 1
_____________ 1. K. Isobe et al (2010) Enzyme Research, 2010 2. R. T. Bossi, A. Negri et al. (2002) Biochemistry, 41, 3018-24
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Immobilization of cells through cryostructuration for biocatalysis
Oksana Zaushitsyna, Harald Kirsebom and Bo Mattiasson Department of Biotechnology, Lund University, Sweden E-mail: Oksana.Zaushitsyna@biotek.lu.se Immobilization of enzymes is widely used in many biotechnological processes due to their reusability and stability during application. Common methods of immobilization like covalent attachment or gel entrapment of purified enzyme into polymeric matrix is rather expensive. Immobilization of whole cells offers certain advantages like avoiding the additional purification of enzyme as well as allowing maintaining a high activity of enzyme. A new technique for preparation of immobilised enzymes with high catalyst density was developed recently[1]. One step cryostructuration process allows formation of biocatalyst from whole cells in so called cryogel. By freezing of cell suspension together with cross-linker and further thawing one obtains a macroporous structure from immobilised cells. It was shown that commonly used crosslinkers like glutaraldehyde (GTA) efficiently killed the microbial cells by penetration through cell membrane. Thereby the cells did not retain the whole spectrum of enzyme activity. The use of macromolecular structures for crosslinking of cells is one of the main way to maintain cellular activity[2,3]. In our study we show successful application of polymeric crosslinkers like oxidized dextran, polyethylenimine crosslinked with GTA and a newly synthesized polymer: poly (4-methylen-6-(1,3-dioxan-2-pentan-5-aldehyde)). Prepared cryogels have macroporous structure with pore size up to 100 m that was confirmed by scanning electron microscopy. Viability test showed that cells saved their metabolic activity after immobilisation. This was confirmed by cultivating pieces of cryogel on LB medium agar plates. Cryostructured biocatalysts were mechanical stable during application due to the crosslinking effect. Enzymatic activity of E.coli that overexpressed -transaminase was retained after immobilization using oxidized dextran and studied in reaction: acetophenone + isopropylamine MBA + acetone
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Human skin microbiota: enzymatic activity and biotransformation of fragrance ingredients
a
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Towards directed evolution and high-throughput screening for enantioselective catalysis
a
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Characterization of trehalose synthase from Deinococcus geothermalis expressed in Escherichia coli.
Anna Paneka, Pawel Filipkowskia, Olga Pietrowa, Jozef Synowieckia a Department of Food Chemistry, Technology and Biotechnology, Faculty of Chemistry, Gdask University of Technology, Gdansk, Poland E-mail: panus85@wp.pl Trehalose (-D-glucopyranosyl-(11)--D-glucopyranoside) a naturally occuring disaccharide which is present in yeast, thermophilic microorganisms, some plants and insects. It can be produced by the intermolecular transglycosylation of maltose. Recombinant, slightly thermophilic trehalose synthase from Deinococcus geothermalis (DSMZ 11300) is one of those enzymes which can catalyze conversion of maltose into trehalose. In this study a trehalose synthase gene from Deinococcus geothermalis (DSMZ 11300) was amplified by PCR and transformed into non-pathogenic Escherichia coli strain for expression. The trehalose synthase gene consisted of 1,668 nucleotides, which encoded 564-amino acid residues. The recombinant enzyme (DgeoTreS) with C-terminal Histag was purified to homogeneity by Co-IDA affinity chromatography and characterized. Comparison of gel filtration and SDS-PAGE results showed that the enzyme is a homodimer whose subunits have a molecular weight of 64.69 kDa. The optimum pH and temperature of reaction catalyzed by DgeoTreS were 7.6 and 40C, respectively. The enzyme was highly stable at pH range from 6.0 to 9.0. The enzyme activity was almost unchanged after 8 h preincubation at 40C and pH 7.6, and retained about 57% of maximal activity after 8 hours of incubation at 55C. After 16 h reaction the yield of conversion of maltose (0.3 M) into trehalose was 59.04%. During this time 7.56% of glucose was produced as by-product. The DgeoTreS was inhibited by Cu2+, Hg2+, Fe3+, Tris, and EDTA but slightly activated by dithiothreitol (DTT). Keywords: trehalose, trehalose synthase, Deinococcus geothermalis, transglukosylation, Escherichia coli cloning, overexpression
Carla Portoa,b, Anita J. Marsaiolia Instituto de Qumica, Universidade Estadual de Campinas, CP 6154,13083-970 Campinas, SP, Brazil. b Natura Inovao e Tecnologia de Produtos, Rod. Anhanguera Km 30.5,07750-000, Cajamar, SP, Brazil. E-mail:carlaporto@natura.net; anita@iqm.unicamp.br The skin is the lrgest organ in the human body and is colonized by diverse microorganisms, most of which are harmless or even beneficial to their host. Skin microbial colonization depends on host intrinsic characteristics as skin location, host metabolism, life style and environmental factors [1]. The skin microbiota is highly and variable and a better understanding of this microbiota is necessary to gain insight of its participation in skin disorders and health [2]. These are major issues in cosmetic industries designing products (including fragrances ingredients) to achieve specific effects on human skin which could be jeopardized by human skin microbiota. Consequently it is mandatory to investigate how these products shift the ecological skin balance or how the skin microbiota biotransform (degradate) some cosmetic ingredients [3]. In this context, this work aims to study the key enzymatic activities of the human skin microbiota and to evaluate the role of these in the degradation of fragrance materials, widely used in cosmetic industry. Microbiota samples were obtained from 55 healthy volunteers [FR-1085/2008] from both genders, resulting in 55 bacteria consortia, 55 yeast consortia and 81 isolated filamentous fungi. Enzymatic activity was assayed by High Throughput Screening (HTS) using fluorogenic probes to detect epoxide hydrolases, Baeyer-Villiger monooxygenases, esterases and lipases [4]. Multibioreaction experiments were conducted with the samples which showed higher enzymatic activity in HTS assays, in order to evaluate the biotransformations of fragrance ingredients possessing diverse chemical structures and belonging to distinct olfactive families. Enzymatic activity assays revealed the presence of hydrolases in all samples (bacteria, yeasts and fungi). But in general all these showed high hydrolytic activity towards esters, top note fragrance ingredients and could be a serious drawback which is minimized by the esters volatility thus undergoing microbial action for a short time. Additionally the presence of oxidoreductase enabled the oxidation of sulfur-containing compounds and reduction or oxidation of cyclic and aliphatic ketones. New experiments are under investigation to evaluate conversion and enantioselectivity of the biotransformations. Most active isolated fungi were submitted to identification by molecular methods their activities will be tested more closely. These findings can provide important informations to cosmetic industry and reinforcing the need of further investigation on the role of human skin microbiota and the skin ecological balance.
Elisa S. Nogueiraa, Yvonne M. Wilsona, Marc Drrenbergera, Thomas R. Warda Department of Chemistry University of Basel, Spitalstrasse 51 CH-4056 Basel Switzerland E-mail: elisa.nogueira@unibas.ch Artificial metalloenzymes are created by incorporation of a catalytically active organometallic moiety within a host protein.[1] By variation of the catalytic metal and ligands or mutagenesis of the protein scaffold (chemogenetic protein engineering), [3, 6] the binding of the catalytic precursor can be improved and the enantioselectivity influenced. [4] In this project, the approach is to use transition metals in hydrogen transfer reactions and to anchor the metal into a protein scaffold to achieve enantioselectivity. The Ward group has an extensive knowledge on the creation and optimisation of artificial metalloenzymes for white biotechnology applications. [5] For this project, biotinylated active redox active metal moieties are combined with streptavidin (Sav) isoforms to afford artificial ADH's based on the biotin-(strept)avidin technology. In this context, new approaches are being investigated in order to perform enantioselective catalysis on cell-free extracts and/or supernatants from E. coli and P. pastoris protein expressions systems, respectively.
Figure 1.Artificial transfer hydrogenases for the enantioselective reduction of cyclic imines [2]
________________ [1] Collot, J., Gradinaru, J., Humbert, N., Skander, M., Zocchi, A., & Ward, T. R. (2003). Artificial Metalloenzymes for Enantioselective Catalysis Based on Biotin-Avidin. 125, 9030. [2] Drrenberger, M., Heinisch, T., Wilson, Y. M., Rossel, T., Nogueira, E. S., Knrr, L., et al. (2011). Artificial Transfer Hydrogenases for the Enantioselective Reduction of Cyclic Imines. Angew. Chem. , 123, 3082. [3] Letondor, C., Pordea, A., Humbert, N., Ivanova, A., Mazurek, S., Novi, M., et al. (2006). Artificial transfer hydrogenases based on the biotin-(strept)avidin technology: fine tuning the selectivity by saturation mutagenesis of the host protein. J. Am. Chem. Soc. , 128 (25), 8320. [4] Pordea, A., & Ward, T. R. (2008). Chemogenetic Protein Engineering: An Efficient Tool for the Optimization of Artificial Metalloenzymes. Chem. Comm. , 4239. [5] Steinreiber, J., & Ward, T. R. (2009). Artificial metalloenzymes for enantioselective catalysis based on the biotin-avidin technology. Organometallic Chem. , 25, 93. [6] Thomas, C. M., & Ward, T. R. (2005). Artificial Metalloenzymes: Proteins as Hosts for Enantioselective Catalysis. Chem. Soc. Rev. , 34, 337.
The biocatalyst successfully works in production of methylbenzylamine (MBA) in phosphate buffer and maintained its initial activity during at least 5 cycles.
__________________ [1] Kirsebom H. et al., Langmuir 2009, 25(15), 8462-8465 [2] Hoffman B. et al., J. Mater Sci: Mater Med. 2009, 20:1495-1503 [3] Hao L. et.al Current Opinion in Cell Biol. 2005, 9:217-226
________________ [1] Grice, E. A. and Segre, J. A. Nature Rev. Microbiol., 2011, 9, 244-253. [2] Grice, E. A. et al. Science, 2009, 324, 11901192. [3] Holland, K.T.; Bojar, R.A. Am. J. Clin. Dermatol., 2002, 3, 445-449. [4] Porto, C.; Aquino, P.A.C.; Crucello, A. and Marsaioli, A.J. Detection of Enzymatic Activity of the Skin Microbiota. In: Biotrans 2009, Berne, Switzerland.
October 2-6, 2011
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Properties and usefulness of proteases from Meiothermus ruber.
Olga Pietrow, Anna Panek, Jzef Synowiecki Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, ul. Gabriela Narutowicza 11/12, 80-233 Gdask, Poland E-mail: 1olga@poczta.fm The aim of the research is isolation and preliminary characterization of proteases derived from extremophilic bacteria Meiothermus ruber. Extremophiles are the source of enzymes (extremozymes) that were shown to be extremely high stable and therefore their use as industrial biocatalysts is very attractive. Currently it is estimated that an average of 40% of the enzymes used in industry sectors such as the production of detergents, foods, pharmaceuticals, leather products, diagnostics, waste management and silver recovery are proteases. The dominance of proteases on the market is still growing. This implies a need to seek new sources of such potentially useful biocatalysts. Leading research allowed to establish that Meiothermus ruber produce extracellular proteases that exhibited highest activity at 80C and pH 9. Maximum cell growth and extracellular proteolytic activity was attained after cultivation in a medium containing 6g/L of beef extract at temperature 55C and pH 8. Activity of postcultivation broth obtained in mentioned conditions was 26,23 U/g cells. The enzymes produced by Meiothermus ruber cause degradation of hemoglobin as well as asocasein and indicate small keratinolytic activity. The study of inhibitors impact suggests that enzyme produced by Meiothermus ruber belong to serine proteases. This was proved by activity decrease to 27,43% of initial value caused by phenylmethylsulfonyl fluoride (PMSF) with minimal, parallel activity decrease (about 5,67%) made by iodoacetamide. Slight activity decrease by EDTA and pepstatine showed that investigated protease not belongs to metallo- and aspartic family. In this case the activity decrease was only 10% and 2,8% of the initial value, respectively. The enzyme is activated by Ca2+, Mn2+ and inhibited by Zn2+, Hg2+, Fe3+, Cu2+, Co2+, Ba2+, Al3+ ions. In case of 1 mM concentration of Ni2+ and Mg2+ no impact was noticed. The study of chemical compounds impact like SDS and Triton X-100 suggests that enzymes produced by Meiothermus ruber could be used as a component of detergents. Triton X-100 do not affected on catalytic properties of proteases and SDS caused only 20% decrease of activity. Keywords: Extremophiles; Enzymes; Thermophiles; Proteases

Variation in nitrile hydratases and their properties
Justin Perrya, Gary Blacka, Meng Zhanga, Sander Van Peltb, Roger Sheldonb, Fred van Rantwijkb a School of Life Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST b Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands E-mail: justin.perry@northumbria.ac.uk Nitrile hydratases (E.C. 4.2.1.84) selectively hydrate nitriles to primary amides without hydrolysis of the C-N bond. They are two-subunit enzymes which have either an iron or a cobalt ion in their active site. We have produced a range of nitrile hydratases which have been produced via recombinant means in E.coli using sequence data from microbial genomes. Using purified samples of these nitrile hydratases (which are free of amidase activity) we have been able to show that they show differing selectivities and activities occur not only between enzymes exhibiting different metal centres and but also between enzymes with the same metal centre but with ~60% primary sequence identity [1]. The great wealth of online genomic data from all kingdoms of organisms is a tremendous resource for the discovery and classification of new variants within an enzyme class and nitrile hydratases are no different [2]. As of June 2011, there were over 100 proteins which could be ascribed nitrile hydratase activity with the majority containing cobalt-specific sequences. Searching the genome record has also revealed the presence of single subunit nitrile hydratases from eukaryotic sources such as the marine organisms Monosiga brevicollis MX1 [3] and Salpingoeca sp. ATCC 50818. We have produced recombinantly the nitrile hydratase from Monosiga brevicollis in E.coli and will be reporting initial results regarding its properties.
________________ [1] (a) GW Black, T Gregson, CB McPake, JJ Perry, M Zhang (2010) Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009. Tetrahedron Letters 51, 1639-1641; (b) S van Pelt, M Zhang, LG Otten, J Holt, DY Sorokin, F van Rantwijk, GW Black, JJ Perry, RA Sheldon (2011) Probing the Enantioselectivity of a Diverse Group of Purified Cobalt-Centred Nitrile Hydratases. Organic and Biomolecular Chemistry 9, 3011 [2] C Haddow, JJ Perry, MC Durrant and J Faith, Predicting functional residues of protein sequence alignments as a feature selection task. International Journal of Data Mining and Bioinformatics, in press [3] KU Foerstner, T Doerks, J Muller, J Raes, P Bork (2008). A nitrile hydratase in the eukaryote Monosiga brevicollis. PLoS ONE 3:e3976

Oriented irreversible immobilization of a glycosylated Candida antarctica B lipase on heterofunctional organoborane-aldehyde support
Gutarra, M. L.E.a,b, Mateo, C.a, Freire, D.M.G. b, Torres, F.A.G.c, Castro, A.M. d, Guisan, J.M.a, Palomo, J.M.a a Departamento de Biocatlisis, Instituto de Catlisis (CSIC), c/marie curie 2, cantoblanco campus UAM, 28049, Madrid, Spain; bInstituto de Qumica, Universidade Federal do Rio de Janeiro,Rio de Janeiro, Brazil; cLaboratrio de Biologia Molecular, Universidade de Braslia, Brazil; dGerncia de Energias renovveis, Centro de Pesquisas e Desenvolvimento, PETROBRAS, Rio de Janeiro, Brazil E-mail: gutarra@gmail.com The immobilization of enzymes represents an exciting goal in the field of enzyme biotechnology as it is a powerful tool to improve enzyme properties such as stability, activity, specificity, selectivity or reduction of inhibition [1]. Enzyme immobilization comprises different strategies including ionic exchange, covalent attachment or hydrophobic adsorptions and more recently, others strategies based on orientation of the enzyme on the support by different groups [2]. Here, it is proposed the design of a tailor-made heterofunctional support containing a combination of boronic acid groups and aldehydes, allowing a specific and oriented irreversible multi-point covalent attachment of glycosylated enzymes in aqueous media. For this purpose Candida antarctica (fraction B) lipase expressed in Pichia pastoris, a selective glycosylated protein at Asn 74 has been employed. The immobilization method occurs via a two step mechanism: first orientation by organoborane interaction at neutral pH and a consecutive multi-point covalent attachment by aldehyde reaction at alkaline pH. The enzyme was specifically immobilized on this support in 70% yield at pH 7, oriented by the reaction of the hydroxyl groups on the sugar moiety with boronic acid on the support, whereas commercial CAL-B from Novozymes (non-glycosylated) was hardly immobilized at this pH (<10%). The consecutive incubation at pH 10 permitted the reaction of amine groups of the protein with aldehyde groups on the support. After a reductive amination, an irreversible immobilization methodology on organoborane-aldehyde support was possible with 99% final immobilization yield. The Borald-CAL-B preparation was a very stable biocatalyst in the presence of high amount of solvent or high temperature (e.g. more than 10 fold in the presence of 60% (v/v) acetonitrile). An improvement of the specific activity up to 5 fold for example in the hydrolysis of methyl phenyl acetate was obtained compared with a one-point covalent preparation. An ee of 89% towards R isomer was achieved with this new immobilized biocatalyst in the enantioselective hydrolysis of methyl mandelate. In conclusion, the glycosylated CAL-B expressed in P. Pastoris has been specifically immobilized by oriented and irreversible method on heterofunctional organoborane-aldehyde support and this methodology produces a biocatalyst with enhanced properties when compared to one-point covalent preparation.
_____________________ [1] J. M. Palomo. Lipases enantioselectivity alteration by immobilization techniques. Curr. Bioact. Compd., 2008, 4,126138. [2] C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan and R. Fernandez-Lafuente, Improvement of enzyme activity, stability and selectivity via immobilization techniques Enzyme Microb. Technol., 2007, 40,14511463.
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Stabilization of H. jeronica EG1 by a semirational approach
Dirk Heesela, Fabrizio Sibillab, Rainer Fischera,c and Ulrich Comandeura a Department of Molecular Biotechnology RWTH-Aachen, Worringerweg1 52074 Aachen, Germany b Department of Biotechnologie, Worringerweg1 52074 Aachen, Germany c Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstrae 6 52074 Aachen, Germany E-mail: dirk.heesel@rwth-aachen.de The catalytic domain of EG1 (Cel7B) from Hypocrea jeronica (Trichoderma reesei) has been modified by site directed mutagenesis utilizing degenerated primer mixes. The aim of the described work was thermal stabilization of EG1. Selection of residues for mutagenesis was based on atomic displacement parameters (b values)[1], derived from xray diffraction data. Expression and high throughput screening (HTS) of mutant libraries revealed 6 thermostable mutants. Individual mutations have been recombined by staggered extension process and the resulting library was subjected to another round of HTS. The Tm50 of the best variant was determined in assays and revealed to be 4C higher than the Tm50 of the wild type enzyme. Further more it was analyzed if mutants of the staggered library also show an increase in stability under high salt conditions, in ionic liquids and in EtOH. There was no effect on stability in ionic liquids and under high salt conditions for any of the evolved variants. But for one mutant a strikingly increase stability in EtOH has been observed. Therefore the b value method is not only suitable for thermal but also for EtOH-stabilization. Stabilized cellulases can be of potential interest for the hydrolysis of Organosolv pretreated cellulose and for enzymatic simultaneous saccharification and fermentation (SSF) processes. __________________
[1] Parthasarathy, S. and M. R. Murthy (2000). "Protein thermal stability: insights from atomic displacement parameters (B values)." Protein Eng 13(1): 9-13.
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Expression of a branched chain aminotransferase (BCAT) in Pichia pastoris: high throughput screening and process development
Andrea Camattaria,b, Katrin Weinhandla and Anton Gliedera a Austrian Centre of Industrial Biotechnology, Petersgasse 14/5,Graz (Austria) b Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, Graz (Austria) E-mail: andrea.camattari@tugraz.at Aminotransferases (ATs) are crucial enzymes for prokaryote and eukaryote metabolism, involved in both amino acid catabolism and anabolism. Due to their broad substrate specificity and high enantioselectivity, ATs have attracted industrial attention for large scale synthesis1 of amino acids and chiral amines. A disadvantage of transamination bioprocesses for industrial use lies in the disfavourable reaction equilibrium constant, that results in a typical conversion of substrate to product of 50% or less: to achieve higher conversions through equilibrium displacement, various enzyme-coupled reactions are documented in the literature. Important requirements for aminotransferase process development include the expression of TAs in large quantities, possibly avoiding expensive enzymatic co-reactions or time-consuming down-stream processing: the methylotrophic yeast Pichia pastoris is a good candidate to fulfill such requirements, since its capabilities in protein expression, high-density growth and convenient downstream processing are well known2. We have expressed in P. pastoris a branched chain aminotransferase (BCAT), encoded in E. coli by the ilvE gene, under the control of either a constitutive (GAP) or an inducible promoter (AOX1). A high throughput screening procedure, based on glutamate oxidase and horse radish peroxidase, has been put in place to identify optimal expression. Several rounds of screening have allowed us to identify high-expression clones; BCAT specific activity in P. pastoris and E. coli cell-free extracts has been compared. Pichia pastoris expressing BCAT have been tested for whole cell bioconversion of an industrially relevant substrate under process conditions. Enzyme engineering, made possible by recent insights of the 3D structure of BCAT3, consisted of manipulating the active site to accommodate bulkier substrates; our high throughput procedure allowed us to screen for a large number of clones, to select for higher reaction rates on non-natural substrates. Mutant selection, together with permeabilization optimization and strain development, represent the next steps toward a whole-cell biocatalyst for transaminase reactions based on Pichia pastoris.
__________________________________ [1] J.S. Shin and B.G. Kim, Biotechnology and Bioengineering, vol. 65, Oct. 1999, pp. 206-211. [2] J.M. Cregg et al., Molecular Biotechnology, vol. 16, Sep. 2000, pp. 23-52. [3] M. Goto et al., Biochemistry, vol. 42, Apr. 2003, pp. 3725-3733.
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Serine decarboxylases: new enzymes for the bio-based production of ethanolamine
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Improved catalytic properties of mutant penicillin acylases from Escherichia coli in acylation of amino compounds
Nikolay Panina, Irina Shapovalovaa, Dorel Gurandab, Vytas vedasa,b Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Vorobjev hills 1-73, Moscow 119991, Russia b Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Vorobjev hills 1-40, Moscow 119991, Russia E-mail: panin@belozersky.msu.ru
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240
Engineering phenylalanine aminomutase (PAM) for the production of -amino acids
a
Rosario Mdicia, Pablo Domnguez de Marab, Linda G Ottenc, Adrie J J Straathofa Bioseparation Technology Group, Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands; bInstitute of Technical and Macromolecular Chemistry RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; cBiocatalysis and Organic Chemistry Group, Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628 BL, Delft, The Netherlands. E-mail: r.medici@tudelft.nl
Matthew M. Heberlinga, Bian Wua, Sebastian Bartscha, Dick B. Janssena University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands E-mail: m.m.heberling@rug.nl -amino acids harbor many applications in their free form and as robust polymeric derivatives, such as -peptides. Although biocatalytic routes have been explored for the production of -amino acids, these eco-friendly routes are far from optimal because they are based on kinetic resolutions. The asymmetric syntheses of enantiomerically pure amino acids and chiral amines have become a prevalent route using biocatalysts, such as aminotransferases, ammonia lyases, and aminomutases[1]. This work presents our contributions to making a phenylalanine aminomutase (PAM) from Taxus chinensis an attractive lead to synthesizing enantiopure, aromatic -amino acids.
Amino acid decarboxylation is a key (bio)chemical step from which many practical uses can be derived. Applications can be found in pharmaceutical [1] or food industry [2]. Likewise, amino acid decarboxylases are gaining increased interest in the production of bio-based commodities, via proteins or amino acids [3,4]. For example, monoethanolamine (MEA) can be obtained by direct decarboxylation of L-serine catalyzed by serine decarboxylases (SDC). The development of sustainable strategy that replaces the current MEA petrochemical synthesis has gained increasing importance, due to its wide range of applications as building blocks in diverse industries and the scale that is produced (>600 000 tons/year). SDCs of Arabidopsis thaliana, spinach and rapeseed have been characterized [5], but their application as biocatalysts was never studied. Three SDCs genes, two from A. thaliana (SDCat and SDCat58) and one from the alga Volvox carteri (SDCvc)) were expressed in E. coli strains and evaluated for MEA synthesis. Even though the highest SDC activity was observed using the truncated enzyme from A. thaliana, MEA production using E. coli cells containing the activity in a fermentation or biotransformation configuration, was still not sufficient for large scale applications. To improve the activity of the enzyme towards L-serine using directed evolution techniques, a high-throughput screening (HTS) method was developed. A selective discrimination of MEA in the presence of L-serine based on the reaction reported by Roth [6], and was shown to be outstanding for an ample number of amine / amino acid pairs, broadening its scope to other biocatalytic strategies involving decarboxylases, to important clinical applications, like drug design or the development of enzyme inhibitors. The results of the SDCat58 library of mutants, combined with a homology model of the enzyme based on other amino acid decarboxylases and docking experiments, allowed the identification of key amino acids in the protein sequence.
________________ [1] Valastro, B.; Dekundy, A.; Tennigkeit, F.; Russ, H.; Franke, L. WO2010/063486 (2010). [2] Klee, H.J., Tieman, D. WO2005/035752 (2005). [3] Lammens, T.M.; De Biase, D.; Franssen, M.C.R.; Scott, E.L.; Sanders, J.P.M. Green Chem. 11, 15621567 (2009). [4] Verseck, S.; Hger, H.; Karau, A.; Eggeling, L.; Sahm, A. WO2008/092720 (2008). [5] D. Rontein, I. Nishida, G. Tashiro, K. Yoshioka, W. Wu, D. Voelker, G. Basset, J. Biol. Chem., 276, 35523-35529 (2001). [6] Roth, M. Anal. Chem. 43, 880-882 (1971).
Penicillin acylase from Escherichia coli (EC 3.5.1.11) is one of the most studied Ntn-hydrolases and is known mainly as a catalyst in antibiotic industry. At the same time enzyme was shown to possess high enantioselectivity in hydrolysis of N-phenylacetylated derivatives of amino acids as well as in acyl transfer to amino acids in aqueous medium. These properties of penicillin acylase from Escherichia coli have been successfully used to resolve racemates of non-conventional alpha-, beta- and gamma-amino carboxylic acids as well as aminophosphonic and aminophosphinic acids. At the same time enantioselectivity to amino alcohols and primary amines of the wild type enzyme is low in both deacylation and acylation reactions and therefore we attempted to improve it by protein engineering. Two mutants of penicillin acylase from Escherichia coli were shown to have improved catalytic properties for resolution of amino alcohols and primary amines: mutation S149R leads to 4-fold improvement of enantioselectivity compared to wild type enzyme in acylation of 2-aminobutanol using amide of S-mandelic acid as acyl donor in aqueous medium. Mutation F71L, in addition to 3-fold increased enantioselectivity in acylation of 2-aminobutanol using amide of R-mandelic acid as acyl donor in aqueous medium and 5-fold increased enantioselectivity in hydrolysis of N-phenylacetylphenylalaninol, leaded to substantially (5-times) increased effectivity of enzymatic acyl transfer to nucleophile (i.e. increased synthesis/hydrolysis ratio). Both characterized penicillin acylase mutants had higher catalytic activity to the chromogenic substrate 2-nitro-5-phenylacetylamidobenzoic acid and quite different substrate profiles compared to wild type enzyme. Experimental studies demonstrated that major catalytic properties of penicillin acylase from Escherichia coli can be remarkably improved by protein engineering. This work was supported as a joint EC-Russian FP7-KBBE-2008-2B project IRENE by the European Commission (grant agreement 227279) and the Russian Ministry for Science and Education (contract 02.740.11.0866).
______________________ [1] Wu, B., Szymaski, W., Heberling, M. M., Feringa, B. L., Janssen, D. B., Trends Biotechnol. In Press. [2] Wu, B., Szymaski, W., Wietzes, P., de Wildeman, S., Poelarends, G. J., Feringa, B. L., Janssen, D. B., Chembiochem 2009, 10, 338-344. [3] Szymaski, W., Wu, B., Weiner, B., de Wildeman, S., Feringa, B. L., Janssen, D. B., J. Org. Chem. 2009, 74, 9152-9157. [4] Wu, B., Szymaski, W., Wijma, H. J., Crismaru, C. G., de Wildeman, S., Poelarends, G. J., Feringa, B. L., Janssen, D. B., Chem. Commun. 2010, 46, 8157-8159.
Figure 1. PAM-catalyzed ammonia addition to trans-cinnamic acid for the synthesis of enantiomerically pure (R)--Phe and (S)--Phe [2]. PAM catalyzes the reversible, asymmetric addition of ammonia to cinnamic acid (Figure 1) yielding a 50/50 mixture of the aromatic amino acids - and -Phe (a key component of the anticancer drug taxol). The active site cofactor 5-methylene-3,5-dihydroimidazol-4-one (MIO) group of PAM assists in this asymmetric ammonia addition. Although the homologous phenylalanine ammonia lyase (PAL) displays higher amination rates for the production of amino acids, the -regioselectivity of PAL hinders its application as a biocatalytic tool for the synthesis of aromatic -amino acids. Furthermore, there is no -lyase known to date. Therefore, our goal is to engineer PAM as a -lyase for the production of enantiopure aromatic -amino acids by manipulating its regioselectivity, while increasing its amination rate to make PAM an attractive biocatalyst with industrial potential. We showed that PAM can add ammonia to cinnamic acid derivatives[2,3]. Using our crystal structure, a focused library of the carboxylate binding pocket within the active site was screened and PAM variants with enhanced -regioselectivity and activity were obtained that retained the high enantioselectivity of wild-type. In addition, a single active-site mutant (C107S) was rationally-engineered to switch PAM to a novel enantioselective tyrosine aminomutase (TAM) for the synthesis of -Tyr[4].
October 2-6, 2011
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Characterizing thermostable nucleoside phosphorylases for their use as biocatalysts
Kathleen Szekera, Xinrui Zhoua, Igor A. Mikhailopulob, Peter Neubauera a Department of Bioprocess Engineering, Institute of Biotechnology, Technische Universitt Berlin, Ackerstrae 71-76, 13355 Berlin, Germany; bInstitute of Bioorganic Chemistry, National Academy of Sciences of Belarus, 220141 Minsk, Acad. Kuprevicha 5/2, Belarus E-mail: k.szeker@mailbox.tu-berlin.de Nucleoside analogues have emerged as an important class of pharmaceutical agents for the treatment of viral infections and cancer. Moreover they have broad applications in molecular biological techniques and diagnostics. The preparation of modified purine nucleosides by chemical methods remains especially challenging - often characterized by multistage processes with low yield. Alternative synthetic routes include chemo-enzymatic approaches in which nucleoside phosphorylases (NPs) catalyze the enzymatic transglycosylation of nucleosides [1] (see Fig. 1). However, the use of biocatalysts is often limited by their instability under harsh reaction conditions. For example, high temperatures are typically used for the preparation of nucleosides to increase the solubility of poorly soluble substrates.

Phosphorothioate-based DNA Recombination: an enzyme-free and sequence-independent method for the combinatorial assembly of multiple DNA fragments
Jan Marienhagena, Alexander Dennigb, Michael Botta, Ulrich Schwanebergb Institut fr Bio- und Geowissenschaften IBG-1: Biotechnologie Forschungszentrum Jlich, 52425 Jlich, Germany; bLehrstuhl fr Biotechnologie RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany E-mail: j.marienhagen@fz-juelich.de
a

In silico discovery of R-selective amine-transaminases
Sebastian Schtzlea, Matthias Hhnea, Helge Jochensa, Fabian Steffen-Munsberga, Ahmad Thontowia, Karen Robinsb, Uwe T. Bornscheuera a Institut of Biochemistry, Dept. of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany; b Lonza AG, Valais Works, Visp, Switzerland E-mail: matthias.hoehne@uni-greifswald.de One key requirement for biocatalytic production of optically active compounds is the availability of an enzyme, which converts the desired substrate with an appropriate enantioselecitivity and enantiopreference. We were searching for R-selective amine transaminases (R-ATA), which would enable asymmetric synthesis of various (R)-amines starting from the respective prochiral ketone substrates. Unfortunately, virtually all of the known ATA were S-selective. Only one R-ATA was commercially available, but no amino acid sequence had been reported [1]. The most common sources of suitable enzymes are nature's own reservoir of microorganisms, which can be accessed by screening strain collections or metagenome libraries. Protein engineering methods have also been developed, which mimic evolution in vitro in the laboratory. We have developed an in silico strategy for a sequence based prediction of substrate specificity and enantiopreference within PLP-fold type IV proteins [2]. First, we used rational protein design to identify key amino acid exchanges in L-branched chain amino acid TA, which could lead to the desired R-ATA activity. Instead of constructing these mutants in the laboratory, we searched protein databases for proteins already carrying these mutations. From 21 hits in the database, 17 proteins could be expressed in sufficient amounts and showed R ATA activity. Hence, before creating mutants or libraries based on rational predictions in the laboratory, it is worthwhile to investigate whether nature has already designed the mutants and optimized them during evolution. The amino donor spectrum of these enzymes was characterized with a fast conductometric assay: During the conversion of an amine and pyruvate to the ketone and alanine, the conductivity of the reaction medium decreases, as charged substrates are converted into a non-charged ketone and the zwitterionic alanine [2,3]. For seven of the most promising enzymes we optimized their production and the reaction conditions. These enzymes were then applied in the asymmetric synthesis of a range of aliphatic, aromatic and arylaliphatic (R)-amines starting from the corresponding prochiral ketones [4]. By using the lactate dehydrogenase/ glucose dehydrogenase system to shift the equilibrium [5], at least one enzyme was found allowing complete conversion for each of the substrate ketones, leading to the corresponding optically pure (R)-amine (>99%ee) [4].
________________ [1] Hhne, M., Bornscheuer, U.T., 2009, ChemCatChem, 1, 42-51 [2] Hhne, M., Schtzle, S., Jochens, H., Robins, K., Bornscheuer, U.T., 2010, Nature Chem. Biol., 6, 807-813 [3] Schtzle, S., Hhne, M., Robins, K., Bornscheuer U.T., 2010, Anal. Chem., 82, 2082-2086 [4] Schtzle S., Steffen-Munsberg, F., Hhne, M., Robins, K., Bornscheuer, U.T., submitted [5] Koszelewski, D., Lavandera, I., Clay, D., Rozzell, D., Kroutil, W., 2008, Adv. Synth. Catal., 350, 2761-2766
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Characterization of novel transaminases for a multi-step biocatalysis
Maria F Villegas-Torresa, Frank Baganza and John Wardb a The Advanced Centre for Biochemical Engineering, University College London, Department of Biochemical Engineering, Torrington Place London, WC1E 7JE, United Kingdom. b Research Department of Structural and Molecular Biology, The Darwin Building, University College London, Gower Street London, WC1E 6BT, United Kingdom. E-mail: maria.torres.09@ucl.ac.uk Abstract Chiral amino alcohols and amino diols are building blocks of sphingolipids, antibiotics such as chlor- and thiamphenicol and antiviral glycosidase inhibitors such as the deoxynojirimycin family. Their chemical synthesis presents several limitations; thus biocatalysis employing a de novo metabolic pathway using a transketolase coupled with a transaminase step, have been reported showing up to a 90% conversion in a one-pot whole cell system [1]. One of the requirements to obtain a high yield is the use of hydroxypyruvate (HPA) as substrate for the transketolase as it drives the reaction towards completion. This compound is commercially available but at high cost limiting its applicability at industrial scale. HPA is the keto acid of serine, thus through a transaminase reaction it could be easily produced. The aim of this study is to identify possible candidates for the synthesis of HPA through a transamination using serine as amino donor. A previously cloned library of transaminases from different organisms were screened for serine usage, 12 enzymes were selected to quantify their reaction yield after a two hour reaction with pyruvate as amino acceptor. 4 out of 12 synthesized more than 1 mM of HPA from 10 mM serine. These transaminases were kinetically characterized and their amino acceptor profiles determined. Two of them were 2-times more active towards aliphatic aldehydes and 3-times faster using erythrulose as amino acceptor compared to the transaminase from Chromobacterium violaceum previously used for chiral amino alcohol synthesis [2]. As a proof of concept these new transaminases were coupled with a transketolase step for the successful synthesis of (2S,3R)-2-amino-1,3,4-butanetriol replacing HPA with serine. This is the first report on the study of transaminase activity using serine as amino donor and their application on chiral amino alcohol synthesis employing a recycling coupled pathway. Key words: Transaminase, transketolase, de novo pathway, chiral amino alcohol synthesis, recycling mechanism.
____________ [1] Rios-Solis et al. A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a mix and match E. coli expression system with microscale experimentation. Biocat and Biotrans. In press. [2] Ingram et al (2007) One-pot synthesis of amino-alcohols using a de-novo transketolase and -alanine:pyruvate transaminase pathway in Escherichia coli. Biotech Bioeng. 96. 559-569
Figure 1. Schematic view of the enzymatic transglycosylation, transferring a pentofuranose moiety from pyrimidine to purine nucleoside. Aim of this work is to overcome the instability of NPs at high temperatures and thus make their use in biocatalytical applications more attractive. For this purpose a set of 6 nucleoside phosphorylases from thermophilic microorganisms (Deinococcus geothermalis, Geobacillus thermoglucosidasius, Thermus thermophilus, Aeropyrum pernix) was selected and successfully overexpressed in E. coli. The selected enzymes include 3 NPs with specificity for pyrimidine nucleosides (PyNP) and 3 NPs with specificity for purine nucleosides (PNP). Preliminary results show that - as expected - half of the recombinant NPs are stable at moderate temperatures (45 - 65 C), while the other half appear to be stable at extremely high temperatures (> 95 C). We have started to purify the thermostable NPs to characterize them in detail. In particular we address i) thermostability, ii) reaction rate dependency on temperature and pH and iii) substrate specificities. The results will allow us to evaluate the potential application of this set of novel biocatalysts in enzymatic transglycoylation for the synthesis of modified nucleosides.
________________ [1] Mikhailopulo, I.A. and A.I. Miroshnikov, 2011. Mendeleev Communications, 21(2)
Rational guided generation of protein chimeras has developed into an important field of protein biochemistry and protein engineering for tailoring catalytic and biophysical properties of enzymes [1]. Combinatorial recombination of structural elements or whole domains however, remains to be technically challenging due to sequence dependent biases which diminish the overall quality of resulting chimeric libraries. Since most methods for generating such libraries are often limited by a low frequency of crossover points and suffer from challenges in handling [2, 3], we developed the Phosphorothioate-based DNA Recombination method (PTRec). This new approach for recombining secondary structure elements, motifs or domains of proteins is unique, since enzymatic steps during fragment generation are avoided and neither a ligation step nor a final assembly PCR is required. Furthermore, PTRec is homology- and sequence-independent, and demands only a short stretch of four identical amino acids for efficient recombination. Key to the simplicity of PTRec is the highly efficient chemical cleavage of multiple phosphorothiodiester bonds generating single-stranded 12 nt-overhangs for hybridization [4]. As proof of principle, PTRec was employed to recombine five domains of three phytases (sequence identity: 45% - 53%) using four cross-over points in the targeted enzymes. In the chimeric phytase library generated within a single day, 88 % of 42 randomly picked and sequenced genes were efficiently recombined [5]. 1. Ostermeier, M. and Benkovic, S. J. (2000). Evolution of protein function by domain swapping. Adv. Protein Chem. 55, 29-77. 2. Sieber, V., Martinez, C. A. and Arnold, F. H. (2001). Libraries of hybrid proteins from distantly related sequences. Nat. Biotechnol. 19, 456-460. 3. Hiraga, K. and Arnold, F. H. (2003). General method for sequence-independent sitedirected chimeragenesis. J. Mol. Biol. 330, 287-296. 4. Blanusa, M., Schenk, A., Sadeghi, H., Marienhagen, J., and Schwaneberg, U. (2010). Anal. Biochem. 406, 141-146. 5. Marienhagen, J., Dennig, A. and Schwaneberg U. (2011). Phosphorothioate-based DNA Recombination: an enzyme-free and sequence-independent method for the combinatorial assembly of multiple DNA fragments (submitted)
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Biocatalysis of epoxides by novel recombinant Epoxide hydrolases identified by genome analysis
Dipti Sareen, Ranjai Kumar, Priya Saini and Shadil Ibrahim Wani Department of Biochemistry, Panjab University, Chandigarh-160014, India. E-mail: diptsare@pu.ac.in EH as a biocatalytic tool : Epoxide hydrolases (EH; E.C.3.3.2.3) from microbial sources have been recognized as a versatile biocatalytic tool for the synthesis of enantiomerically pure epoxides and vicinal diols. The ubiquitous distribution of epoxide hydrolases and hence a wide range of substrate specificity and enantioselectivity makes these enzymes an ideal choice for biotechnological applications [1]. EH from microbial sources have found industrial applications in biocatalysis due to the easier scale up of the enzyme produced there from. Keeping in mind the potential of enantiopure epoxides & diols in the pharmaceutical, agrochemical and flavour industries, a range of substrates have been analyzed using epoxide hydrolase as the catalyst. Most microbial epoxide hydrolases identified till today, accept only a limited range of substrates and enantioselectivity is often low. Hence, research efforts have been on (a) to engineer known EHs for better enantioselectivity and (b) to find novel enantioselective EHs with a wide substrate scope. A successful bioinformatic approach that has been explored by several groups, is the mining of genomic database for identifying putative EH-encoding genes, using EH-specific sequence motifs, and their expression in the surrogate host cells. Though most of the known EHs have been found to have a low sequence identity (19-35%), yet the presence of some conserved sequence motifs, like nucleophillic elbow (Sm-X-Nu-X-Sm-Sm), the G-X-Sm-X-S/T motif, the H-G-X-P oxyanion hole and the Asp-His-Asp catalytic triad, have helped to annotate a / hydrolase fold gene as an EH. By using these conserved EH sequence motifs, for the bioinformatic analysis of the vast microbial genome databases available, the other structurally and mechanistically related / hydrolase gene sequences such as those of esterases and dehalogenases can easily be separated out. Using this bioinformatic approach in our lab, we have successfully identified, cloned and overexpressed a few novel EH bacterial genes and have also characterized one of them in detail [2]. ________________ [1] Dipti Sareen and Ranjai Kumar (2011) Prospecting for efficient enantioselective epoxide hydrolases. Indian Journal of Biotechnology 10, 161-177. [2] Ranjai Kumar, Shadil Ibrahim Wani, Nar Singh Chauhan, Rakesh Sharma and Dipti Sareen (2011) Cloning and characterization of an Epoxide hydrolase from Cupriavidus metalidurans CH34. Protein Expression and Purification doi: 10.1016/j.pep.2011.04.007
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In-situ peracetic acid production using CE-7 perhydrolase variants
Robert DiCosimo, John E. Gavagan, Mark S. Payne, Raymond R. Zolandz DuPont, Central Research & Development, Experimental Station, Wilmington, DE, USA E-mail: Robert.DiCosimo@usa.dupont.com Peracetic acid (PAA) is widely-used as a biocide, having broad efficacy towards bacteria, viruses, spores and other pathogens. This disinfectant is also environmentally sustainable, producing acetic acid, oxygen and water as reaction byproducts. It is used in a number of applications such as food processing, institutional cleaning and disinfection, clinical disinfection and medical instrument sterilization. Peracetic acid is often preferred to alternative disinfectants such as quats (quaternary ammonium cations) and aldehydes (formaldehyde, glutaraldehyde, and phthalaldehyde), but issues related to shelf life stability and odor have limited the wide-scale use of peracetic acid in many applications. By using enzyme catalysis to reproducibly generate an efficacious concentration of peracetic acid in situ and on demand, one can mitigate or eliminate storage stability and odor issues. Enzymes belonging to the structural family of CE-7 esterases, such as cephalosporin C deacetylases and acetyl xylan esterases, have significant activity and selectivity for catalyzing the perhydrolysis of acetyl esters by hydrogen peroxide in aqueous solution, producing efficacious concentrations of peracetic acid. These CE-7 perhydrolases share a conserved structural motif, as well as superior perhydrolysis activity when compared to other /-hydrolases. The wild-type thermophilic perhydrolase isolated from Thermotoga maritima has excellent stability at temperatures of up to 90 C, making possible a simple and economical purification and isolation process for enzyme production. Variants of a Thermotoga maritima perhydrolase have been prepared with improved specific activity, and improved selectivity for perhydrolysis rather than peracid hydrolysis, when compared to the wild-type enzyme.
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Investigations of a highly selective artificial transfer hydrogenase for the reduction of cyclic imines
Marc Drrenberger, Yvonne Wilson, Thibaud Rossel, Annette Mutschler, Tillmann Heinisch, Thomas R. Ward University of Basel, Department of Inorganic Chemistry, Spitalstrasse 51, Switzerland E-mail: marc.duerrenberger@unibas.ch E-mail: yvonne.wilson@unibas.ch Incorporation of biotinylated piano stool transition metal complexes [ArM(biot-p-L)Cl] into streptavidin (SAV) affords artificial metallohydrogenases for the transfer hydrogenation of ketones and cyclic imines. The combination of different biotinylated complexes (M = Ru, Rh, Ir; Ar = benzene, cymene, Cp*) with a library of SAV mutants resulting from saturation mutagenesis at position 112 allowed optimization of the reduction of 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline 1 to salsolidine 2 in terms of activity and selectivity. This screening led to the identification of [Cp*Ir(biot-p-L)Cl] SAVS112A 3 as the most active (TON up to 4000) and selective (ee up to 96%) hybrid catalyst whose crystal structure was published recently [1]. The presence of cellular components severely affects catalysis, requiring a SAV purification protocol involving numerous dialysis steps. We discuss practical measures taken to reduce the number of steps involved in the purification procedure, thus allowing the more efficient screening of hybrid catalysts.
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Altering the spectral properties of high-redox potential laccases by directed evolution: from blue to yellow
Diana Mata, Sergey Shleevb and Miguel Alcaldea Department of Biocatalysis, Institute of Catalysis, CSIC, 28049, Cantoblanco, Madrid, Spain; bBiomedical Laboratory Science, Health and Society, Malm University, 20506, Malm, Sweden. E-mail: diana.mate@icp.csic.es During the directed evolution of the high-redox potential laccase PM1 for functional expression in Saccharomyces cerevisiae [1], the characteristic absorption band of the T1 copper site was quenched, turning the typical colour of the enzyme from blue to yellow. To find out the molecular bases for this change, the evolved laccase and several intermediate mutants of the evolutionary process have been deeply characterized. Whilst peptide printing of the evolved variant addressed the lack of traces that could mask the signal at 610 nm at the T1 site, the redox potential of native PM1 and evolved mutants were similar (+750 mV). Variants were capable to oxidize a broad array of substrate (ABTS, Guaicol, DMP, synapic acid) also including high-redox potential dyes indicating that the enzyme did not lose catalytic performance. It was detected an N-terminal tail containing 4-extra amino acids, which may affect the spectral characteristics of the enzyme. The laccase over-expression by yeast suggests an alternative processing based on the fail of the STE13 protease at the Golgi compartment [2]. A truncated version of the mutant was engineered displaying similar spectroscopic features, but with a drastic reduction in the secretion levels. Mutations introduced in the first cycles of evolution at the T1 environment seem to be responsible for the spectral alteration by affecting the coordination of the T1 copper.
_______________ [1] Mat, D., Garca-Burgos, C., Garca-Ruiz, E., Ballesteros, A.O., Camarero, S. and Alcalde, M. (2010). Chem. Biol. 17, 1030-1041. [2] Zsebo, K.M., Lu, H.S., Fieschko, J.C., Goldstein, L., Davis, J., Duker, K., Suggs, S.V., Lai, P.H., and Bitter, G.A. (1986). J. Biol. Chem. 261, 5858-5865.
Scheme 1. Perhydrolysis of triacetin to produce peracetic acid.

____________________ [1] M. Drrenberger, T. Heinisch, Y.M. Wilson, T. Rossel, E. Nogueira, L. Knrr, A. Mutschler, K. Kersten, M.J. Zimbron, J. Pierron, T. Schirmer, T.R. Ward, Angew. Chem. Int. Ed. Engl. 2011, 50, 13.
October 2-6, 2011
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New strategies for the production of medicinal compounds: Tropane alkaloids
Alejandra B. Cardillo, Julin Rodriguez Talou, Ana M. Giulietti Microbiologa Industrial y Biotecnologa, Facultad de Farmacia y Bioqumica, Universidad de Buenos Aires. Argentina E-mail: acardillo@ffyb.uba.ar Plants are able to produce a large diversity of natural products of vast interest for pharmaceutical purposes. Tropane alkaloids, such as hyoscyamine and scopolamine, are secondary metabolites traditionally applied in medicine according to their anticholinergic activity [1]. Hyoscyamine is converted by Hyoscyamine 6-hydroxylase (H6H) into anisodamine and scopolamine [2]. Recently, potential medical applications were also described for anisodamine [3]. Nowadays, these compounds are obtained from natural producer plants due to the cost and complexity of the chemical synthesis of them. For this reason, tropane alkaloids production by biotransformation processes is an attractive strategy for the pharmaceutical industry [4]. In the present work we explored the development of an alternative strategy for the production of the most valuable alkaloids, anisodamine and scopolamine, using the H6H as biocatalyst. For this purpose the h6h gene was amplified from total RNA preparations obtained from immature anthers of the South American tropane alkaloid producer plant, Brugmansia candida and cloned into different vectors in order to produce tagged and untagged enzymes. The H6H enzyme was expressed fused at its C-terminus to a V5 epitope and a His-tag (H6H-V5-6His) [4]. On the other hand, it was fused at its N-terminus to a cellulose binding domain (CBD-H6H) in order to combine the purification step with the enzyme immobilization on cellulose, a low cost matrix [5]. The constructions were introduced by chemical transformation in S. cerevisiae CEN PK2. In order to explore the different strategies, crude protein extracts of the induced yeast strains were assayed for the enzyme activity at 30C for 15hs. The analysis of the alkaloids was carried out by HPLC with UV detection. The mobile phase used was octanesulfonic acid 0.01M pH3/methanol (65:35), flow rate 1ml/min [4]. The results showed that the tagged and untagged enzymes were able to transform hyoscymine, showing a functional expression of the h6hcDNA. However, the products obtained were different when the reaction was performed by the different enzyme constructions. The untagged H6H and the CBDH6H were able to convert hyoscyamine into anisodamine and scopolamine while H6HV5-6His only produced anisodamine. Surprisingly, the cellulose binding domain did not negatively affect the catalytic activity of the H6H as the V5 epitope and the Histidine tag affected it. These facts are encouraging for the development of a biocatalytic process using immobilized enzymes.
________________ [1] Hashimoto, T., Yamada, Y. Plant Physiol. 1986, 81, 619625. [2] Hashimoto, T., Matsuda, J., Yamada, Y. FEBS Lett 1993, 329, 35-39. [3] Poupko, J. M., Baskin, S. I., Moore, E. J Appl Toxicol 2006, 27, 116-121. [4] Cardillo, A. B., Rodriguez Talou, J., Giulietti, A. M. Microb Cell Fact 2008, 7, 17-23. [5] Engel, S., Vyazmensky, M., Berkovich, D., Barak, Z., et al. Biotechnol Bioeng 2005, 89, 733-740.

Application of microbial 2-oxoglutarate-dependent dioxygenase, unique and practical enzyme for amino acid hydroxylation
Ryotaro Haraa, Kuniki Kinoa, b a Research Institute for Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan; bDepartment of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan E-mail: ryo_h@aoni.waseda.jp Optically active hydroxyamino acids are very useful compounds for the synthesis of fine chemicals. To produce them, an enzymatic direct hydroxylation of commercially available amino acids is the most efficient reaction because highly selective and simple reaction is able to achieve under moderate conditions. 2-Oxoglutarate-dependent dioxygenase catalyzes diverse oxidation reactions, such as hydroxylation, epoxidation, desaturation, and ring expansion. Conveniently, electron transfer proteins and expensive cofactors such as NAD(P)H are not necessary for the enzyme reaction. Here, we demonstrate an application of 2-oxoglutarate-dependent dioxygenases to synthesize optically active hydroxyamino acids (Fig. 1).

Characterization of a thermostable methylaspartate ammonia lyase from carboxydothermus hydrogenoformans
Hans Raja, Vinod Puthan Veetila, Wiktor Szymanskib, Frank J. Dekkerc, Wim J. Quaxa, Ben L. Feringab, Dick B. Janssend, and Gerrit J. Poelarendsa Departments of aPharmaceutical Biology and cPharmaceutical Gene Modulation, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands; bStratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; dDepartment of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands E-mail: h.raj@rug.nl Methylaspartate ammonia lyase (MAL; EC 4.3.1.2) catalyzes the reversible addition of ammonia to mesaconate to give (2S,3S)-3-methylaspartic acid and (2S,3R)-3-methylaspartic acid as products [1]. MAL is of considerable biocatalytic interest because of its potential use for the asymmetric synthesis of substituted aspartic acids, which are important building blocks for synthetic enzymes, peptides, chemicals, and pharmaceuticals [2]. Here, we have cloned the gene encoding MAL from the thermophilic bacterium Carboxydothermus hydrogenoformans Z-2901. The enzyme (named Ch-MAL) was overproduced in Escherichia coli and purified to homogeneity. Ch-MAL is a dimer in solution, consisting of two identical subunits (~49 kDa each), and requires Mg2+ and K+ ions for maximum activity. The optimum pH and temperature for the deamination of (2S,3S)-3-methylaspartic acid are 9.0 and 65C, respectively. Heat inactivation assays showed that Ch-MAL is stable at 50C for >4 h, which is the highest thermal stability observed among known MALs. Ch-MAL accepts fumarate, mesaconate, ethylfumarate and propylfumarate as substrates in the ammonia addition reaction. The enzyme also processes methylamine, ethylamine, hydrazine, hydroxylamine and methoxylamine as nucleophiles in the addition to mesaconate, which results in the corresponding N-substituted methylaspartic acids with excellent diastereomeric excess. Ch-MAL appears to be an attractive biocatalyst for the selective synthesis of substituted aspartic acid derivatives on large scale.
_____________________ [1] Goda SK, Minton NP, Botting NP, Gani D. (1992) Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding -methylaspartase and characterization of the recombinant protein. Biochemistry 31: 10747-10756. [2] Gulzar MS, Akhtar M, Gani D. (1997) Preparation of N-substituted aspartic acids via enantiospecific conjugate addition of N-nucleophiles to fumaric acids using methylaspartase: synthetic utility and mechanistic implications. J. Chem. Soc., Perkin Trans. 1: 649-655.
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Occurrence and characterization of novel imine reductase
Koichi Mitsukuraa, Toyokazu Yoshidaa, Toru Nagasawaa a Department of Biomolecular Schience, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan E-mail: mitukura@gifu-u.ac.jp Chiral amine is a useful constituent molecule for the synthesis of various pharmaceuticals and agrochemicals. In biocatalytic syntheses of chiral amine, enzymes such as hydrolase, transaminase, amine oxidase and amine dehydrogenase, and whole cells showing enzymatic activity have been studied. Enzymatic imine reduction is a promising method for the synthesis of chiral amine. However, no information about imine reductase acting on nonnatural imine has been reported yet. This might be attributable to the remarkable instability of most of imines, which are easily decomposed into carbonyl compound and amine in water. We thus focused on waterstable cyclic imine, 2-methyl-1-pyrroline (2-MPN), and surveyed imine-reducing microorganisms using 2-MPN (Figure 1). After incubation of 2-MPN with whole cells, the reduction activity and enantioselectivity were checked by TLC and HPLC analyses. We found several Streptomyces strains exhibit 2-MPN-reducing activity for the first time among bacteria, actinomycetes, molds and yeasts stocked in our laboratory. Streptomyces sp. GF3587 catalyzed asymmetric reduction of 2-MPN to 2-methylpyrrolidine (2-MP) with excellent (R)-selectivity (99%e.e.), and Streptomyces sp. GF3546 was the only strain showed high (S)-selectivity (81%e.e.). We optimized the reaction conditions using these whole cells. The production of (R)-and (S)-2-MP has been achieved with high optical purity in an over 90% molar conversion [1].
Figure 1. Amino acid hydroxylation using 2-oxoglutarate-dependent dioxygenase. 1) Hydroxyproline cis-4-Hydroxy-L-proline, which is a stereoisomer of common trans-4-hydroxy-L-proline, is used as antitumor drug or pharmaceutical intermediate. We found a novel enzyme that catalyzes the hydroxylation of L-proline to form cis-4-hydroxy-L-proline using genomic information [1]. Actually, this enzyme is applied to commercial use for bioproduction of cis-4-hydroxy-L-proline by KYOWA HAKKO BIO Co., Ltd. (Tokyo, Japan) from 2011. 2) Hydroxyaspartic acid L-threo-3-Hydroxyaspartic acid is used as a material for functional polymer, and itself shows antibiotic activity. We constructed a novel strategy for the synthesis of L-threo3-hydroxyaspartic acid using asparagine hydroxylase [2] followed by asparaginase. 3) Hydroxy aliphatic amino acids Hydroxy aliphatic amino acids, including 4-hydroxy-L-isoleucine which is natural antiobesity drug, are useful compounds for pharmaceutical intermediate. We found aliphatic amino acid hydroxylases that can hydroxylate wide range of aliphatic amino acids (e.g., D- and L-isoleucine, leucine, valine, norleucine, and norvaline). Toward practical amino acid hydroxylation, these findings from this study will be contribute to the development of a platform for the utilization of 2-oxoglutarate-dependent dioxygenases in the fine chemical industry.
Figure 1. Biocatalytic asymmetric reduction of 2-MPN. (R)-Imine reductase (RIR) of Streptomyces sp. GF3587 was purified to be homogeneously from cell-free extract. The RIR was found to be a NADPH-dependent enzyme (homodimer) consisting of 32 kDa subunit. The characterization of RIR has been carried out [2].
________________ [1] Mitsukura, K., Suzuki, M., Tada, K., Yoshida, T., Nagasawa, T., Org. Biomol. Chem., 2010, 8, 45334535 [2] Mitsukura, K., Suzuki, M., Shinoda, S., Kuramoto, T., Yoshida, T., Nagasawa, T., Biosci. Biotech. Biochem., in press
________________ [1] R. Hara and K. Kino, Biochem Biophys Res Commun, 2009, 379(4), 882-886. [2] M. Strieker, et al., ACS Chem Biol, 2007, 2(3), 187-196.
251
Cloning and expression of alcohol hydrogenases from Bacillus Subtillis for reduction of ketones
Araujo, Yara J. K., Canduri, Fernanda, Mouad, A. M., Porto, Andr L. M. Instituto de Qumica de So Carlos, Universidade de So Paulo, Av. Trabalhador Socarlense, 400, 13560-970, So Carlos, So Paulo Email: yarajaque@iqsc.usp.br; almporto@iqsc.usp.br Bacillus subtilis isolated from red marine algae Botrychia tenella was used as biocatalyst, demonstrating high selectivity for the reduction of iodoacetophenone derivatives (figure 1).[1] Chiral alcohols are important building blocks for the synthesis of optically active compounds,[2,3] and (S)-iodophenylethanols are used to obtain chiral biphenyl compounds,[3] which are versatile intermediates in organic synthesis.[5] The present study shows the cloning and expression of the alcohol dehydrogenase (ADH) from B. subtilis. PCR amplification of DNA fragment of ADH was separated using a gel electrophoresis in 0.8% agarose (wt/vol), exhibiting a broad band between 1000-1200 bp that corresponds to the ADH DNA with 1137 bp. The DNA fragment was purified by QIAGEN protocol and included the pGEM-T easy cloning vector (Invitrogen). The transformation into DH5 competent cells was successful, and the PCR reaction of the colonies was obtained with the purification of plasmidial DNA. The next experiment involves the cleavage of the DNA and its insertion into an expression vector (pET28a) for subsequent testing of expression. The isolated ADH will be tested in the reduction of iodoacetophenones to compare the activity of the isolated protein against that present in native bacteria.
O native Bacillus subtilis I ortho-I meta-I para-I 32oC, 130 rpm, 4-8 days I (S)-iodoalcohols >99 % ee c up to 50% OH
252
Obtaining chimeric laccases by directed evolution
Isabel Pardo a, Diana Mate b, Miguel Alcalde b, Susana Camarero a a Centro de Investigaciones Biolgicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid; bInstituto de Catlisis y Petroleoqumica, CSIC, Marie Curie 2, 28049 Madrid, Spain. iparmen@cib.csic.es In recent years we have developed new platforms for the functional expression of basidiomycete laccases in S. cerevisiae using molecular directed evolution methods (1-4). At this moment, it is possible to undertake new challenges in laccase engineering based on these platforms by means of directed evolution. The objective of this work is the tuning of chimeragenesis methods to join fragments from different laccases into the same protein sequence so as to combine the best laccase properties in a single chimera. The starting points of this work were two high-redox potential laccases from the ligninolytic basidiomycetes PM1 and Pycnoporus cinnabarinus, sharing 76% sequence homology, which functional expression in S. cerevisiae and enhancement of their catalytic properties were previously achieved in our lab by directed evolution (1-4). In this process, the replacement of the native signal sequences by the S. cerevisiae -factor pre-proleader and their combined evolution with the codifying sequences was crucial. Two chimeragenesis methods were employed, using in vitro and in vivo DNA recombination techniques. The first strategy is based on in vivo family shuffling, which consisted of recombination of the parental sequences and their ligation into an episomic vector inside the yeast. The high frequency of homologous and homeologous recombination in S. cerevisiae favors crossovers between related DNAs, and its gap repair mechanisms allows in vivo cloning of the insert into the linearized vector by designing spacer sequences on both ends of the insert that overlap with those of the plasmid. In the second strategy, named CLERY, both in vitro and in vivo DNA recombination is used (5). In vitro DNA shuffling included random fragmentation of parental genes by DNase I digestion, fragment reassembly by a primer-less PCR (recombination takes place during the annealing of high-identity sequence regions), and a final amplification by PCR of the chimeric sequence using primers that allow its cloning into the expression vector. Subsequently, the ligation of the in vitro generated chimeric inserts into the linearized vector would take place in the yeast, giving place to a second round of recombination by in vivo DNA shuffling.
(1) Camarero, S., Alcalde, M., Caas, A. I., Martnez, A. T., Martnez, M. J., Plou, F. J., Ballesteros, A., Record, E., and Asther, M. High redox potential laccses engineered by directed evolution. Patent (Spain) application 2008, P200803322. PCT/ES2009/070516. (2) Camarero, S., Caas, A. I., Molina, P., Pardo, I., and Alcalde, M. Directed evolution of a high redoxpotential laccase for functional expression in Saccharomyces cerevisiae. University of Santiago de Compostela.School of Engineering. 2010, ISBN 13:978-84-614-2824-3. (3) Mat, D., Garca-Burgos, C., Garca-Ruiz, E., Ballesteros, A., Camarero, S., and Alcalde, M. Laboratory evolution of high-redox potential laccases. Chem. Biol. 2010, 17, 1030. (4) Mat, D., Garca-Ruiz, E., Camarero, S., and Alcalde, M. Directed evolution of fungal laccases. Current Genomics 2011, 12, 113. (5) Arnold, F. H. and Georgiou, G. (2003) Directed evolution library creation. Methods and protocols, Methods in Molecular Biology, 2003, 231. Humana Press, Totowa, New Jersey.
255
Catalophore: Mining of structural databases using 3D-templates
Georg Steinkellnera,b, Christian C. Grubera,b, Tea Pavkov-Kellera,b, Andrzej yskowskia,b, Orsolya Schwambergerb, Kerstin Steinera,d, Christoph Winklerc, Helmut Schwabd, Kurt Faberc, Karl Gruberb a ACIB GmbH, Petersgasse 14, 8010 Graz, Austria; bInstitute of Molecular Biosciences, Humboldtstrae 50, 8010 Graz, Austria, cDepartment of Chemistry, Heinrichstrae 28, 8010 Graz, Austria ,dInstitute of Molecular Biotechnology, Petersgasse 14, 8010 Graz, Austria E-mail: georg.steinkellner@acib.at, christian.gruber@acib.at Structural databases contain a vast amount of information about enzymes and their active sites. Structural genomics projects increasingly provide large amounts of structural data, also of enzymes where nothing or little is known about their function. As the need for novel biocatalysts with different functionality also increases, the mining of these structural databases can identify enzymes with desired catalytic properties. We propose an approach using three-dimensional motifs reflecting specific active site arrangements (catalophore) which does not depend on overall protein similarity and therefore enables the search across enzyme families and the detection of potential catalytic promiscuity. We present an example where this catalophore approach led to the discovery of two novel enzymes with enoate reductase activity. Enzymes of this family have recently been shown to possess a great potential for (industrial) biotransformations. Neither the amino acid sequence of these two enzymes nor their overall structure are related to the well known old yellow enzymes (OYE). However, they do show significant similarities regarding the position and nature of active site residues.(Figure 1)
256
Characterization and engineering of cupin dioxygenases
Ivan Hajnal, Kerstin Steiner, Helmut Schwab ACIB GmbH (Austrian Centre of Industrial Biotechnology), c/o TU Graz, Petersgasse 14, A-8010 Graz, Austria. E-mail: ivan.hajnal@acib.at The cupin superfamily comprises small -barrel proteins which mostly contain a tightly bound divalent metal at their core. The name itself derives from the latin cupa for small barrel. Cupins display many different functions, both enzymatic such as dioxygenases, hydroxylases, isomerases, epimerases, oxidases and decarboxylases, as well as non-enzymatic, either as monocupins or in combination with additional cupin or non-cupin domains [1]. The potential versatility of cupins as biocatalysts has prompted us to look into the rationale behind some of the different activities found in cupins, with special emphasis on monocupins from microbial origins. A set of modestly homologous proteins predicted to have a cupin fold were cloned and expressed in E. coli BL21 (DE3) Gold, in order to characterise the proteins. Most of the chosen cupins already have a solved crystal structure in the protein database, but no known biological or biochemical function. One protein of this set shows high sequence homology to a known 3-hydroxyanthranilate-3,4-dioxygenase (HAO) from Ralstonia metallidurans., the structure of which has been solved by Zhang et al. (PDB: 1YFU) [2]. This activity was confirmed by measuring the increase of absorbance at 360 nm, attributed to the oxidative cleavage of 3-hydroxyanthranilate to yield 3-carboxymuconic semialdehyde. In addition to the cupin Fe centre both bacterial proteins comprise a C-terminal FeS centre which consists of four cysteines presumed to coordinate a Fe2+ cation. This additional metal centre is missing from the mammalian homologs, including the human HAO protein, while at the same time the cupin domain seems to be highly conserved. The enzyme may be used as a model to understand the basis for oxidative ring-cleavage activity found in cupins. The catalytic properties of this enzyme regarding substrate scope and the residues responsible for selectivity are not yet fully elucidated. This knowledge is important in order to make cupin dioxygenases better accessible to engineering and in further course to biocatalytic applications. ________________ [1] Dunwell J.M., Purvis A., and Khuri S. (2003) Cupins: the most functionally diverse protein superfamily?. Phytochemistry 65:7-17 [2] Zhang Y., Colabroy K.L., Begley T.P., and Ealick S.E. (2005). Structural Studies on 3-Hydroxyanthranilate-3,4-dioxygenase: The Catalytic Mechanism of a Complex Oxidation Involved in NAD Biosynthesis. Biochemistry 44 (21): 76327643
Fig 1. Reduction of iodoacetophenones by native B. subtilis[1]

_______________ [1] Mouad, A.M.; Martins, M.P.; Debonsi, H.M.; De Oliveira, A.L.L; De Felcio, R.; Yokoya, N.S.; Fujji, M.T.; De Menezes, C.B.A.; Fantinatti-Garboggini, F.; Porto, A.L.M. Helv. Chim. Acta. 2011, doi: 10.1002/hlca.201000434 (accepted). [2] Kamaruddin, A.H.; Uzir,M.H.; Aboul-Enein, H.Y.; Halim, H.N.A. Chirality 2009, 21, 449-467. [3] Malhotra, S. V. Methodologies in Asymmetric Catalysis, Amer. Chem. Soc., 2004, Vol. 880, ISBN13: 9780841238350. [4] Rocha, L.C.; I.G. Rosset; Luiz, R.F.; Raminelli, C.; Porto, A.L.M. Tetrahedron Asymmetry, 2010, 21, 926-929. [5] Baudoin, O.; Cesario, M.; Gunard, D.; Guritte, F. J. Org. Chem. 2002, 67, 1199-1207.
Figure 1. Active site comparison of the identified enzyme with enoate-reductase activity and Old Yellow Enzyme (OYE). The OYE active site is shown in red the active site of the novel enzyme is shown in blue. The structures are aligned with respect to their FMN cofactors. The two active sites show an approximate mirror-image symmetry.
October 2-6, 2011
136 257
Artificial alcohol dehydrogenases and lyases based on the biotinstrept(avidin) technology
Tommaso Quinto, Thomas R. Ward Department of Chemistry, University of Basel, Spitalstrasse 51 CH-4056 Basel, Switzerland E-mail: tommaso.quinto@unibas.ch The creation of an artificial enzyme is a very fascinating challenge. To create an artificial metalloenzyme, an organometallic complex is incorporated within a host protein. For this purpose we utilize the biotin-avidin technology. This technique exploits the exceptionally high affinity of biotin for the streptavidin protein (Sav) (Ka =1015M-1). The complex is linked to biotin in order to ensure its positioning in Sav. [1] The design, the synthesis and the optimization of new organometallic catalyst was carried out for: a) carbon-carbon bond forming reaction (allylic alkylation), and b) selective oxidation of secondary alcohols and amines. For this purpose, two different catalysts have been identified in the literature: a) [Cp*Ru(COD)Cl] 1 is known to catalyze C-C bond formation reaction in living cells;[2] b) [Cp*Ir(C^N)] 3 is known to selectively oxidize secondary alcohols.[3] The ultimate aim of the project is to optimize artificial lyases and alcohol dehydrogenases by directed evolution protocols. For this purpose, organometallic catalysts that are compatible with cell debris are indispensable. We report on our efforts to synthesize biotinylated analogs of these d6-pianostool complexes which are expected to be amenable to the implementation of directed evolution protocols (Figure 1).

-Glycosyl azide as donors of -glycosynthases in oligosaccharide synthesis
Beatrice Cobucci-Ponzanoa, Carmela Zorzettia, Andrea Strazzullia, Emiliano Bedinib, Maria Michela Corsarob, Gerlind Sulzenbacherc, Mos Rossia and Marco Moraccia a Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy. bDipartimento di Chimica Organica e Biochimica, Universit di Napoli Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy.c rchitecture et Fonction des Macromolcules Biologiques, UMR6098 CNRS, Universit de Provence, Universit de la Mditerrane, Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France. E-mail: m.moracci@ibp.cnr.it The great structural variety of carbohydrates makes sugar molecules suitable for mediating many biological processes, but it complicates greatly their production. This challenge currently motivates the efforts in developing chemo-enzymatic methods for large-scale production of oligosaccharides. Glycosynthases, mutant glycosidases derived from glycoside hydrolases, are interesting engineered catalysts that synthesize sugars by promoting transglycosylation reactions to an acceptor with almost quantitative yields. (Figure 1) [1].

Optimized refolding and characterization of S-peroxidase (CWPO_C of Populus alba) expressed in E. coli
Le Thanh Mai Phama, Su Jin Kimb, Bong Keun Songb, Yong Hwan Kima* Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-Dong, Nowon-Gu, Seoul 139-701, Republic of Korea; bKorea Research Institute of Chemical Technology, Sinseong-no 19, Yuseong-gu, Daejeon 305-600, Republic of Korea E-mail: metalkim@kw.ac.kr
a
137 262
High-level expression and characterization of Rhizopus Chinensis mature lipase in Pichia Pastoris
Xiaowei Yu, Yan Xu, Rui Wang, Sa Chong, Jiayou Xie, Le-Le Wang Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, Jiangnan University, Wu xi 214122, P.R.China E-mail: bioyuxw@yahoo.com.cn Lipases (EC 3.1.1.3) can catalyze not only hydrolysis of ester bonds under aqueous conditions but also synthesis of ester bonds under micro-aqueous conditions, which make them the most widely used class of enzymes in the manufacture of food ingredients, pitch control in the pulp and paper industry, and biocatalysis of stereoselective transformations in organic chemistry [1]. Rhizopus sp. are an important lipase-produce strains [2]. Several Rhizopus lipases have been cloned and expressed in E. coli or yeast, such as R. oryzae, R. arrhizus, R. javanicus [3,4]. In our studies, a new lipase (proRCL) from R. chinensis CCTCC M20102, a strain isolated from Da Qu, a kind of traditional leaven for the production of Chinese liquor, showed very high ability of esterification of ethyl esters from short-chain fatty acids [5]. The lipase gene (GenBank no. EF405962) was cloned from the genomic DNA. Compared with other Rhizopus sp. lipase the amino acid sequence identity was 73~76%. In order to enhance its thermostability and performance in industrial applications, directed evolution was applied by successive steps of two rounds of ep-PCR, followed by two rounds of DNA Shuffling and a round of site-directed mutagenesis. A novel method, named PDM to construct mutant pool based on in-vivo homologous recombination was also developed to generate mutants with large capacity and abundance. S4-3 and S5-2 were the most thermostable variants from the mutant pool. S5-2 possessed seven protein substitutions: E107G, A129T, S151N, L180H, S234F, Q363R and V329A, and S4-3 carried three mutations: T218S, L180H and A230F. S4-3 and S5-2 conferred 46 and 42-fold increase in half-life at 65C, compared to that of the wild-type enzyme. The Tm of the S4-3 and S5-2 were increased 15.5 C and 12.5 C respectively without losing enzyme activity. The lipase (proRCL) was high-level expressed extracellularly in Pichia pastoris in pilot scale fermentation (30m3) at the activity of 21 000 U/mL. The lipase was successfully applied in the baking industry. The lipase significantly affected the thermomechanical properties of the dough and enhanced bread quality. Comparing with other commercial lipase, proRCL not only significantly increased the specific volume, but also improved the gumminess and cohesiveness of bread. ______________ [1] Hasan, F., Shah, A.A. and Hameed, A. 2006. Industrial applications of microbial lipases. Enzyme
Figure 1: Reaction mechanisms of (A) - and (B) -glycosynthases Unfortunately, -glycosidases are not prone to be engineered in -glycosynthases hampering so far the access to the synthesis of a large class of -oligosaccharides of biotechnological interest. We report here on a new glycosynthetic methodology for the production of -glycosynthases exploiting -glycosyl azide derivatives. This approach allowed the production of two retaining -fucosynthases and a -galactosynthase with transglycosylation yields up to 91% [2, 3]. These enzymes follow the classical reaction mechanism proposed for -glycosynthases, but utilize -glycosyl azide as donor substrate. The general applicability of this approach open new perspectives in the use of azide derivatives for the production of novel -glycosynthases for the synthesis of oligosaccharides of biotechnological interest.
________________ [1] Mackenzie et al. (1998) J Am Chem Soc 120, 5583 [2] Cobucci-Ponzano et al. (2009) Chem Biol. 16, 1097 [3] Cobucci-Ponzano et al. (2011) Glycobiology 21, 448
Lignin plays a vital role in plant growth and development by providing mechanical support to bind plant fibers together, enhancing the strength of fibrous tissues and limiting the spread of pathogens in plant tissues [1]. The formation of these linkages between monolignols is catalyzed by class III plant peroxidases (EC 1.11.1.7). Most of the peroxidases fully characterized in flowering plants, such as ATP A2 and HRP A2, can only oxidize coniferyl alcohol, and are termed G-peroxidases. Studies on the presence of peroxidases that could oxidize derivatives of sinapyl alcohol, termed S-peroxidases [2], were first reported over 20 years ago. However, reports have only recently appeared concerning the molecular characterization and cloning of peroxidases capable of oxidizing sinapyl alcohol in vascular plants, both in herbaceous and woody angiosperms, and also in woody gymnosperms, which lack S-type lignins. Cationic cell wall peroxidase (CWPO_C) from poplar tree (Populus alba L) was heterologously expressed in Escherichia coli as an inclusion body. The insoluble inclusion body was solubilized and reactivated via a refolding procedure. The condition for this procedure was optimized by varying the refolding pH, and the concentrations of the oxidizing agent (GSSG), denaturing agent (GmdCl), and hemin, respectively. The optimal conditions for refolding CWPO_C were 100 mM TrisHCl at pH 8.5, 0.6 mM GSSG, 5 M hemin, 0.6 M GmdCl and 5mM CaCl2. The oxidation rate for sinapyl alcohol was almost seven times higher than that for coniferyl alcohol catalyzed by refolded CWPO_C, which was a very unique property of S-peroxidase. The optimal pH and reaction temperature for the oxidation of sinapyl alcohol by refolded CWPO_C were 6.4 and 37 C, respectively. The successful expression of CWPO_C in Escherichia coli provides a valuable tool to elucidate the structure and functional relationship of S-peroxidase, which plays an important role in the lignification of angiosperm woody plant cell walls.
Figure 1: Biotynylated d6 pianostool complexes 2, 4 for the creation of artificial alcohol dehydrogenases and lyases based on the biotin-(strep)avidin technology.
__ [1] T. R. Ward, Acc. Chem. Res. 2011, 44, 47-57 [2] C. Steau, E. Meggers, Angew. Chem. Int. Ed. 2006, 45, 5645-5648 [3] B. L. Feringa, J. G. de Vries et al. J. Am. Chem. Soc. 2008, 130, 13509
Fig. 1. (A) Expression vector pET-CWPO_C including the CWPO_C cDNA sequence. (B) SDS-PAGE of different fractions from the extraction of inclusion bodies and refolded CWPO_C.
Microb. Technol. 39, 235-251. [2] Sharma, R., Chisti, Y. and Banerjee, U.C. 2001. Production, purification, characterization, and applications of lipases. Biotechnol. Adv. 19, 627-662. [3] Ueda, M., Takahashi, S., Washida, M., Shiraga, S. and Tanaka, A. 2002. Expression of Rhizopus oryzae lipase gene in Saccharomyces cerevisiae. J. Mol. Catal. B-Enzym. 17, 113-124. [4] Di Lorenzo, M., Hidalgo, A., Haas, M. and Bornscheuer, U.T. 2005. Heterologous production of functional forms of Rhizopus oryzae lipase in Escherichia coli. Appl. Environ. Microbiol. 71, 8974-8977. [5] Xu, Y., Dong W., Mu X.Q., Zhao G.A. and Zhang K.C. 2002. Biosynthesis of ethyl esters of shortchain fatty acids using whole-cell lipase from Rhizopus chinensis CCTCC M201021 in non-aqueous phase. J. Mol. Catal. B-Enzym. 18: 29-37
259
Enantioselective biocatalysis for the preparation of optically pure tertiary alcohols
Giang-Son Nguyen[a,b], Robert Kourist[a,c], Monica Paravidino[b], Mark L. Thompson[a], Fabian Steffen-Munsberg[a], Susanne Herter[d], Ulf Hanefeld[b], Uwe T. Bornscheuer[a] [a] Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis, Felix-HausdorffStr. 4, D-17487, Greifswald, Germany [b] Gebouw voor Scheikunde, Afdeling Biotechnologie, Technische Universiteit Delft, Julianalaan 136, 2628 BL, Delft, The Netherlands [c] Institute of Chemistry of Biogenic Resources, TU Mnchen, Schulgasse 16, D-94315 Straubing, Germany [d] Institute of Microbiology, Department of Applied Microbiology, University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, 17489, Greifswald, Germany E-mail: g.s.nguyen@tudelft.nl Tertiary alcohols have become interesting targets for organic synthesis themselves or as building blocks for valuable pharmaceutical compounds.[1] Enzymes containing the GGG(A)X motif in the active site region have been known to show activity towards these sterically demanding substrates.[2]
260
Generation of highly catalytic inclusion bodies in living Escherichia Coli cells using cellulose-binding domain from cellulomonas fimi as a fusion partner
Seung-Goo Lee*, Su-Lim Choi, Sang Jun Lee, and Eugene Rha Korea Research Institute of Bioscience and Biotechnology (KRIBB) Gwahak-ro 125, Yuseong-gu, Daejeon, 305-806, Korea E-mail: sglee@kribb.re.kr The cellulose-binding domain (CBD) of an exoglucanase from Cellulomonas fimi was introduced as the fusion partner to induce the formation of catalytic inclusion bodies. Two enzymes, Escherichia coli -glucuronidase and Thermus caldophilus -glycosidase, were expressed in the form of inclusion bodies in Escherichia coli, and then isolated by centrifugation of the cell lysates. Catalytic analyses of the isolates exhibited that approximately 92% of both enzymes were localized in the insoluble fraction, and the specific activity of -glucuronidase in the inclusion body reached 210.8 units per mg protein. The protein particles were then treated with 0.1% glutaraldehyde for 1 hour and applied as a biocatalyst for the hydrolysis of -glycoside substrates. As a result, the CBD inclusion bodies could be used for three batch operations without significant loss of their initial activity, indicating that CBD inclusion bodies have potential as a new preparation for immobilized biocatalysts.
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Noble substrate specificity of UDP-galactose-4-epimerase and improvement of its monosacchride 4-epimerization using mutagenesis
Hye-Jung Kima, Sueng Yeun Kanga, Pil Kima a Department of Biotechnology, Catholic University of Korea, Bucheon, Gyeonggi 420743, Korea, TEL +82-2-2164-4964. FAX +82-2-2164-4964. rain320@catholic.ac.kr Uridine diphosphogalactose-4-epimerase (UDP-galactose-4-epimerase, GalE, EC 5.1.3.2) mediates the 4-epimerization of nucleic acid-activated galactose into UDP-glucose. To date, no enzyme is known to mediate 4-epimerization of free monosaccharide substrates. To determine the potential activity of GalE for free monosaccharide, Escherichia coli GalE was expressed and purified using Ni-affinity chromatography, and its ability to mediate 4-epimerization of a variety of free keto- and aldohexoses was assessed. Purified GalE was found to possess activity for free galactose, glucose, fructose, tagatose, psicose, and sorbose at 0.47, 0.31, 2.82, 9.67, 15.44, and 2.08 nmol/mg protein/min, respectively. No 4-epimerization activity was found for allose, gulose, altrose, idose, mannose, and talose. The kinetic parameters of 4-epimerization reactions were Km=26.4 mM and kcat=0.0155 min-1 for D-galactose and Km=237 mM and kcat=0.327 min-1 for D-tagatose. The 4-epimerization of tagatose, a reaction of commercial interest, was enhanced 2-fold (19.79 nmol/mg protein/min) when asparagine was exchanged with serine at position 179 by resional digsin mutation. To find the related more residues for the 4-epimerization of tagatose, random mutations were introduced into GalE by error-prone PCR and clones showing fructose 4-epimerization activity higher than 115% were selected. Two clones carrying five mutation points (D58E, N100S, P193S, I196N and T317S) were selected among 3,060 mutant clones. To investigate the effect of the mutation points on the desired characteristics, 5 mutant clones carrying single mutation were constructed and the variant proteins were assessed. Among the enhanced mutant clones, mutant D58E and N100S were found to contribute the twice and three times as much activity enhancement (63nmol/mg-protein, 101nmol/mg-protein) respectively. The novel activity of GalE for free monoaccharide may be beneficial for the production of rare sugars using cheap natural resources.
____________ [1] Hye-Jung Kim & Sueng Yeun Kang & Jong Jin Park and Pil Kim, (2010) Appl Biochem Biotechnol, 163(3), 444-451. [2] Ran-Young Yoon, Soo-Jin Yeom, Hye-Jung Kim and Deok-Kun Oh (2009) Journal of Biotechnology, 139, 26-32 [3] James B. Thoden, and Hazel M. Holden (1998) Biochemistry, 37, 11469-11477 [4] James B. Thoden, Adrian D. Hegeman, Gary Wesenberg, Marie C. Chapeau, Perry A. frey, and Hazel M. Holden (1997) Biochemistry, 36, 6294-6304 [5] Yijeng Liu, James B. Thoden, Jeong min Kim, Elizabeth Berger, Andrew M. Gulick. Frank J. Ruzicka, Hazel M. Holden, and Perry A. Frey (1997) Biochemistry, 36, 10675-10684
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Directed evolution of versatile peroxidases
Eva Garcia-Ruiza, David Gonzalez-Pereza, Francisco Javier Ruiz-Dueasb, Angel Toms Martnezb, Miguel Alcaldea. a Department of Biocatalysis, Institute of Catalysis, CSIC, Cantoblanco, 28049 Madrid, Spain; bCentro de Investigaciones Biolgicas, CSIC, 28040 Madrid, Spain E-mail: malcalde@icp.csic.es. Versatile peroxidases (VPs) secreted by white-rot fungi are high-redox potential ligninolytic enzymes which combine most of the catalytic features of other ligninolytic peroxidases (i.e. manganese and lignin peroxidases) as well as plant peroxidases resulting in a huge catalytic promiscuity. Hence, VPs show a remarkable biotechnological potential finding applications in paper and textile processing, bioremediation, organic synthesis, nanobiotechnology and more. The main hurdle for their industrial implementation is the difficulty of attaining functional expression. Moreover, stability is a desirable property for its practical use. Here, a fusion gene containing the VP from Pleurotus eryngii was subjected to six rounds of directed evolution. Secretion levels of 21 mg/L were achieved in Saccharomyces cerevisiae, the highest yet reported for any ligninolytic peroxidase. The signal leader processing at the Golgi changed as consequence of over-expression, remaining an extra Nterminal tail that was helpful for secretion [1][2]. Additional cycles of evolution shifted upwards the T50 8C (up to 66C) [3] and increased the stability at alkaline pH (retaining over 50% of its activity after incubation at pH 9.0 for 120 h). In the course of artificial selection, the Km for H2O2 was enhanced up to 15-fold and a notorious improvement in the oxidative stability was detected [2]. The evolutionary platform describe in this work permits to produce VPs highly active, soluble and stable being a suitable vehicle for synthetic biology studies
Figure 1: Enzymatic synthesis of enantiopure tertiary alcohols Our study aims at providing a better understanding of enantiorecognition of GGG(A)X motif hydrolases in the enzymatic synthesis of enantiomerically enriched tertiary alcohols. Kinetic resolution of a wide range of tertiary alcohols using hydrolases provided insights on factors that can influence enantioselectivity of GGG(A)X motif enzymes. [3] Additionally, a newly proposed chemoenzymatic method to synthesize protected ,-dialkyl--hydroxycarboxylic acids has broadened the application of these enzymes to synthesize optically pure tertiary alcohols.[4] Novel biocatalysts through functional screening,[5] database mining[6] approaches provide a better enzyme platform for optically pure tertiary alcohol resolution.
________________ [1] P. Cozzi, R. Hilgraf, N. Zimmermann, Eur. J. Org. Chem. 2007, 59695994. [2] R. Kourist, U.T. Bornscheuer, Appl. Microbiol. Biotechnol. 2011, in press. [3] G.S. Nguyen, R. Kourist, M. Paravidino, A. Hummel, J. Rehdorf, R. Orru, U. Hanefeld, U. Bornscheuer, Eur. J. Org. Chem. 2010, 2010, 27532758. [4] R. Kourist, G. Nguyen, D. Strbing, D. Bttcher, K. Liebeton, C. Naumer, J. Eck, U. Bornscheuer, Tetrahedron-Asymmetry 2008, 19, 18391843. [5] S. Herter, G. Nguyen, M.L. Thompson, F. Steffen-Munsberg, F. Schauer, U.T. Bornscheuer, R. Kourist, Appl. Microbiol. Biotechnol. 2011, 90, 929939. [6] G.S. Nguyen, M.L. Thompson, G. Grogan, U.T. Bornscheuer, R. Kourist, J. Mol. Catal. B:Enzym 2011, 70, 8894.
_______________________ [1] Garcia Ruiz E, Martinez MJ, Ruiz-Dueas FJ, Martinez AT, Alcalde M.(2010) Peroxidas de elevado potencial redox diseadas por evolucin dirigida. Patent: PCT/ES2010/070316. [2] Garca Ruiz, E, Gonzalez-Perez, D, Ruiz-Dueas, FJ, Martinez A.T. and Alcalde M. (2011). Directed evolution of versatile peroxidase. Submitted. [3] Garca-Ruiz E, Mat D, Ballesteros A, Martinez AT, Alcalde M. (2010) Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast.. Microb Cell Fact.; 9:17.
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Novel applications from the evolution of the substrate specificity of glycine oxidase
Gianluca Molla, Elena Rosini, Mattia Pedotti, Loredano Pollegioni The Protein Factory, Universit degli studi dellinsubria Varese and Politecnico di Milano, Italy E-mail: gianluca.molla@uninsubria.it Glyphosate is the most used herbicide in modern agricolture; accordingly, approximately 80% of the total worldwide area devoted to transgenic crops has been planted with glyphosate resistant crops. Currently, the sole suitable mechanism providing resistance to this herbicide involves glyphosate-resistant forms of 5-enolpyruvylshikimate-3-phosphate synthase, the plant enzyme inhibited by the herbicide [1]. Glycine oxidase from Bacillus subtilis (GO, EC 1.4.3.19) is a flavoprotein oxidase containing non-covalently bound FAD that catalyses the oxidative deamination of various amines and D-amino acids [2, 3]. GO is also active on the herbicide glyphosate but with low efficiency due to a higher Km as compared to that for the reference substrate glycine: 87 mM vs. 0.7 mM, respectively. We improved the oxidative activity of GO on glyphosate by means of a combined approach of rational design (based on in silico molecular docking analysis) and in vitro evolution (by site saturation mutagenesis). A variant enzyme possessing three point mutations (G51S/A54R/H244A) was isolated. This GO variant showed a dramatically increased affinity for glyphosate as suggested by the 170-fold decrease of Km (up to 0.5 mM for the GO triple variant). The resulting 210-fold increase in catalytic efficiency and 15000-fold increase in specificity constant (the kcat/Km ratio between glyphosate and glycine) as compared to WT GO, allows the efficient oxidation of the herbicide by the triple variant. The resolution of the 3D structure of the GO triple variant [4] shows that this remarkable substrate specificity change mainly results from new favorable electrostatic interactions between the guanidinium group of the introduced Arg54 and the phosphonate group of the herbicide and the simultaneous displacement of the negatively charged side chain of Glu55 from the active site. The new improved efficiency of the GO triple variant on glyphosate paves the way to new applications: e.g., the use of this variant as a biotool for the analytical determination of the herbicide or for its degradation. In fact, and in contrast to the existing commercial glyphosate-resistant crops, resistance to the herbicide in GO transgenic plants is achieved through its degradation to nontoxic compounds. For this reason, GO-based transgenic plants could be used in bioremediation processes. ____________________________
[1] L. Pollegioni, E. Schonbrunn, D. Siehl, Molecular basis of glyphosate resistance: different approaches through protein engineering. FEBS J (2011) in press. [2] V. Job, G.L. Marcone, M.S. Pilone, L. Pollegioni, Glycine oxidase from Bacillus subtilis. Characterization of a new flavoprotein. J Biol Chem 277 (2002) 6985-6993. [3] V. Job, G. Molla, M.S. Pilone, L. Pollegioni, Overexpression of a recombinant wild-type and Histagged Bacillus subtilis glycine oxidase in Escherichia coli. Eur J Biochem 269 (2002) 1456-1463. [4] M. Pedotti, E. Rosini, G. Molla, T. Moschetti, C. Savino, B. Vallone, L. Pollegioni, Glyphosate resistance by engineering the flavoenzyme glycine oxidase. J Biol Chem 284 (2009) 36415-36423.

High throughput screening for cellulolytic activity in deep eutectic solvents (DES)
Christian Lehmanna, Fabrizio Sibillaa, Zaira Maugerib, Pablo Domnguez de Marab, Wolfgang Streitc, Ulrich Schwanenberga a Institute of Biotechnology, RWTH Aachen University, Germany; bInstitute of Technical and Macromolecular Chemistry (ITMC), RWTH Aachen University, Germany; cInstitute of Microbiology and Biotechnology, University Hamburg, Germany E-mail: c.lehmann@biotec.rwth-aachen.de In the last decades Ionic liquids (ILs) have attracted increasing interest, due to their large potential for solvent engineering in biocatalysis, electrochemistry and downstream processing. ILs have a low vapour pressure and are non-flammable. In the field of technical biocatalysis practical use of ILs has been hampered because of high costs and environmental impact [1]. Deep Eutectic Solvents (DESs) are a new class of solvents that connect the advantages of ILs but with reduced economic costs and environmental concerns. The first generation of DESs was based on a mixture of quaternary ammonium salts combined with an organic hydrogen donor. Due to their nontoxicity, biodegradability and low cost production (in the range of traditional organic solvents) the DESs can play a pivotal role for bulk and fine chemicals production via biocatalysis. In order to gain a deeper understanding of the behavior of enzymes with DESs a directed evolution study is currently underway. As a prerequisite for directed evolution a reliable High Throughput Screening assay (HTS) is needed to find improved DES resistant variants. For these experiments the conversion of the substrate (4-Methylumbelliferyl beta D cellobioside) by a newly discovered cellulase was online-monitored via increase of a cleaved fluorescence product (4-Methylumbelliferone). The fluorescence assay was established in a 96-well microtiterplate format with an excellent standard deviation (with DES: 12 %; without DES: 9 %). In Figure 1 the conversion over the time with and without the use of DES is shown.

Cyanide hydratase activity of recombinant nitrilase from Aspergillus niger K10
A.Ringelov, A.B.Vesel, L.Martnkov Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vdesk 1083, CZ-142 20 Prague, Czech Republic Cyanide hydratases and nitrilases gain increasing popularity due to their utility in the mild hydrolysis of inorganic cyanide and organic cyanides (nitriles), respectively. Hence, cyanide hydratases are a promising alternative to chemical degradation of cyanide wastes. Despite a significant degree of amino acid sequence homology between the two enzymes groups, activity of cyanide hydratases for organic cyanides is rare and insignificant, and the same holds for the transformation of inorganic cyanide by nitrilases (OReilly and Turner 2003). This is also the case of native nitrilase (Nit-ANigWT), which was recently purified and characterized by us in Aspergillus niger K10 [1]. The enzyme was an aromatic nitrilase and exhibited only low activities towards HCN. Following heterologous expression using E. coli, the recombinant enzyme (Nit-ANigRec) exhibited a different substrate specifity towards an array of organic nitriles [2]. Moreover, the cyanide hydratase activity of Nit-ANigRec was higher compared to Nit-ANigWT. The specific activity of purified Nit-ANigRec towards HCN was approximately 300 U/mg of protein, that is the ratio of its nitrilase activity for benzonitrile and its cyanide hydratase activity was approximately 1:700. The purified enzyme or E. coli cells expressing the enzyme were used to degrade KCN at initial 20 mM concentration into formamide as the only product. The differing properties of Nit-ANigRec and Nit-ANigWT may be ascribed to different post-translational modification, that is a missing cleavage of C-terminal peptide from the recombinant enzyme [2]. Financial support from projects P504/11/0394, 305/09/H008 (Czech Science Foundation), IAA500200708 (Grant Agency of the AS CR, Czech Republic), LC06010, OC09046 (Ministry of Education of the Czech Republic), TA01021368 (Technology Agency of the Czech Republic) and Institutional Research Concept AV0Z50200510 (Institute of Microbiology).
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Molecular cloning and overexpression of the NADPH-dependent 7- and 7-hydroxysteroiddehydrogenase from Clostridium absonum
Erica E. Ferrandia, Daniela Montia, Giulia Bertolesia, Sergio Rivaa a ICRM-CNR, via Mario Bianco 9, 20131 Milano, Italy; E-mail: erica.ferrandi@icrm.cnr.it NADPH-dependent 7- and 7-hydroxysteroid dehydrogenase (7-HSDH and 7-HSDH) from Clostridium absonum, a perfringens-like Clostridium, catalyze the epimerization of primary bile salts through a 7-keto intermediate (Figure 1) and may be suitable as biocatalyst for the synthesis of bile acids derivatives of industrial interest [1]. C. absonum 7-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman degradation. Cloning of the gene (795 nt) has been carried out in three steps. A 269 nt fragment has been amplified by PCR using chromosomal DNA as a template and a pair of degenerate primers on the basis of the experimentally determined N-terminal sequence and of the comparative alignment of gene sequences codifying for 7-HSDHs from other Clostridia strains. A second fragment (170 nt) has been amplified by Inverse-PCR. Finally the 3 termini of the target gene (350 nt) has been obtained by direct sequencing of the C. absonum genomic DNA, using as a primer an oligonucleotide complementary to the 3 termini of the previously sequenced gene fragment. The sequence coding for the 7-HSDH (783 nt) has been obtained by direct genomic DNA sequencing of the region flanking the 5 termini of the 7-HSDH gene, the two genes being part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in E. coli.
Figure 1. Fluorescence assay with and without DES (choline chloride-glycerol). The newly developed HTS will allow us to identify DES resistant cellulases and will help us shedding a light on the molecular effect of DESs on enzymatic activities.
________________ [1] Domnguez de Mara and Maugeri (2011) Curr. Opin. Chem. Biol. 15, 220-225.
[1] Kaplan, O., Vejvoda, V., Martnkov, L. et al.: Purification and characterization of a nitrilase from Aspergillus niger K10. Appl Microbiol Biotechnol 2006, 73:567-575 [2] Kaplan, O., Bezouka, K., Martnkov, L. et al.: Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10. BMC Biotechnology 2011, 11:2
Figure 1. Epimerization of chenodeoxycholic acid to ursodeoxycholic acid catalyzed by the 7-HSDH and 7-HSDH from C. absonum.
________________ [1] Monti D. et al. (2009) Adv. Synth. Catal. 351:1303-1311
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A microbial chymotrypsin-like enzyme useful for specific hydrolytic bioconversions
Federica Volonta, Ines Pisanellia, Paola DArrigoa, Fiorenza Vianib, Gianluca Mollaa, Stefano Servia, Loredano Pollegionia a Centro di Ricerca Interuniversitario in Biotecnologie Proteiche The Protein Factory, Politecnico di Milano and Universit degli studi dellInsubria, via J.H. Dunant 3, 21100 Varese, Italy; bCNR - Istituto di Chimica del Riconoscimento Molecolare (ICRM) - Sezione Adolfo Quilico , via Mancinelli 7, 20131 Milano, Italy. E-mail: federica.volonte@uninsubria.it Serine proteases represent relevant enzymes in biotechnology, which have been employed in a number of fields. In order to overcome the European limitations related to the use of enzymes from animal sources and to simultaneously develop an efficient hydrolytic enzyme active on compounds of pharmaceutical and industrial interest, we planned the production of a microbial chymotrypsin-like enzyme as recombinant protein in E. coli. In particular, the enzyme CHY1 from the deuteromycete Metarhizium anisopliae [1] was produced as a fully soluble and mature active enzyme in E. coli Origami 2(DE3) cells. CHY1 was synthesized as 37.5 kDa pro-protein which acquired the native and active conformation by an autoproteolytic cleavage. Under standard conditions, an overall production of ~ 0.1 mg/L fermentation broth was achieved. The expression of CHY1 chymotrypsin as soluble protein was improved acting on different experimental parameters: the host strain, the inducer concentration, the temperature and time of growth after induction resulted crucial parameters. Indeed, the optimization of the medium composition allowed a further increase in the enzyme productivity, up to ~ 1.6 mg of protein/L of fermentation broth [2]. The recombinant enzyme was purified by a single chromatographic step on HiTrap chelating column bacause of a six His-tag introduced at the C-terminal end of CHY1. The purified recombinant CHY1 was active on classical substrates of chymotrypsin such as caseine, azocaseine and N-succinyl-L-phenylalanine-p-nitroanilide (SPNA). Noteworthy, CHY1 showed a 15-fold lower Km for SPNA as substrate than bovin chymotrypsin and an higher specific activity on azocasein than the enzyme from animal pancreas. Subsequently, the activity was assayed on amino acid methylesters. The highest activity was with L-phenylalanine methylester while the highest kinetic efficiency (kcat/Km ratio) was observed for L-tyrosine methylester. The recombinant enzyme was also strictly stereospecific: D-phenylalanine-, D-tryptophan- and D-tyrosine-methylesters are not hydrolyzed by CHY1. In conclusion, recombinant CHY1 overexpressed in E. coli represents a suitable alternative to animal chymotrypsin for the resolution of racemic mixtures of natural (and nonnatural) amino acids and thus an innovative biotool for biopharmaceutical and industrial processes.
____________ [1] Screen S.E. and St. Leger R.J., (2000) J. Biol. Chem., 275, 6689-6694. [2] Volont F. et al., (2011) Enzyme Microb Technol., in press.
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Identification of novel monooxygenases
Anke Hummel, Uwe T. Bornscheuer Greifswald University, Institute of Biochemistry, Dept. of Biocatalysis and Enzyme Catalysis, Felix-Hausdorff-Str. 4, D-17487 Greifswald, Germany E-mail: anke.hummel@uni-greifswald.de Although there are numerous biocatalysts already described in literature, there is constant need for new enzymes with improved or novel properties regarding substrate scope, stability or enantioselectivity and -preference. There are basically three ways to discover novel enzymes: (a) the classical approach of enrichment cultures or isolation from wildtype microorganisms; (b) the in silico approach of database mining for sequences with homology towards already describes enzymes; or (c) the metagenome approach in which the complete DNA from an environmental sample is extracted and cloned without cultivation of the host organisms. Monooxgenases are a promising group of enzymes as they catalyse diverse oxidations, amongst them hydroxylation, epoxidation or Baeyer-Villiger reactions - often with high regio- and enantioselectivities. However, these enzymes are often instable, especially in isolated form, have low conversion rates or form undesired by-products. Therefore the discovery of new enzymes is highly important. In contrast to enzyme classes like hydrolases (i.e. lipases and esterases), finding oxidative enzymes in metagenome libraries is much more demanding. Monooxygenases often consist of several subunits, which need to act jointly and the assays are not as straightforward as for hydrolytic enzymes. Here we compare different methods to access new monooxygenases by the metagenome approach, in silico database mining and the isolation of monooxygenases from wild type strains.
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Laboratory evolution of an epoxide hydrolase towards an enantioconvergent biocatalyst
Michael Kotika, Alain Archelasb, Veronika Famrova, Pavla Oubrechtova, Vladimr Kena a Laboratory of Biotransformation, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vdesk 1083, 142 20 Prague 4, Czech Republic; bInstitut des Sciences Molculaires de Marseille, Biosciences, UMR CNRS 6263, University of Aix-Marseille, Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20, France E-mail: kotik@biomed.cas.cz We performed a laboratory evolution study with the epoxide hydrolase from Aspergillus niger M200. This enzyme shows no enantioconvergence with the substrates styrene oxide or para-chlorostyrene oxide, i.e. racemic vicinal diols are produced from the racemic substrates. After saturation mutagenesis, screening by chiral gas chromatography revealed enzyme variants with improved enantioconvergence as manifested by an increased enantiomeric excess for the diol product. Nine amino acid exchanges accumulated in the active site and the substrate access tunnel in the course of 5 productive rounds of iterative saturation mutagenesis, resulting in an enantioconvergent epoxide hydrolase variant. During directed evolution a steady and pronounced increase in the regioselectivity coefficient S was observed, while the value for R remained low (Figure 1). As a consequence, the enantioconvergence of the EH increased considerably from 3% eep for the wild-type EH to 70% eep, i.e. the final mutant enzyme transformed racemic styrene oxide and parachlorostyrene oxide to diol enantiomers of R-configuration with enantiomeric excesses of 70%. Sequential bi-enzymatic reactions using the wild-type EH and/or its evolved variants enabled preparation of the chiral building blocks (R)-phenyl-1,2-ethanediol and (R)para-chlorophenyl-1,2-ethanediol from inexpensive racemic epoxides with enantiomeric excesses of 91% and 89%, respectively.
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Omnichange: the sequence independent method for simultaneous site-saturation of up to five codons
Alexander Denniga, Amol Shivangea, Jan Marienhagenb, Ulrich Schwaneberga a Lehrstuhl fr Biotechnologie RWTH Aachen University, Worringerweg 1 52074 Aachen, Germany; bInstitut fr Bio- und Geowissenschaften IBG-1: Biotechnologie Forschungszentrum Jlich, 52425 Jlich, Germany E-mail: a.dennig@biotec.rwth-aachen.de Diversity generation through focused mutant libraries became a standard approach for rational or semi-rational protein engineering to improve mainly localizable enzyme properties such as activity and selectivity [1, 2]. Multi site-saturation mutagenesis allows exchanging multiple residues in close proximity simultaneously offering the opportunity to explore synergistic substitutions. Several methods for multi site-saturation mutagenesis have been developed, often requiring subsequent PCR steps and/or enzymes for DNA modifications such as restriction or ligation [3, 4]. As a consequence the number of generated variants and positions which can simultaneously be targeted are limited. Here, we report on the multiple site-saturation mutagenesis method named OmniChange which simultaneously and efficiently saturates five independent codons. As proof of principle five DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot reaction without additional PCR-amplification or application of restriction enzymes or ligases [5, 6]. Sequencing of 48 randomly picked clones revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4 % of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, not requiring a minimal distance between mutated codons and diverse focused mutant libraries can be generated within a single day.
Figure 1. The regioselectivity coefficients R() and S() as a function of the laboratory evolution process. The values of the best EH variant obtained in each productive randomization round (site A site B site C site D site H) are indicated. This research was supported by the Czech Science Foundation (grant P207/10/0135) and the Institutional Research Concept No. AV0Z50200510.
1. Reetz, M.T., Bocola, M., Carballeira, J.D., Zha, D., and Vogel, A. (2005). Angew. Chem., Int. Ed. Engl. 44, 4192-4196. 2. Reetz, M.T., Prasad, S., Carballeira, J.D., Gumulya, Y., and Bocola, M. (2010). J. Am. Chem. Soc. 132, 9144-9152. 3. Shivange, A.V., Marienhagen, J., Mundhada, H., Schenk, A., and Schwaneberg, U. (2009). Curr. Opin. Chem. Biol. 13, 19-25. 4. Wong, T.S., Roccatano, D., and Schwaneberg, U. (2007). Environ. Microbiol. 9, 2645-2659. 5. Blanusa, M., Schenk, A., Sadeghi, H., Marienhagen, J., and Schwaneberg, U. (2010). Anal. Biochem. 406, 141-146. 6. Dennig, A., Shivange, A., Marienhagen, J., and Schwaneberg, U. (2011). Submitted to Chemistry & Biology
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Directed evolution of nitrobenzene dioxygenase for enantioselective synthesis of chiral sulfoxides
Janna Shainsky, Kalia Bernath-Levin, Sivan Isaschar and Ayelet Fishman Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel E-mail: jannash@tx.technion.ac.il The recent years have witnessed increased utilization of enzymes as industrial biocatalysts for the synthesis of fine chemicals, particularly in the production of chiral compounds. In industry today there are approximately 150 implemented biocatalytic processes, the majority of them being in the pharmaceutical sector. The advantage of biocatalysis over chemical synthesis is that enzyme catalyzed reactions are often highly enantioselective and regioselective. Some of the industrially essential chemical elements are chiral sulfoxides. As natural products, they possess a wide range of biological activities. In addition, they are efficient auxiliaries that lead to essential asymmetric transformations. Furthermore, one of the most significant applications of chiral sulfoxides is in the pharmaceutical industry. For example, modafinil, that is marketed under the name Provigil by Cephalon and prescribed for the treatment of excessive daytime sleepiness, is a drug that contains a chiral sulfoxide. Nitrobenzene dioxygenase (NBDO) from Commamonas sp. strain JS765 is a multi component enzyme which is a member of the Rieske non-heme dioxygenase family. NBDO has a relatively relaxed substrate preference and is able to oxidize a wide range of substrates including nitrotoluene, naphthalene and dinitrotoluene. This is the first report on the enantioselective oxidation of pro-chiral sulfides by NBDO. The main objective of this research is to investigate structure-function correlations of NBDO and pro-chiral sulfides. An additional goal is to use protein engineering to obtain variants with improved activity and enantioselectivity which will enable its implementation in industrial processes. In order to enable easy manipulation of the -oxygenase gene containing the active site, two new expression systems were designed, one based on pET28a and the other on pET-Duet. Eleven saturation mutagenesis libraries at pre-determined positions were created using NNK codon degeneracy requiring the screening of 94 mutants for a 95% coverage of the protein sequence space. Target positions were chosen using three approaches: (i) based on previous data with other substrates and on the crystal structure of NBDO (PDB code 2BMO) (e.g. N258); (ii) based on alignment with a relatively distant enzyme aniline dioxygenase (19% similarity) (position V207); (iii) based on the HotSpot Wizard created by Damborsky and co-workers (e.g. F222 and N295). Four model substrates were screened (thioanisole, methyl-p-tolyl sulfide, bromo-thioanisole and chloro-thioanisole) (Figure 1) providing very interesting yet suprising results.

Towards improved aldolases for organic synthesis
Irene Kullartz, Jrg Pietruszka Heinrich-Heine-University Dsseldorf, Institute for Bioorganic Chemistry I.Kullartz@fz-juelich.de Nowadays the synthesis of natural products is a major research interest of many scientists. Their isolation especially from marine organisms is often challenging and the amount of the substance obtained is rather low. [1] However, the variety of their biological activities is often remarkable, so that the development of efficient syntheses of the natural products and of key-building blocks towards them is a matter of particular interest. Over the past years the usage of enzymes as catalysts for nearly all kind of reactions got more and more popular. A very promising enzyme is desoxyribose-5-phosphat-aldolase (DERA) which catalyses the aldol reaction between two aldehydes, a challenging reaction when utilising conventional chemical methods.[2,3] In this project DERA should ultimately be used to synthesise tetrahydropyrans which are a common structural motif in many natural products, including polyketides such as psymberin or bryostatin that show remarkable selective cytotoxicity.[4-6] A main part of the project is therefore to optimise a) the purification of DERA and b) the biocatalytic usage of the enzyme. Since known DERAs are preferentially D-selective, a general application in the synthesis of differently configured compounds is not feasible. Therefore, we aim at altering the enantioselectivity for the synthesis of selected precursors. En route it will be essential to have an efficient assay at our disposal. Current results of the project, especially concerning DERA optimisation will be presented.

Stereoselectivity of the Michael hydratase MhyADH
Jianfeng Jina, Sanjib K. Karmeea, Maarten Gorselinga, Adrie J. J. Straathofb, Ulf Hanefelda a Gebouw voor Scheikunde, Afdeling Biotechnologie, Technische Universiteit Delft, Julianalaan 136, 2628 BL Delft, The Netherlands; bBioseparation Technology, Afdeling Biotechnologie, Technische Universiteit Delft, Julianalaan 67, 2628 BC Delft, The Netherlands. E-mail: u.hanefeld@tudelft.nl Recently we have isolated a Michael hydratase/alcohol dehydrogenase enzyme: MhyADH. This catalyses the Michael addition of water and the subsequent oxidation of the formed 3-hydroxy ketone [1-3]. The oxidation step only takes place in the presence of an electron acceptor such as methylene blue. Earlier studies demonstrated that the oxidation catalysed by MhyADH occurs in an enantioselective fashion [1,2]. Only the R-alcohol is oxidised. Here we show that the water addition is enantioselective too. MhyADH catalyses the Michael addition of water to cyclohexenone in an enantioselective manner. After extensive extraction of the reaction mixture we isolated the hydroxyketone. Derivatisation led to the acetate that was analysed and compared to a standard. In this way it was demonstrated that the water addition is R-selective, just like the oxidation reaction (Scheme 1).
141 278
Purification of oxidoreductase from Arthrobacter sp. for the synthesis of chiral alcohols
Leandro H. Andradea, Lidiane S. Arajoa, Edna Kagoharaa a Instituto de Qumica, Universidade de So Paulo, Av. Prof. Lineu Prestes 748, SP 05508900, So Paulo, Brazil E-mail: lidianepiaui@yahoo.com.br Biocatalytic transformation, an enzyme-catalyzed reaction, is one of the techniques that have been rapidly developed in the last years for the synthesis of chiral alcohols. The most employed enzymes for this process are alcohol dehydrogenases, a subclass of oxidoreductases [1,2]. In this study we were involved in the purification and characterization of oxidoreductase from Arthrobacter sp. The crude extract was carried out the cell disruption with French pressure cell that leaves the target enzyme in the supernatant, followed by the precipitation with ammonium sulfate (30%). Enzyme activity was measured in the supernatant using the oxidation of (R,S)-1-(4-methylphenyl)ethanol with the addition of NAD+ or NADP+ (3-24 hours of reaction). After 3 h, the enzyme activity was excellent, using both cofactors. The enzyme solution was concentrated by ultrafiltration (10 kDaltons) and loaded onto an Octyl FF column (Hi-Trap, 1 mL) previously equilibrated in 20 mM phosphate buffer containing 1 M ammonium sulfate. The enzyme was eluted with a decreasing gradient of ammonium sulfate (1M - 0 M in 20 mM phosphate buffer). Its purity was determined by SDS-PAGE, which showed a single type of subunit with a molecular mass of 42 kDa (Figure 1).
Figure 1.: project outline

_______________ [1] S. V. Bhat, B. A. Nagasampagi, M. Sivakumar, Chemistry of Natural Products, Springer, 2004. [2] A. Heine, J. G. Luzb, C.-H. Wong, I. A. Wilson, J. Mol. Biol. 2004, 343, 1019-1034. [3] H. J. M. Gijsen, L. Qiao, W. Fitz, C.-H. Wong, Chem. Rev. 1996, 96, 443-473. [4] X. Huang, N. Shao, R. Huryk, A. Palani, R. Aslanian, C.Seidel-Dugan, Org. Lett., 2009, 11, 867-870. [5] G. R. Pettit, C. L. Herald, D. L. Doubek, D. L. Herald, E. Arnold, J. Clardy, J. Am. Chem. Soc. 1982, 104, 6846-6848. [6] R. H. Cichewicz, F. A. Valeriote, P. Crews, Org. Lett. 2004, 6, 1951-1954.
Scheme 1. MhyADH catalyses the enantioselective addition of water to cyclohexenone. In the presence of and oxidation reagent the subsequent oxidation is also enantioselective. Again the R-enantiomer is preferred.
________________ [1] J. Jin, P. C. Oskam, S. K. Karmee, A. J. J. Straathof and U. Hanefeld, Chem. Commun. 2010, 46, 85888590. [2] J. Jin, A. J. J. Straathof, M. W. H. Pinkse, and U. Hanefeld, Appl. Microbiol. Biotechnol. 2011, 89, 18311840. [3] J. Jin and U. Hanefeld, Chem. Commun. 2011, 47, 25022510.
Figure 1. SDS-PAGE-Line 1, BenchMarkTM Protein Ladder (Invitrogen); Line 2, partially purified fraction; Line 3, pure fraction. The purified oxidoreductase was used to catalyze the (S)-enantiomer oxidation from (RS)1-(phenyl)ethanol, (RS)-1-(4-methyl-phenyl)ethanol and (RS)-1-(4-chloro-phenyl)ethanol to their corresponding ketones which gave good conversions.
________________ [1] Matsuda, T.; Yamanaka, R.; Nakamura, K. Recent progress in biocatalysis for asymmetric oxidation and reduction. Tetrahedron: Asymmetry 2009, 20, 513557. [2] Voss, C.V.; Gruber, C.C.; Kroutil, W. Deracemisation of secondary alcohols via biocatalytic stereoinversion. Synlett 2010, 7, 991998.
Figure1. The model reaction chosen for this study. R=H, CH3, Br, or Cl, for methyl phenyl sulfide (thioanisole), methyl p-tolyl sulfide, bromo-thioanisole or chloro-thioanisole, respectively.
275
Esterification of oleic acid with glycerol and amyl alcohol by basidiomycetes from semi-arid region of Brazil
Tassia C. Ramosa, Wesle S. Gamaa, Alini T. Fricksb , Ivan S. C. Gonzleza, Heiddy M. Alvareza*, Angelica M. Lucchesea* a Departamento de Cincias Exatas, Universidade Estadual de Feira de Santana, km 03, BR 116, Campus da UEFS, Feira de Santana, BA 44031-460, Brasil. bUNIT - Universidade Tiradentes. Av. Murilo Dantas, 300, 49032-490, Farolndia, Aracaju, SE, Brasil. E-mail: marquezheiddy@gmail.com The monoacylglycerol (MG) and diacylglycerol (DG) via enzymatic synthesis could be obtained by esterification of fatty acids with glycerol [1,2] or through vegetable oils glycerolysis reactions [3]. These compounds are widely used as emulsifiers in the food, cosmetic and pharmaceutical industries. In this work we investigated the presence and the catalytic activity of lipases of seventeen basidiomycetes fungi isolated in semi-arid region of Bahia, Brazil. The fungi were cultured at 28C for 7 days on solid medium. The detection of lipase activity was based on the halo formed by the reaction between Rhodamine B and the fatty acid generated in the enzymatic process, Figure 1. Only two fungi showed lipolytic activity, the fungus 404 e 413.
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Reactivity of a recombinant esterase from Thermus thermophilus HB27 in aqueous and organic media
a Pablo Fuciosa, aRoberto Gonzlez, aM. Luisa Ra, bOlalla Lpez-Lpez, bM. Isabel Gonzlez-Siso, bM Esperanza Cerdn, cRamn Rodrguez, cBegoa Pampn a Department of Analytical and Food Chemistry, University of Vigo, Campus of Ourense, As Lagoas, 32004 Ourense, Spain. bDepartamento de Bioloxa Celular e Molecular, Facultade de Ciencias, Universidade da Corua, 15071 A Corua, Spain. cGalChimia, Cebreiro s/n, O Pino, 15823 A Corua, Spain. E-mail: mlrua@uvigo.es
279
Using a thermostable Baeyer-Villiger monooxygenase for the creation of chimeric enzymes
Hugo L. van Beeka, Gonzalo de Gonzalob and Marco W. Fraaijea a Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands; b Departamento de Qumica Orgnica e Inorgnica, Instituto de Biotecnologa de Asturias, Universidad de Oviedo, c/ Julin Clavera 8, 33006 Oviedo, Spain. E-mail: h.l.van.beek@rug.nl Baeyer-Villiger monooxygenases (BVMOs) have been studied during the last decades for their ability to perform Baeyer-Villiger oxidations, N-oxidations and sulfoxidations, often with high chemo-, regio-, and/or enantioselectivity [1,2]. Numerous BVMOs have been identified and characterized but with the exception of one BVMO (phenylacetone monooxygenase, PAMO), they are all relatively unstable [3]. Unfortunately, PAMO has a narrow substrate specificity as conversion is only observed for phenylacetone, similar ketones and small aromatic sulfides. Together with other BVMOs, PAMO has been the subject of protein engineering to alter its substrate specificity and enantioselectivity [4]. This has revealed that PAMO tolerates multiple mutations. A loop close to the putative substrate binding site has been target of several enzyme engineering studies. Mutations in or near this loop affect enantioselectivity and Figure 1. Model of one of the created chimeric BVMOs. substrate specificity [1]. In this study, PAMO was used as a scaffold to introduce the substrate specificity deterThe PAMO part is in green while the exchanged part is in mining part of another BVMO. For this, the C-terminal blue. Based on PAMO crystal part of PAMO was replaced, including the substrate binding loop and many second-sphere residues. structure [5] Two of the created chimeric enzymes show increased stability compared to the less-thermostable parent, and convert several new substrates, sometimes with reversed enantioselectivity. This illustrates that the swapping of subdomains between different enzymes can be a successful approach to alter substrate specificity while maintaining a high thermostability.
________________ [1] G. de Gonzalo, M. D. Mihovilovic, M. W. Fraaije, Chembiochem, 2010, 11, 2208. [2] G. Gonzalo, D. E. Torres Pazmio, G. Ottolina, M. W. Fraaije, G. Carrea, Tetrahedron: Asymmetry, 2005, 16, 3077. [3] M. W. Fraaije, J. Wu, D. P. Heuts, E. W. van Hellemond, J. H. Spelberg, D. B. Janssen, Appl. Microbiol. Biotechnol., 2004, 66, 393. [4] M. T. Reetz, Angewandte Chemie International Edition, 2010, 50(1), 138.
280
NMR-based high-throughput screening of carbohydrate-active enzyme specificity: application to dextransucrase evolution
Romain Iraguea-c, Laurence Tarquisa-c, Claire Moulisa-c, Isabelle Andra-c, Stphane Massoua-c, Jean-Charles Portaisa-c, Olivier Saureld, Alain Milond, Agns Sabate, Alain Bulone, Pierre Monsana-c, Magali Remaud-Simona-c, Gabrielle Potocki-Vronesea-c. a Universit de Toulouse; INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France ; bINRA, UMR792, Ingnierie des Systmes Biologiques et des Procds, F-31400 Toulouse, France ; cCNRS, UMR5504, F-31400 Toulouse, France ; dCNRS,UPS, Institut de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, 31077 Toulouse Cedex 4, France ; eINRA, UR1268, Biopolymres Interactions Assemblages, F-44300 Nantes, France E-mail: Claire.Moulis@insa-toulouse.fr The development of glycoenzyme-based processes for the production of novel glycans, oligosaccharides or glycoconjugates has gained interest for applications in human health, food and feed industries, fine chemistry and white biotechnologies. Transglycosidases and glycosyltransferases, in particular, constitute very efficient tools for glycodiversification [1]. In addition, protein engineering has shown to be extremely powerful to expand their substrate or product specificities and adapt them to synthetic substrates [2]. However, searching for carbohydrate-active enzymes (CAZymes) showing required specificities in libraries of thousand mutants is still a challenge. This is principally due to the high structural complexity and diversity of carbohydrates and to the difficult acquisition of carbohydrate structural information via rapid and automated methods. Recently, we have developed a straightforward, sensitive and quantitative NMR-based method for high-throughput characterization of carbohydrate structure and screening of carbohydrate active enzyme specificity [3]. This innovative strategy was applied to screen a library of transglucosidases variants obtained by combinatorial engineering of the Leuconostoc mesenteroides NRRL-B512F dextransucrase DSR-S, the most efficient dextransucrase known to date. This enzyme is classified in the family 70 of Glycoside-Hydrolases [4]. From sucrose, a readily available and natural renewable resource, it catalyzes the synthesis of high molecular weight polymers of D-glucosyl units linked at 95% by -1,6 osidic bounds. These dextrans found several commercial applications since sixty years, especially in therapeutic or analytical fields, but is also used as texturing agent in food industry [5]. DSR-S combinatorial engineering allowed to isolate more than 300 clones with altered linkage specificity compared to the parental enzyme. Among them, 7 were selected, that are able to efficiently produce a panel of dextrans of innovative structure and properties, contaning various -1,3 linkage contents. Analysis of structure-function relationships of these variants and detailed physico-chemical characterization of the -glucans they produce will be discussed.
________________ [1] Monsan, P et al., 2010, Curr. Opin. Microbiol. 13: 293; [2] Homann, A. and Seibel, J., 2009, Nat. Prod. Rep. 26: 1555; [3] Irague, R. et al. 2011, Anal Chem 83: 1202; [4] Cantarel, B.L et al., 2009. Nucl. Acids Res. 37: D233; [5] Vandamme, E et al. 2002, US Patent 6,627,235.
A novel membrane-associated esterase (E34Tt) with thermoalkalophilic properties was purified from wild-type Thermus thermophilus HB27. Gene sequence indicated very low identity to any known esterases or lipases [1]. The gene sequence coding for the thermophilic enzyme was successfully cloned and extracellularly expressed in Kluyveromyces lactis NRRL Y-1140 using the integrative expression vector pKLAC1 [2]. The recombinant enzyme was active and stable in a wide range of pH and temperature (pH 5.59 and 35 70C), although optimal activity was determined at pH 7.5 and 47.5C with p-nitrophenyl dodecanoate as a substrate, and maximal stability at pH 8.1 and 65 C. The half-life of the enzyme at 85C and pH 7.0 was over 3.8 h [2], and no apparent decrease in activity was observed after 7 h at 70C. A study on the enzyme reactivity was performed. In aqueous media, KLEST-3S was active in the presence of 10% organic solvents and 1% detergents. The esterase activity was enhanced by methanol, and inhibited by 2-propanol, whereas no significant effect was found in the presence of dimethyl sulfoxide (DMSO). Detergents inhibited the activity to various extents. 55% activity was retained with Tween-20 and 29% with CHAPS. Activity was completely inhibited in the presence of SDS. KLEST-3S showed activity towards a wide range of p-nitrophenyl esters, but had preference for medium-length acyl chains (C10). Among triglycerides, the enzyme catalyzed the hydrolysis of saturated substrates with acyl chain lengths ranging from 2 to 10 carbons. KLEST-3S displayed interfacial activation towards triacetin over the solubility limit, and highest activity was observed with tributyrin. The recombinant enzyme was also tested as biocatalysts in a number of reactions (alcohol and amine acetylation, ester hydrolysis, and acid esterification) in organic media using unnatural substrates and different reaction conditions to produce intermediate bioactive compounds for the pharmaceutical industry. The recombinant esterase provided high yields for the acetylation of alcohols and amines (99% conversion). In addition KLEST-3S showed stereoselective hydrolysis of ibuprofen methyl ester (87% ee). These properties indicate that KLEST-3S could be a robust and efficient biocatalysts for application in industrial bioconversion.
________________ [1] Fucios, P., L. Pastrana, A. Sanromn, M.A. Longo, J.A. Hermoso, M.L. Ra, J. Mol. Catal. B Enzym. 70 (2011) 127-137. [2] Fucios, P., E. Atanes, O. Lpez-Lpez, M. Esperanza Cerdn, M. Isabel Gonzlez-Siso, L. Pastrana, M. Luisa Ra, Prot. Expr. Purif. 78 (2011) 120-130.
Figure 1. Halo after 72 h. The catalytic activity of the lipases present in these fungi was evaluated by the esterification of oleic acid with isoamyl alcohol and glycerol. The estimation of the degree of these syntheses was made by titrating the material in alcoholic medium with sodium or potassium hydroxide solution. The reactions products were extracted from each reaction mixture with ethyl acetate and identified by thin layer chromatography through comparison with authentic standards. The fungus 404 was efficient in catalyzing the esterification of isoamyl alcohol meanwhile the fungus 413 only synthesized the glycerides. In the case of glycerides the production of TG (triacylglycerol) was higher than MG and DG.
_______________ [1] D. G. Hayes, E. Gulari. Biotechnology and Bioengineering 38(50 (1991) 507-517. [2] V. Tripathi, R. Trivedi, R. P. Singh.Journal of Oleo Science 55(2) (2006) 65-69. [3] P. B. L. Fregolente, G. M. F. Pinto, M. R. Wolf-Maciel, R. M. Filho, C. B. Batistella. Quim. Nova 32(6) (2009) 1539-1543.
October 2-6, 2011
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Fine enzymes for organic synthesis
a

A platform for the fast access to high-performance enzymes for biocatalysis
Hedda Merkens c-LEcta GmbH, Deutscher Platz 5b, 04103 Leipzig, Germany E-mail: hedda.merkens@c-LEcta.de Despite the availability of enzymes from different suppliers for biocatalytic purposes, there is still a huge demand for customized enzymes with improved performance. Another bottleneck is fast access to larger enzyme amounts required for preparative synthesis. The addressable sources of biocatalysis enzymes include new enzymes from biodiversity and improved variants from enzyme engineering approaches. c-LEcta established a technology platform covering the whole value-chain for the fast access to enzymes from both sources and for the rapid scale-up of development projects into commercial scale. c-LEctas proprietary Cluster Screening enables the rapid screening of libraries from metagenomic biodiversity as well as from artificial diversity in engineering projects. New enzymes are identified by activity in a throughput of a few 10,000 to several 100,000 clones per screening run. In this talk we will report on a screening program in which we could rapidly identify new wild-type alcohol dehydrogenases (ADHs) from biodiversity. Most of them are new sequences displaying only weak homology to enzymes in the database. Cluster Screening was also used for selection of ADH-variants with novel properties by a structure-based engineering. The collection of enzymes from both sources shows high R- and S-stereoselectivity and a diverse substrate profile. A set of 24 ADH enzymes, exhibiting diverse substrate spectra and high stereoselectivities are combined in the ADH c-LEction and are ready-toscreen for customer applications. For the rapid implementation of customized enzymes for biocatalysis c-LEcta has set up a streamlined process to rapidly establish and scale-up enzyme production processes up to the commercial supply of the biocatalysts. Enzyme production processes at industrial scale were already established for several enzymes. We will present examples for preparativescale processes established with our ADH enzymes.

Diverse enzyme panels via rational sequence space sampling
Meng Zhang, Vatsala Malik, Justin J Perry, Gary W Black School of Life Sciences, Northumbria University, Newcastle upon Tyne, UK E-mail: gary.black@northumbria.ac.uk The lead enzyme in a novel biocatalytic synthesis can often be sourced from an offthe-shelf biocatalysis enzyme panel, which offers the ability to investigate a range of enzymes which perform the same chemical transformation but with different specificities. A common approach to obtain such diversity within an enzyme panel has been to subject a single enzyme to laboratory-based evolution and search through the many evolved proteins for those with improved performance [1]. However, we have taken an approach to short-cut the search for a specific biochemical outcome that involves rational sampling of the diversity that already exists within the sequence space that Nature provides. Through mining of the ever-expanding sequence databases available online using existing bioinformatic tools, it is possible to choose naturally occurring sequences with a defined level of sequence diversity (Fig. 1) which are suitable for hyperexpression in host such as Escherichia coli. We will present data on how this approach has been applied to the production of panels of nitrile hydratases [2], carbonyl reductases [3] and glycosyltransferases [4], delivering small panels of enzymes which offer different chemo-, regio- and enantioselectivities.
143 286
Laboratory evolution of dehalogenase from esterase
Santosh Kumar Padhi, Uwe T. Bornscheuer Dept. of Biotechnology & Enzyme Catalysis, Institute of Biochemistry, Greifswald University, Felix-Hausdorff Strasse 4, Greifswald, D-17487,Germany E-mail: skp_77@yahoo.com Enzyme catalysis plays a vital role in the current century as it brings clean and green technologies important for the society, for example, ranging from the synthesis of enantiopure pharmaceutical molecules to bioremediation. In the last decade protein engineering was largely used to make tailor made enzymes with improved biocatalytic properties.[1-2] However, with the increasing demand of enzymes to catalyze nonnatural reactions, to provide solutions to the recent chemical problems in the society, it still remains a challenge for protein engineers to produce enzymes with new activity and only a handful of examples have been described.[3-4] Enzymes from the /-hydrolase structural super family consists of esterases, epoxide hydrolases, hydroxynitrile lyases, dehalogenases and C-C bond hydrolases etc.[5] These group of enzymes have a common structural fold but diverse catalytic functions, wide substrate specificity, high stereoselectivity and for many of them their crystal structure data is available. Therefore they are ideal targets for designing new catalytic activity. We aim to engineer a common esterase to catalyze an industrially important reaction particularly hydrolysis of a carbon-halogen bond, which a haloalkane dehalogenase (HLD) does.[6] This project therefore will provide insights how to design and create new enzymes with completely different activity and to understand the molecular basis of converting an esterase to haloalkane dehalogenase. The engineered esterase variants with new substrate selectivity to catalyze the hydrolysis of carbon-halide bond will be explored. This provides an alternative route to organic reactions for the biodegradation of environmental toxic halide compounds. We have used different protein engineering approaches in the Pseudomonas fluorescens esterase gene and a systematic study was made to generate HLD activity. The various protein engineering approaches and the results will be presented.
________________ [1] S. Lutz, U. T. Bornscheuer (Eds.), Protein Engineering Handbook (2009), Wiley-VCH, Weinheim [2] R. J. Kazlauskas, U. T. Bornscheuer, Nature Chem. Biol 2009, 5, 526-529 [3] H. Jochens, K. Stiba, C. Savile, J. G. Yu, T. Gerassenkov, R. J. Kazlauskas, U. T. Bornscheuer, Angew. Chem. Int. Ed. 2009, 48, 3532-3535. [4] S. K. Padhi, R. Fujii, G. Legatt, S. L. Fossum, R. Berchtold, R. J. Kazlauskas, Chem. Biol. 2010, 17, 863-871. [5] M. Holmquist, Curr. Protein Pept. Sci. 2000, 1, 209-235. [6] S. Keuning, D. B. Janssen, B. Witholt, J. Bacteriol. 1985, 163, 135-139.
Michael Pulsa, Christian Leggewiea, Thorsten Eggerta evocatal GmbH, Merowingerplatz 1a, 40225 Duesseldorf, Germany E-mail: m.puls@evocatal.de The molecular tools of industrial or White Biotechnology are enzymes that catalyze specific reactions inside living cells. Enzymes usually exhibit high substrate specificity and enantioselectivity, and, at the same time, work in aqueous environment and under mild reaction conditions. These properties suggest using them as biocatalysts in the chemical industry to conduct "green chemistry". Currently, regardless of a possible ecological advantage, biotechnologically synthesized fine chemicals have to be price competitive with traditionally produced ones and have to meet the market price. Therefore, there is a demand for novel and improved enzymes that have to be produced inexpensively. In addition, tailor-made biocatalytical processes have to be developed to reduce the total costs of the product. Therefore, we developed holistic solutions that take advantage of a range of biotechnological methods to produce fine chemicals very efficiently. This includes the search for novel enzymes and the improvement of biocatalysts as well as their efficient expression and production. Furthermore we find a customized process for each synthesis. Based on case studies we present the efficient production of various chiral intermediates like enantiopure amines, alcohols or cyanohydrins which are all of industrial interest. The reactions are mainly catalyzed by optimized Transaminases, Alcoholdehydrogenases and Hydroxynitrilases. Besides these enzymes, which perform very well in organic synthesis at all scales, we generated focused variant libraries (panel plates) which are ready to screen for the conversion of unusual substrates, increased enantioselectivity or feasibility for specific process conditions. Moreover, all optimization technologies are transferable and suitable for industrial scale processes which is a prerequisite to compete with traditionally produced fine chemicals.
________________ [1] AM Sawayama, M MY Chen, P Kulanthaivel, M-S Kuo, H Hemmerle, FH Arnold (2009) A Panel of Cytochrome P450 BM3 Variants to Produce Drug Metabolites and Diversify Lead Compounds. Chemistry - A European Journal 15, 1172311729. [2] GW Black, T Gregson, CB McPake, JJ Perry, M Zhang (2010) Biotransformation of nitriles using the solvent-tolerant nitrile hydratase from Rhodopseudomonas palustris CGA009. Tetrahedron Letters 51, 1639-1641; S van Pelt, M Zhang, LG Otten, J Holt, DY Sorokin, F van Rantwijk, GW Black, JJ Perry, RA Sheldon (2011) Probing the Enantioselectivity of a Diverse Group of Purified Cobalt-Centred Nitrile Hydratases. Organic and Biomolecular Chemistry 9, 3011-3019. [3] GW Black, JJ Perry, M Zhang, Tom Moody, Chirality through reduction with carbonyl reductasesdiversity of action through diversity of sequence?, in preparation. [4] V Malik, JJ Perry, LG Dover, T Flinn, J Northen, E Ludkin, GW Black, Glycosyl- transferases for the synthesis of sterol glycosides, in preparation.
Figure 1. Rational sampling of sequence space as visualised via a dendogram. Each branch of the dendogram represents an enzyme sequence and the distance between each branch is indicative of the level of similarity. Those branches closer together are more similar than those branches further apart.
283
Hyperthermophilic Aldolases as Biocatalyst. Characterization of Rhamnulose 1-phosphate aldolase from Thermotoga maritima
Isabel Oroz-Guineaa, Israel Snchez-Morenoa and Eduardo Garca-Juncedaa a Departamento de Qumica Bio-Orgnica, Instituto de Qumica Orgnica General, CSIC. 28006 Madrid, Spain. E-mail: eduardo.junceda@csic.es Recently, we have reported the engineering of a new bifunctional enzyme which presents the aldolase and the kinase activities in the same protein.[1] The covalent union of both enzymes allows that some of their properties are transferred to the fusion protein. Thus, we have demonstrated that the thermal stability of the fused enzyme is higher than that of the multi-enzyme system and that the physical association of the parent enzymes produces an increase in the aldol reaction rate of 20-folds.[2] We are involved now in extending this strategy to other DHAP-dependent aldolases. A bioinformatic screening for aldolase enzymes revealed that the TM1072 gene from Thermotoga maritima codifies for a putative form of rhamnulose-1-phosphate aldolase (Rha1PA). The crystal structure of the codified protein is known (PDB:1PVT) indicating that is a monomeric protein. Furthermore, Thermotoga maritima is a hyperthermophilic eubacterium that grows at 80 C, this is an advantage because enzymes from thermophilic organism offer intriguing possibilities both for practical biocatalysis and for understanding structure-function relationship in enzyme mechanism.[3] Therefore, Rha1PA from T. maritima is an ideal candidate for the engineering of a new bifunctional aldolase/kinase enzyme. In this communication we describe the biochemical characterization of this enzyme and its evaluation as a new biocatalyst for C-C bond formation. Acknowledgements: We thank the Spanish Ministerio de Ciencia e Innovacin (Grant CTQ2010-15418) and Comunidad de Madrid (Grant S2009/PPQ-1752) for financial support. I. Oroz-Guinea is a JAEPredoc fellow from CSIC.
________________ [1] L. Iturrate, I. Sanchez-Moreno, E. G. Doyaguez, E. Garcia-Junceda, Chem. Commun. 2009, 17211723. [2] L. Iturrate, I. Snchez-Moreno, I. Oroz-Guinea, J. Prez-Gil, E. Garca-Junceda, Chem. Eur. J. 2010, 16, 4018-4030. [3] C. Vieille, G. J. Zeikus, Microbiol. Mol. Biol. Rev. 2001, 65, 1-43.
284
Exploiting the catalytic promiscuity of 4-oxalocrotonate tautomerase for carbon-carbon bond formation
Ellen Zandvoorta, Bert-Jan Baasa, Edzard M. Geertsemaa, Wim J. Quaxa and Gerrit J. Poelarendsa a Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands E-mail: e.zandvoort@rug.nl The enzyme 4-oxalocrotonate tautomerase (4-OT) is a member of the tautomerase superfamily, a group of homologous proteins that are characterized by a -- structural fold and a catalytic amino-terminal proline [1]. In the mechanism of 4-OT, Pro-1 is a general base that abstracts the 2-hydroxyl proton of 2-hydroxy-2,4-hexadienedioate for delivery to the C-5 position to yield 2-oxo-3-hexenedioate. In the present study, 4-OT was explored for nucleophilic catalysis based on the mechanistic reasoning that its Pro-1 residue has the correct protonation state (pKa ~6.4) to act as a nucleophile at pH 7.3. Inhibition studies and mass spectrometry experiments demonstrated that 4-OT can use Pro-1 as a nucleophile to form an imine/enamine with various aldehyde and ketone compounds. The chemical potential of the smallest enamine, that generated from acetaldehyde, was explored for further reactions using a set of selected electrophiles. This systematic screening approach led to the discovery of two new promiscuous activities in wild-type 4-OT. First, 4-OT catalyzes the aldol condensation of acetaldehyde (1) with benzaldehyde (2) to form cinnamaldehyde (3) (kcat/KM = 8.5*10-4 M-1 s-1) (Scheme 1) [2]. Rational design resulted in a single-site 4-OT mutant with strongly improved aldolase activity (600-fold in kcat/KM).
O H 12 H O H 34 O OH -H2O H O
287
Design of artificial enzymes as new highly selective biocatalysts
Jose M. Palomo* Department of Biocatalysis, Institute of Catalysis, CSIC, Campus UAM .Madrid. Spain E-mail: josempalomo@icp.csic.es Natures mastery in creating complex molecules from simpler ones through highly effective and selective bond-forming reactions in a highly predictable manner is unparalleled, being enzymes the principal actors (natural asymmetric catalysts). Enzymes best perform in aqueous media usually and have high catalytic efficiency and selectivity. However, they tend to be unstable, highly substrate specific, among other constrains. These constrains limit the use of enzymes as catalysts at industrial scale to some specific well-optimized application. In this way, the development of new strategies to design new enzymes as robust and selective biocatalysts by specific incorporation of novel functionalities is a great challenge being an outstanding issue in the actual modern chemistry. Different kind of techniques for protein derivatization on aqueous solution based on synthetic organic chemistry such as chemical amination, maleimide cysteine conjugation, lysine modification or click ligation have focused on modifying natural enzymes to impart new or altered catalytic function [1]. Physical modification by polymer and peptides coating have been also applied for improvement of enzymes functionality, stability and selectivity. Nevertheless, the most promising alternative to create new artificial enzymes is based on the combination of chemical tools with genetic engineering. Tailor-made molecules could be incorporate on specific position on the protein surface to study their effects [2]. The selective incorporation of organometallic complexes on proteins could represent another interesting field for the creation of new artificial enzymes focus on the improvement of the selectivity for the heterogeneous catalyst by using the tridimensional space of the enzyme as modulator [3]. New highly active and selective artificial biocatalysts have been prepared and used in green biotransformations: glycerin transformation, desymmetrization of prochiral diesteres, regioselective deprotection of carbohydrates or kinetic resolution of racemic esters.
288
Characterisation of bacterial hydroxynitrile lyases with high homology to cupin superfamily proteins
Kerstin Steiner, Ivan Hajnal, Andrzej Lyskowski, Georg Steinkellner, Karl Gruber, Helmut Schwab ACIB GmbH (Austrian Centre of Industrial Biotechnology) c/o TU Graz, Petersgasse 14, 8010 Graz, Austria kerstin.steiner@acib.at Hydroxynitrile lyases (HNLs) or oxynitrilases catalyse the cleavage of cyanohydrins in vivo. They are one of the few enzyme classes that are able to biocatalytically form carboncarbon bonds in vitro and catalyse the enantioselective condensation of hydrocyanic acid with aldehydes and ketones yielding -hydroxynitriles. These cyanohydrins are versatile building blocks for a variety of chemical and enzymatic follow up reactions yielding a broad product range for industrial applications[1]. In an activity-based screen for hydroxynitrile lyases in gene libraries of endophytes, a 132 aa candidate protein from Pseudomonas mephitica was identified. This first bacterial HNL showed high homology to proteins of the cupin superfamily[2], a fold which has not been reported for HNLs before. Based on sequence and structural homology a set of cupins with unknown function was chosen for further investigation of HNLs with cupin fold. The proteins were successfully expressed in E. coli in high yield. They exhibit activity in the cleavage reaction of mandelonitrile. More importantly, some of the bacterial HNLs were also able to perform the synthesis of mandelonitrile from benzaldehyde and HCN, exhibiting up to 74% conversion of benzaldehyde after 60 minutes and an enantiomeric excess of 89% for the (R)-enantiomer of mandelonitrile. Comparison of the structures or models of the different HNLs revealed amino acids in the putative catalytic domain, which might be involved either in substrate binding or conversion. These amino acids were mutated in the putative HNLs. Several of the mutants showed either increased or reduced HNL activity. Currently co-crystallisation experiments with mandelonitrile and benzaldehyde are performed to gain an insight in the catalytic mechanism of bacterial HNLs with cupin fold.
_______________ 1.Purkarthofer T., Skrane W., Schuster C., and Griengl H. (2007). Appl. Microbiol. Biotechnol. 76:309320 2.Hussain Z., Wiedner R., Hajek T., Avi M., Hecher B., Krenn B., and Schwab H., submitted to AEM
Scheme 1. Aldol condensation catalyzed by 4-OT. Second, the Michael addition of 1 to trans-nitrostyrene (5) is catalyzed by 4-OT (kcat/ KM = 66 M-1 s-1) (Scheme 2). Chiral-HPLC analysis of enzymatically produced 4-nitro3-phenylbutanal (6) showed that this 4-OT-catalyzed reaction is highly enantioselective and yields predominantly S-6 (90% ee). Interestingly, para-substituted analogs of 5 are also accepted as substrates by 4-OT. The corresponding nitro-aldehydes are important building blocks for pharmaceuticals like Phenibut and Baclofen [3].
O O H 15 6 NO2 H NO2 +
Scheme 2. Michael addition catalyzed by 4-OT.

________________ [1] Poelarends, G.J., Puthan Veetil, V., Whitman, C.P., Cell. Mol. Life Sci. 2008, 3606-3618 [2] Zandvoort, E., Baas, B.-J., Quax, W.J., Poelarends, G.J., ChemBioChem 2011, 602-609 [3] Maltsev, O.G., Kucherenko, A.S., Beletskaya, I.P., Tartakovsky, V.A., Zlotin, S.G., Eur. J. Org. Chem. 2010, 2927-2933
___________ [1] a) Palomo, J.M. Eur. J. Org Chem.2010,33,6303;b) Palomo, J.M. Curr. Org. Synth. 2009, 6. [2] a) Godoy, C. et al. Process Biochem. 2010,45, 534-541; b) Rakuba, D. Curr Opin Chem Biol. 2010, 14, 790. [3] a) Roelfes G et al. . Angew Chem, Int Ed 2009, 48:5159;b) T. Heinisch and T.R. Ward. Curr Opin Chem Biol. 2010, 14, 184.
October 2-6, 2011
144 289
Engineering Phenylacetone Monooxygenase by Semi Random Mutagenesis
Hanna M. Dudek, Marco W. Fraaije Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands E-mail: h.dudek@rug.nl Baeyer-Villiger monooxygenases (BVMOs) comprise a group of enzymes catalyzing Baeyer-Villiger oxidations in addition to heteroatom oxidations [1,2]. Phenylacetone monooxygenase from Thermobifida fusca is an example of an exceptionally robust enzyme in this family [3]. It can be produced at high levels in a heterologous expression system. Furthermore, it is resistant to high temperatures and unnatural environments such as organic cosolvents or ionic liquids [3-5]. Lastly, it can tolerate a high load of mutations. All of these features in combination with the availability of the crystal structure [6], make PAMO an ideal candidate for enzyme engineering. Redesign studies of BVMOs have been limited so far due to, among other reasons, the lack of a generic and high-throughput screening assay. Recently, we have developed a novel screening method for detecting BVMO activity, which is based on three features. First, PAMO is exported to the periplasm via the twin arginine translocation (Tat) system. Second, phosphite dehydrogenase is utilized to provide the reduced coenzyme for the Baeyer-Villiger reaction. Phosphite dehydrogenase regenerates NADP+ to NADPH while it oxidizes phosphite to phosphate. In this way, the BVMO activity is coupled to the formation of phosphate. Thus, the amount of produced phosphate corresponds to BVMO activity. This leads to the third feature of the screening assay, namely the detection of phosphate by the reaction with ammonium molybdate, resulting in a blue product. Our newly developed and generic assay offers the advantage of screening for activity with any desired substrate. Here we report on the application of the assay in screening sitesaturation libraries of PAMO. We have identified several variants of PAMO that are able to convert substrates not or poorly accepted by the wild-type enzyme. This demonstrates the applicability of this phosphate-based screening assay in the redesign of PAMO for an expanded substrate scope.
________________ [1] G. de Gonzalo, M. D. Mihovilovic, M. W. Fraaije, Chembiochem, 2010, 11, 2208. [2] G. Gonzalo, D. E. Torres Pazmio, G. Ottolina, M. W. Fraaije, G. Carrea, Tetrahedron: Asymmetry, 2005, 16, 3077. [3] M. W. Fraaije, J. Wu, D. P. Heuts, E. W. van Hellemond, J. H. Spelberg, D. B. Janssen, Appl. Microbiol. Biotechnol., 2004, 66, 393. [4] F. Secundo, S. Fial, M. W. Fraaije, G. de Gonzalo, M. Meli, F. Zambianchi, G. Ottolina, Biotechnol. Bioeng., 2011, 108, 491. [5] C. Rodriguez, G. de Gonzalo, M. W. Fraaije, V. Gotor, Green Chemistry, 2010, 12, 2255. [6] E. Malito, A. Alfieri, M. W. Fraaije, A. Mattevi, PNAS, 2004, 101, 13157.

Protein solubility enhancement by directed evolution: characterization of wild-type and mutant S-hydroxynitrile lyase from Manihot esculenta expressed in Escherichia coli and comparative expression in several hosts
Mohammad Dadashipour, Yasuhisa Fukuta, Mizue Yamazaki, and Yasuhisa Asano* Laboratory of Enzyme Chemistry and Engineering, Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan Corresponding author (Email: asano@pu-toyama.ac.jp) Hydroxynitrile lyases are mainly plant enzymes catalyze the addition of cyanide to aldehyde or ketones resulting in chiral cyanohydrins which are valuable intermediates in industry. MeHNL was primarily expressed in E. coli as a low in vivo soluble enzyme therefore; protein engineering strategies were employed to improve its solubility in this versatile host. Mutagenesis reactions resulted in mutations in some hot spots on the surface and inside the protein and changed the solubility of the enzyme drastically at 37C, so that the expression profiles of the mutants in soluble and insoluble fractions were completely different. Saturation mutagenesis of the buried residue His provided more soluble mutants with hydrophobic substitutions [1], while causing only minor structural changes, proved by biophysical evaluations. Specific activity of highly soluble mutants was elevated several times in the cell-free extract, while the catalytic efficiency of the mutants was affected only slightly in their purified form. The stability of the mutants differed from that of the wild-type at high temperatures and at pH>8 [2]. As a part of attempts to clarify the mechanism of this phenomenon, we have studied the possibility of expression of a highly active and soluble mutant as well as wild-type enzyme in several expression systems ranging Pichia pastoris, Leishmania tarentolae and two cell-free translations including an E. coli lysate and wheat germ translation system. Two distinguishable protein expression patterns were observed in prokaryotic and eukaryoticbased systems: The wild-type and mutant enzyme showed high activity for both genes in eukaryotic hosts, while those of E. coli exhibited totally different levels, indicating the phenomenon is specific to the E. coli system [3].
______________ [1] Asano, Y., T. Akiyama, F. Yu, F. Sato, Patent WO/ 4562006/041226. [2] Asano, Y., M. Yamazaki, M. Dadashipour, N. Doi, H. Komeda, submitted. [3] Dadashipour, M., Y. Fukuta, Y. Asano, Pro. Expr. Puri., 2011, 77 (1): 92-97

Directed Evolution of P450cin for Mediated Electron Transfer
Ketaki D. Belsarea, Amol V. Shivangea, Dirk Holtmanb, Jens Schraderb, Ulrich Schwaneberga a Lehrstuhl fr Biotechnologie, RWTH Aachen University, Worringerweg 1, 52074 Aachen Germany; bDECHEMA e.V.,Karl- Winnacker-Institut, Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, Germany. E-mail: k.belsare@biotec.rwth-aachen.de Cytochrome P450 monooxygenases catalyze regio- and stereo-selective hydroxylations and epoxidations of a broad range of industrially relevant compounds by using molecular oxygen [1, 2]. Cytochrome P450cin is isolated from Citrobacter braakii and catalyzes the oxidation of 1,8-cineole to 2-hydroxy-1,8-cineole [3]. P450cin uses a multicomponent flavodoxin reductase system for the transfer of electrons from NADPH. The three component system of P450cin consists of a FAD containing cindoxin reductase (CinB) which has not yet been expressed functionally, a FMN containing cindoxin (CinC) and the heme monooxygenase P450cin (CinA). The presence of these multicomponent protein domains limits the electron transfer efficiency. Here we report the fusion of the CinA and CinC domain comparable to fusion protein of P450BM-3. Fusion of CinA and CinC domains with variable linker length is achieved through the PLICing method [4]. The flexibility of the two protein domains is essential for holding the domains in a position for efficient electron transfer. Various linkers were therefore designed based on the computational prediction for flexibility of the fused protein domains. Linker insertion was validated by sequencing and a high throughput screening system based on TLC robot was developed for screening of P450cin fusion libraries. Site saturation mutagenesis on Y81 position yielded an indole converting variant. The P450cin fusion variants were screened using an indole based detection system (production of indigo from indole) for identification of functionally fused P450cin enzyme. The fused CinA-CinC variant will further be evolved for mediated electron transfer. The use of mediators like phenosafranine or zinc/cobalt sepulchrate instead of expensive cofactor NADPH will further widen synthetic options of P450cin [5].
145 294
Towards property dependent directed evolution by modulating mutational bias, mutational load and diversity through SeSaMPD
Hemanshu Mundhadaa, Amol V. Shivangea, Jan Marienhagenb and Ulrich Schwaneberga a Lehrstuhl fr Biotechnologie, RWTH Aachen University, Aachen, Germany bInstitut fr Bio- und Geowissenschaften, Forschungszentrum Juelich, Juelich, Germany E-mail: h.mundhada@biotec.rwth-aachen.de Elucidating structure-function relationships in proteins and adapting biocatalysts to nonconventional substrates and production conditions are of high academic and economical interest. Independent from any structural information, directed evolution can steer protein properties in iterative cycles of diversity generation on the gene level and screening for improved variants on the protein level. Achieving chemically diverse substitutions and combinatorial complexity of the mutations are the key challenges for generating quality mutant library. Generally applied random mutagenesis methods (epPCR) are constrained in generating functional diversity due to a transition biased mutational spectra and lack of introducing consecutive mutations. [1] Furthermore flexibility to modulate mutational bias and load is limited in these methods and therefore do not offer a solution to combinatorial complexity challenge of directed evolution. SeSaMPD has been developed for addressing above mentioned challenges. This method is an advancement of SeSaM-Tv-II [2] protocol and also involves four steps for construction of mutant library. (i) Generation of DNA fragments by random incorporation of cleavable dNTPS in the gene by PCR followed by cleavage and purification of the fragments, (ii) Enzymatic addition of universal base/s, (iii) Elongation of fragments (iv) Replacement of universal bases by standard nucleotides. Mutational flexibility in the SeSaM was adjusted by the choice of dNTPS used to generate pool of DNA fragments and consecutive mutations are created by addition of more than one universal base. As a proof of concept, mutational bias flexibility of SeSaM was revealed by constructing three types of libraries of eGFP gene. Variable numbers of cycles in step 3 PCR were used to tune the mutation load. Sequencing of 40 to 45 clones from each library revealed enhanced diversity, 9 to 11 fold enrichment of desired sets of transversions and expected tunable mutational load. SeSaMPD method therefore can offer tunable mutational bias and mutational load which will impart complete flexibility to experimentalist to design high quality mutant libraries with respect to desired properties and screening systems. SeSaMPD also promises unparallel diversity which can be tuned towards property dependent evolution. _____________ [1] AV. Shivange, J. Marienhagen, H. Mundhada, A. Schenk, U. Schwaneberg, 2009, Advances in generating functional diversity for directed protein evolution. Curr. Opin. Chem. Biol., 13(1):19-25. [2] H. Mundhada, J. Marienhagen, A.Scacioc, A. Schenk, D. Roccatano and U.Schwaneberg, SeSaM-Tv-II generates a protein sequence space that is unobtainable by epPCR. ChemBioChem., 12 : 2011 (In press).
________________ [1] Tee K., Schwaneberg U., Directed Evolution of Oxygenases: Screening Systems, Success Stories and Challenges. Combinatorial Chemistry and High Throughput Screening, (2007) 10, 197-217. [2] Nazor J., Schwaneberg U., Laboratory Evolution of P450 BM-3 for Mediated Electron Transfer. ChemBioChem, (2006) 7, 638-644. [3] Hawkes D., Adams G., Burlingame A., Montellano P., Voss J., Cytochrome P450cin(CYP176A), Isolation, Expression and Characterization. The Journal of Biological Chemistry (2002) 277, 27725-27732. [4] Blanusa M., Schenk A., Sadeghi H., Marienhagen J., Schwaneberg U., Phosphorothioate- based ligase independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method. Analytical Biochemistry (2010) 406, 141-146. [5] Cekic S., Holtmann D., Gven G., Mangold K., Schwaneberg U., Schrader J., Mediated Electron Transfer with P450cin. Electrobiochemistry Communications (2010) 11, 1547-1550.
291
Amino acids acylation in aqueous medium using amino acylase activity from Streptomyces ambofaciens
Naudot Mariea, Ferrari Florenta, Martins Evandroa, Aymes Arnauda, Kapel Romaina, Humeau Catherineb, Delaunay Stphanea, Chevalot Isabellea a Laboratoire Ractions et Gnie des Procds, Nancy-Universit, CNRS, 2 avenue de la Fort de Haye, B.P. 172 F-54505 Vanduvre ls Nancy, France; bLaboratoire dIngnierie des Biomolcules, Nancy-Universit, 2 avenue de la Fort de Haye, B.P. 172 F-54505 Vanduvre ls Nancy, France E-mail: Catherine.Humeau@ensaia.inpl-nancy.fr The cosmetic and therapeutic potentials of amino acids and peptides can be restricted by their polarity that can limit their transport through biological membranes, and their sensibility to endogenous proteases. To overcome these constraints, structural modifications such as O- or N-acylation with fatty acids can be used. The performances of enzymatic acylation of a polar dipeptide, carnosine, using either lipase B from Candida antarctica in organic medium or acyl-transferase from Candida parapsilosis in aqueous medium were previously compared [1]. Low conversion yield and initial rate were observed in organic media owing to the poor solubility of the peptide. Best performances were obtained with acyl-transferase, due to the increased solubility of carnosine in the aqueous medium. Unfortunately, up to now, very few enzymes are available to perform such acylation reactions in aqueous medium. The identification of new enzymes allowing the acylation of amino acids or peptides in aqueous medium is therefore an important challenge. Recently, we identified an amino-acylase activity from a culture supernatant from Streptomyces ambofaciens which was able to catalyze carnosine acylation in a buffered solution [2]. We then investigated the acylation potential of this enzyme activity with various amino acids using oleic acid, lauric acid, ethyl laurate or vinyl laurate as acyl donors. The acylation of lysine (mono-acetylated in or position), cysteine, tyrosine and methionine was achieved suggesting that the amino acylase activity from S. ambofaciens was able to catalyze the O-, N- and even S-acylation of amino-acids. As Streptomyces mobaraensis has been previously shown to produce several amino-acylases [3-5], a purification of the enzyme(s) responsible for this acylation reaction was undertaken from the culture supernatant of the microorganism. Combining anion exchange chromatography and size exclusion chromatography, two amino-acylase activities were detected and separated. The regioselectivity of the hydrolytic activity of both was evaluated toward and acetyl-lysine. Both enzyme activities catalyzed the hydrolysis of the amide bond in position whereas only one was able to catalyze the hydrolysis of the amide bond in position. This result suggests that S. ambofaciens might possess at least two amino-acylases exhibiting different regiospecificities. The evaluation of the synthetic potential of these enzymes is underway
________________ [1] Husson et al., Process Biochemistry, 2011, 46(4), 945-952 [2] Delaunay et al., 9th international symposium on biocatalysis and biotransformations, 5-9 juillet 2009, Berne, Suisse. [3] Koreishi et al., JOACS, 2005, 82(9), 631-637. [4] Koreishi et al., Journal of Agricultural and Food chemistry, 2006, 54, 74-78. [5] Koreishi et al., Bioscience, Biotechnology and Biochemistry, 2009, 73(9), 1940-1947.
292
Development of a solvent-tolerant amino acid racemase for application in racemate to single enantiomers processes
a
295
Redesigning the active site of arylmalonate decarboxylase variants for improved racemising and decarboxylating activity
Robert Kourista,b, Yusuke Miyauchia, Daisuke Uemuraa, Kenji Miyamotoa a aDepartment of Biosciences and Informatics, Keio University,3-14-1 Hiyoshi, Yokohama 223-8522, Japan; b Group of Biochemistry and Enzymology, Chair of Chemistry of Biogenic Resources, Technische Universitt Mnchen, Schulgasse 16, D-94315 Straubing (Germany) E-mail: kourist@tum.de Arylmalonate decarboxylase from Bordatella bronchoseptica is a promising catalyst for the enantioselective decarboxylation of -aryl--methylmalonates to give optically pure -aryl-propionates (profens). While the wild-type produces the non-desired (R)-enantiomers, double mutant G74C/C188S gives (S)-profens, albeit with a greatly reduced activity [1]. Herein we present an approach to recover this activity: By three rounds of structureguided directed evolution the decarboxylating activity of the mutant could be increased up to 920-fold. The best variant had a 220-fold improved activity in the production of (S)-naproxen with excellent enantioselectivity (>99%ee).
296
Novel enzymes for fatty acid transformation to conjugated fatty acids and hydroxy fatty acids
Shigenobu Kishinoa,b, Kenzo Yokozekia, Sakayu Shimizub, and Jun Ogawab a Laboratory of Industrial Microbiology, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakcho, Sakyo-ku, Kyoto 606-8502, Japan; b Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan E-mail: kishino@kais.kyoto-u.ac.jp Conjugated fatty acids have attracted much attention as a novel type of biologically beneficial functional lipid. In particular, the unique activities of conjugated linoleic acid (CLA) have been intensively studied, showing that CLA is expected to be a potential material for pharmaceuticals and dietary supplements. We screened the ability to produce CLA from linoleic acid within lactic acid bacteria, and selected Lactobacillus plantarum AKU 1009a as a potential strain. This strain produces two CLA isomers, i.e., cis-9,trans-11 and trans-9,trans-11-CLA, from linoleic acid with 10-hydroxy-cis-12-octadecenoic acids (18:1) as intermediates[1]. We tried to characterize the enzymes involved in CLA production from linoleic acid and identified three proteins (CLA-HY, CLA-DH, and CLA-DC) in the cell-free extracts of L. plantarum AKU 1009a [2] (Fig. 1). CLA-HY was found as a membrane bound protein, and CLA-DH and CLA-DC as soluble proteins. The CLA-HY of L. plantarum AKU 1009a was overexpressed in E. coli, purified with Ni column, and characterized. CLA-HY catalyzed the reaction forming 10-hydroxy-cis-12-18:1 from linoleic acid in the presence of FAD and NADH (Fig. 2). The enzyme showed high enantioselectivity towards (S)-isomer (>99% e.e.) and showed strict substrate specificity toward free form of C18 unsaturated fatty acids with delta-9 double bonds in cis configuration. This enzyme is expected to produce hydroxy fatty acids useful for industry, such as resins, waxes, nylons, plastics, cosmetics and so on.
________________ [1] Kishino, S. et al. (2009) Metabolic diversity in hydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production Appl. Microbiol. Biotechnol., 84, 87-97. [2] Kishino, S. et al. (2011) Linoleic acid isomerase in Lactobacillus plantarum AKU 1009a proved to be a multi-component enzyme system requiring oxidoreduction cofactors Biosci. Biotechnol. Biochem, 75, 318-322.
Christian Femmera, Markus Fueredera, Matthias Bechtolda, Sven Pankea Bioprocess Laboratory, ETH Zurich, Switzerland E-mail: christian.femmer@bsse.ethz.ch The conversion of racemic mixtures into single enantiomers in 100% yield constitutes a very attractive process route for fine chemicals[1]. Next to coupling an enantioselective transformation with an in-situ racemisation step (usually referred to as dynamic kinetic resolution)[2] integration of a physical chiral separation such as simulated moving bed (SMB) technology and a mild enzymatic racemisation has been identified as an attractive process variant. However, direct coupling of SMB, the technical realization of continuous chromatography, and bioreactor requires the use of the same solvent in both units. Amino acids, arguably the single most important molecule class in fine chemistry, constitute an intriguing example for such a process integration since many amino acid racemases are established1 and chromatographic amino acid enantioseparations based on macrocyclic glycopeptides using potentially enzyme compatible solvents have been reported.[3] However, a detailed investigation of the SMB separation conducted for different amino acids revealed that the productivity is strongly increasing with methanol content indicating that for efficient operation of the integrated process high concentration of methanol (MeOH), approx. 40 % v/v, are required. Since wild-type amino acid racemase (AAR) from P. putida DSM 3263, the most relevant racemase due to its broad substrate spectrum, is neither stable nor sufficiently active at this organic solvent concentration we developed a protein engineering approach to simultaneously improve both properties. Specifically, we selected the racemisation of L-Ornithine (L-Orn) as model system. For identification of an improved variant an in vitro microtiter plate (MTP) based assay (in 40% MeOH) was developed that relies on the quantification of conversion of L-Orn to D-Orn. However, of the two developed quantification procedures, one based on HPLC analysis the other using a coupled enzyme assay, neither qualified for high throughput screening with approximated rates of 500 and 3000 variants per day, respectively. In order to reduce the number of variants in this selection step an additional screening step that allowed exclusion of all non-functional variants from the original error-prone PCR library and automatic sorting of active variants into MTPs was performed. This was achieved by engineering an L-Ornithine auxotrophic strain with a chromosomally integrated fluorescent marker (sfGFP) that is amenable to sorting with the COPAS technology upon growth in alginate beads (with D-Orn supplied in the medium). It is worth noting that the applied screening strategy can be generically applied to other amino acid racemases.
________________ [1] M. Bechtold, S. Makart, J. of Biotechnology, 2006, 124, 146. [2] Schnell, B.; Faber, K.; Kroutil, W. Adv. Synth. Catal. 2003, 345, 653-666. [3] Berthod, A.; Yu, T.; Kullman, J. P.; Armstrong, D. W.; Gasparrini, F.; D'Acquarica, I.; Misti, D.; Carotti, A. J. Chromatogr. A 2000, 897, 113-129.
Scheme 1: AMDase variants catalyze the formation of both profen enantiomers by decarbox-ylation (left) and the recycling of the undesired enantiomer after kinetic resolution (right) Alternatively, AMDase variants can also be used for the recycling of the undesired enantiomer in the resolution of profens. The racemase-like catalytic machinery of AMDase mutant G74C conveys it a unique racemising activity, which makes AMDase G74C an interesting object for the mechanistic investigation of cofactor-independent racemases. By rational protein design, a variant with a 20fold shift towards the promiscuous racemisation was achieved, based on a reduced activity in the decarboxylation and a two-fold increase in racemisation activity [2]. The mutant showed an extended substrate scope, including a 30fold increased activity in the racemisation of ketoprofene. Both examples outline the usefulness of protein engineering for the production of pharmaceutical relevant compounds.
________________ [1] Y. Miyauchi, R. Kourist, D. Uemura and K. Miyamoto, submitted. [2] R. Kourist, Y. Miyauchi, D. Uemura and K. Miyamoto, Chem. Eur. J., 2011, 17, 557-563
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Development of high throughput screening methods for a directed evolution study of transketolase.
Adeline Ranoux a, Isabel C.W.E. Arendsa, Ulf Hanefelda a Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, The Netherlands. E-mail: a.c.m.ranoux@tudelft.nl Transketolase catalyses the asymmetric formation of C-C bonds. This is of great interest for chemical synthesis, especially for the synthesis of biologically active compounds. The natural substrates of this enzyme are phosphorylated polyols (Figure 1) but some examples of non-phosphorylated short chain substrates are described in the literature.[1-3]

A novel combination of the plasmid/host permits to increase the level of production of the recombinant human IFN- in E. coli.
Luiza Chojnacka-Puchta, Jolanta Kuthan-Stycze, Anna Wjtowicz-Krawiec, Agata Jagieo, Diana Mikiewicz, Grayna Pcienniczak, Andrzej Pucienniczak Institute of Biotechnology and Antibiotics, Staroscinska 5, 02-516 Warsaw, Poland e-mail: chojnackal@iba.waw.pl In modern biotechnology the most useful vectors are the so-called expression vectors, which facilitate efficient synthesis of proteins encoded by the genes introduced into the vector. Obtaining new vectors which may be used in commercial projects is nowadays extraordinarily difficult but particularly desirable goal. Most expression vectors for the highlevel production of proteins are patented as well as elements for their construction. Here we demonstrated validation of stable expression vector for expression of recombinant protein IFN-, based on the pIGAL1 plasmid (Accession No AY424310). The new vector comprise new, strong, constitutive pms promoter and synthetic transcriptional terminators. Also we compared the same IFN- sequence in vector pDB with deoP1P2 promoter and pT7RS vector with T7 promoter. The Gram-negative Escherichia coli bacteria (laboratory strains: DH5, NM522, BL21 DE3) as a host for highlevel expression of cloned gene were used. We show that efficient expression of human gene IFN- from these vectors in Escherichia coli cells requires adaptation to the optimal E. coli codon usage. We determined the stability of clones with regard to two parameters: level of recombinant protein expression and the frequency of ampicilin resistant clones in general population cultivation in the absence of ampicilin selection. Identification of expression level was analysed by SDS- PAGE (12%). Determination of the plasmid/ host combination may be applied for maximizing the recovery of therapeutically important protein IFN- expressed as inclusion bodies in E.coli.
Enzyme structure & mechanism, bioinformatics and modelling 301

Multiple Domain Old Yellow Enzyme
a
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Surface-hydrophobic residues of a xylanase : impacts on biochemical properties and interaction with lignin
Rmond Carolinea, Rakotoarivonina Harivonya, Agui Vroniquea, Renau-Ferrer Shania, Chabbert Brigittea, Gabrielle Zeder-Lutzb, Danile Altschuhb a UMR Fractionnement des Agroressources et Environnement, INRA-Universit de Reims Champagne-Ardenne, Reims, France b UMR 7242 CNRS-Universit de Strasbourg, ESBS Illkirch, France E-mail: caroline.remond@univ-reims.fr The development of a biorefinery based on the transformation of lignocelluloses for biofuel and molecules of interest production requires to fractionnate cellulose and hemicelluloses such xylans. Depending on plant species, xylans represent 20-40% of plant dry matter. The depolymerization of these polymers implies the hydrolytic action of endo--(1,4)-xylanases (EC 3.2.1.8). One important challenge to develop enzyme-based processes for fractionation of lignocellulosic plant cell walls is the use of robust, efficient and cheap biocatalysts. The xylanase Tx-Xyn11 from Thermobacillus xylanilyticus, which belongs to the family 11 of glycoside hydrolases, represents an interesting biocatalyst in the context of biorefinery of lignocelluloses. Indeed, Tx-Xyn11 is a thermostable enzyme and is active in a large pH range [1]. Our previous studies indicate that Tx-Xyl11 hydrolyze 50% and 20 % of xylans respectively from wheat bran and wheat straw [2-3]. Modest action on lignocellulosic biomass as wheat straw could be attributed to xylanase-lignin interactions that limit the progression of the enzyme into the cell wall matrix [3]. Negative role of non specific adsorption of Tx-Xyn11 on lignin has also been recently suggested by results indicating that hydrolytic efficiency of Tx-Xyn11 is affected by the presence of lignin in model systems mimicking plant cell walls [4]. In order to get new insights concerning the non specific interactions between lignin and Tx-Xyn11, direct mutagenesis was performed on hydrophobic residues forming sticky patches on the surface of Tx-Xyl11. Impact of the mutations on the biochemical properties of Tx-Xyn11 was investigated. The thermostability of some mutants drastically decreased compared to the native xylanase. Furthermore preliminary data concerning measurement of interactions between the mutants of Tx-xyn11 and some model lignins will be presented. This work is financially supported by the french ANR Bioenergy Program ANR 08 BIOE 004 and by the french cluster Industries des Agro-Ressources.
________________ [1] Debeire-Gosselin, M., et al., in Xylans and Xylanases, J. Visser, et al., Editors. 1992, Elsevier: Amsterdam. p. 463-466. [2] Benamrouche, S., et al. Journal of Cereal Science, 2002. 36(2): p. 253-260. [3] Zilliox, C. and P. Debeire. Enzyme and Microbial Technology, 1998. 22(1): p. 58-63. [4] Boukari, I., et al. Biomacromolecules, 2009. 10(9): p. 2489-2498.
Suzanne Litthauera, Esta van Heerdena, Frank Hollmannb, Dirk J Oppermana University of the Free State, Department of Biotechnology, Bloemfontein, South Africa; b Delft University of Technology, Delft, The Netherlands E-mail: litthauers@ufs.ac.za
Old Yellow Enzymes (OYEs) have drawn much attention recently due to the realization of their biocatalytic potential in the asymmetric reduction of various compounds [1]. OYEs have also been the focus in directed evolution studies to increase their substrate scope even further [2]. OYEs are typically characterized as monomers, dimers or tetramers consisting of identical subunits which all adopts a similar (,)8-barrel (TIM-barrel) fold. This single domain contains the substrate binding pocket above a flavin co-factor to which both the reductant [NAD(P)H] and oxidant binds in a bi-bi ping-pong catalytic mechanism. 2,4-Dienoyl-CoA reductase is an iron-sulphur flavoenzyme containing FMN, FAD and a 4Fe-4S cluster. This two dinucleotide binding domain flavoprotein binds the oxidant and reductant in separate active sites, catalyzing the reduction of double bonds during the -oxidation of unsaturated fatty acids [3]. The N-terminal domain of the 2,4-dienoylCoA reductase also adopts a TIM-barrel fold, however with an alternative substrate binding architecture and mechanism. Here we report a new member to the OYE family consisting of monomers predicted to contain two dinucleotide binding domains linked via a 4Fe-4S cluster, with the Nterminal (,)\-barrel having a typical OYE binding site architecture. The 63 kDa enzyme exhibits typical enoate reductase activity using NAD(P)H as reductant. An added advantage is the thermostability of the enzyme, allowing for rapid purification through selective precipitation of surrogate host proteins. We propose this novel OYE to be a good starting point for future directed evolution studies, as the NADPH and oxidants are bound in separate sites, allowing for more evolutionary flexibility in the oxidant binding site, without influencing NAD(P)H binding.
________________ [1] Toogood HS, Gardiner JM & Scrutton NS (2010) Biocatalytic Reductions and Chemical Versatility of the Old Yellow Enzyme Family of Flavoprotein Oxidoreductases. ChemCatChem 2, 892-914. [2] Bougioukou D-, Kille S, Taglieber A & Reetz M (2009) Directed Evolution of an Enantioselective Enoate-Reductase: Testing the Utility of Iterative Saturation Mutagenesis. Advanced Synthesis & Catalysis 351, 3287-3305. [3] Hubbard P a, Liang X, Schulz H & Kim J-JP (2003) The crystal structure and reaction mechanism of Escherichia coli 2,4-dienoyl-CoA reductase. The Journal of biological chemistry 278, 37553-60.
Figure 1. Example of natural substrates coupling, ribose and xylulose 5-phosphate. To expand the scope of transketolase by directed evolution, the development of efficient and sensitive screening methods is a key step.4, 5 Therefore, we developed new high throughput screening methods based on a GC analysis enabling the screening for activity and stereoselectivity of a wide range of non-phosphorylated polyol substrates (Figure 2).
R' R O H + O LiOOC OH
CO2
Transketolase Mg2+, TPP
O R OH OH R'
Figure 2. General scheme of the coupling of non-phosphorylated compounds Thus, we developed and adapted for high-throughput screenings a GC analysis after derivatization of non-phosphorylated polyol substrates. Indeed, this analysis brings four major advantages: high accuracy and sensitivity, the possibility of studying the stereoselectivity of the enzymatic reaction, and the possibility to be generalized to any substrate. Moreover, this GC screening has been shown to detect even low bioconversion, >1%, much lower than previously described4. An automatized protocol was implemented with success. It is currently used as assay for high throughput screenings of a mutant library of more than 1000 mutants.
_____________ [1] Sukumaran, J.; Hanefeld, U., Chem. Soc. Rev. 2005, 34, 530 - 542. [2] Wohlgemuth, R., J. Mol. Catal. B 2009, 61, (1-2), 23-29. [3] Hailes, H.; Dalby, P.; Lye, G.; Baganz, F.; Micheletti, M.; Szitab, N.; Ward, J., Curr. Org. Chem. 2010, 14, 1883-1893. [4] Smith, M..; Kaulmann, U.; Ward, J.; Hailes, H., Bioorg. Med. Chem. 2006, 14, 7062-7065. [5] Sevestre, A.; Hlaine, V.; Guyot, G.; Martin, C.; Hecquet, L., Tetrahedron Lett. 2003, 44, (4), 827830.
299
Enzimatic activity oxidation-reduction from human skin fungi.
Dvila Zampieria, Caroline Gonalvesa, Carla Portoa,b, Lara D. Settec, Rafaella C. Bonugli-Santosc Anita J. Marsaiolia a Instituto de Qumica, Universidade Estadual de Campinas, CP 6154,13083-970 Campinas, SP, Brazil. b Natura Inovao e Tecnologia de Produtos, Rod. Anhanguera Km 30.5,07750-000, Cajamar, SP, Brazil. c Diviso de Recursos Microbianos, CPQBA, Universidade Estadual de Campinas, CP 6171, 13083-970 Campinas, SP, Brazil. E-mail: anita@iqm.unicamp.br Chemical biotransformation by fungi isolated from human skin has an extraordinary appeal to fragrance and perfumery industries. Furthermore, it can reveal new biocatalysts with high specificity and selectivity for several important oxidation-reduction reactions. Thus, this work proposed evaluation of the enzymatic reduction and oxidation of some ketones structurally diverse by skin fungi and modulation of these activities. Fifteen fungi strains isolated from human skin were identified by molecular method (18S rDNA) as belonging to 11 genera: Alternaria, Aureobasidium, Cladosporium, Epicoccum, Fusarium, Massarina, Penicillium, Phoma, Rhodotorulla, Scolecobasidium, and Simplicillium. Enzymatic activities assays were performed in a small scale biocatalysis (resting cells) using nineteen ketones (Figure 1), in order to analyse the respective biotransformations by oxidation-reduction reactions. Best strains expressing oxidoreductase activites were selected for enzyme modulation studies by optimizing substrate/product concentration, cell concentration, aeration and cosubstrate addition [2].
300
Biochemical characterization of the novel thermostable protease from Coprothermobacter proteolyticus
a
303
Exploring the conformational states and rearrangements of yarrowia lipolytica lipase
Florence Bordesa,b,c, Sophie Barbea,b,c, Guillaume Narsa,b,c, Lionel Moureyd, Isabelle Andra,b,c, Alain Martya,b,c, and Samuel Traniere a Universit de Toulouse; INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France ; bINRA, UMR792, Ingnierie des Systmes Biologiques et des Procds, F-31400 Toulouse, France ; cCNRS, UMR5504, F-31400 Toulouse, France;dIPBS,CNRS, Toulouse, France. Universit de Toulouse, eUniversit Paul Sabatier, IPBS, F-31077 Toulouse, France E-mail: florence.bordes@insa-toulouse.fr Yarrowia lipolytica Lip2 lipase is a very promising lipase from a biotechnological point of view. It presents interesting activities and selectivities for a wide range of substrates[1]. Our work is focused on the tailoring of this lipase by molecular engineering to improve its selectivity toward a class of pharmaceutical intermediates[2], the 2-bromo-arylacetic esters. The knowledge of the structural determinants involved in selectivity is clearly essential to design enzymes for specific applications. Here, we report the 1.7 resolution x-ray structure of this lipase in its closed form, with the so-called lid region covering the enzyme active site. The Lip2 structure is highly homologous to fungal lipases. However, it also presents some unique structural features. As the catalytic activity of lipases is strongly dependent on this structural rearrangement, molecular dynamics studies were performed to obtain a better understanding of the lid opening mechanism under the influence of solvent or substrate binding. Results suggest a two-step opening mechanism mediated in a first step, by water/octane interface (and leading to a semi-open conformation) and in a second step, by substrate binding (leading to a complete opening of the lid). This study[3] which combined crystallographic and molecular dynamics studies, allowed us to obtain a detailed description of conformational rearrangements at the atomic level and provide key information to guide the design of lipase variants with improved properties for industrial needs.
304
The evolution of triazine hydrolases: intramolecular epistasis and the emergance of new enzyme function
Sajid Noora,b, Colin Jacksonb, Robyn Russella, John Oakeshotta, Colin Scotta a CSIRO Ecosystem Sciences GPO Box 1700, Canberra, ACT 2601, Australia; bResearch School of Chemistry, Australian National University, Acton, ACT, 0200, Australia E-mail: colin.scott@csiro.au We have developed a suite of enzymes for the detxoification of pesticides, herbicides and fungicides in environmental flows [1,2]. The enzymes are sourced from a wide variety of organisms and usually require improvment before they are application ready. Here, the in vitro improvement of the atrazine dechlorinase (AtzA) [3] is used to exemplify this (fig 1A). Investigation of the evolutionary relationship between atrazine dechlorinase and the 98% identical melamine deaminase [4](fig. 1B) have allowed us to reconstruct a plausable evolutionary trajectory beween these two naturally occuring enzymes, revealing the direction of evolution and the role that intramolecular epistasis plays in constraining this trajectory within the fitness landscape.
Ana Toplaka, Fabrizia Fusettira, Dick B. Janssena Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands E-mail:a.toplak@rug.nl Subtilases are subtilisin-like proteins found in various sources like Archaea, Bacteria, Fungi and higher Eukaryotes [1]. Subtilases in extremophiles might add an additional value to subtilisins particularly in thermal stability as well as in potentially new/wide substrate specificities. A novel thermostable and organic solvent-resistant protease from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and actively expressed in Escherichia coli. Amino acid sequence comparison and phylogenetic analysis classifies the enzyme in the sub-group of thermitase. Hereby, we report an enzyme activation and purification in a single-heating step. Cop is a proteolytic enzyme with broad pH range and optimum at 90 C. Kinetic parameters were determined for several substrates. The S1 site specificity was determined by mass spectroscopy. Cop protease hydrolysis of oxidized insulin B chain was performed, followed by MALDI-MS [2]. Several substrate to enzyme ratio were tested, as well as the effect of temperature and cosolvent on primary cleavage site. The enzyme has preference for hydrophobic residues in S1 binding pocket.
________________ [1]R. J. Siezen, J. A. M. Leunissen, Protein Science, 1997, 6:501-523. [2]Palmieri et al., Enzyme and Microbial Technology, 2005, 37:745-749
Figure 1. Structure of ketones selected for testing of enzymatic activities. Preliminary results indicate that skin fungi have a higher reduction potential however with better oxygen availability these fungi responded increasing oxidation of 4-methylcyclohexanone to 98 % conversion and over 96% ee (R). This study provides novel and relevant information on human skin fungi enzymatic activity and which these can be modulated by the availability of oxygen, cell and substrate concentration.
________________ [1] Porto, C.; Aquino, P. A., Crucello, A. and, Marsaioli, A.J. Detection of Enzymatic Activity of the Skin Microbiota. In: Biotrans 2009, Berne, Switzerland. [2] Law, H.E.M.; et al. Chem. Eng. Sci., 2006, 61, 6646-6652.
Figure 1. A) Atrazine dechlorinase ativity from atrazine dechlorinase and a semi-rationally designed varient wit reduced KM; the altered amino acid residues are shown in green (wild-type) and gold (variant). B) Active site amino acids that differ in atrazine dechlorinase and the 98% identical melamine deaminse (shown in pink). Figure 1. : Conformational rearrangements of Lip2 lipase inferred by molecular dynamics simulations starting from the x-ray structure.
________________ [1] Fickers et al., Biotechnology Advances, 2011, In press [2] Cambon et al, Biotechnology and Bioengineering, 2010, 106 (6), 852 [3] Bordes at al., Biophysical journal, 2010, 99, 2225 ________________ [1] Russell et al. (2011) Evolut. Appl. 4: 225-248. [2] Scott et al. (2008) Ind. J. Microbiol. 48: 65-79. [3] Scott et al. (2009) Appl. Environ. Microbiol. 75: 2184-2191. [4] Seffernick et al. (2001) J. Bacteriol. 183: 2405-2410.
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Construction of a 3D model of the lipase/acyltransferase CpLIP2 from Candida parapsilosis
Herv Nozac'h, Maeva Subileau, Vronique Perrier and Eric Dubreucq Montpellier SupAgro, UMR IATE, 2 Place Viala, F-34060 Montpellier cedex, France Maeva.subileau@supagro.inra.fr CpLIP2 from Candida parapsilosis and CaLA from Candida antarctica exhibit 31% of identity and a low sequence similarity to other members of the / hydrolases. A model structure of the lipase/acyltransferase CpLIP2 was thus constructed by comparative modelling to the available 3D structure of CaLA. It was tested by site-directed mutagenesis, limited proteolysis and confronted to molecular docking. Enzyme variants were created for amino acid residues predicted to play key roles for catalytic activity and substrate selectivity. The results from activity measurements of the overproduced and purified mutant enzymes indicated a structure where the active site consists of the classical Ser/Asp/His catalytic triad. Other mutation consequences were well correlated to the predicted position of the corresponding residues: substitutions of the residues located at the outer part of the protein had limited consequences on the functionality of the protein, while modifications of residues predicted to be close to the active site or important for the structure stability resulted in important to complete losses of specific activity. Limited proteolysis resulted in the identification of restrictions sites located in loops at the periphery of the protein 3D model. The model was strengthened by molecular docking of a triolein molecule in the active site. This 3D model was thus well confirmed by experimental data and is currently used for targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability or higher enantioselectivity.

Molecular characterization of a thermostable L-Fucose isomerase from Dictyoglomus turgidum
Seung-Hye Hong, Deok-Kun Oh Department of Bioscience and Biotechnology, Konkuk University, Hwayang-Dong, Gwangiin-Gu, Seoul 143-701, Republic of Korea E-mail: deokkun@konkuk.ac.kr A recombinant L-fucose isomerase from Dictyoglomus turgidum was purified by heat treatment and His-trap affinity chromatography with a specific activity of 33 U mg-1. The native enzyme was a 68-kDa hexamer with a molecular mass of 470 kDa. The maximum activity for L-fucose isomerization was observed at pH 7.0 and 80C in the presence of 1 mM Mn2+. The enzyme activity with aldose substrates was highest for L-fucose, with a kcat of 15,487 min-1 and a Km of 72 mM, followed by D-arabinose, D-altrose, and L-galactose. The twelve putative active-site residues within 5 of the substrate L-fucose in the homology model based on the previously solved crystal structure were individually replaced with alanine. The significantly decreased kcat of the alanine-substituted mutant enzymes at E349, D373, and H539 as well as homology modeling suggested that these three residues are not only metal-binding residues but also catalytic residues. The significantly decreased Km of the alanine-substituted mutant enzymes at Q314, E349, S405, and N538 suggested that these four residues are substrate-binding residues. Alanine substitutions of Y451, W510, and F452 resulted to decrease of kcat, but no change of Km. Although the three residues may not interact directly with substrate or metal, the rings, which may provide hydrophobic environment and then stabilize the formation of cyclic sugar, was essential for enzyme activity.
_________________________ [1] Boulter, J. R. & Gielow, W. O. (1973) Properties of D-arabinose isomerase purified from two strains of Escherichia coli. J. Bacteriol. 113, 687-696 [2] Joachim E. S. & Georg E. S. J. Structure and mechanism of L-fucose isomerase from Escherichia coli. Mol. Biol. (1997) 273, 256-268

Bioinformatic analysis and molecular modeling to predict mutations widening substrate specificity of D-aminopeptidase from Ochrobactrum anthropi
Ilyas Khaliullina,b, Kosuke Tokunanc, Mariko Himic, Dmitry Suplatova,d, Daria Shalaevaa, Yasuhisa Asanoc, Vytas vedasa,d a Faculty of Bioengineering and Bioinformatics, bDepartment of Chemistry, dBelozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Lenin Hills 1/73, Moscow, 119991, Russia; cBiotechnology Research Center, Toyama Prefectural University, Imizu, Toyama, 939-0398, Japan E-mail: ilyas@belozersky.msu.ru D-amino acids and their derivatives are important building blocks for synthesis of physiologically active compounds. D-aminopeptidase from Ochrobactrum anthropi catalyzes hydrolysis of D-amino acid derivatives with high enantioselecivity. However substrate specificity of the D-aminopeptidase is limited to D-alanine derivatives [1]. Yet, it was shown that this enzyme can catalyze acyl transfer to 3-aminopentane using D-alanine amide or D-alanine methyl ester as acyl donors and possess high stereoselectively to acyl donor [2]. In this work we performed bioinformatic analysis of the family of penicillin-binding proteins which includes D-aminopeptidases, D-amino acid amidases, DD-carboxypeptidases and -lactamases classes A and C. Certain positions in the substrate binding pocket of the D-aminopeptidase were identified as responsible for limited substrate specificity of the enzyme. Based on results of bioinformatic analysis a library of 2100 virtual mutants was generated by molecular modeling using native crystallographic structure 1EI5 of Daminopeptidase from Ochrobactrum anthropi [3] and subjected to in silico screening. Substrate specificity of the generated mutants was studied by molecular docking with 38 potential substrates (D- and L-amino acid amides and other amino acid derivatives). Resulting 79800 enzyme-substrate complexes were analyzed to retrieve the mutants with enhanced substrate specificity. Finally, mutations of three active site residues of D-aminopeptidase from Ochrobactrum anthropi were suggested to extend substrate specificity towards bulky amino acid derivatives while preserving D-stereospecificity. Mutants possessing the most promising catalytic properties were recommended for experimental investigation, prepared, expressed and purified. Subsequent experimental studies demonstrated extended substrate profile of selected mutants. As a result bioinformatic analysis of the family of penicillin-binding proteins allowed to design and produce mutants of Daminopeptidase from Ochrobactrum anthropi with extended substrate specificity. This work was supported by the Russian Ministry for Science and Education (contract 02.740.11.0866).
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Identifying rate limitations in the enzymatic hydrolysis of cellulose: insights from experimental and computational studies
Prabuddha Bansal1, Bryan J. Vowell1, Mlanie Hall1,3, Yuzhi Kang1,4, Matthew J. Realff1, Jay H. Lee1,5, Andreas S. Bommarius1,2 1 School of Chemical and Biomolecular Engineering, 2School of Chemistry and Biochemistry, 4 Paper Science and Engineering program; Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology 315 Ferst Drive, Atlanta, GA 30332-0363, USA 3 Current Address: Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, 8010 Graz, Austria 5 Current Address: Dept of Chemical & Biomolecular Engineering, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-roYuseong-gu, Daejeon, Korea 305-701 E-mail: andreas.bommarius@chbe.gatech.edu Enzymatic hydrolysis of cellulose, a key step in the production of biofuel from lignocelluloses, is currently limited by long residence times for breaking down cellulose into glucose, especially at high cellulose conversions. The complex nature of substrate-enzyme interactions in this heterogeneous bio-catalytic process has made it difficult to determine the exact causes for rate retardation [1-2]. In this work, we present: a) the use of results from experiments as well as simulations to screen the different hypotheses in literature, and b) results from model-guided experiments that quantify the contributions of various rate limiting factors. Over-parameterization of models, which happens when only time-conversion data is taken into consideration, is avoided by fitting our model to data from adsorption and re-suspension experiments. Specifically, we investigate the behavior of the determined model parameters over the conversion profile. When product inhibition is alleviated with excess beta-glucosidase, we find completely amorphous cellulose to fit a first-order reaction curve, implying limitations only due to substrate depletion. Crystalline cellulose, however, did not fit a first-order curve and the apparent rate order varied with time. We have found that the hypotheses of crystallinity increase with conversion, and enzymes getting stuck on the cellulose surface when considered a first-order reaction, are not plausible reasons for decelerating rates [3-4]. Modeling studies will be shown at two levels 1. Micro-kinetic scale where events such as finding a reactive site, getting stuck at an obstacle, and reacting are investigated, and 2. Macroscopic scale where changes in substrate and enzyme properties as a function of conversion are incorporated.
________________ [1] Asano, Y., Nakazawa, A, Kato, Y., Kondo, K. (1989) Properties of a novel D-stereospecific aminopeptidase from Ochrobactrum anthropi. Journal of Biological Chemistry, 264(24), 14233-14239. [2] Kato, Y., Asano, Y., Nakazawa, A., Kondo, K. (1989) First stereoselective synthesis of D-amino acid N-alkyl amide catalyzed by D-aminopeptidase. Tetrahedron, 45(18), 5743-5754. [3] Bompard-Gilles, C., Remaut, H., Villeret, V., Prang, T., Fanuel, L., Delmarcelle, M. et al. (2000) Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the penicillinrecognizing enzyme family. Structure, 8(9), 971-980.
________________ [1] A.S. Bommarius, A. Katona, S.E. Cheben, A.S. Patel, A.J. Ragauskas, K. Knudson, Y. Pu,Cellulase kinetics as a function of cellulose pretreatment, Metab. Eng. 2008, 10, 370-81 [2] P. Bansal, M. Hall, M.J. Realff, J.H. Lee, A.S. Bommarius, Modeling Cellulase Kinetics On Lignocellulosic Substrates, Biotechnology Advances 2009, 27, 833-848 [3] M. Hall, P. Bansal, J.H. Lee, M.J. Realff, and A.S. Bommarius, Cellulose Crystallinity: a Key Predictor of Enzymatic Hydrolysis Rate, FEBS Journal 2010, 277, 1571-1582 [4] P. Bansal, M. Hall, M.J. Realff, J.H. Lee, and A.S. Bommarius, Multivariate statistical analysis of X-ray data from cellulose: A new method to determine degree of crystallinity and predict hydrolysis rates, Bioresources Technology 2010, 101, 4461-4471 [5] M. Hall, P. Bansal, J.H. Lee, M.J. Realff, A.S. Bommarius, Biological pretreatment of cellulose: Enhancing enzymatic hydrolysis rate using cellulose-binding domains from cellulases, Biores. Technol., 2011, 102, 2910-15
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Improved expression vector for Ralstonia eutropha H16
Petra Kfingera, Zalina Magomedovaa, Daniel Schwendenweina, Steffen Grubera, Helmut Schwaba a Institute of Molecular Biotechnology, Graz University of Technology E-mail: petra.koefinger@tugraz.at Ralstonia eutropha is a Gram-negative, strictly respiratory facultative chemolithoautotrophic bacterium which can use H2 and CO2 as sole sources of energy and carbon in the absence of organic substrates. It has attracted great interest for biotechnology for its ability to degrade a large list of chloroaromatic compounds and chemically related pollutants. Moreover the production of biodegradable polymer polyhydroxyalkanoates on an industrial scale has already been applied [1]. R. eutropha serves as a model organism for the mechanisms involved in the control of autotrophic carbon dioxide fixation, hydrogen oxidation and denitrification. In our project we are going to use Ralstonia eutropha as an organism for establishing specialized cell factories by genetic engineering. Therefore we constructed the new inducible vector pKR-Tac for gene expression in Ralstonia eutropha. This new plasmid has several advantages: (i) it is smaller in size; 5,7 kb; (ii) it carries the tac promoter for inducible expression; and (iii) it can be transformed via electroporation.
_______________ [1] Steinbchel, A., Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example; Macromol Bioscience 1, 2001: 124
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KeySIDE: iterative key-residues interrogation of Yersinia mollaretii phytase with thermostability increasing substitutions identified in directed evolution
Amol V. Shivangea,b, Danilo Roccatanob, and Ulrich Schwaneberga,b Lehrstuhl fr Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; bSchool of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany E-mail: a.shivange@biotec.rwth-aachen.de
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Structural studies of aminotransferase enzymes of commercial interest
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Radical resculturing of an enzyme active site for enantioselective and fast hydrolysis of ibuprofen esters
Ylva Wikmarka, Anders G. Sandstrma, Karin Engstrma, Jonas Nyhlna and Jan-E. Bckvalla a Department of Organic Chemistry, Stockholm University, Arrhenius laboratory, SE-106 91, Stockholm, Sweden E-mail:ylva@organ.su.se By introducing nine mutations simultaneously into the active site of CalA, a variant was created that is highly enantioselective and active towards hydrolysis of 1, a substrate that we never succeeded to access by previously used directed evolution techniques. The variant, with an E-value of 100, was found when producing an extremely condensed library, allowing only for a reduced set of amino acids at each site. In this quintuple mutant, all mutations contribute to the increased activity and enantioselectivity, as proved from investigating all possible double mutants from the five residues.
Christopher Sayera, Martin Boomerb , Misha Isupova, John Wardb , Jennifer Littlechilda Exeter Biocatalysis Centre, Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, EX4 4QD,UK bDepartment of Structural and Molecular Biology, University of London, Darwin Building, Gower Street, London WC1E 6BT, UK E-mail: Presenting author J.A.Littlechild@exeter.ac.uk A programme of structural studies is being carried out on series of transaminase enzymes of potential commercial interest for the production of chiral amines. It is hoped that this information will provide an insight into the stability and substrate specificity of these important pyridoxal phosphate dependent enzymes. The -aminotransferase enzymes from Chromobacterium violaceum and Pseudomonas aeruginosa in addition to a serine aminotransferase from Sulfolobus solfataricus have been cloned and over-expressed to high levels in Escherichia coli. A studies have been carried out regarding the stability and substrate specificity of these enzymes [1]. This has been followed by the crystallisation and successful structural determination of each of these enzymes including the enzymes in a number of complexes which have given insight into their mechanism of action and specificity. The structure of the recombinant Chromobacterium enzyme has been determined in holoenzyme, apoenzyme, gabaculine bound enzyme and pyruvate bound enzyme forms. These structures represent detailed X-ray structural analysis of an aminotransferase enzyme which accepts amine substrates containing no carboxyl group. A comparison of these structures reveals movement of the loop regions around the active site of the enzyme. A comparison of the new data with the previously published homology based model of the Vibrio fluvialis -aminotransferase has revealed some significant differences. Site directed mutants of the active site residues of the C. violaceum AT have also been constructed. Residues phenylalanine 22 and tryptophan 60 were mutated to glycine residues and initial characterisation of these mutant enzymes will be discussed [2, 3]. The structure of the recombinant Pseudomonas enzyme has been determined in the holoenzyme form. A comparison to the Chromobacterium enzyme reveals some interesting differences in the active site between the two enzymes which define their differences in substrate specificity [3]. The structure of the recombinant Sulfolobus aminotransferase enzyme has been determined in the holoenzyme, gabaculine bound enzyme and phenylpyruvate bound enzyme forms. These results represent the first report of a serine:pyruvate aminotransferase structure. A comparison to the related alanine:glyoxylate aminotransferase enzymes has explained their subtle differences in substrate specificity [4].
___________________ [1] Kaulmann, U., Smithies, K., Smith, M. E. B., Hailes, H. C. and Ward, J. M. (2007) Enzyme Microb. Tech. 41, 628-637. [2] Sayer, C., Isupov, M. N. and Littlechild, J. A. (2007) Acta Crystallog. sect. F 64, 696-699 [3] Sayer, C PhD thesis University of Exeter, 2009. [4] Sayer, C. Bommer,M., Isupov,M., Ward,J. and Littlechild. J. (2011) J.Mol.Biol. submitted.
Phytase improves as a feed supplement the nutritional quality of phytate rich diets by hydrolyzing indigestible phytate. Bacterial phytases have attracted industrial interest due to their high catalytic efficiency. Prerequisites for phytase applications in industry are high activity and sufficient thermostability necessary for feed pelleting. Only a single study has explored the structure-function relationships of phytase activity [1]. Here we report the cloning, characterization, directed evolution and structure-function analysis of the Yersinia mollaretii phytase (Ymphytase). A screening system was developed comprises a prescreening step (filter paper based or in 384 well microtiter plates) and subsequent screening in 96-well microtiter plates. Directed Ymphytase evolution after mutant library generation (SeSaM method) and two step screening (in total ~8400 clones) yielded a first thermally improved variant (SM2P3E4: D52N, T77K, K139E, G187S, V298M) with ~20% improved thermostability (58C for 20 min) with a Figure 1: Structural superimposition of slight loss of specific activity (993 U/mg) compare 10 configurations obtained by projecting to wild-type (1073 U/mg) which is ~10 times higher the MD simulation trajectories onto the first five principle component of wild-type than widely used fungal phytases. A KeySIDE (An iterative Key-residue interrogation (a) or M6 variant (b), configurations are of the wild-type with Substitutions Identified in Di- separated by 1 ns. Cartoon representation rected Evolution) approach was employed for Ym- shows a most abundant conformation obphytase thermostabilization. The key-beneficial four tained by cluster analysis. Flexible loops shown positions were identified in two directed phytase in Ymphytase aredepictedin red color and active site loop is by orange color. evolution experiments and combined iteratively by site directed mutagenesis to elucidate the contribution of each position. Out of the five positions in SM2P3E4 variant two turned out to contribute to an improved thermal resistance. The best combination (M6) resulted in a ~54% improvement in thermostability (58C for 20 min) and 3C increase in melting temperature without a loss of specific activity and preserving its tetrameric structure. Molecular dynamics (MD) simulations revealed a decreased overall Ymphytase flexibility in the thermostable variant M6 with a slight increase in the active site loop flexibility (Figure 1). Reduced flexibility was found in the loops located next to helices (B, F and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in loops might on a molecular level be caused by intra-protein interactions which strengthen hydrogen bonds (e.g. G187S, K289E/ K289Q) and a salt-bridge (T77K).
________________ [1] Shivange AV, Schwaneberg U, Roccatano D. Biopolymers 2010,93(11):994-1002.
Figure 1.
October 2-6, 2011
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Characterization of Lipase Pf200160 from Pyrococcus furiosus expressed in Pichia pastoris and Escherichia coli
Marcelo Victor Holanda Mouraa, Gabriela Coelho Brdaa, Bianca Cruz Nevesa, Fernando Araripe Gonalves Torresb, Rodrigo Volcan Almeidaa a UFRJ Insto de Qumica Laboratrio de Microbiologia Molecular e de Protenas Rio de Janeiro - RJ, BRAZIL; bUnB- Instituto de Biologia Grupo de Gentica Molecular e Biotecnologia de Leveduras - Braslia DF BRAZIL E-mail: volcan@iq.ufrj.br The importance of biocatalysts in the global scenario, particularlly enzymes, is increasing. They present some advantages over traditional catalysts, like the capacity to act effectively in less extreme conditions of temperature, pressure and pH, often having characteristics of regiospecificity and stereospecificity. On the other hand, two major limitations for large-scale use of enzymes are the lack of stability and the cost of those catalysts. The search for more stable and cost-effective biocatalysts has led to the study of enzymes from extremophile organisms, naturally able to withstand extreme conditions. The gene pf200160, which encodes for the lipase Pf200160 from the Archaea Pyrococcus furiosus was previously cloned and expressed in Escherichia coli, producing an enzyme with optimal temperature of 60C. It also retained 90% of its activity when incubated for 2 hours at 75C[1]. The enzyme was then purified and characterized in the presence of triton X-100. The optimal temperature was 80C and the enzyme retained 95% of activity after being incubated for 6 hours[2]. In the present study, the enzyme was cloned and expressed in the methylotrophic yeast Pichia pastoris. This study aimed to compare the lipasic extracts produced using both microorganisms as heterologous hosts, P. pastoris and E. coli, in terms of optimal temperature and pH, activity towards p-nitrophenyl-esters and thermostability. Optimal temperature and pH were determined using factorial design of experiments. The extract produced by E. coli showed an optimal temperature of 68 C and optimal pH of 6.8, while the extract produced by P. pastoris presented higher activity in values of pH >8 and in temperatures higher than 70C. The lipase expressed by P. pastoris was more stable than the enzyme expressed by E. coli after being incubated at 60C, 70C and 80C for 6 hours. The extract produced by Pichia pastoris presented higher activity towards low- length chain ester substrates (C2), while the E. coli extract presented higher activity towards medium-chain length ester substrates (C8). Though more studies remain necessary, the presence of post-translational modifications may be a possible explanation for the differences between the enzyme expressed in P. pastoris and E. coli.
________________ [1] ALMEIDA et al., (2006). Cloning, expression, partial characterization and structural modeling of a novel esterase from Pyrococcus furiosus. Enzyme and Microbial Technology, 39(5), 1128-1136 [2] ALQURES et al., (in press). Characterization of the recombinant thermostable lipase (Pf2001) from 1 Pyrococcus furiosus: effects of thioredoxin fusion tag and Triton X-100 . Enzyme Research.

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1
Alexander Seifert and Jrgen Pleiss Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany E-mail: alexander.seifert@itb.uni-stuttgart.de Abstract Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene limonene. It has a high potential as anti-tumor substance, but is of limited availability. In principle cytochrome P450 monooxygenases such as self-sufficient CYP102A1 are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild type enzyme converts (4R)-limonene to four different oxidation products, however, terminal hydroxylation at allylic C7 is not observed[1]. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed but chemically less reactive primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contains variants with improved or strongly shifted regio-, stereo- and chemoselectivity as compared to wild type[1]. From this library the variant with the highest perillyl alcohol ratio was fine-tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)-limonene to perillyl alcohol with a selectivity of 97%[2]. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for an efficient screening for enzyme selectivity.
___________________ [1] Seifert, A., et al., Rational design of a minimal and highly enriched CYP102A1 mutant library with improved regio-, stereo- and chemoselectivity. Chembiochem, 2009. 10(5): p. 853-61. [2] Seifert, A., et al., Efficient Route to Selective Biooxidation Catalysts: Iterative Approach Comprising Modeling, Diversification and Screening Based on CYP102A1. 2011: DOI: 10.1002/cbic.201100067.

Crystal structure of Est112 isolated from Korean intertidal at sediment metagenome
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MAP 2.03D sever: a structure based substitution spectra analyses of mutagenesis methods
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Koushik Guchhaita,b, Tae-Kwang Oha, Myung Hee Kima,b Division of Biosystems Research, Korea Research Institute of Bioscience and Biotechnology,Daejeon 305-806, Korea; bBiosystems and Bioengineering Program, University of Science and Technology, Daejeon 305-333, Korea. E-mail: mhk8n@kribb.re.kr A novel esterase with desirable biochemical properties is potentially useful for industrial processes. The increasing demand for novel biocatalysts stimulates exploration of different resources. Metagenomics, a culture independent approach, represents a sheer unlimited resource for discovery of novel biocatalysts from uncultured microorganisms. Such an attempt has been made by generating a metagenomic library from Korean intertidal flat sediment. A gene encoding a 339-residue long esterase (Est112) has been isolated from the metagenomic library. The C-terminal His-tagged Est112 was purified and crystallized under conditions of 1.5 M ammonium phosphate and 0.1 M Tris-HCl (pH 7.5). The crystal diffracted to a maximum resolution of 1.8 on beamline 4A at PAL. The native crystal belongs to the space group P43212. Repeated experiments aimed at the preparation of single SeMet-substituted crystals of the Est112 protein have failed. Accordingly, tag-free SeMet-substituted Est112 protein was purified and crystallized in the presence of 1.7 M ammonium phosphate and 0.1 M bicine (pH 7.0). SAD data were collected at 2.8 resolution on the same beamline. The SeMet-crystal belongs to the space group I4122. The structure was determined by utilizing the anomalous signals from Se atoms using program SOLVE. On the other hand, the structure of the C-terminal His-tagged Est112 was solved by molecular replacement using the partially refined model of the SeMet-Est112. Structure refinement is in progress. ________________ [1] Lee MH, Hong KS, Malhotra S, Park JH, Hwang EC, Choi HK, Kim YS, Tao W, Lee SW:A new esterase EstD2 isolated from plant rhizosphere soil metagenome. Appl Microbiol Biotechnol. 88(5):1125-34, 2010. [2] Kim MH, Kang BS, Kim S, Kim KJ, Lee CH, Oh BC, Park SC, Oh TK: The crystal structure of the estA protein, a virulence factor from Streptococcus pneumoniae. Proteins. 70(2):578-83,2008.
Rajni Vermaa,b, Ulrich Schwanebergb, Danilo Roccatanoa School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany; bLehrstuhl fr Biotechnologie, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany. E-mail: ra.verma@jacobs-university.de Mutagenesis Assistant Program (MAP) server [1] analyses the consequences of mutational bias on amino acid substitution patterns in proteins [2]. The server performs statistical analysis on nucleotide sequence for 19 commonly used random mutagenesis methods [3]. MAP2.03D server is the new improved version of our currently running MAP server. It extends the sequence based MAP analysis by correlating it with global and local structural properties of the target protein, which generally influences the acceptance of amino acid substitution. The structural properties take in account are local structural component (e.g. hydrogen bonds. hydrophobic contacts), which contribute defining protein secondary structure, residue flexibility and solvent accessibility. The new server provides the results of the analysis in both tabular format and mapping the information on the 3D structure of the target protein. Target based query system is implemented in this version which facilitates to fetch the statistical analysis of amino acid substitution probability with structural information of the specified region in the protein. It helps to explore the random mutagenesis methods in a structure guided and property specific manner by focusing on the effect of mutational spectra on a set of amino acid residues responsible for the stability and activity of the protein. In addition, the new server allows to extend the analysis to user defined mutagenesis methods and to change the default parameters to customize the structural analysis. The MAP2.03D server is a very useful tool for the protein engineers to help designing directed enzyme evolution campaigns by the in silico benchmarking of different random mutagenesis method. In this poster, examples of analysis of different proteins are reported. The beta version of the web-server is accessible with the link: http://map.jacobs-university.de/map3d.html.
________________ [1] http://map.jacobs-university.de/MAP.html. [2] Wong T.S., Roccatano D., Zacharias M. and Schwaneberg U. 2006. A statistical analysis of random mutagenesis methods used for directed protein evolution. J. Mol. Biol. 355: 858-871. [3]Wong T.S., Zhurina D. and Schwaneberg U. 2006. The diversity challenge in directed protein evolution. Comb. Chem. High Throughput Screen. 9: 271-288.
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The molecular basis of substrate specificity: modeling hydrolases at water-substrate interfaces
Sven Bensona, Christian Grubera, Paolo Tessaroa, Axel Lehmanna, Juergen Pleissa a Institute of Technical Biochemistry, Allmandring 31, University of Stuttgart, Germany E-mail: sven.benson@itb.uni-stuttgart.de The spatial proximity of an enzyme to its substrate is a necessary requirement for any catalytic reaction. We seek to identify distinctive amino acid residues on enzyme surfaces and explore their influence on the substrate recognition process. The focus hereby lies on hydrolytic enzymes and their water-insoluble substrates, which form interfaces under physiological conditions. For different hydrolases, the catalytic residues are identical (catalytic triad) and the morphology of the active site is similar, yet distinctive substrate specificity can be observed, which raises the principle question whether interfacial binding affinity directly contributes to substrate specificity. In this context, the interaction of Candida antarctica lipase B with a tributyrin interface and of Penicillium funiculosum PHB (polyhydroxybutyrate) depolymerase with a PHB interface were modeled by molecular dynamics simulation. It was shown that these enzymes bind in a definite orientation to their respective interfaces, with the crevice to the active site facing towards the substrate molecules. Energy of binding for both hydrolase/interface systems was analyzed by steered molecular dynamics to identify residues relevant for binding. Mutagenesis of these residues on both hydrolases serves to distinguish the differences in binding characteristics and to establish the basis of transferring binding specificity from one enzyme to the other.
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Bioinformatic analysis of /-hydrolase fold enzymes reveals subfamilyspecific positions responsible for discrimination of amidase and lipase activities
Vytas vedasa,b, Dmitry Suplatova,b, Werner Besenmatterc, Daria Shalaevaa, Allan Svendsenc a Faculty of Bioengineering and Bioinformatics and bBelozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Vorobjev hills 1-73, Moscow 119991, Russia, cNovozymes A/S, Krogshoejvej 36, 2880 Bagsvaerd Denmark E-mail: vytas@belozersky.msu.ru Superfamily of /-hydrolases is one of the largest groups of structurally related enzymes with diverse catalytic functions [1]. Bioinformatic analysis was used to understand why lipases do not hydrolyze amides despite their similarity to peptidases in organization of the active site and the reaction mechanism. Multiple alignment of Candida antarctica lipase B with lipases, carboxypeptidases, dipeptidyl peptidases IV and prolyl oligopeptidase with /-hydrolase fold was performed using sequence and structural information. Comparative analysis revealed a major structural similarity between lipases and peptidases in active site regions with the most significant fit of the catalytic triad residues Ser-His-Asp [2]. Bioinformatic analysis revealed subfamily-specific positions (SSPs) that define functional variability between enzymes with different properties - that is residues with a tendency to be conserved within groups of lipases and peptidases, but different between them. The most statistically significant subfamily-specific positions were selected as templates for mutagenesis to generate in silico library of 12000 Candida antarctica lipase B variants introducing the amino acid types of homologous peptidases, e.g. from carboxypeptidases, into Candida antarctica lipase B. Molecular docking and molecular dynamics were used to evaluate the efficiency of mutated Candida antarctica lipase B variants in binding and catalytic conversion of amide substrates. Structural filtration was implemented as a post-docking tool to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of near-to-attack conformation. Three sets of filters were used to evaluate: i) substrate interaction with catalytic serine (distance, angle and dihedral angle of nucleophilic attack [3]); ii) substrate interaction with oxyanion hole residues; iii) substrate interaction with catalytic His to consider its involvement in protonation of the substrates leaving group. The enzyme-substrate complexes formed by generated mutants corresponding to above mentioned criteria were subjected to in silico screening in order to reveal the most promising lipase variants with amidase activity and the selected mutations were recommended for experimental production and evaluation of catalytic activity. As a result bioinformatic analysis of /-hydrolase fold enzymes allowed to design and produce Candida antarctica lipase B mutants with improved amidase activity.
________________ [1] M.Holmquist // Alpha Beta-Hydrolase Fold Enzymes Structures, Functions and Mechanisms // Current Protein and Peptide Science, 2000, 1, 209-235; [2] D.A.Suplatov, V.K.Arzhanik, V.K.vedas // Comparative bioinformatics of active site structures in remote homologs from alpha-beta hydrolase enzymes superfamily // Acta Naturae, 2011, 3(1), 99-105; [3] E.S.Radisky, D.E.Koshland // A clogged gutter mechanism for protease inhibitors // PNAS, 2002, 99, 16, 1031610321 This work was supported as a joint EC-Russian FP7-KBBE-2008-2B project IRENE by the European Commission (grant agreement 227279) and the Russian Ministry for Science and Education (contract 02.740.11.0866).
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Rational design of the regioselectivity of the -galactosidase from Thermotoga maritima
Georgy N. Rychkova, Anna S. Borisovaa, Kirill S. Bobrova, Alexander M. Golubeva, Elena V. Eneyskayaa, Natalia L. Rongjinaa, Konstantin A. Shabalina, Anna A. Kulminskayaa a Petersburg Nuclear Physics Institute, Russian Academy of Science, Molecular and Radiation Biology Division, Gatchina, St. Petersburg 188300, Russia E-mail: kulm@omrb.pnpi.spb.ru Detailed biochemical studies of alpha-galactosidases from clan D (GHF 27 and 36) confirmed the homology of the active sites structures and similar mechanism of action [1]. We found that -galactosidase from Thermotoga maritima is suitable for the syntheses of valuable galacto-oligosides. This enzyme is known to possess transglycosylating activity and capable of working at elevated temperatures. Single amino acid substitutions predicted to change the transglycosylation patterns of a GH 36 -galactosidase were identified on the basis of molecular modeling. The covalent enzyme-galactosyl intermediate was constructed in silico for each mutant, and interactions with possible acceptors calculated. On the basis of simple assumptions, residues were identified whose mutation could yield increased 1,4-regioselectivity of transglycosylation. Site-directed mutagenesis of the residues so identified was carried out: native and mutant galA genes were expressed in E. coli, and the corresponding enzymes were purified and checked for transglycosylating activity. The regioselectivity of wild type and mutant -galactosidases was confirmed by NMR analysis of the transglycosylation products. Three mutants showed both an increase in total transglycosylation and increased of 1,4-regioselectivity. This was especially marked with one mutant, which produced, optimally, 16 times the yield of the 1,4-isomer of wildtype, accompanied by lower absolute yields of other regioisomers. The work was supported by the European Community's Seventh Framework Program [FP7-KBBE-2008-2B grant agreement n 227279] and by the Russian Federal Agency for Science and Innovation [contract n. # 02.527.11.0001].
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Structure-activity relationship of commercial enzymes in aqueous and micellar environments
Karen M. Gonalves a,b, Ivana C. R. Leal a,b, Rodrigo O. M. A. Souzaa And Yraima Cordeirob a boss Group - Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, Fax:+552125627001, CEP22941-909. bFaculdade de Farmcia, Universidade Federal do Rio de Janeiro, Centro de Cincias da Sade, Ilha do Fundo, Cidade Universitria, 21.941-590, Rio de Janeiro,Brazil E-mail: karen.goncalves@yahoo.com.br Lipases are triacyl glycerol acyl hydrolases which catalyses hydrolysis of esters, esterification and transesterification [1]. These enzymes are alpha/beta hydrolases with a catalytic triad of ser-his-asp (glu) residues covered by an alpha-helical mobile surface loop [2]. These enzymes act at an interface between aqueous and non-aqueous phases [1] and have widespread applications in biotechnology and in biomedical sciences. In the present work, we investigate conformational changes of lipases in different environments, as AOT/isooctane reverse micelles. These nanosystems provide a high interfacial area of contact [3] and can promote interfacial activation, important to the enzymes catalytic activity. The stability of the enzymes upon thermal and guanidine hydrochloride denaturation was investigated by circular dichroism (CD) and intrinsic fluorescence spectroscopy. The first investigated enzyme in aqueous solvent was Thermomyces lanuginosus (TL) lipase with a hydrodynamic radius obtained by hplc of 28.8 and of 31 by small-angle xray scattering (saxs) measurements. Moreover, the ab initio molecular envelope calculated from the scattering curve indicates that the protein is dimeric at the analyzed concentration, corroboration previous x-ray diffraction data (pdb: 1dt3). Gdnhcl induced unfolding yielded a G1/2 of 3.4 0.02 M. Far-uv cd spectra presented dominance of characteristic signals of alpha-helices. Analysis of tl thermal stability (heating up to 90oc) showed that the enzyme recovered almost all secondary structure content of the native state after cooling; however, only 19% of the initial activity could be recovered. Although a gradual loss in secondary structure was observed when temperature was increased, the high thermostability was evidenced by a tm of 73.6 0.76C. However, no activity was detected at 75c. Another investigated enzyme was the lecitase ultra from Thermomyces lanuginosus (TL), a new enzyme preparation with phospholipase a1 activity. Due to the nature of lecitase ultra, some authors found that this enzyme displays both lipase and phospholipase properties in a single active site [4]. Gdnhcl-induced unfolding of this phospholipase yielded a G1/2 of 3.6 0.03 M. Analysis of thermal stability by far-uv cd showed Tm of 59.5 0.7C. When incorporated into aot/isooctane systems the esterification activity was 32% (24h) for lecitase and 96% (3h) for TL in reverse micelles, showing that this system is more suitable for tl incorporation than for Lecitase.
_______________ [1] Fernandez-Lafuente, R., Journal of Molecular Catalysis B: Enzymatic, 62, 197212(2010) [2] brzozowski, A. M., Savage, H., Verma, C. S., Turkenburg, j. P., Lawson, D. M., Svendsen, A. And patkar, S., Biochemistry, 39, 15071-15082 (2000) [3]Fernandes, M.L.M., Krieger, N., Baron, A.M., Zamora, P.P., Ramos, L.P., Mitchell, D.A., Journal of Molecular Catalysis B: Enzymatic 30, 4349 (2004) [4] mishra, M.K., Kumaraguru, T., Sheelu, G., Fadnavis, N.W., Tetrahedron: Asymmetry, 20, Issue 24, 2854-2860 (2009).
Figure 1. Attachment of PHB Depolymerase of P. funiculosum to a PHB (polyhydroxybutyrate) interface determined by specific residues (red)
Figure 1. Docking of p-nitrophenyl -galactopyranoside within the active site of a mutant.

________________ [1] Comfort et al., Biochemistry, 2007 46:3319-3330.
October 2-6, 2011
152 321
Unraveling the catalytic mechanism of Aspartate Ammonia Lyase
Vinod Puthan Veetila, Guntur Fibriansahb, Andy-Mark W.H. Thunnissenb, and Gerrit J. Poelarendsa a Department of Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands b Department of Biophysical Chemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands E-mail: v.puthan.veetil@rug.nl Aspartate ammonia lyases (also referred to as aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. They belong to the aspartase/fumarase superfamily, members of which show a highly conserved homotetrameric fold and a common active site architecture. Although used as an industrial biocatalyst and studied extensively for several decades, the precise catalytic mechanism of aspartases has remained vague. We did a comprehensive analysis of all the putative active-site residues of the structurally characterized aspartase from Bacillus sp. YM55-1 (AspB) by site-directed mutagenesis, pH-rate profiles and inhibition studies to elucidate their role in either substrate-binding or in catalysis [1]. To further corroborate our findings we solved the crystal structures of AspB in an unliganded state and in complex with its natural substrate (Laspartate) at 2.4 and 2.6 resolution, respectively [2]. A comparison of the two AspB structures reveals that substrate binding induces a large conformational change of the SSloop (residues G317SSIMPGKVN326) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS-loop residue Ser318 is at a suitable position to act as a catalytic base and likely abstracts the C-proton of the substrate in the first step of the reaction mechanism. The resulting enediolate intermediate is then stabilized by an extensive network of hydrogen bonds between the substrates -carboxylate group and residues Thr101, Ser140, Thr141 and Ser319. In the final step, the enediolate intermediate collapses releasing ammonia and fumarate. This work sets the stage for engineering of AspB towards expanding its substrate scope.
_____________________ [1] Puthan,Veetil,V; Raj,H.; Quax,W.J.; Janssen,D.B. and Poelarends,G.J. Site-directed mutagenesis, kinetic and inhibition studies of aspartate ammonia lyase from Bacillus sp. YM55-1. FEBS J. 276(11):2994-3007 [2] Guntur Fibriansah; Vinod Puthan Veetil; Gerrit J. Poelarends and Andy-Mark W.H. Thunnissen. Structural Basis for the Catalytic Mechanism of Aspartate Ammonia Lyase Accepted in Biochemistry, DOI: 10.1021/bi200497y

Indicating protein-protein interaction by uorescence colocalization to artificial protein body in living Escherichia Coli cells
Sang Jun Lee, Su-lim Choi, Su-Jin Kim, Seung-Goo Lee Korea Research Institute of Bioscience and Biotechnology (KRIBB) Gwahak-ro 125, Yuseong-gu, Daejeon, 305-806, Korea E-mail: leesj@kribb.re.kr Detecting protein-protein interaction (PPI) in living cells has been an important subject in modern biosciences. To detect PPI in Escherichia coli cells, this study established a new analysis tool based on colocalization of interacting proteins to inclusion bodies (IBs), which is produced in E. coli by the expression of cellulose-binding domain (CBD) from Cellulomonas fimi. When a PPI between antiparallel leucine zippers of Kd = 20uM were tested in the fusion forms of LZ-GFP-CBD and LZ'-RFP, both the green and red fluorescence clearly localized to the same IB spots on microscopic imaging of the cells. On the contrary, the red fluorescence stayed in the dispersed states in cytosol while the GFP-CBD without LZ was used instead of the LZ-GFP-CBD, proving the molecular interaction between LZ and LZ'. The fluorescence colocalization was detected indeed in flow cytometric analyses of E. coli cells, putatively enabling the single cell-level screening of PPIs in living E. coli. The reliability of flow cytometric analyses was also confirmed in vitro by SDS-PAGE analyses of sorted positive cells.

Automatic evolution of mutants by means of modeFRONTIER software
Lorena Knapica, Valerio Ferrarioa, Paolo Braiucaa,b, Marco Foscatoa, Cynthia Eberta, Lucia Gardossia a Dipartimento di Scienze Chimiche e Farmaceutiche, Universit degli Studi di Trieste, Piazzale Europa 1, 34127 Trieste, Italy b SPRIN S.p.A., Technologies for Sustainable Chemistry, via Flavia 23/1, 34148 Trieste, Italy E.mail: lorena.knapic@phd.units.it This work is part of the EU-FP7 IRENE project The development of an automated approach for generation of virtual mutants and virtual screening for amidase will be presented. This approach would like to be complementary and integrate theoretical approaches by designing a framework embracing different computational methods, such as docking and molecular dynamics, QM-QM/MM methods and QSAR optimization strategies. A computational infrastructure is used for integrating all the software employed for the different steps of the mutant design, modeling and scoring, within an optimization environment able to learn, generation after generation, the correlation between mutations and their efficiency, thus accelerating the evolution of the system. The framework relies on the software modeFRONTIER that organizes a flexible and versatile work-flow. These feature makes the approach highly tunable and conversely distant from the concept of black box.
153 326
First structures of an active bacterial tyrosinase and of variants with altered selectivity
Mor Sendovski Goldfedera, Rita Kanteevb, Vered Shustera, Noam Adirb and Ayelet Fishmana a Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel; bSchulich Department of Chemistry, Technion-Israel Institute of Technology, Haifa 32000, Israel E-mail: mors@tx.technion.ac.il Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. Crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 1.7-2.3 . The enzyme crystallized as a dimer in the asymmetric unit, and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding caddie protein. Two Cu(II) ions, serving as the major co-factors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions, show varying occupancy and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Residues R209 and V218, situated in a second shell of residues surrounding the active site, were suggested to play a role in substrate binding orientation, on the basis of their flexibility and position. Indeed, mutations introduced in these positions resulted in variants with higher monophenolase/diphenolase activity ratios compared to wild-type. Structures of variants R209H, V218G and V218F were determined and provided additional information on the importance of these positions. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site (Figure 1).
The software makes use of a genetic algorithm for the optimization implemented in a computer simulation in which a population of abstract representations (called chromosomes) of candidate solutions (called individuals; enzyme mutants in this specific case) for an optimization problem evolves toward better solutions. The evolution usually starts from a population of randomly generated individuals and happens in generations. In each generation, the fitness of every individual in the population is evaluated, multiple individuals are selected from the current population (based on their fitness), and modified (recombined and mutated) to form a new population. The new population is then used in the next iteration of the algorithm. Commonly, the algorithm terminates when either a maximum number of generations has been produced, or a satisfactory fitness level has been reached for the population.
________________ This work is part of the EU-FP7 IRENE project and it has received funding from the European Community's Seventh Framework Programme under the FP7-KBBE-2008-2B grant agreement n 227279
_______________ [1] M. Sendovski, M. Kanteev, V. Shuster, N. Adir, A. Fishman, (2010) Crystallization and preliminary Xray crystallographic analysis of a bacterial tyrosinase from Bacillus megaterium, Acta Crystallographica F: Structural Biology and Crystallization Communication, 66 (9), 1101-1103. [2] M. Sendovski, M. Kanteev, V. Shuster, N. Adir, A. Fishman, (2011) First structures of an active bacterial tyrosinase reveal copper plasticity, Journal of Molecular Biology, 405 (1), 227-237.
Figure 1. The structure of tyrosinase from Bacillus megaterium in complex with kojic acid; view of the active site.
323
Protein engineering of yeast Saccaromyces cerevisiae reductase for the production of pharmaceutical chiral building block
Jihye Jung, Hyung Kwoun Kim Division of Biotechnology, the Catholic University of Korea, Bucheon 420-743, Republic of Korea E-mail: airgreen13@hanmail.net The asymmetric reduction of ketones into optically pure alcohols is frequently conducted with a variety of microbial reductase enzymes. Although these enzymatic reactions are efficient processes to produce useful chiral alcohols, the cost of cofactor of the enzymes prevents this process from being utilized widely in organic synthesis industry. Moreover, most reductases usually prefer NADPH to NADH as their cofactors in spite that NADPH is much more expensive and unstable in comparison with NADH. In the previous literature, yeast YDL124W reductase was reported to produce (S)-ethyl-4-chloro-3-hydroxybutanoate, a pharmaceutical chiral building block. In that reaction, NADPH and glucose dehydrogenase were used as cofactor and coupling enzyme, respectively. In this research, we found several amino acids located nearby 2-phosphate group of adenosine ribose of NADPH by homology model and docking model. Through rational design and site-directed mutagenesis we made two mutant reductases showing high enzyme activity toward NADH. Subsequent kinetic study verified that the mutant reductases had dramatically decreased Km values toward NADH. These two mutant reductases produced optically pure chiral alcohol efficiently in the presence of NADH and formate dehydrogenase as cofactor and coupling enzyme, respectively.
324
Conformational changes of lipases: a comparative computational study and experimental implications
Valerio Ferrario, Marco Foscato, Lorena Knapic, Cynthia Ebert, Diana Fattor, Lucia Gardossi Dipartimento di Scienze Chimiche e Farmaceutiche, Universit degli Studi di Trieste, Piazzale Europa 1, 34127 Trieste, Italy E-mail: gardossi@units.it Conformational changes occurring to the open form of five different lipases were studied by means of molecular dynamic simulations in explicit water. The study analyses changes occurring at three levels: a) surface properties with special regards to variation of the exposure of the hydrophobic area in the proximity of the active site; b) conformational changes of the lid domain and other domains possibly involved in the occlusion of the hydrophobic active site; c) conformational changes occurring at the catalytic machinery of the lipases. The objective was to observe different phenomena that might cause a decrease of activity of lipases when exposed to aqueous media. The methodology was validated by comparing the calculated closed conformations with the crystal structures obtained in water.
327
Protein engineering of a avoprotein oxidase based on a computational study that predicts the O2-diffusion
Elena Rosinia, Gianluca Mollaa, Jan Saamb, Sandro Ghislaa, Loredano Pollegionia a Centro di Ricerca Interuniversitario in Biotecnologie Proteiche The Protein Factory, Politecnico di Milano and Universit degli studi dellInsubria, via J.H. Dunant 3, 21100 Varese, Italy; bBeckman Institute, University of Illinois, USA E-mail: elena.rosini@uninsubria.it Molecular oxygen participates in numerous enzymatic reactions, e. g. flavooxidases use O2 to directly oxidize the reduced flavin cofactor. In this class of enzymes, a key, open question regards the control of O2-reactivity with the reduced flavin cofactor. There are several examples for channels in flavoproteins that guide O2 to the reaction site. In this context several questions emerge: i) to what extent are these channels controlling O2-reactivity? ii) do they have a relevant O2 affinity? In this work we combined a computational (MD simulations and implicit ligand sampling) and an experimental approach (based on site-saturation mutagenesis and kinetic studies) to investigate the O2-diffusion and reactivity in the flavoprotein D-amino acid oxidase (DAAO) [1]. Several interconnected paths (corresponding to narrow channels in the protein structure) leading dioxygen from the solvent to several O2 high affinity sites close to the active site were identified by the computational analysis (Figure 1) [2]. In particular, site A is adjacent to the reactive N(5)-C(4a) locus on the si side of the flavin, a well suited position to speed up the flavin reoxidation process. Based on this, residues that flank the putative O2 high affinity sites have been exchanged with bulky residues in order to introduce steric constrains. In Gly52Val DAAO, the valine side chain occupies the site that in the wild-type enzyme has the highest O2 affinity. As expected, the reactivity of the reduced Gly52Val DAAO variant with O2 is decreased 100-fold and the turnover number is 1000-fold lower. On the other hand, the T201L variant (a residue close to site B, Figure 1) shows a 3-fold increase in O2-reactivity [3]. The present work demonstrates the great potential of joint computational and experimental research. The results of MD simulations, supported by the experimental data, provide a detailed mechanistic picture for the existence of functional oxygen access channels in DAAO and are of great interest for designing new flavoenzymes for biotechnological applications.
328
Enzyme-Catalyzed Kinetic Resolution of Racemic Clopidogrel
Yasser Gaber1,2, M. Ismail3 M. Takwa1, Magdy Ali Amin4, Rajni Hatti-Kaul1 1 Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden 2 Faculty of Pharmacy, Microbiology Department, Beni Suef University, Beni Suef, Egypt 3 Faculty of science, Helwan University, Egypt 4 Faculty of Pharmacy, Microbial Biotechnology Center, Cairo University, Kasr El-Aini, Cairo, Egypt E-mail: Yasser.Gaber@biotek.lu.se Biocatalysis based approach is evaluated for asymmetric hydrolysis of Clopidogrel, one of the most effective antiplatelet aggregators [1,2]. Screening a selected number of hydrolases (lipases, esterases and proteases) has resulted in inactive enzymes (ex. Ps. fluorescence esterase), active enzymes with no selectivity (Novozym 435, Lipo TL, Lipo RM), and a selective enzyme: pig liver esterase (PLE). PLE was found to selectively hydrolyze the undesired R ester in the clopidogrel racemic mixture. The enantioselectivity of PLE was rather low (E = 8.2), but the recorded enantiomeric excess was high (95- 98%). Molecular modelling of the R and S enantiomers inside the active site of PLE model was performed to understand the possible geometric hindrances for formation of the enzyme-substrate tetrahedral intermediate. Glu203 in the vicinity of the active site was found to form hydrogen bonds with the catalytic His449 and GGG(X) motif residues. The hydrogen bonding of these residues should be available for the stabilization of the tetrahedral intermediate (Fig.1). Hence, Glu203 would be a potential site for a site directed mutation in the PLE gene expressed in Escherichia coli.
Figure 1. Superimposition of crystal structures of the closed lipase from Humicola lanuginosa (HL) in red with the calculated closed form of HlL in blue. Lid domain highlighted in the red box. Figure 1. Enantioselective production of (S)-ECHB using two coupling reactions.
__________________ [1] J. Jung, H. J. Park, K. N. Uhm, D. Kim, and H. K. Kim. 2010. Asymmetric synthesis of (S)-ethyl4-chloro-3-hydroxybutanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme-substrate docking studies. Biochem. Biophys. Acta 1804: 1841-1849. [2] M. Katzberg, N. Skorupa-Parachin, M. F. Gorwa-Grauslund, and M. Bertau. 2010. Engineering cofactor preference of ketone reducing biocatalysts: A mutagenesis study on a diketone reductase from the yeast Saccharomyces cerevisiae serving as an example. Int. J. Mol. Sci. 11: 1735-1758.
The conformational changes indicate remarkable differences among the lipases considered, not only in terms of accessibility of the active site but also of modification of the geometry of the catalytic machinery. Lipase B from Candida antarctica undergoes no conformational change at either level. Other lipases undergo the most relevant conformational variations both at the level of the catalytic triad and the residues involved in the oxyanion stabilization, suggesting that its interfacial activation is not simply related to a variation of the accessibility of the active site.
_______________ This work has received funding from the European Community's Seventh Framework Programme under the FP7-KBBE-2008-2B grant agreement n 227279
Figure 1. Molecular modelling of the tetrahedral intermediate of R-clopidogrel inside the Pig liver estearse model. Glu203 forms undesirable hydrogen bond with the important His449, G124 and G125 representing a potential site for mutation. The model was created by Yasara [3].
________________ [1] Pollegioni L., et al. (2007) Cell. Mol. Life Sci. 64, 1373-1394. [2] Saam J., et al. (2010) J. Biol. Chem. 285, 24439-24446. [3] Rosini E., et al. (2011) FEBS J. 278, 482-492.
Figure 1. Overview of O2 access pathways to the active site of DAAO [2].
________________ [1] McQuaid, K.R. and L. Laine, Amer J Med, 2006. 119(8): p. 624-638. [2] Tao, J. and J.H. Xu, Curr Opin Chem Biol, 2009. 13(1): p. 43-50. [3] www.Yasara.com,
October 2-6, 2011
154 329
Mechanistic insights into regioselective enzymatic Baeyer-Villiger oxidation of diketones by empirically supported docking studies
Florian Rudroff,* Michael Fink, David Biedermann, Thomas Fischer, Marko D. Mihovilovic Vienna University of Technology Institute of Applied Synthetic Chemistry Getreidemarkt 9/163-OC, A-1060 Vienna, Austria *email: frudroff@ioc.tuwien.ac.at Baeyer-Villiger monooxygenases (BVMOs) are natures mimicry of the chemical BV reaction. These enzymes are flavin and NADPH dependent proteins that are capable of nucleophilic oxygenation of a wide range of linear, cyclic or aromatic ketones, yielding optically pure esters or lactones. By using molecular oxygen as the oxidant, and the formation of water as the only byproduct. BVMOs represent a perfect source for environmentally friendly and green catalysts. Due to their highly chemo-, regio- and enantio-selectivities they are highly valuable bioreagents in asymmetric catalysis. [1]

Characterization of -galactoside phosphorylases with diverging acceptor specificities
Chen Chao, Soetaert Wim, Desmet Tom Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium E-mail: Chao.Chen@UGent.be Glycoside phosphorylases are a special group of carbohydrate-active enzymes, with characteristics in between those of glycoside hydrolases and glycosyl transferases[1]. The phosphorylases from family GH-112 are exceptional because they employ galactose-1-phosphate instead of glucose-1-phosphate as glycosyl donor. Different acceptor specificities have been observed in this family, ranging from L-rhamnose to GlcNAc, GalNAc and a combination of the latter. Three new phosphorylases from previously unexplored branches of the phylogenetic tree of family GH-112 have now been characterized to shed more light on this divergence in acceptor specificity. The enzymes from Erysipelothrix rhusiopathiae and Streptobacillus moniliformis were found to prefer GalNAc as acceptor, while that from Anaerococcus prevotii displays similar activities on GalNAc and GlcNAc[2]. These results confirm the correlation between the amino acid residue at position 162 and the enzymes specificity, i.e. a threonine in the former group and a valine in the latter. However, mutagenesis of residue 162 did not allow the rational transformation of the substrate preference, as the substitution of valine by threonine in the enzyme from Bifidobacterium longum did not tighten its specificity towards GalNAc. Furthermore, substituting valine with isoleucine generated a LNBP, while activity towards L-rhamnose was expected. Although position 162 thus clearly has been shown to influence the enzymes specificity, it can by itself not fully explain the behavior of the various -galactoside phosphorylases in family GH-112. Interestingly, the phosphorylase from E. rhusiopathiae displays a kcat/Km for GalNAc that is approximately 115 times higher than that for GlcNAc, which results in the strictest specificity reported to date. It is, therefore, a suitable candidate for the practical preparation of galacto-N-biose, a core structure of O-linked glycoproteins in mucin [3].
________________ [1] Kitaoka M, Hayashi K. Trends Glycosci Glycotechnol 2002;14(75):33-50. [2] Chen C, Soetaert W, Desmet T. Enzyme Microb Technol 2011;49(1):59-65. [3] Nishimoto M, Kitaoka M. Carbohydr Res 2009;344(18):2573-2576.

Functional analysis of UDP-sugar: sterol glucosyltransferases
Justin Perrya, Lynn G. Dovera, Vatsala Malika,Tony Flinnb, Julian Northenb, Edwin Ludkina and Gary W. Black a, Tony Flinnb, Julian Northenb a Department of Biomedical Sciences, School of Life Sciences, Northumbria University, Newcastle-Upon-Tyne, NE1 8ST, U.K; Onyx-Scientific, Sunderland, SR5 2TQ, U.K. E-mail: vatsala.malik@northumbria.ac.uk Glycosyltransferases (GTs) are essential for the biosynthesis and diversification of many therapeutically important natural products. Of these, UDP-sugar: sterol glucosyltransferases (UGTs) (2.4.1.173) catalyse the synthesis of therapeutically important steryl glycosides (SGs) [1-2]. Guided by the sequence similarity with a previously characterised N-terminally truncated UGT from Saccharomyces cerevisiae (UGT51) [3], this study reports the cloning of the gene fragment encoding the C-terminal catalytic domains from related yeasts and the expression and characterisation of their encoded products produced. N-terminally histidine tagged proteins were purified for in vitro assays against a panel of sterol and steroidal acceptors. Liquid chromatography-mass spectrometry (LC-MS) and kinetic analysis led to the successful characterisation of two novel UGTs from Pichia angusta and Kluyveromyces lactis. In addition, testosterone was shown to be utilized by all UGTs, including the previously characterised S. cerevisiae UGT51. Random mutagenesis of UGTs and homology modelling of the S. cerevisiae UGT revealed structural similarities with family 1 bacterial glycopeptide GTs. Given the structural and mechanistic similarities among GT family 1 UGTs, this approach may provide a template for genetic manipulation of novel UGTs from other members of the GT superfamily with a better understanding of catalytic domains and for broadening their scope in drug development. It may also aid the development of a generic process in the synthesis of SGs.
________________ [1] Osmani, S. A., Bak, S. & Lindberg, M. (2009) Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling, Phytochemistry 70, 325-347 [2] Coutinho, P. M., Deleury, E., Davies, G. D. & Henrissat, B. (2003) An Evolving Hierarchical Family Classification for Glycosyltransferases, J. Mol. Biol. 328, 307-317 [3] Warnecke, D., Erdmannn, R., Fahl, A., Hube, B., Muller, F., Zank, T., Zahringer, U. & Heinz, E. (1999) Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum, J. Biol. Chem. 274, 13048-13059
155 334
Structural and molecular characterisation of the first inverting sec-alkylsulfatase (pisa1) from Pseudomonas sp.
T. Knausa, M. Schoberb, U. Wagnerc, K. Faberb and P. Macherouxa a Institute of Biochemistry, Graz University of Technology, Austria; bDepartment of Chemistry, University of Graz, Austria; cInstitute of Molecular Biosciences, University of Graz, Austria E-mail: tanja.knaus@tugraz.at Hydolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize sulfur. Three classes of sulfatases divided according to their reaction mechanism have been discovered so far. These enzymes catalyse the cleavage of the sulfate ester bond yielding the corresponding alcohol and hydrogen sulfate. In contrast to the majority of hydrolases, which do not alter the stereochemistry of the substrate during catalysis, the stereochemical course of sulfate ester hydrolysis may proceed via cleavage of the S-O or the C-O bond of a sec-alkyl sulfate going in hand with retention or inversion of the stereogenic carbon atom, respectively. Recently a novel enzyme was identified in Pseudomonas DSM 6611 (termed Pisa1) that is able to convert secondary alkylsulfates in contrast to the only known primary alkylsulfatase SdsA1 from Pseudomonas aeruginosa, und reacts under inversion of configuration at the carbon atom. Current structural studies of Pisa1 show that both proteins largely share the same activesite architecture, such as a sulfate binding site (composed of two Arg), a nucleophile site composed of a binuclear Zn2+-cluster typical for metallo--lactamases and an Asn/Thrhydrogen binding network for substrate positioning. Although Pisa1 shares 44 % sequence identity with SdsA1 and features a closely related active site, the substrate preference of both proteins differs significantly. Whereas SdsA1 has a (150-fold) dominant affinity for the prim-sulfate ester octylsulfate, Pisa1 has a pronounced (190-fold) opposite preference for the sec-sulfate ester analog (R)-2-octylsulfate. Here, we provide a biochemical and structural characterisation of the first inverting secalkylsulfatase, Pisa1.
________________ [1] P. Gadler and K. Faber (2007) Eur. J. Org. Chem., 5527-5530 [2] P. Gadler and K. Faber (2007) Trends Biotechnol. 25, 83-88 [3] G. Hagelueken, T.M. Adams, L. Wiehlmann, U. Widow, H. Kolmar, B. Tuemmler, D.W. Heinz and W.D. Schubert (2006) Proc. Natl. Acad. Sci. USA 103, 7631-7636
Figure 1 Regioselective Baeyer-Villiger oxidation of diketones. In the last decades many different molecular scaffolds were tested for their acceptance by BVMOs, but only a small number of studies were conducted on substrates with more than one carbonyl moiety.[2] Whereas these studies investigated the general possibility of BVMOs to oxidize bi- and polycyclic ketones to the desired lactones we are going to present a more systematic approach based on experimental data and mathematical simulations to obtain deeper mechanistic insights into the regio- and stereoselective preference of BVMOs on multiketones with cyclic as well as exocyclic carbonyl functionalities. Oxidation of simple model diketones by various BVMOs lead to interesting and completely unexpected regiodivergency (figure 1). Based on these results we will conduct an enzyme-substrate docking approach to unravel mechanistic aspects of the regiodivergent transformation of BVMOs.
__________________ [1] Mihovilovic; M.D.; Bianci, D.A.. Asymmetric Organic Synthesis with Enzymes. 2008 WILEY-VCH Verlag, Weinheim [2] a.) Ottolina, G.; Gonzalo, G.; Carrea, G.; Danieli, B. AdvSynthCatal. 2005, 347, 1035-1040 b.) Beneveti, E.; Ottolina, G.; Carrea, G.; Panzeri, W.; Fronza, G.; Lau, P.C.K. JMolCatalB. 2009, 58, 164-168
331
Increasing the reaction rate of aminomutases by rational protein engineering
Sebastian Bartscha, Matthew Heberlinga, Bian Wua, Dick B. Janssena a University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands E-mail: s.bartsch@rug.nl The family of MIO-dependent ammonia lyases catalyzes the reversible, non-oxidative deamination of aromatic amino acids to the corresponding / unsaturated acids and ammonia.[1] For synthetic purposes, it is of special interest that the equilibrium of the naturally occurring deamination reaction can be shifted towards the amination direction by using high ammonia concentrations (Fig 1).[2] Such amination reactions on double bonds are very interesting alternatives for the classical organic synthesis of chiral amines. These asymmetric reactions with direct addition of ammonia to an unsaturated substrate proceed without requiring further activation, protection or regeneration steps and can be carried out under mild conditions in water. Although aromatic ammonia lyases such as phenylalanine ammonia lyase (PAL) show high amination rates, the major limitation is their exclusive -regioselectivity (Fig 1). Recently, a homologous phenylalanine aminomutase (PAM) was discovered to show activity with cinnamic acid derivatives to produce optically pure -amino acids (Fig 1), which are interesting building blocks for the synthesis of bioactive compounds like the anticancer drug taxol.[3-5] The major limitation of this enzyme is the low reaction rate.
332
Thermal stabilization of Baeyer-Villiger monooxygenases via a structure guided consensus approach
Saima Feroza, Hanna M. Dudekb, Marco Fraaijeb, Andreas S. Bommariusc and Marko D. Mihovilovica a Institute of Applied Synthetic Chemistry, Vienna University of Technology, Getreidemarkt 9/163, 1060 Vienna, Austria b Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands c Georgia Institute of Technology School of Chemical & Biomolecular Engineering,N.W. Atlanta saima.feroz@ias.tuwien.ac.at Oxygenation reactions of ketones to esters or lactones were discovered more than a century ago by Adolf von Baeyer and Victor Villiger[1]. Since then, Baeyer-Villiger monooxygenases (BVMOs) were recognized as highly versatile biocatalysts for this reaction. A prominent transformation of such enzymes is the stereoselective oxidation of cyclic and/ or aliphatic ketones to chiral lactones/esters which are interesting building blocks for the synthesis of bioactive- and natural compounds[2-3]. However due to number of reasons, large-scale application was not enforced on a satisfactory level so far. Cyclohexanone monooxygenase (CHMO) originating from Acinetobacter NCIB 9871 was chosen for this study. Mutations to stabilize helical structure motifs in particular were predicted by sequence comparison with the structure of phenylacetone monooxygenase (PAMO) from Thermobifida which was recently established by X-ray diffraction[4]. Certain combinatorial and rational protein design approaches were applied for the development of several generations of mutants. Parallel screening methods both on whole-cells as well as crude cell extracts were utilized to assess biocatalytic performance. Herein we report the development of CHMO mutants with increased thermal and kinetic stability without compromising the substrate specificity and selectivity. Shelf life of mutant enzymes at 30C can be improved from 80 hours (WT) to 722 hours (mutant). An increase of more than 1400 % in the yield during the life time of enzyme, over the wild type is achieved. In summary, the generality of the consensus based approach for the improvement of thermal stability of biocatalysts could be successfully applied to a flavin monooxygenase for the first time.
______________________ [1] A. von Baeyer and V. Villiger. Ber. Dtsch. Chem. Ges 32 (1899) 3625 [2] J.D. Stewart. Curr. Org. Chem. 2 (1998) 195 [3] M.D. Mihovilovic, B. Muller and P. Stanetty. Eur.J.Org.Chem. (2002) 3711 [4] E. Malito, A. Alfieri, W. Fraaije Marco and A. Mattevi. PNAS 101 (2004) 13157
335
Diversity of lipases/acyltransferases in yeasts
Pisey M. Neang, Vronique Perrier, Eric Dubreucq, Maeva Subileau UMR IATE, Montpellier SupAgro, 2 Place Viala, F-34060 Montpellier, France E-mail: Pisey.Neang@supagro.inra.fr Candida parapsilosis is known to secrete CpLIP2 [1], a particular lipase/acyltransferase which catalyzes transesterification preferentially to hydrolysis even in media with high thermodynamic activity of water (aW>0.9) [2]. This yeast has phylogenetic relationships with other species in which enzyme expression and activity remained unstudied. The present work involved the pre-selection of phylogenetically related yeast strains and the characterization of their potential production of lipases/acyltransferases. Among the 17 strains studied, six exhibited interesting lipase activity. The analysis of protein extracts from culture supernatants by SDS-PAGE followed by the sequencing of the protein bands with an apparent molecular weight close to 50 kDa confirmed the presence of peptides homologous to CpLIP2 for two strains. The catalytic behaviour and the substrate specificity of these new enzymes were compared to that of CpLIP2. In parallel to this functional characterization of enzymes from wild strains, a functional genomics study based on gene sequences is undertaken. ________________ [1] Neugnot V., Moulin G., Dubreucq E. et Bigey F. (2002) The lipase/acyltransferase from C. parapsilosis: molecular cloning and characterization of purified recombinant enzymes. Eur. J. Biochem. 269, 1734-1745. [2] Briand D., Dubreucq E. and Galzy P. (1995) Functioning and regioselectivity of the lipase of C. parapsilosis (Ashford) Langeron and Talice in aqueous media. New interpretation of regioselectivity taking acyl migration into account. Eur. J. Biochem., 228, 169-175
336
Probing the relationship between substrate structure and cofactor dependency by mapping substrate profile of a stereospecific carbonyl reductase from Candida parapsilosis
Yao Niea, Yan Xua,b, Rong Xiaoc School of Biotechnology and Key laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; bState Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; cCenter for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08854, USA. E-mail: nieybird@hotmail.com
a
Figure 1. Reaction catalyzed by PAL (left) and PAM (right). Based on computational studies, we could show that the low activity of PAM is due to a more tightly closed lid structure compared to PAL. While PAM was crystallized in the closed conformation[6], homologous ammonia lyases are crystallized in an open conformation or the lid structure is not visible because of excessive flexibility[7]. By using molecular dynamics simulations, the conformational change from the closed to the open state was simulated and yielded information about the key residues influencing this structural change. Through rational protein design, different mutants were identified that were shown to have a significant increase in the reaction rates of PAM. These mutations are based on changed hydrophobic interactions influencing the equilibrium between the closed and open state, as well as on a changed hydrogen bond pattern.
Stereospecific carbonyl reductases have been widely used in synthesis of optically active alcohols as important precursors for pharmaceuticals and fine chemicals, from various carbonyl compounds [1]. The stereospecific carbonyl reductase CPADH from Candida parapsilosis [2], in this study, was evaluated by mapping the substrate profile to different carbonyl compounds including aliphatic ketones, aryl ketones, and - and -ketoesters. The enzyme performed specific activities and presented a broad substrate preference toward the tested substrates. Of the involved carbonyl compounds, the enzyme showed higher activities to ketoesters than to aryl and aliphatic ketones. Additionally, the substrates with electron-withdrawing groups neighbouring the carbonyl group, such as -hydroxyl-substituted acetophenone derivatives and ethyl 4-chloroacetoacetate, were more suitable for the enzyme-catalyzed reduction. Furthermore, the steric effect occurred in the hydride transfer reaction, relating to substrate accommodation in catalytic domain of the enzyme and cofactor dependency performed from specific activity of the enzyme-mediated reaction. That observation corresponded to the docking studies on enzyme-cofactor complex (Figure 1), indicating that the conformation of substrate-binding pocket formed from cofactor and enzyme has a steric effect on the interaction between cofactor and substrate, and thus there is a relationship between cofactor dependency of the stereospecific carbonyl reductase and chemical structure of substrate. The present study also showed that the enzyme possesses a broad substrate specificity and has an application potential in catalyzing ketones reduction.
________________ [1] S. Bartsch, U. T. Bornscheuer, Angew. Chem. Int. Ed. 2009, 48, 3362-3365. Corrigendum: Angew. Chem. Int. Ed. 2010, 49, 3860 [2] S. Bartsch, U. T. Bornscheuer, Prot. Eng. Des. Sel. 2010, 23, 929-933. [3] B. Wu, W. Szymanski, P. Wietzes, S. De Wildeman, G. J. Poelarends, B. L. Feringa, D. B. Janssen, ChemBioChem 2009, 10, 338-344. [4] W. Szymanski, B. Wu, B. Weiner, S. De Wildeman, B. L. Feringa, D. B. Janssen, J. Org. Chem. 2009, 74, 9152-9157. [5] B. Wu, W. Szymanski, S. de Wildeman, G. J. Poelarends, B. L. Feringa, D. B. Janssen, Adv. Synth. Catal. 2010, 352, 1409-1412. [6] L. Feng, U. Wanninayake, S. Strom, J. Geiger, K. D. Walker, Biochem. 2011, 50, 2919-2930. [7] S. Pilbak, A. Tomin, J. Rtey, L. Poppe, FEBS J. 2006, 273, 1004-1019.
Figure 1. Model structure of the substrate-binding pocket generated from enzymecofactor docking with NADPH (a) and NADH (b), respectively, illustrated by the PyMOL program.
________________ [1] G.W. Huisman, J. Liang, A. Krebber. Curr. Opin. Chem. Biol., 2010, 14:122129. [2] Y. Nie, Y. Xu, X.Q. Mu, et al. Appl. Environ. Microbiol., 2007, 73:37593764.
October 2-6, 2011
156 337
Structure-performance relationships of lipolytic enzymes immobilized on polymeric nanoparticles
Laura Chronopouloua, Gihan Kamelb, Federico Bordib, Marco Diociaiutic, Stefano Lupib, Cleofe Paloccia a Department of Chemistry, Universit degli Studi di Roma "La Sapienza", Piazzale Aldo Moro 5, 00185 Rome, Italy; bDepartment of Physics, Universit degli Studi di Roma "La Sapienza", Piazzale Aldo Moro 5, 00185 Rome, Italy; cDipartimento di Tecnologie e Salute, Istituto Superiore di Sanita`, viale Regina Elena 299, 00161 Rome, Italy E-mail: laura.chronopoulou@uniroma1.it The intersection between biotechnology and nanotechnology is opening the way to the development of hybrid materials, that can sum the catalytic and highly selective recognition properties of biological molecules to the peculiar characteristics of nanoparticles. Moreover, we have evidenced that polylactic acid (PDLLA) nanoparticles can influence Candida rugosa lipase (CRL) catalytic performance, enhancing dramatically its activity in aqueous medium [1]. PDLLA was processed using an innovative patented methodology [2] that permitted to obtain spherical nanoparticles with a mean diameter of 200 nm, that were used as carrier for the physical adsorption of CRL preparations. Enzymatic activity and stability before and after conjugation to the nanopolymeric support were evaluated in different conditions (pH, T, organic solvents), evidencing the higher specific activity and stability to denaturing agents of the bioconjugates. FTIR characterization of free and immobilized enzymes showed a reproducible modification in the conformational features of CRL in different media (solid state and aqueous solutions). In an attempt to assess the structure-performance relationship of the immobilized CRL, based on biochemical studies and FTIR characterization, we propose that the enhancement of the enzyme performance is closely related to the conformational changes of the protein structure due to the immobilization on nanocarriers.

Simulation of C.antarctica lipase B in hydrophobic environments: the critical role of water
Tobias Kulschewskia, Christian C. Gruberb, Jrgen Pleissa a Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany; bAustrian Centre of Industrial Biotechnology, University of Graz, Humboldtstr.50/III, 8010 Graz, Austria E-mail: Juergen.Pleiss@itb.uni-stuttgart.de Candida antarctica Lipase B (CALB) is an efficient biocatalyst for hydrolysis of waterinsoluble esters and for esterification in organic solvents. However, in organic solvent it is essential to add small amounts of water to maintain stability and flexibility of the enzyme. It has been proposed that protein-bound water serves as a lubricant, but the interplay between protein, the protein-bound water, and the organic solvent is not yet fully understood. It has been reported that enzymatic activity can be correlated with the solvent polarity, other reports correlated it with thermodynamic activity of water. To investigate the molecular mechanism of protein-solvent interactions, two protein systems were studied by molecular dynamics simulations: (1) CALB in a mixture of toluene and short-chain alcohols, and (2) CALB binding to a tributyrin-water interface. The inactivation of many enzymes by short-chain alcohols is considered as major problem in lipase-catalyzed reactions at low water content. In organic solvents, water binds tightly to the surface of CALB [1], and the flexibility of the protein depends on the logP value of the solvent and the hydration level [2]. Extensive molecular dynamics of CALB and other lipases in organic solvents at varying concentrations of water and short-chain alcohols were performed to study the interactions between the solvents and the protein, as well as the effect of solvent composition to structure and flexibility of the enzymes. The molecular basis of short-chain alcohol sensitivity of lipases was studied and critical parameters such as the thermodynamic activity of water were identified. Even though CALB does not show classical interfacial activation, stable binding to a hydrophobic tributyrin-water interface was observed, and the crucial role of water was confirmed during the binding process. Upon binding to the interface, CALB is directed and oriented by three hydrophobic anchor residues (L147, L219, V272). The anchor residues are located near the entrance to the active site, but are not part of the substrate binding site. Upon binding, a slight conformational change is observed and bulk water is expelled. During extended simulations for several hundreds of ns, spontaneous binding of single tributyrin molecules in the active site were observed, replacing most of the remaining water molecules trapped in the active site and leading to productive near-attack-complexes.
________________ [1] Trodler, P., Pleiss, J., 2008. Modeling structure and flexibility of Candida antarctica lipase B in organic solvents. BMC Struct Biol 8: 9 [2] Branco, R.J.F., Graber, M., Denis, V., Pleiss, J., 2009. Molecular mechanism of the hydration of Candida antarctica lipase B in gas phase: water adsorption isotherms and molecular dynamics simulations. ChemBioChem 10: 2913-291

Prediction of (enantio)selectivity of Pharma PLETM mutants by 3D-QSAR modeling
Paolo Braiucaa, Elly Raemakers-Frankenb, Bas Rossiusb, Linda Vermoteb, Oliver Mayb a SPRIN S.p.A., Viale XX Settembre 17, 34100 Trieste, Italy; bDSM Innovative Synthesis B.V., Urmonderbaan 22, Geleen, The Netherlands E-mail: braiuca@sprintechnologies.com; E-mail: elly.raemakers@dsm.com It is widely recognized that biocatalysis is vital for the development of sustainable processes in the fields of both life sciences as well as material sciences. To find a biocatalyst for a target bioconversion, high throughput screening of enzyme platforms is often applied. Although the process of high-throughput screening ensures fast hit-finding in most cases, precise prediction of enzyme mutations to achieve higher activities and (enantio)selectivities than the initial hit is still very challenging. At present, mutations are often predicted on basis of molecular modelling after which mutant libraries are constructed and screened. Often several mutation rounds are needed to achieve the desired high activity and/or enantioselectivity. For the prediction of enantioselectivity several approaches have been used in the past. For example, an early empirical model for pig liver esterase (PLE), where the active site was represented by box-shaped hydrophobic and hydrophilic pockets, which formed the boundaries of the available substrate binding volume, helped rationalise the specificity of PLE towards a structurally diverse range of substrates [1]. More sophisticated computational models have been described for the prediction of e.g. substrate selectivity (kcat/Km), which are based on free energy calculation of the transition state of the reaction [2]. However, such methods often are too computationally demanding or suffer from over-simplifications and hence poor quantitative predictive power. However, three-dimensional quantitative structure-activity relationship (3D-QSAR) models, avoid free energy calculations, thereby reducing computation time. In this approach, statistical analysis is used to correlate suitable molecular descriptors of substrates to enzymatic reaction parameters, such as enantiomeric ratio (E) or specific activity. Construction of such models requires training sets of experimental data and 3D structure models of the concerning enzymes. The substrates are docked into the enzymes active site and the most optimal conformers are selected by a docking algorithm. After a short molecular dynamics simulation, GRID-based molecular descriptors are generated from those conformers with the lowest potential energy. Using statistical analysis, these descriptors are then correlated with experimental data, which ultimately gives rise to the predictive model [3]. At SPRIN Technologies a 3D-QSAR model, based on differential molecular interaction fields (DMIF), has been developed [4, 5]. Here, the results are presented for quali-quantitative prediction of specific activity and enantioselectivity of two of DSMs Pharma PLETM mutants, important enzymes for the production of pharma intermediates [6].
________________ [1] Toone, E.J., Worth, M.J., and Jones, J.B. J. Am. Chem. Soc. (1990), 112, 4946-4952. [2] Kazlauskas, R.J.. Curr. Opin. Chem. Biol. (2000), 4, 81-88. [3] Tomic, S., Kojic-Prodic, B. J. Mol. Graph. Model. (2002), 21, 241-252. [4] Braiuca, P., Boscarol, L., Ebert, C., Linda, P., and Gardossi, L. Adv. Synth. Catal. (2006), 348, 773-780. [5] Braiuca, P., Lorena, K., Ferrario, V., Ebert, C., and Gardossi, L. Adv. Synth. Catal. (2009), 351, 1293-1302. [6] May, O. and Rusnak, M. (2008). Specialty Chemicals Magazine (2008), 26-27.
157 342
3DM: protein engineering super-family systems
Henk-Jan Joosten Bio-Prodict, Dreijenplein 10, Wageningen, The Netherlands. E-mail: joosten@bio-prodict.nl A powerful method to gain biological insights in the functioning of a protein is to use data available for the protein (super)-family. The large amounts of different data types can be used to detect correlations that reveal the function of amino acids, but collecting and analyzing the data is difficult and time consuming. Therefore, protein super-family specific databases are needed. It is shown that such systems can help to predict protein interaction sites, active site residues, residues important in enzyme specificity/activity, residues involved in discrimination between recognition of agonist and antagonist binding, etc. These systems are therefore powerful tools in drug design and protein engineering studies. Therefore, 3DM was developed, a system that can automatically build such protein superfamily databases. 3DM collects many different data types, such as sequences, structures, mutation data (collected from different sources, such as mutation databases and literature), structure derived data (e.g. protein-protein interactions, ligand-protein interactions, hbridges, salt-bridges, solvent accessibility, etc), and alignment derived data (conservation, correlated mutations, etc). All the different data-types are connected to each other via a unified numbering scheme, called 3D-numbers, which is applied to all sequences, structures and to the alignments. This connectivity of the data enables detection of hidden correlations, transfer of different data types between family members, and easy visualization of the data in the alignment and in all super-family structures (fig. 1). 3DM has successfully been applied in a series of protein engineering studies all published in peer review articles. 3DM was used to chance enzyme specificity, increase enzyme activity, reveal an unknown reaction mechanism, predict specific double mutants that were more active than the wildtype protein, reveal the different binding modes of inhibitors and activating compounds, and 3DM was used for smart libraries design for improved thermostability and enantioselectivity.
Figure 1. CRL immobilization on PDLLA nanoparticles ________________ [1] L. Chronopoulou et al. Soft Matter (2011) 7: 26532662. [2] L. Chronopoulou et al. Langmuir (2009) 25: 11940-46.
Figure 1. 3D-numbers (white) enable automatic visualization of different data-types (correlated mutations (purple), conserved amino acids (yellow)) in any super-family structure. Ref: www.bio-prodict.nl/EN/publications
This poster is on display in the exhibitors room.
339
Molecular mechanisms of the effects of organic solvents on activity of haloalkane dehalogenases
Veronika Stepankovaa, Radka Chaloupkovaa, Morteza Khabirib, Jan Brezovskya, Zbynek Prokopa, Rudiger Ettrichb, Jiri Damborskya a Loschmidt Laboratories, Department of Experimental Biology and Centre for Toxic Compounds in the Environment, Masaryk University, Kamenice 5/A13, Brno, Czech Republic b Institute of Systems Biology and Ecology, AS CR, Zamek 136, Nove Hrady, Czech Republic E-mail: stepankova.veronika@gmail.com The use of enzymes in media-containing organic solvents significantly expands possibilities of their biotechnological applications [1]. This approach is particularly interesting for enantio- and regioselective catalysis used for the production of fine chemicals and optically active compounds. However, the catalytic activity of enzymes generally decreases in the presence of organic solvents. An understanding of molecular mechanisms governing the solvent effects on enzyme activity can be useful for selection of appropriate type and concentration of solvent. In the present study, we focused on the resistance of three haloalkane dehalogenases towards organic solvents. These enzymes are valuable catalysts due to their broad substrate specificity and enantioselectivity [2, 3]. Extensive activity screening of LinB from Sphingobium japonicum UT26, DhaA from Rhodococcus rhodochrous and DbjA from Bradyrhizobium japonicum USDA110 in the presence of 16 water-miscible protic and aprotic organic solvents demonstrates that the effect is different for individual enzymes, even though they belong to the same protein family. DbjA is activated in the presence of most tested organic solvents whereas activities of DhaA and LinB sharply decrease with increasing solvent concentration. Circular dichroism and fluorescence spectroscopy revealed that enzyme deactivation is connected with the conformation changes. The crystal structures of LinB, DhaA and DbjA were subjected to molecular dynamics simulations in the presence of three representative organic solvents, to elucidate the mechanisms of interactions between solvents and enzymes at the molecular level. Solvent molecules were found to enter the active site during the simulations. Moreover, the volume of the active site occupied by solvent differs from one enzyme to another and correlates with activity data. The steady-state kinetic parameters of LinB, DhaA and DbjA obtained in the same representative organic solvents indicate, that the activation of enzymes is attributed to solvent-induced stabilization of the transition state (increase of kcat), while the inactivation is caused by promotion of substrate inhibition (decrease of Ksi). Moreover, the improvement of affinity for tested substrate (decrease of Km) was observed for each studied case. Systematic analysis conducted with three well-characterised enzymes is useful for prediction of enzyme activity in organic solvents and for identification of the regions responsible for their resistance towards organic solvent. Selected regions can be targeted by protein engineering to design enzymes with higher stability in organic solvents.
________________ [1] Klibanov, A.M., Nature 409 (2001) 241-246 [2] Pieters, R.J., et al., Tetrahedron Lett. 42 (2001) 469-471 [3] Prokop, Z., et al., Angew. Chem. Int. Ed. 49 (2010) 6111-6115
340
First structures of an active bacterial tyrosinase and of variants with altered selectivity
Mor Sendovski Goldfedera, Rita Kanteevb, Vered Shustera, Noam Adirb and Ayelet Fishmana a Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel; bSchulich Department of Chemistry, Technion-Israel Institute of Technology, Haifa 32000, Israel E-mail: mors@tx.technion.ac.il Tyrosinase is a member of the type 3 copper enzyme family involved in the production of melanin in a wide range of organisms. Crystal structures of a tyrosinase from Bacillus megaterium were determined at a resolution of 1.7-2.3 . The enzyme crystallized as a dimer in the asymmetric unit, and was shown to be active in crystal. The overall monomeric structure is similar to that of the monomer of the previously determined tyrosinase from Streptomyces castaneoglobisporus, but it does not contain an accessory Cu-binding caddie protein. Two Cu(II) ions, serving as the major co-factors within the active site, are coordinated by six conserved histidine residues. However, determination of structures under different conditions, show varying occupancy and positions of the copper ions. This apparent mobility in copper binding modes indicates that there is a pathway by which copper is accumulated or lost by the enzyme. Residues R209 and V218, situated in a second shell of residues surrounding the active site, were suggested to play a role in substrate binding orientation, on the basis of their flexibility and position. Indeed, mutations introduced in these positions resulted in variants with higher monophenolase/diphenolase activity ratios compared to wild-type. Structures of variants R209H, V218G and V218F were determined and provided additional information on the importance of these positions. The determination of a structure with the inhibitor kojic acid, the first tyrosinase structure with a bound ligand, revealed additional residues involved in the positioning of substrates in the active site (Figure 1).
Figure 1. The structure of tyrosinase from Bacillus megaterium in complex with kojic acid; view of the active site.
_______________ [1] M. Sendovski, M. Kanteev, V. Shuster, N. Adir, A. Fishman, (2010) Crystallization and preliminary Xray crystallographic analysis of a bacterial tyrosinase from Bacillus megaterium, Acta Crystallographica F: Structural Biology and Crystallization Communication, 66 (9), 1101-1103. [2] M. Sendovski, M. Kanteev, V. Shuster, N. Adir, A. Fishman, (2011) First structures of an active bacterial tyrosinase reveal copper plasticity, Journal of Molecular Biology, 405 (1), 227-237.
October 2-6, 2011
158 343
The role of particle properties for IL-assisted enzymatic degradation of wood for biofuel production.
Helene Wulfhorsta, Tony Trieua, Carsten Flakea, Matthias Hoffmanna, Martin Falkenberga, Anita Ogolonga, Johann Hospital a, Jrn Viellb, Antje C. Spiea a AVT Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany; bAVT - Process Systems Engineering, RWTH Aachen University, Turmstr. 30, 52064 Aachen, Germany E-mail: Helene.Wulfhorst@avt.rwth-aachen.de Wood is a rich source for biofuel, materials and chemicals production, but it has a complex and recalcitrant structure. This hinders the enzymatic degradation of the main component cellulose to sugar monomers. Therefore, effective pretreatment is needed to destroy the high ordered structure of wood fibres and to improve the cellulose accessibility for cellulases.The various pretreatment methods result in characteristic structural properties of cellulosic substrate and affect its enzymatic degradability. The influence of those structural effects on the cellulose hydrolysis is still not sufficiently understood and quantified. In this contribution the effect of most important particle properties, particle size, crystallinity, and porosity on the hydrolysis reaction was analysed. Three cellulolytic model substrates, Sigmacell 101,-cellulose and Avicel were hydrolysed using the commercial cellulase preparation Celluclast (Novozyme, Novo Nordisk, DK) and at different substrate to enzyme ratios in aqueous system. The reduction of solid cellulose mass was followed online in shaken micro titer platesusing the BioLector (m2p labs, Aachen, DE) scattered light signal. The particle size distributions were analysed using a coulter counter. Two different porosity measurement techniques, differential scanning calorimetry (DSC) and Simons stain were applied for the analysis of the porosity of cellulose and lignocellulose particles. The software Parsival (CiT Rastede, DE) was used to model and simulate the particle population. The results were then transferred from model substrate to real system, beech and spruce, each pretreated with the ionic liquids EMIM Ac and BMIM Cl. The particle analysis shows that both the particle size and number of particles decrease during the enzymatic hydrolysis, leading to a decrease of the total surface area available for the enzyme adsorption. The reaction rate reduces proportionally to the amount of adsorbed cellulase. This phenomenon formed the basis for a simplified mechanistic model, whose parameters were determined from the hydrolysis data. Thus, potential limitations in the hydrolysis of cellulose can be detected and avoided. Highest reaction rates were detected for the most amorphous substrate with the smallest particle size. Moreover, this substrate also has the highest porosity, leading to the high available surface area. Accordingly, the hydrolysis experiments of wood particles pretreated with ionic liquids show that already short pretreatment times are sufficient for an efficient biomass hydrolysis. The porosity analysis indicates a significant increase of total surface area with pretreatment time in ionic liquids. The higher porosity correlates with increase of hydrolysis rates and sugar release of pretreated wood compared to untreated wood. Consequently, the dissolution in ionic liquid improves the degradability of wood due to the higher surface area available for enzyme adsorption.
New application of biocatalysis 344

In planta expression of hyperthermophilic -glucanase
a

Searching for new sources of ligninolytic enzymes for directed plant biomass decomposition
Alicja Kaczmarek, Rita Py, Milena Stpczyska, Tomasz Ramiga, Radosaw Wchaa, Mirosawa Szczsna-Antczak, Tadeusz Antczak Technical University of Lodz, Institute of Technical Biochemistry, Stefanowskiego 4/10, 90-924 Lodz, Poland; E-mail: alicja.kaczmarek@p.lodz.pl Abundance and diversity of lignocellulosic materials renders them excellent raw materials for energetics and chemical industry. Selected waste lignocellulosic materials can be also used to fabricate cellulose nanofibers with defined structural parameters that in turn can be applied to modify polymer materials and manufacture functional composites. One of objectives of presented project is the development of chemo-enzymatic procedure of cellulose nanoparticles releasing from selected plant biomass. Biotechnological tools for this purpose will be obtained within the frames of task 2.1 realized at ITB TUL. These tools are based on multienzyme preparations containing cellulases, hemicellulases and pectinases [1,2] in proportions adjusted to a particular biomass as well as ligninolytic enzymes making easier the access to cellulose fibers. Presented study aimed at selection of microbial sources of oxidoreductases (e.g. laccase, lignin peroxidase and Mn-peroxidase) capable of directed lignin degrading in various waste lignocellulosic materials. Microorganisms subjected to screening either originated from pure culture collection at ITB TUL or were isolated from waste plant biomass (wheat, oat, and hemp straws) potential sources of cellulose nanofibers. Screening procedure consisted of 2 steps. In the first of them, producers of laccase and other ligninases were selected in plate tests on solid media supplemented with guaiacol and alkaline lignin. The selected, most efficient producers of extracellular ligninolytic enzymes were subjected to further screening in liquid culture media. The laccase and lignin peroxidase synthesizing (12 and 29 u/ml, respectively) strain Trichoderma sp. QM9123 was found to be their best producer among 39 tested strains. Laccase biosynthesis conditions were optimized by mathematical method of Box and Wilson which increased laccase synthesis yield by 4.5-fold. The obtained enzyme preparation was used in directed degradation of wheat and oat straws. The study was realized within the scope of the project POIG 01.01.02-10-123/09 Application of biomass in production of environmentally friendly polymer materials task PZ 2.1., co-financed from the funds of European Fund of Regional Development within the frames of Operation Program Innovative Economy 2007-2013.
________________ [1] Patent RP, 209163 (2011) [2] Patent RP, 209161 (2011)
159 348
Chemo-enzymatic conversion of oils into polyols components of polymers
Mirosawa Szczsna-Antczak1, Agnieszka Borowska1, Magorzata Rzyska1, Dariusz Hiler1, Julia Gibka2, Tadeusz Antczak1 1 Institute of Technical Biochemistry, ( 2Institute of Food Chemistry Fundamentals) Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland; E-mail: miroslawa.szczesna-antczak@p.lodz.pl An objective of a task of a project with acronym Biomass, which is realized at the Institute of Technical Biochemistry TUL, is the development of a method of conversion of oleaginous biomass (mainly rapeseed oil) into oligo-/polyols which will be used in production of biodegradable alkyl-aryl co-polyesters. The concept of this procedure is based on chemo-enzymatic conversion of triacylglycerols (TAGs) into oligomers which will be converted by enzymes into oligo-/polyols [1]. Oligomers of triacylglycerols are obtained by chemical method from enzymatically modified plant oils in which unsaturated fatty acids (UFAs) are partly substituted with saturated FAs (stearic and/or palmitic). Lipases with sn-1,3 specificity are used in two enzyme-catalyzed steps of the procedure. One of them is immobilized preparation of intracellular Mucor circinelloides lipase which is predestined to catalysis in non-aqueous media [2,3]. Presented study focused on optimization of conditions of enzymatic structurization of trioleate (or rapeseed oil) with saturated FA in acidolysis process (time, temperature, molar ratio of substrates, type and amount of a solvent, water content in reaction mixture). The course of reaction was controlled by quantification of free FAs (by copper soaps method, [4]) in FAs fraction isolated from reaction mixture (on aminopropyl SPE mini-columns) and quantification of UFAs by sulpho-phospho-vanilin method [5]. The mathematical model was developed and used to calculate the degree of enzymatic modification of TAGs. The course of acidolysis reaction was also monitored by HPLC analysis of TAGs fraction (RP C18 column, J.T. Baker, detector NQAD from Quant Technologies) and by GC. Production of structured TAGs in continuous processes was also tested. The study was realized within the scope of the project POIG 01.01.02-10-123/09 Application of biomass in production of environmentally friendly polymer materials task 3.2., co-financed from the funds of European Fund of Regional Development within the frames of Operation Program Innovative Economy 2007-2013.
________________ [1] P. Kiatsimkul, G.J. Suppes, W. R. Sutterlin, Ind. Crops & Prod., 25, 2007, 202209 [2] M. Szczesna-Antczak, T. Antczak, M. Rzyska, Z. Modrzejewska, J. Patura, H. Kalinowska, S. Bielecki:, J. Mol. Cat. B, Enzymatic, 29, 2004, 163-171 [3] Antczak T., Graczyk J., Szczesna-Antczak M., Bielecki S. J. Mol. Cat. B, Enzymatic 19-20, 2002, 287-294, [4] R. R. Lowry, I. J. Tinsley, J Am. Oil Chem. Soc, 1976, 53, 470-472 [5] J. A. Knight, S., Anderson, J. M. Rawle, Clin. Chem. Soc. 1972, 18, 199-202
H. Klosea, J. Rdera, M. Girfoglioa R. Fischera,b & U. Commandeura RWTH Aachen University, Institute for Molecular Biotechnology, Worringerweg 1, 52074 Aachen, Germany; bFraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, 52074 Aachen, Germany E-mail: holger.klose@rwth-aachen.de Lignocellulosic biomass derived from plant cell wall is one of the most abundant resources for renewable energy. For this reason, increasing efforts are made to develop a convenient processes to breakdown the main constituents of the cell wall to fermentable free sugars and to use these as starting material for the production of biofuels. Common approaches for cell wall deconstruction consist of physico-chemical pretreatment steps followed by enzymatic hydrolysis of the cell wall polysaccharides. In planta heterologous expression of cellulolytic enzymes with very good catalytic performances at harsh conditions could be a promising approach to greatly improve these processes. In this study, we could demonstrate that the expression of the sso1354 gene coding for an endoglucanase from the hyperthermophilic Archaeon Sulfolobus solfataricus in tobacco plants results in the production of a functional endoglucanase. The recombinant enzyme is active at high temperatures with an optimum at 90C and retains activity in high concentrations of cellulose dissolving ionic liquids. Due to its intrinsic features, the S. solfataricus endoglucanase could be an ideal candidate for an improved process of biomass conversion to fermentable sugars utilizing plant produced cellulolytic enzymes.
356
Lipase/acyltransferase-catalyzed alcoholysis of raw coconut oil emulsion in uidized- and packed-bed bioreactors
Vronique Perrier, Nakry Pen, Benot Moreau, Eric Dubreucq UMR IATE, Montpellier SupAgro, 2 Place Viala, F-34060 Montpellier, France E-mail: Vronique.Perrier@supagro.inra.fr The ability of the lipase/acyltransferase CpLIP2 from Candida parapsilosis to catalyze alcoholysis in aqueous emulsions with high thermodynamic activity of water (aW>0.9) allows the use of aqueous solutions instead of dehydrated alcohols [1, 2]. Reaction can be performed without the use of organic solvent and no control of aW. It also allows an easy separation of polar vs. lipid products. These conditions permit the development of intensified and environmentally friendly transesterification processes [3]. In the work presented here, recombinant CpLIP2 [4] was immobilized and used to perform the methanolysis at 30C of an emulsion containing 75 % v/v raw coconut oil in a 1L-scale fluidized bioreactor and in a two-step continuous packed-bed bioreactor. The emulsion was obtained by mechanical stirring without the addition of emulsifier. A 90% conversion into methyl esters, without net release of free fatty acids, was achieved in 9h in the fluidized bed reactor (97% in 24h). In the packed-bed reactor, 90% conversion was obtained with a residence time of 1.5h. No significant loss of activity was observed after 10 batches of 24h in the fluidized bed reactor and in 14 days of operation in the packedbed reactor.
________________ [1] Briand D., Dubreucq E., Galzy P. (1995) Functioning and regioselectivity of the lipase of C. parapsilosis (Ashford) Langeron and Talice in aqueous media. New interpretation of regioselectivity taking acyl migration into account. Eur. J. Biochem., 228, 169-175 [2] Dubreucq E., Bigey F., Moulin G., Weiss, A. Lipase/Acyltransferase. Int. Pat. WO03006644, EP1275711, US7247463 [3] Dubreucq E.,Weiss A., Gutsche B., Fabry B., Moulin G. Process for the production of fatty acid alkyl esters. Int. Pat. WO07140862, EP07004377, JP 2007502103, US20100234458 [4] Brunel L., Neugnot V., Landucci L., Boze H., Moulin G., Bigey F., Dubreucq E. (2004) Cloning and overexpression of lipase/acyltransferase from C. parapsilosis in P. pastoris. J. Biotechnol. 111, 41-50
346
Biological oxidation and amination from renewables to diamines
Jan Pfeffer, Steffen Schaffer, Thomas Haas Evonik Degussa GmbH, Paul-Baumann-Strae 1, 45764 Marl, Germany E-mail: jan.pfeffer@evonik.com The aim of the project is the establishment of a technology platform for the conversion of cheap and readily available renewable resources to functional amines as monomer building blocks. Functional amines, e.g. diamines or aminocarboxylic acids are important starting compounds for polymers, e.g. polyamides, polyurethanes or epoxides. Today functional amines are usually produced from petrochemical resources via complex chemical processes. The production of functional amines via biotechnological routes or a combination of biotechnological and chemical processes has two advantages: on the one hand complex processes leading to known and established amines can be replaced partially or entirely by new processes that preserve resources. On the other hand the variety of natural amino-metabolites can be converted easily in few biological or chemical steps into new multifunctional amines which are not accessible via pure chemical routes. The feasibility of the concept of this project is first demonstrated using isosorbide as substrate. After conversion with cognate oxidoreductases and aminotransferases the corresponding diamines will be formed. Using new as well as previously characterized aminotransferases we were able to synthesize these new-to-the-world diamines starting from an intermediate based on isosorbide. Conversion of the di-alcohol to the corresponding diamines will be performed in a whole-cell biocatalyst producing the relevant enzymes.
349
One-pot enzymatic depolymerization of cellulose in [BMIm][Cl]
Paola DArrigoa,b, Lorenzo Ceriolia, Andrea Melea,b, Loredano Pollegionib,c, Chiara Signa, Stefano Tamborinib,c, Davide Tessaroa,b a Politecnico di Milano, Department of Chemistry, Materials, and Chemical Engineering, Piazza Leonardo da Vinci 32, 20133 Milano, Italy b The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universit degli Studi dellInsubria, via Mancinelli 7, 20131 Milano, Italy c Dipartimento di Biotecnologie e Scienze Molecolari, Universit degli Studi dellInsubria, Via Dunant 3, 21100 Varese, Italy E-mail: loredano.pollegioni@uninsubria.it Lignocellulosic biomass is a renewable, low cost, non-food and abundant feedstock for biofuel production and a source of C atoms for chemistry alternative to fossil resources. The most important factor hampering massive exploitation of cellulose, the main component of plant biomass, is its well known low reactivity and recalcitrance to chemical processing. The usual steps in producing biofuels from cellulosic sources are: pretreatment followed by cellulose precipitation, enzymatic hydrolysis and fermentation. Pretreatment of cellulose is a necessary step in the production of ethanol from cellulosic material since it makes the recalcitrant cellulosic biomass more accessible to enzymatic hydrolysis. A possible greener alternative for the activation of cellulose fibrilles towards facile hydrolysis and/or derivatization passes through an emerging chemical pretreatment step using ionic liquids (ILs) [1]. Unlikely, cellulases have been reported to be inactivated by ILs [2]. In the present work we present a study on a single-batch, homogeneous phase enzymatic hydrolysis of cellulose using a commercial IL, 1-butyl-3-methylimidazolium chloride ([BMIm][Cl]), which is a well known good solvent for celluloses [3]. We have tested two native proteins from Trichoderma reesei and Humicola insolens and two engineered proteins from T. reesei and Streptomyces sp.. The [BMIm][Cl] does not denature the cellulases used, increases their operational stability (at 75C) as compared to standard buffer solutions and facilitates the dissolution of cellulose. Interestingly, the stability of the four cellulases in the presence of the IL allows to set-up a procedure lacking of the cellulose pretreatment step. We believe that this strategy could be amenable of scale-up and innovative industrial applications for the efficient one-batch conversion of inexpensive cellulosic materials into derivatives (biofuels, derivatized cellulose, monosaccharides for fine chemicals, etc.) with high potential commercial interest and in the framework of environmentally friendly chemistry.
________________ [1] Dadi AP, Varanasi S, Schall CA. Biotechnol Bioeng 2006, 95 (5), 904-910. [2] Turner MB, Spear SK, Huddleston JG, Holbrey JD, Rogers RD. Green Chem 2003, 5(4), 443-447. [3] Zhu SD, Wu YX, Chen QM, Wang C, Jin S, Ding Y, Wu G. Green Chem 2006, 8, 325-327.
350
Design of an enzymatic FAEE-biodiesel process
Mathias Nordblada, Yuan Xua, Jesper Braskb and John M. Woodleya a Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark; bNovozymes A/S, Krogshjvej 36, DK-2880 Bagsvrd, Denmark E-mail: man@kt.dtu.dk Biodiesel is receiving increasing attention as a promising alternative to liquid fossil fuel for vehicles, being a direct replacement for petrochemical diesel. Compared to the conventional biodiesel production process, enzymatic synthesis offers several advantages such as lower reaction temperature and eliminated soap formation, as well as greater flexibility with respect to oil feedstocks. However, compared to the homogeneous alkaline catalysts that are used in the conventional process, the cost of the enzyme-based catalysts is considerably higher and it is therefore critical that their reuse is considered, both in the formulation of the catalyst and in the reactor design. The process design is complicated by the multi-phasic nature of the biodiesel reaction system, which can result in inhibiting or inactivating conditions for the enzyme catalyst, thereby reducing its productivity (amount of product/kg catalyst). To avoid this, well-mixed conditions and controlled substrate supply should be ensured. Furthermore, it is highly desirable to maximize the yield of biodiesel from the oil feedstock through near-complete conversion. To achieve the level of conversion required to meet the very strict specifications on biodiesel transportation fuel, it is necessary to employ methods for separation of both by-products of the process, water and glycerol. Finally, it is also important that the process is designed with continuous operation in mind, if it is to be operated cost-effectively at scale. This work presents an overview of the process characteristics and requirements for large-scale enzymatic biodiesel production, together with options for process design, equipment choice and operating conditions.
October 2-6, 2011
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Immobilization of endo-xylanase for xylo-oligosaccharides production from xylan oligomers extracted from sugarcane bagasse using alkaline pretreatment
Anny Manrich1, Viviane Marcos Nascimento1, Roberto de Campos Giordano1, Raquel de Lima Camargo Giordano1* 1 Federal University of So carlos- Chemical Engineering Department, Rodovia Washington Lus, km 235 CEP: 13565-905, So Carlos, SP, Brazil *E-mail: raquel@ufscar.br XOS, small oligomers of xylose (2-5 units), are estimable ingredients for special diet food and pharmaceuticals and can be obtained through controlled enzymatic hydrolysis of xylan, one of the main hemicelluloses polymers. In this work, the commercial endoxylanase (NOVOZYMES 22036) was immobilized for XOS generation from the xylan oligomers obtained after alkaline/NaOH pretreatment of sugarcane bagasse. This raw material, abundant and available in Brazil, contains circa 30% of hemicelluloses (w/w). Generation of xylose must be avoided in the both steps extraction and hydrolysis, since its separation from XOS is expensive. Enzymes are potent and specific catalysts, but also are expensive and fragile. Their immobilization and stabilization may reduce the hydrolysis costs. Xylanase was immobilized in agarose 6BCL activated with glyoxyl groups, at pH 10.05, 25C, for 24 hours. Immobilization yield was around 100% and 90% of offered activity was measured after immobilization. Thermal inactivation studies indicated stability improvement of 55 times after immobilization, being the half-live of the immobilized enzyme 9.5 hours, at 70C, while the soluble xylanase presented half-live of 10 minutes. Xylan alkaline extraction was performed in autoclave at 121C, with NaOH concentrations of 1, 4 and 7% (m/v)), dry solid/liquid relation 1/10 (m/m) and reaction time from 15 to 90 minutes. Best treatment condition was found to be 60 minutes, 4% NaOH, which led to 55% of xylan extraction, without monomers production. In order to produce XOS, soluble and immobilized xylanases were used to hydrolyze commercial birchwood xylan (as control) and the extracted sugarcane bagasse xylan oligomers, at 50C, with 20 UI/mL. Soluble and immobilized enzymes were effective to hydrolyze the both substrates.

Production of ethyl esters via enzymatic catalysis with different canola oil / alcohol molar ratios
Joo Henrique Dantasa, Leandro Daniel de Parisa, Carlos Eduardo Baroa, Pedro Augusto Arroyoa, Flvio Faria de Moraesa, Gisella Maria Zanina. a State University of Maringa, Chemical Engineering Department, Avenida Colombo 5790, PR-Brazil. E-mail: gisella@deq.uem.br Diesel derived from petroleum can be replaced by ethyl esters produced from vegetable oil. Vegetable oil may be applied directly into engines for the replacement of diesel but some adverse effects normally occur. To circumvent these problems a transesterification reaction can be performed with the vegetable oil and ethanol producing ethyl esters. Transesterification is a reaction that involves a triglyceride and an alcohol in a minimum molar ratio of 1:3 (oil:alcohol) forming esters of that alcohol and glycerin. This reaction can be performed by chemical route, supercritical, catalytic and enzymatic. The enzymatic route is important due to environmental appeals of the green chemistry, the ability to esterify oils high in fatty acids and also carry out the reaction under mild conditions of temperature and pH, but the high reaction time, high operating cost and difficulty of using high molar ratios of oil:alcohol are problems to be surmounted. In reactions using chemical catalysts, excess alcohol is used to ensure a high conversion and minimize the effects of diffusional restrictions due to high viscosity. However, in enzymatic synthesis, excessive levels of alcohol can inhibit the enzyme and decrease its catalytic activity during reaction. According to Salis et al. (2005) [1], a high ratio alcohol:substrate means a higher polarity of the medium that may be associated with inactivation of the biocatalyst or the possibility of destabilizing the layer of water essential to the catalytic site of these biocatalysts [2]. This work carried out transesterification reactions using canola oil and ethanol added in stages (three equal portions, one at the beginning, and two others at 12 and 24 h). The reactions were conducted in batch reactors at 37 C using 5% (w/v) of lipase from Thermomyces lanuginosus in relation to oil and reaction time of 72 h. The molar ratios of oil:alcohol were 1:3, 1:6, 1:9 and 1:12. The production of ethyl esters and triacylglycerol consumption were analyzed by HPLC. The results are shown in Table 1 Table 1. Enzymatic activities and esters yield

Biodiesel Synthesis from the Cyanobacteria non-microcystin producer Microcystis aeruginosa NPCD-1 by Enzymatic Route
Da Rs P. C. Ma, Silva C. S. Pb, Giraldelli1, M. Ga, Fiore M. Fb, De Castro H. Fa a Engineering School of Lorena, University of So Paulo, P.O. Box 116, 12602-810 Lorena, So Paulo, Brazil; bCenter for Nuclear Energy in Agriculture, University of So Paulo, 3400-970, Piracicaba, So Paulo,Brazil. E-mail: patriciadaros@dequi.eel.usp.br Cyanobacteria have significant potential for producing a wide range of products including lipids, carotenoids, pigments, vitamins and aromatic compounds. The production of lipids is of particular interest because of their potential to be used as feedstock for biodiesel synthesis. In a previously work, the cyanobacteria Microcystis aeruginosa NPCD-1, strain isolated from sewage treatment plant and characterized as non-microcystin producer[1], was found to be a potential source of lipid when cultivated in ASM-1 medium at 251C under constant white fluorescent illumination (109 mol photon m2 s1). In these conditions, biomass productivity of 46.92 5.84 mg.L1.day1, lipid content of 28.10 3.47% and lipid productivity of 13.20 0.20mg.L1day1 were obtained. Qualitative analysis of the fatty acid methyl esters demonstrates high proportion of saturated fatty acids (50%) among which stands out palmitic (24.34%) and lauric (13.21%) acids. The others 50% are constituted by unsaturated fatty acids, with higher concentrations for oleic (26.88%) and linoleic (12.53%) acids. Such composition is similar to that one found in vegetable oils, such as palm oil a feedstock that showed potential to be converted in biodiesel using different catalysts, including also biocatalyst (immobilized lipase)2. To check the feasibility to produce biodiesel from the cyanobacterium lipid, enzymatic transesterification runs were performed using 1:12 molar ratio (biomass oil/ethanol) at 50C assessing isooctane and tert-butanol as solvents and immobilized lipase (Novozym 435) as catalyst, for a maximum periodic of 48h. Batch runs were also carried out under the same reaction conditions using palm oil instead of microbial biomass oil. Results showed similarity on the main ethyl esters formed for both feedstocks. As expected, the highest ethyl ester concentrations were in regard to palmitate and oleate esters followed by laurate and linoleate esters. However, both the reaction rates and ester yields were dependent on the solvent tested. Total ethyl ester concentrations varied in the range from to 44.24 to 67.84%, corresponding to ester yields from 80 to 100 %. Iso-octane provided better solubility and miscibility of the starting materials and ester yield of 98.10% was obtained at 48 h for reaction performed with the lipid material and full conversion was achieved in 12h for reaction carried out with palm oil. These results are favorable compared with those reported using oil from microalgae and cyanobacteria as feedstocks. Further research is still necessary in this fast-moving field, particularly for cyanobacteria strains which potential as a source of raw material for biodiesel has not been deeply studied.
Acknowledgements: The authors gratefully acknowledge the financial assistance from Brazilian Agencies (FAPESP and CAPES). ______________________________ [1] Silva-Stenico et al, Non-ribosomal peptides produced by Brazilian cyanobacterial isolates with antimicrobial activity. Microbiol. Res. v. 166, p. 161-175, 2011. [2] Moreira et al, Biodiesel synthesis by enzymatic transesterification of palm oil with ethanol using lipases from several sources immobilized on silica-PVA composite. Energ. Fuel. v. 21, p. 3689-3694, 2007.
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Immobilized lipases catalyze the conversion of oils from coffee waste into biodiesel
Diana Fattora, Elisabetta De Angelisb, Patrizia Spizzoc, Luciano Navarinib, and Lucia Gardossia,c a Dipartimento di Scienze Chimiche e Farmaceutiche, Universit degli Studi di Trieste, Piazzale Europa 1, 34127 Trieste, Italy b Illycaff S.p.A., via Flavia 110, 34147 Trieste, Italy c SPRIN S.p.A., Technology for Sustainable Chemistry, via Flavia 23/1, 34148 Trieste, Italy E-mail: diana.fattor@phd.units.it Here we report on the development of a sustainable process for the enzymatic conversion of oils from coffee waste into biodiesel. Lypases (EC 3.1.1.3) were immobilized and applied as heterogeneous bio-catalyst for the transesterification of the waste oils. Mild conditions and stoichometric amounts of substrates (oil and methanol) are required to perform the enzymatic synthesis of biodiesel. Since lipases can catalyze also the esterification reaction the removal of free fatty acids is not required before the starting of the reaction. This represents one of the major advantages as compared to the basic catalysts, which reacts with the fatty acids giving the corresponding salts. Moreover, the glycerol obtained shows higher purity so that its commercial value is improved. In this study different samples of oils from coffee wastes were tested and the activity and stability of two immobilized lipases assessed. The results indicate that coffee waste oils can be converted quantitatively within hours. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. Keywords: coffee waste oil, enzymatic synthesis of biodiesel, lipases
AT Enzymatic activity of triacylglycerol consumption, AEST Enzymatic activity of ethyl esters formation
The best yield was observed in the molar ratio of 1:12 oil:alcohol. However, the best result of enzymatic activity in the production of esters was observed for molar ratio of 1:3. This suggests that a greater amount of alcohol promotes the formation of products, but can accelerate the deactivation of the lipase.
_____________________ [1] Salis, A.; Pinna, M.; Monduzzi, M.; Solinas, V.. J. Biotechnol.,119, 291-299, 2005. [2] Kose, O., Tuter, M., Aksoy, H.A. Bioresource Technology, 83, 125-129, 2002.
353
Process design for continuous biodiesel production
Hemalata Ramesha, Yuan Xua, Mathias Nordblada,*, Anders Rancke-Madsenb and John M. Woodleya a Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark b Novozymes A/S, Krogshjvej 36, DK-2880 Bagsvrd, Denmark *Corresponding author: man@kt.dtu.dk Tel: 0045-45252804 Fax: 0045-45932906 Lipases are well-established catalysts for many reactions, and have recently been shown to have great potential for large scale production of biodiesel . Batch operation is straightforward and efficient for such reactions at small scale. However, for large-scale production of biodiesel, continuous operation is an attractive alternative as it enables efficient use of manpower and capital assets including equipment and raw materials. Continuous operation has been extensively carried out in tubular reactors such as the packed bed reactor (PBR). However, the formation of glycerol, which separates into a second bulk phase during the reaction, poses a problem as it clogs the enzyme, leading to loss of catalyst activity and consequently a lower reaction rate and shorter enzyme life-time. For this reason, a well-mixed continuous stirred tank reactor (CSTR) system is particularly interesting for transesterification reaction. The disadvantage of this system is that it cannot achieve equilibrium, since CSTRs operate at leaving conditions. This also reduces the volumetric efficiency compared to batch STR. However, the efficiency of the reactor system can be significantly improved by using reactors in series. The scope of this work was to study the effects of operating parameters on the performance of a CSTR using immobilized lipases, and eventually determine the optimal conditions. This investigation involved carrying out batch reactions in a STR and using the reaction data to determine the rate at which glycerides are converted to define CSTR operation. Experimental set points, specifically the residence time and feed conditions, were then calculated based on this reaction profile and validated in a CSTR. A comparison of a batch STR and a 3-stage CSTR system in terms of residence time and oil processed show that they are similar in performance. In an attempt to further facilitate the conversion of glycerides to biodiesel, several process alternatives have been evaluated. This involved using a combination of reactors (PBR and batch STR) to react the CSTR effluent further. Similar equilibrium conversions were achieved when using PBR and STR at different conversion levels, meaning that a completely continuous operation is not only feasible but is also equally efficient as a batch system. These findings make continuous biodiesel production an attractive option.
_____________ [1] Nielsen et.al., (2008) Enzymatic biodisel production: Technical and economical considerations. Eur. J. Lipid Sci. Technol, 692-700
354
Screening of oleaginous yeasts and obtaining of their oils to produce biodiesel, using residual glycerol from the process as carbon source.
a
357
Microwave activation of immobilized lipase for transesterification of vegetal oils
Monna Lisa B. Queiroza, Heiddy M. Alvarezb, Cleide M. F. Soaresa,c, lvaro S. Limaa,c, Montserrat Fortuny Herediaa,c, Claudio Darivaa,c, Alini T. Fricksa,c* a *UNIT - Universidade Tiradentes. Av. Murilo Dantas, 300, 49032-490, Farolndia, Aracaju, SE, Brasil. bDepartamento de Cincias Exatas, Universidade Estadual de Feira de Santana, km 03, BR 116, Campus da UEFS, Feira de Santana, BA 44031-460, Brasil. cITP - Instituto de Tecnologia e Pesquisa, Av. Murilo Dantas, 300, 49032-490, Farolndia, Aracaju, SE, Brasil E-mail: alinitf@yahoo.com.br Microwave energy can promotes a significant effect on esterification and transesterification enzymatic reactions rates [1,2]. This work investigated the effect of microwave irradiation (MI) on the rate of transesterification of soybean and sunflower oils with ethanol catalyzed by supported enzyme, Novozyme 435 (Candida antarctica) and compared with transesterification activity under conventional heating (CH). All the experiments were performed at 60 C. Transesterification using CH was investigated employing soybean oil, with 3.36 % (w/w) of enzyme, evaluating the effects of: percentage of tert-butanol, water addition and oil:ethanol molar ratio. Under the optimum conditions (oil:ethanol molar ratio 1:3, tert-butanol 10% (w/w), without water addition) the formation of biodiesel from soybean and sunflower oils in CH was monitored up to 24 h of reaction to determine the conditions of initial velocity of reaction. The investigated variables under MI (50 W) were: reaction time (5.0 - 90 min) and mode of reactor operatorion (fixed power, dynamic and cycles) in the absence and presence of tert-butanol (10 % (w/w)). The measured response was reaction conversion which was connected to the catalytic activity: one unit (U) of enzyme specific activity was defined as the amount of enzyme that was necessary to produce 1 mmol fatty acid ethyl esters per minute. The results indicated that the use of microwave improves the activity at fixed power and dynamic modes. It was observed a significant effect of the association of tert-butanol and microwave irradiation on the catalytic activity. Compared to CH, the enzymatic reaction rate was increased under microwave heating for soybean and sunflower oils using tert-butanol as a cosolvent. (Figure 1).
358
Biodiesel waste-glycerol to added-value metabolites. broadening the biorefinery
a
Niehus Xochitla,b, Sandoval Georginaa, Gschaedler Annea, Pelayo Carlosb Centro de Investigacin y Asistencia en Tecnologa y Diseo del Estado de Jalisco (CIATEJ), 800 Normalistas Ave. Guadalajara, Jalisco, Mexico. bUniversidad de Guadalajara, Centro Universitario de Ciencias Exactas e Ingenieras (CUCEI), 1421 Marcelino Garcia Barragan Blvd. Guadalajara, Jalisco, Mexico. E-mail: xniehus@hotmail.com / georgina@confluencia.net Oleaginous microorganisms are considered to be those able to accumulate more than 20% of their dry cell weight as oils, sometimes up to 70% [1]. These oils are also called Single Cell Oils (SCO). They are mainly triacylglycerols which can be compared, in terms of their chemical composition, to the oils and fats obtained from plant oilseeds. The SCO contain mostly oleic (18:1), palmitic (16:0), steatic (18:0), and linoleic (18:2) fatty acid residues [2]. The quality and quantity vary from specie to specie. Among the oleaginous microorganisms, yeasts have many advantages: 1) high biomass yield, 2) they are able to grow up on a wide variety of carbon sources, 3) simpler cellular walls which makes lipid extraction easier, 4) in high C/N ratio media they can convert excess carbon into lipid, and 5) their physiology and genetics are well known. Biodiesel is a common liquid bio-fuel that can be used as a substitute for petroleum based diesel. The conventional raw materials to produce biodiesel are plant oils which represent 70 to 90% of the costs. Since microbial oils have similar chemical composition, they can also be used as alternative raw materials for biodiesel production, and in this case, using cheap carbon sources, the costs can be diminished. In the biodiesel production process, glycerol is a byproduct; in which for every 10 kilograms of biodiesel, 1 kilogram of residual glycerol is also produced. This means that, with the rapid development of the global biodiesel industry, more and more crude glycerol will be available. In this study, more than 30 yeasts from CIATEJs collection were tested for their oil production capacity using firstly anhydrous glycerol and secondly crude glycerol from the production of biodiesel. The objective was to select, at least, one yeast able to grow on medium with crude glycerol as the only carbon source, with the highest lipid yield. Lipase activity was also monitored in the selected strains, since many lipase production microorganisms coincide with those oleaginous.
Sergi Abada, Ilaria Mannazzub, Xavier Turona IQS-Institut Qumic de Sarri, Ramon Lllul University, via Augusta 390,Barcelona, Spain; b Universit degli Studi di Sassari,Viale Italia 39, Sassari, Italy E-mail: xavier.turon@iqs.edu Biodiesels waste-glycerol is used as a carbon source in fermentations. The metabolites obtained are added-value products, typically nutraceuticals as poly unsaturated fatty acids (PUFA) or Beta-carotenes. The results presented are from fermentations based on heterotrophic micro algae and yeast. The added value of such molecules are contributing to the economic viability of a biodiesel biorefinery, broadening the platform as includes chemicals to the liquid fuels obtained.
________________ [1] C. Ratledge, "Microorganisms for lipids," Acta Biotechnologica, vol. 11, pp. 429-438, 1991. [2] C. Ratledge and J. P. Wynn, "The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms," in Advances in Applied Microbiology. vol. Volume 51, ed: Academic Press, 2002, pp. 1-44.
_______________ [1] H.V. Kamath, I. Regupathi, M.B. Saidutta. Fuel Processing Technology 92 (2011) 100105. [2] B. M. Nogueira, C. Carretoni, R. Cruz, S. Freitas, P. A. Melo Jr, R. Costa-Flix, J. C. Pinto, M. Nele. Journal of Molecular Catalysis B: Enzymatic 67 (2010) 117121.
Figure 1. Residual activity of Novozyme 435 under microwave compared with CH (7). 1. fixed power, tert-butanol absent; 2. fixed power with tert-butanol; 3. Cycles, tert-butanol absent; 4. Cycles with tert-butanol; 5. Dinamic, tert-butanol absent; 6. Dinamic with ter-butanol.
October 2-6, 2011
162 359
Immobilization of Pseudomonas lipases for biodiesel production
Cesarini Silvia, Martinez Marta, Javier Pastor, Pilar Diaz University of Barcelona, Faculty of Biology, Department of Microbiology E-mail: silviacesarini@ub.edu Immobilization of enzymes plays an important role within applied catalysis and biotechnology. The main reason for immobilizing enzymes is the ability to isolate the biocatalyst from the reaction product and to re-use it for cost reduction and productivity increase. Moreover, immobilization enhances enzyme properties such as stability and activity under harsh conditions of temperature, pH or presence of organic solvent [1]. Lipases (triacylglycerolhydrolases) combine a broad substrate specificity with a high enantio and regioselectivity. For this reason they are used in different industrial applications as modification of fats and oils, production of intermediates for organic synthesis or in the resolution of racemic mixtures. In lipase catalyzed reactions, immobilization can also help in providing non-aqueous conditions necessary for ester synthesis and transesterification of the triacylglicerides contained in vegetable oils, as in the synthesis of biodiesel. In this work we performed the immobilization of three novel lipases isolated from Pseudomonas strains: Lip1.3, an extracellular lipase from Pseudomonas sp CR-611 (close to P. fluorescens), and LipA and LipC, two lipases isolated from Pseudomonas sp 42A2 (close to P. aeruginosa). In previous works Lip1.3 was expressed in E. coli DH5 and LipA and LipC were overexpressed with their specific foldase (LipH) in a mutant strain of P. aeruginosa PAO1 lacking the genomic copy of lipA and lipH. For LipI.3 immobilization, solubilised inclusion bodies were used, whereas direct culture supernatants were used for LipA and LipC, thus skipping several time-consuming steps of protein purification. Since adsorption is still one of the most commonly used immobilization approaches due to its easy use and low price, two different supports for immobilization of each enzyme were tested: a highly porous polypropylene matrix (Accurel EP100), and diatomaceous beads composed of silica (Celite 545) [2]. Because their chemical inertness and unique interconnected pore structure, these supports are very suitable for physical adsorption of lipases, mostly based on hydrogen bonding, Van der Waals forces and hydrophobic interactions. The immobilization conditions for all three enzymes were set up for each support (temperature, ph and enzyme/support ratio), and the resistance to lyophilization and drying methods of each enzyme immobilized onto both supports was evaluated. Also the possibility to store the immobilized enzymes for long time was investigated.
________________ [1] Christensen M. W., Andersen L., Husum T. L., Kirk O.(2003) Eur. J. Lipid Sci. Technol. [2] Ferrer M., Plou F.J., Fuentes G., Cruces M.A., Andresen L., Kirk O., Christensen M., Ballesteros A. (2002) Biocatalysis and Biotransformation

Immobilization of photobacterium lipolyticum lipase by cross-linked enzyme aggregate method for the production of Biodiesel
Jin Yee Han, Hyung Kwoun Kim Division of Biotechnology, The Catholic University of Korea, Bucheon 420743, Republic of Korea E-mail: yni8708@naver.com Biodiesel is defined as methyl and ethyl esters of long chain fatty acids produced from vegetable oils or animal fats. Lipase enzymes have been occasionally used to produce this biofuel. Recently, biodiesel production using immobilized lipase has received a great attention. Through the enhanced stability and reusability, immobilized lipase can contribute to reduce the cost for biodiesel production. In this study, methanoltolerant lipase M37 from Photobacterium lipolyticum was immobilized using crosslinked enzyme aggregate (CLEA) method. Lipase M37 has high lysine content (9.7%) in the protein sequence. Most lysine residues located evenly on the surface of the protein except for the region of lid structure, which made the CLEA preparation yield very high (~93%). CLEA M37 showed optimum temperature of 30, and optimum pH of 910. It was stable up to 50 and in a pH range of 4.011.0. Both soluble M37 and CLEA M37 were stable in the presence of high concentration of methanol, ethanol, 1-propanol and n-butanol. That is, their activities were maintained at the solvent concentration higher than 10% (v/v). CLEA M37 could produce biodiesel from olive oil and alcohols such as methanol and ethanol. In addition, CLEA M37 produced biodiesel by both 2step methanol feeding procedures. In view of physical stability and reusability, CLEA M37 can be used as potential catalyst in the organic synthesis including biodiesel production reaction.

Mechanistic insights in enzymatic surface functionalization of polyesters
Doris Ribitscha, Enrique Herrero Aceroa, Georg Steinkellnera, Karl Grubera, Katrin Greimela, Inge Eiteljoerga, Eva Trotschaa, Wolfgang Zimmermannc, Artur CavacoPaulod , Giuliano Freddie, Helmut Schwaba, Georg M. Guebitza,b a Austrian Centre of Industrial Biotechnology ACIB, Petergasse 14, 8010 Graz, bInstitute of Environmental Biotechnology, Graz University of Technology, cInstitute of Biochemistry, University of Leipzig, Leipzig, Germany, dDepartmement of Textile Engineering, University of Minho, Guimaraes, Portugal, eStazione Sperimentale per la Seta, Milano, Italy E-mail: guebitz@tugraz.at The major advantage of enzymatic functionalization of polymeric materials enzymes over chemical processes lies in the surface specific and endo-wise mode of action of enzymes combined with mild reaction conditions. Surface hydrophilisation of polyesters is a crucial step in processing for many applications ranging from electronics and textiles to medical. Limited surface hydrolysis of PET with lipases and cutinases leads to a dramatic increase of the surfacial acid and hydroxyl group content to >5% (XPS) in contrast to conventional exo-wise chemical hydrolysis [1]. The generation of large fragments (MALDI-TOF MS) is typical for endo-type cleavage while exo-type chemical treatment leads to soluble mono- and oligomers (LC-MS). Consequently chemical treatment drastically affects the surface morphology (ESEM) resulting in crater-like structures while enzymes do not affect morphology of fibres or bulk properties such as strength [2]. Interfacial activation of lipases increases turnover by one order of magnitude while the plasticizer N,N-diethyl-2-phenylacetamide (DEPA) enhances both lipase and cutinase action. When compared to esterases and lipases, selected cutinases have been most active on PET [3]. Consequently, several cutinases from Thermobifida cellulosilytica (Thc_Cut1 and Thc_ Cut2) and T. fusca (Thf42_Cut1) were cloned. These enzymes were highly homologous with differences only outside the active site. Yet, they showed different kinetic parameters on water insoluble bis-(benzoyloxyethyl) terephthalate and PET in terms of hydrolysis products, crystallinity index and hydrophilicty changes and number of hydroxyl groups introduced. Modeling indicated that regions of Thc_Cut1 and Thc_Cut2 which differed in electrostatic and in hydrophobic surface properties were able to reach/interact with PET which may explain their different hydrolysis efficiencies.
163 364
Polyester synthesis using enzyme: loading impending groups
Azam Sharif Mohammed Shafioula.b, MD Ashrafuzzamana.b Jung In Pyob,c, Kwan Soo Kimb,c, Chan Seong Cheongb a University of Science and Technology, Daejeon, South Korea; bBiomolecular Functional Research Center, P.O.Box 131, Cheongryang, Seoul, 130-650, South Korea; cDepartment of Chemistry, Yonsei University, Shinchon dong, Seoul, 136-702 E-mail: c2496@kist.re.kr Enzyme catalyzed polycondensation of glycerol and divinyl esters was investigated and xanthorrhizol, one of the natural bioactive product, was loaded as pendent group. Its loaded amount (%) in polymer backbone is a factor of the chain length of divinyl ester. The polymer backbone molecular weight Mw on several other parameters was investigated and relationship was developed. Additionally, the linker which is connected with polymer backbone and pendent group was employed. Enzyme mediated in vitro release of xanthorrhizol from polymer backbone was measured by HPLC. Polymer and its pending group molecular structure were analyzed by GPC, 1H and 13C NMR. This final product was composed of the natural components as renewable and biocompatible component. This material and incorporated technique would be useful for the synthesis of glycerol based polymeric prodrug as drug delivery system.
Figure 1. Synthesis of polymer backbone and its pendent containing polymer. Figure 1. Production of biodiesel using CLEA M37
________________ [1] Kyung Seok Yang, Jung-Hoon Sohn, Hyung Kwoun Kim. Catalytic properties of a lipase from Photobacterium lipolyticum for biodiesel production containing a high methanol concentration. J. Biosci. Bioeng. 2009; 107: 599-604 [2] Suk-Kyeong Jung, Dae Gwin Jeong, Mi Sook Lee, Jung-Kee Lee, Hyung-Kwoun Kim, Seong Eon Ryu, Byoung Chul Park, Jae Hoon Kim, Seung Jun Kim. Structural basis for the cold adaptation of psychrophilic M37 lipase from Photobacterium lipolyticum. Proteins. 2008; 71: 476-484 _______________ [1] Kobayashi, S.; Makino, A. Chem. Rev. 2009. 109, 5289. [2] Hwang, J.K.; Shim, J.S.; Baek, N.I.; Pyun, Y.R. Planta Med. 2000. 66(2), 196. [3] Hu, J.; Gao, W.; Kulshrestha, A.; Gross, R. A. Macromolecules 2006; 39, 6789. [4] Kobayashi, S.; Uyama, H. Macromol. Symp. 1999, 144, 237. [5] Albertsson, A. C.; Varma, I. K. Biomacromolecules 2003, 4, 1466.
Figure 1. Models of Thc_Cut1 and Thc_Cut2

________________ [1] Brueckner et al. 2008, J. Polym. Sci. Polym.Chem. 6435 6443 [2] Eberl et al. 2009. J. Biotechnol. 143:207-212 [3] Korpecka et al. 2010. Macromol. Symp. 296:342346 [4] Ribitsch et al. 2011. Biotechnol. Progr. In press
361
Cellulase activity in imidazolium-based ionic liquids
Paola DArrigoa,b, Franca Castiglionea, Lorenzo Ceriolia, Andrea Melea,b, Loredano Pollegionib,c, Stefano Tamborinib,c, Davide Tessaroa,b a Politecnico di Milano, Department of Chemistry, Materials, and Chemical Engineering, Piazza Leonardo da Vinci 32, 20133 Milano, Italy b The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universit degli Studi dellInsubria, via Mancinelli 7, 20131 Milano, Italy c Dipartimento di Biotecnologie e Scienze Molecolari, Universit degli Studi dellInsubria, Via Dunant 3, 21100 Varese, Italy E-mail: paola.darrigo@polimi.it Green alternatives to fossil-based fuels are very attractive and can be produced from cellulosic materials. Cellulose is the primary product of photosynthesis in plants and has immense importance as a renewable raw material. The production of biofuels starting from cellulose is gaining increasing attention and obviously implies the partial or total hydrolysis of cellulose: enzymatic processes are considered the most promising technology [1]. Cellulases (EC 3.2.1.4) are the enzymes most commonly employed to selectively depolymerize cellulose in buffered aqueous solvents. Because of the very low solubility of cellulose due to its highly organized structure, enzymatic conversions proceed at very slow reaction rates. To improve the yield of fermentable monosaccharides, pretreatments of cellulose, such as thermal, chemical or physical treatment, have been applied to afford a better enzymatic conversion [2]. Ionic liquids have been increasingly recognized as novel solvents for dissolution and pretreatment of cellulose. However, it was previously reported that ionic liquids efficiently solubilise cellulose but also often inhibit the enzymatic activity of native cellulases [3]. The present study provides a useful comparison of the effect on stability and activity of four cellulases in two commercial imidazolium-based ionic liquids, namely 1-ethyl3-methylimidazolium diethylphosphate ([Emim][DEP]) and 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]). The final aim of this investigation is to produce in one-step low molecular mass intermediates from cellulose, which could be successively used for further industrial modifications and/or applications.
________________ [1] Bose S, Armstrong DW, Petrich JW J Phys Chem B 2010, 114 (24), 8221-8227. [2] Kumar R, Wyman CE Biotechnol Prog 2009, 25(3), 807-819. [3] Turner MB, Spear SK, Huddleston JG, Holbrey JD, Rogers RD Green Chem 2003, 5(4), 443-447.
362
Harvesting of novel polyhydroxyalkanaote synthase genes from metagenomic libraries by phenotypic screening
Marcus Schallmeya,f, Anh Lyf, Chunxia Wangb, Gabriela Megleif, Sonja Vogetc, Wolfgang R. Streitd, Brian T. Driscolle, Trevor C. Charlesf a Lehrstuhl fr Biotechnologie, RWTH Aachen University, Germany; bVirginia Bioinformatics Institute, Virginia Tech, VA, USA; cGttingen Genomics Laboratory, University of Gttingen, Germany; dAllgemeine Mikrobiologie und Biotechnologie, University of Hamburg, Germany; eDepartment of Natural Resource Sciences, McGill University, QC, Canada; f Department of Biology, University of Waterloo, ON, Canada E-mail: m.schallmey@biotec.rwth-aachen.de, tcharles@uwaterloo.ca Polyhydroxyalkanoates (PHA) are biodegradable polyesters which are accumulated by a wide range of bacteria as carbon storage compounds. Moreover, PHA have been evaluated since the late 1970s as sustainable biopolymers with a high potential to substitute petrochemically derived plastics. Especially in times of peak-oil, research on PHA metabolism is of considerable interest and might contribute to pave the way to a sustainable bioeconomy-based future. PHA are formed by PHA synthases (PhaC) from various 3-hydroxyalkanoyl-CoA precursors such as (R)-3-hydroxybutyryl-CoA derived from the free acetyl-CoA pool and catalytic activity of two additional PHA metabolism enzymes (Figure 1).
O SCoA O SCoA PhaA O O SCoA PhaB OH O SCoA PhaC O O n
365
Poly(-glutamic acid) (-PGA): a versatile biopolymer for novel biotechnological applications. Production, characterization and functionalization of -PGA from Bacillus subtilis laboratory strains*
Giovanni Borghesea,e, Viola Scoffoneb, Marco Biagiottia, Carlo F. Morellia,e, Daniela Ubialic,e, Alessandra M. Albertinib, Dario Pasinid,e, Cinzia Calviob,e, Giovanna Speranzaa,e a Dipartimento di Chimica Organica e Industriale, Universit degli Studi di Milano, and Italian Biocatalysis Center, Pavia, Italy; bDipartimento di Genetica e Microbiologia, Universit degli Studi di Pavia, Italy; cDipartimento di Scienze del Farmaco, Universit degli Studi di Pavia, and Italian Biocatalysis Center, Pavia, Italy; dDipartimento di Chimica, Pavia, Italy; eINSTM Research Unit, Italy E-mail: giovanna.speranza@unimi.it In recent years the interest for applications of natural biopolymers O H has tremendously increased due to the growing demand of industrial C N processes based on safe raw materials. Poly(-glutamic acid) (-PGA) is a versatile unusual, water-soluble, n edible, anionic polymer composed of glutamic acid monomers conCOOH nected by amide linkages between -amino and -carboxyl groups (Figure 1); its chemical structure resembles that of synthetic polyamFigure 1. -PGA ides, but bears a carboxylic group in the position, which is available for derivatization. This biopolymer is raising considerable industrial interests for the multitude of its potential applications. The chemical synthesis of -PGA is presently unfeasible on an industrial scale; however -PGA is efficiently produced by several species of bacteria, mainly belonging to the genus Bacillus. For the industrial application of -PGA it is necessary to enhance its productivity, to find optimal fermentation conditions and to chemically modify it for the development of usable advanced materials. Recently, by introducing selected mutations, a number of B. subtilis mutant strains with improved and sustained accumulation of the product has been derived from the fully characterized laboratory strain 168.[1] In particular, a new strain able to produce up to 20 g/l of purified -PGA has been obtained. The purification of -PGA from the growth culture medium allowed the obtainment of the polymer in high purity, as determined by NMR analysis. The polymer has been characterized by GPC and its stereochemical composition has been determined. The chemical modification of -PGA has been tackled by improvement of the literature protocols for reactions and purification.[2] Several ester derivatives have been synthesized, purified and characterized, with varying degrees of functionalization on the resulting copolymer. The possibility of transesterification of the ethyl ester -PGA derivative has also been verified with low molecular weight polyethylene glycols. The ultimate goal of this project is to develop innovative biomaterials through the modification of -PGA into macromolecular derivatives with suitable mechanical and processing properties, while preserving the inherent biocompatibility and biodegradability of the native biopolymer.
366
Polymer materials and composites prepared from plant biomass
Gabriela Dziworska1 and Andrzej Okruszek Faculty of Biotechnology and Food Sciences, Technical University of Lodz Stefanowskiego 4/10, 90-924 Lodz, Poland gabriela.dziworska@p.lodz.pl Various kinds of plant biomass are employed for the preparation of biodegradable fibrous polymer materials and composites by biotechnological methods, involving enzymatic or microbial processes. The major intermediates in the preparation of final products are cellulose nanofibres, tactical polylactide and biodegradable aliphatic-aromatic copolyesters. For the preparation of cellulose nanofibres, a cellulose rich plant biomass is being utilized, including grass and straw of various cereals or other agriculture useful plants, as well as waste fibres from textile industry (cotton, linen). The biomass is first pretreated with physical and/or chemical methods including boiling, steam-explosion or treatment with appropriate chemicals. Multienzyme complex obtained from Aspergillus niger mould serves as the main enzymatic tool. Saccharification of other kinds of biomass (patatoes, cereal grains or beet pulp) with Aspergillus niger preparations leads to simple sugars which are being subjected to the fermentation yielding L-lactic acid. The fermentation microorganisms (bacteria) are selected by classical microbiology methods from the environment. L-Lactic acid, after its dimerization to L,L-lactide is chemically polymerized to give tactical polylactide, a substrate for many fibrous materials and thermoforming. The third path involves utilization of various oil-plant biomass, which on sequential treatment with lipase preparations obtained from Mucor circinelloides and Mucor racemosus moulds (structurization, hydrolysis) and appropriate chemical reactions (cycloaddition process, hydrogenation) are transformed into oligodiols/polyols with glyceride backbone. These will be copolymerized with appropriate reagents to produce new biodegradable aliphatic-aromatic copolyesters. The polyesters will be utilized for preparation of fibrous polymers and composites. The fibrous materials and composites prepared on the basis of abovementioned intermediates will be further utilized for obtaining new functional textiles and nonwovens with potential sanitary or technical applications, such as sweat-absorbing textile inserts, sanitary textiles, filtration materials, geotextiles and agrotextiles. The processes of ageing and controlled biodegradation of prepared materials will be studied, as well as the conditions of their recycling and possible use of degradation products in agriculture. The poster presents information about the research project Utilization of biomass for the preparation of environmentally friendly polymer materials which is being realized within the time frame 2010-2013 by the Consortium composed of eight research groups from Poland.
The Project (POIG 01.01.02-10-123/09) is partially financed by the European Union within the European Regional Development Fund
Figure 1. Formation of polydroxybutyrate by the concerted biocatalytic activity of 3-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB) and PHA synthase (PhaC). Previously, we have described the isolation of PHA metabolism genes from various genomic libraries through phenotypic complementation in Sinorhizobium meliloti PHA metabolism mutants [1,2,3]. Recently, we have successfully used a S. meliloti acetoacetyl-CoA reductase mutant (phaB) as an alternative surrogate host for the functional complementation of metagenomic libraries in contrast to the commonly applied Escherichia coli [4]. Here, we applied this principle to harvest novel PHA synthases from a soil metagenome library by phenotypic screening in S. meliloti PHA synthase mutants (phaC). Five unique cosmid clones were able to functionally complement phaC mutants. PHA accumulation was confirmed by GC analysis and transmission electron microscopy. Three of the isolated cosmid clones were partially sequenced and revealed as expected the presence of phaC sequences as well as other PHA metabolism genes. The cloned PHA synthases showed 45% to 60% amino acid sequence identity with other characterized PhaC enzymes. [5] We are convinced that the application of our phenotypic complementation strategy will be useful to collect other PHA metabolism genes from the metagenomes bounty which might lead to the improvement and optimization of industrial PHA bioplastic production.
________________ [1] Aneja and Charles (1999) J. Bacteriol. 181, 849-857. [2] Aneja et al. (2004) FEMS Microbiol. Lett. 239, 277-283. [3] Aneja and Charles (2005) FEMS Microbiol. Lett. 242, 87-94. [4] Wang et al. (2006) Appl. Environ. Microbiol. 72, 384-391. [5] Schallmey et al. (accepted article) FEMS Microbiol. Lett. DOI: 10.1111/j.1574-6968.2011.02324.x
________________ [1] C. Osera, G. Amati, C. Calvio, A. Galizzi, Microbiology 155 (2009) 2282-87 [2] H. Kubota, Y. Nambu, T. Endo, J. Polym. Sci. Pol. Chem. 31 (1993), 2877-78 *This work was supported by INSTM-Regione Lombardia and Fondazione Alma Mater Ticinensis
October 2-6, 2011
164 367
Enzymatic polymerization of rutin and esculin: kinetic and biological activities studies
Ghada BEN RHOUMAa,b, Latifa CHEBILa, Jean-Marc Engassera, Leila CHEKIRGHEDIRAb, Mohamed GHOULa a Laboratoire dIngnierie des Biomolcules, ENSAIA-INPL, 2, Avenue de la Fort de Haye 54500 Vandoeuvre-ls-Nancy, France; b Department of Pharmacognosy, Faculty of Pharmacy, Rue Avicenne, Monastir 5000, Tunisia E-mail: latifa.chebil@ensaia.inpl-nancy.fr Plant phenols have gained attention due to their biological and pharmacological effects. Their anti-inflammatory antioxidant, antimutagenic, anticarcinogenic and antiviral properties have been demonstrated in many in vivo and in vitro studies [1]. However, phenols are sensitive to pH, temperature and light. To increase their stability, enzymatic polymerization was suggested as a promising way [2, 3]. In this context, the enzymatic polymerization of rutin and esculin was carried out in the presence of laccase from Trametes versicolor. Final reaction media enriched with rutin and esculin oligomers was separated, by successive filtration processes on membranes of different porosity (15, 1, 3 and 5 KDa). The aim of this study is: - to investigate the kinetic of esculin and rutin polymerizations; - to evaluate the antiradical activity (ABTS, DPPH and hydroxyl radical, CUPRAC, xanthine oxidase inhibition) of the synthesized oligomers, - to mesure mutagenic and antimutagenic activities against different biological systems (Salmonella typhimurium TA102 and Salmonella typhimurium TA104 strains). The kinetic and the biological activities will be discussed and compared according to the molecular weight distribution of the different fractions.
________________ [1] Yoshino, K., Ogawa, K., Miyase, T. and Sano, M. Journal of Agricultural and Food Chemistry, 2004. 52 (15), 4660-4663 [2] Anthoni J., Linneton, F., Wieruzeski, J. M., Magdalou, J., Engasser, J. M., Chebil, L., Humeau, C. and Ghoul. M. Rasayan Journal of Chemistry, 2008. 1 (4), 718-731. [3] Anthoni, J., Humeau, C., Maia, E., Chebil, L., Engasser, J.-M. and Ghoul, M. European Food Research and Technology, 2010. 231 (4), 571-579.

Allyl acrylates production from waste oils and glycerol via enzymatic catalysis without solvent
Edinson Yaraa, Jordi Erasa, Jonh Jairo Mendezc, Merc Torresb, Ramon Canelaa a Department of Chemistry. University of Lleida. Plaa Vctor Siurana 1. LLeida. Spain; b Department of Food Science and Technology. University of Lleida. Plaa Vctor Siurana 1. Lleida. Spain. c Department of Chemistry. University of the Tolima. Barrio S .Helena. Ibagu. Colombia. E-mail: yara@quimica.udl.cat Polyacrylates are one the most used polymers with important presence in fields like resins, textiles, adhesives, paints, flocculants, etc.[1] Polyacrylates could be prepared as polymer or copolymer depending on the final properties required. The starting material is an acrylate or a mixture of them. Our research group has developed a new procedure to prepare mixtures of allyl acrylate and alkyl acrylate via enzymatic transacylation. The reaction consists in the use of lipases to transform a mixture of alkyl acrylates and allyl fatty esters without solvent. Allyl esters can be prepared in two steps from glycerol and either fats or vegetable oils with high yields. [2,3] Crude glycerol from biodiesel industry, and vegetable oils or fat from agrofood wastes can be used as starting materials to obtain the allyl esters. The product obtained from the enzymatic reaction is a mixture of allyl and alkyl acrylates and allyl and alkyl fatty esters. The acrylates can be recovered by distillation to produce polyacrylates. The remaining product, mainly a mixture of allyl and alkyl esters of fatty acids, could be used as biofuel.

Simple and more sustainable enzymatic resolution-separation of secalcohols based on nanofiltration
a
165 372
Inuence of the use of Aliquat 336 in the immobilization procedure in solgel of lipase from Bacillus sp. ITP 001
Ranyere Lucena de Souzaa, Wagner Charles Siqueira Resendea, Carlos Eduardo Barob, Gisella Maria Zaninb, Heizir Ferreira de Castroc, Onlia Aparecida Andreo dos Santosb, Alini Tinoco Fricksa, lvaro Silva Limaa, Cleide Mara Faria Soaresa a Universidade Tiradentes, Instituto de Tecnologia e Pesquisa. Av. Murilo Dantas, 300, Prdio do ITP, Farolndia, Aracaju Sergipe, Brazil; bUniversidade Estadual de Maring, Avenida Colombo 5790, Zona 07, Maring Paran, Brazil, cEscola de Engenharia de Lorena, Universidade de So Paulo, CP 116, Lorena So Paulo, Brazil. E-mail: cleide.soares@pq.cnpq.br Stabilization of enzymes is the key issue to develop more efficient biocatalysts for industrial, enviromental, or biomedical applications. In respect, entrapment with adsorption in sol-gel matrices has been proved as one of the most efficient immobilization methods. Due to their nano- or microporous structute, sol-gel materials exhibit valuable properties, such as high surface to volume ratio, large surface area and porosity. As protection of the enzyme during the encapsulation process is very important to prevent inactivation caused by gel shrinkage throughout maturation and dryning or by inadequate pore size resulting in slow diffusion rate, several compunds have been tested as immobilization additives. In this work, the stabilizing effect of Trioctylmethylammonium chloride (Aliquat 336) during immobilization using the sol-gel process of lipase obtained submerged fermentation by a newly isolated strain of Bacillus sp. ITP-001 was investigated. Different concentrations of Aliquat 336 (0,5 3,0% w/v) were used for the preparation of the encapsulated Bacillus sp. ITP-001 lipase (EN-Aliquat 0.5 to 3) using tetraethoxysilane (TEOS) as precursor immobilized biocatalyst. For comparison were prepared similarly the encapsulated Bacillus sp. ITP-001 with absence of Aliquat 336 (EN-Bacillus) and pure silica sol-gel (PS) with absence of enzyme and additive. Samples produced (PS, EN-Bacillus and EN-Aliquat 0.5 to 3) were characterized with regard to specific surface area (B.E.T. area), pores volume (Vp) and mean diameter (dp) by nitrogen adsorption (BJH method) and scanning electron microscopy (SEM). Catalytic activities of the immobilized enzyme were assayed by the hydrolysis of olive oil [1]. The process of immobilization of the enzyme in the presence of Aliquat 336 decreased the values of specific surface area, pores volume and mean diameter when compared to pure silica matrices. For pure silica the values observed for B.E.T. area, Vp and dp were respectively 607 m/g, 0.38 cc/g and 18 . However, for the immobilized enzyme in presence and absence of additive, the values were approximately: 125 m/g, 0.26 cc/g and 14 , respectively. Probably, this behavior occurred due the enzyme partial adsorption in the external surface of the hydrophobic matrix (EN-Bacillus and EN-Aliquat) as can be seen in scanning electron microscopy analysis. On the other hand, the results obtained of the catalytic activities suggest that the concentrations of Aliquat 336 in the range of 1 and 2% (w/v) had better substrate affinity. The better result obtained was 399.34 U/g for EN-Aliquat 1 when compared with EN-Bacillus (236.55 U/g).
______________ [1] Pinheiro, C. R.; Soares, C. M. F.; Santos, O. A. A.; Castro, H. F.; Moraes, F. F.; Zanin, G. M. Influence of gelation time on the morphological and physico-chemical properties of the solgel entrapped lipase. Journal of Molecular Catalysis B: Enzymatic 5253 (2008) 2733.
Carlos M. Monteiroa, Nuno M. T. Lourenob, Carlos A. M. Afonsoa iMed.UL, Faculdade de Farmcia da Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal b IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Tcnico, Av. Rovisco Pais,1049-001 Lisbon, Portugal E-mail: carlosmmonteiro@ff.ul.pt Secondary alcohols play an important role in the synthesis of active compounds. Our main goal was to attain attractive, innovative, competitive and more sustainable processes for the enzymatic kinetic resolution of secondary alcohols. In this process, we aim to develop new strategies for the resolution-separation of free sec-alcohols by the use of new acylating agents combined with sustainable separation technology, namely nanofiltration.[1] The nanofiltration is a membrane technology founded on a separation based on the molecular weight. The resolution-separation is carried out on a selective reaction of one alcohol enantiomer with an acylating agent with an high molecular weight, where one enantiomer stays in medium, leaving the other enantiomer free to be removed by nanofiltration. The anchored enantiomer can be isolated by a second enzymatic reversible reaction (Figure 1). The major advantage of this methodology is the possibility to obtain both free enantiomers, recycle of the enzyme and acylating agent. Likewise, this approach circumvents the limitations of the use of chromatography separations, organic solvents and post-chemical transformations for the isolation of free enantiomers.
________________ [1] Agarwal, Y. K.; Kaushik S. D.; Kumar, P. C. Journal of Macromolecular Science. Pure and Applied Chemistry 2007, 44, 877880. [2] Escrib, M.; Eras, J.; Duran, M.; Simon, S.; Butchosa, C.; Villorbina, G.; Balcells, M.; Canela, R. Tetrahedron 2009, 65 (50), 10370-10376. [3] Eras, J.; Escrib, M.; Villorbina, G.; Oromi-Farrus, M.; Balcells, M.; Canela, R.. Tetrahedron 2009, 65 (25), 4866-4870.
Figure 1. Methodology for enzymatic resolution of sec-alcohols

Acknowledgments: We thank Fundao para a Cincia e Tecnologia (POCI 2010) and FEDER (SFRH/BD/48395/2008 and SFRH/BPD/41175/2007) and ACS Green Chemistry Institute (Ref. GCIPRF#49150-GCI for financial support. ________________ [1] Loureno, N.M.T., Afonso, C. A. M. Angew. Chem. Int. Ed., 2007, 46, 8178; Monteiro, C.A.; Loureno N. M. T.; Afonso C.A.M., Tetrahedron: Asymmetry, 2010, 952-956; Loureno N.M.T, Monteiro C.M., Afonso C.A.M, Eur. J. Org. Chem., 2010, 69386943.
369
Oligosaccharide synthesis by the action of glucosyltransferases from different strains of leuconostoc mesenteroides in the presence of diterpenes from salvia officinalis
Ilia Iliev,.*, Tonka Vasileva, Petko Bozov Department of Biochemistry and Microbiology, Biological Faculty, Plovdiv University, Plovdiv, Bulgaria, E-mail: *iliailiev@uni-plovdiv.bg Glucosyltransferases(GTFs) are extracellular and cell associated enzymes mainly synthesized by Leuconostoc mesenteroides and Streptococcus mutans. GTFs catalyze the synthesis of -glucans from sucrose. On the other hand the glucosyltransferase (GTF) and fructosyltransferase (FTF) enzymes play a pivotal role in dental biofilm formation as they synthesize polysaccharides that act as the extracellular matrix of the biofilm [1]. Previously, we had described the use of GTF from Leuc. mesenteroides URE13 to synthesizes glucans and oligosaccharides via acceptor reactions with maltose. We showed that the studied enzyme forms oligosaccharides of DP>5 from maltose as acceptor [2]. The present study reports on the synthesis of a glucans and oligosaccharides from extracellular GTFs by Leuc. mesenteroides URE 13, Lm 17- isolated from Bulgarian fermented products and NRRL B1149 from ATCC. We selected different acceptors for synthesis of oligosaccharides with prebiotic potential. Glucan is formed in major or minor amounts depending on the strength of the acceptor and the reaction conditions. Some of the glucans synthesized contain -(1-6) osidic linkages and were completely hydrolyzed by dextranase. The other glucans, resisted the action of this enzyme. We found that some of polymers, synthesised by GTF from URE13 in the presence of lactulose contain more tan 5% fructose. In the presence of melibiose and lactulose as acceptor molecules, GTF from URE 13 makes mainly an oligosaccharides with DP-3 and DP-4. The behavior of GTFs from different studied strains in the presence of different diterpenes isolated from Salvia officinalis was investigated. The activity of GTF from NRLL B-1149 in solution was effectively inhibited more than 60% by the DMSO solutions of some diterpenes. In contrast the activity of GTF from URE 13 was not inhibited by the same studied diterpenes. Acknowledgements: We would like to express our gratitude for the support of this work by research grant of NSF Bulgaria RNF02/2-2009.
___________________ [1] Andr, I., G. Potocki-Vronse, S. Morel, P.Monsan and M. Remaud-Simon,(2010) Sucrose-utilizing transglucosydases for biocatalysis, Topics in Current Chemistry, 2010, Volume 294/2010, 25-48, DOI: 10.1007/128_2010_52 [2] Vasileva,T., Kirilov, A., Bivolarski, V., Bounaix,, M., Gabriel, V., Robert, H., Fontagne-Faucher, C., Gabriel, B.,Ivanova, I., Iliev, I., (2009) Characterization of glucansucrase activities from Leuconostoc mesenteroides Lm17 and URE 13 strains, Biotechnol. & Biotechnol. Eq., special edition/ on-line, 23: 698-701;
370
Enzyme encapsulation and biocatalysis in mesoporous materials with varying pore sizes
Hanna Gustafssona, Christian Thrnb and Krister Holmberga a Applied Surface Chemistry, Chalmers University of Technology, Gothenburg, Sweden; b Industrial Biotechnology, Chalmers University of Technology, Gothenburg, Sweden E-mail: hanna.gustafsson@chalmers.se Enzymes are biological catalysts and control all reactions inside the cells. They operate in mild conditions and the efficiency and selectivity is much higher compared to synthetic catalysts. However, enzymes free in solution are easily degraded and inactivated, especially under process conditions used in industrial applications, and they are difficult to recover from the reaction mixture. In order to overcome these disadvantages one method is to encapsulate enzymes inside a mesoporous inorganic material, allowing them to be recycled and increasing their stability [1]. Typical characteristics of mesoporous material are high specific surface area, large specific pore volume and narrow pore size distribution with dimensions comparable to many biological molecules, like enzymes.
373
Production of unnatural amino acids from the corresponding -keto acids by the recombinant Corynebacterium glutamicum
Ju-Yeon Kim, Kyung-Mi Yang, Jin-Byung Park Dept. of Food Science & Engineering, Ewha Womans University, Seoul 120-750, Republic of Korea E-mail: jbpark06@ewha.ac.kr The unnatural amino acids can be produced from the corresponding -keto acids and amino donors with amino transferases. The reactions are often limited by inhibition from byproducts (i.e., the -keto acids converted from the amino donors). To reduce the byproduct concentration, additional amino transferases and amino donors were provided into the reaction system. In this study, a new biocatalyst based on Corynebacterium glutamicum was constructed to enable to attenuate the byproduct inhibition in situ and to allow high product concentration (Fig. 1). Trimethyl pyruvate and 2-(3-hydroxy-1-adamantyl)2-oxoethanoic acid were efficiently transformed into L-tert-leucine and 2-(3-hydroxy1-adamantyl)-(2S)-amino ethanoic acid by the recombinant C. glutamicum expressing branched chain amino transferase of Escherichia coli. The both product concentration reached over 35 mM during 60 to 80 h fermentation. The enzyme inhibition of the byproduct (i.e., -ketoglutarate) appeared efficiently attenuated by the metabolic enzymes. Furthermore, the C. glutamicum-based process did not require the amino donor, glutamic acid. The amino donor was in situ produced from glucose and ammonia by the metabolic enzymes. Therefore, the recombinant C. glutamicum is a promising biocatalyst for the production of unnatural amino acids from the corresponding -keto acids.
Glucose
374
Engineering of 2,3/2,6-sialyltransferase using hybrid approach for the production of sialyllactose
Yun-Hee Choia, Joon Ho Parkb, Nahum Leeb, Dae-Hee Kimc, IL-Hyang Parka, Jong Hoon Kima, Byung-gee Kima,b a Interdisciplinary Program for Biochemical Engineering and Biotechnology, Institute of Molecular Biology and Genetics, Seoul National University,151-742, Seoul, South Korea b School of Chemical and Biological Engineering, Seoul National University, 151-742, Seoul, South Korea c GeneChem Inc., Daejeon, 302-804, South Korea E-mail:byungkim@snu.ac.kr Sialyltransferase (ST) transfers N-acetylneuraminic acid (Neu5Ac) from cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) to acceptor substrate such as lactose. Most sialylated motifs consist of Neu5Ac attached to galactose by an 2,3- or 2,6linkage. These two motifs are recognition sites for the avian and human influenza viruses, respectively. Two linked sialyllactose are present in high concentrations in human milk and are considered to protect breast-fed infants from infection. In contrast to human milk, cow milk contains only small amounts of these oligosaccharides and supplementing synthetic human milk oligosaccharides has been considered as a way to improve humans immunity. In this research, 2,3- and 2,6-ST were engineered by hybrid approach to improve production of siayllactose. Hybrid approach is combined with rational design and directed evolution. This method can reduce library size by selecting target region such as substrate binding pocket and functional residues based on alanine scanning and computational mutation analysis. Saturation mutagenesis was done for selected residues to find best hits. First, multifunctional 2,3 ST from Pasteurella multocida was engineered by this approach. We selected non-conserved residues located in substrates binding site by alignment with STs in GT80 family. And we applied alanine scanning for the selected residues. Mutants which show neutral activity were selected for saturation mutagenesis, and a single mutant interacting with lactose showed 168% of increased specific activity. Also, specific activity of a combination mutant was increased 200% compared to wild-type. In addition, 2,6 side reaction was reduced significantly for R313 mutants. Second, 2,6 ST from Photobacterium damselae showed low activity and protein expression level. Therfore, it was engineered to increase catalytic activity. Substrate binding sites were predicted through homology modelling and functional residues were selected by the same method. Several mutants which show higher activity than wild type were screened by color assay method. Among them, single mutant interacting with CMP showed 4-5 times higher activity than wild type. Thus, 2,3- and 2,6-ST mutants obtained by hybrid approach will be an efficient tool for the improvement of production of sialyllactose
________________ [1] Biochemistry 2007, 46, 6288-6298 [2] Glycobiology 2008, 18, 66-73
Figure 1. Enzymes encapsulated inside the pores of hexagonal mesoporous silica. The objective of this work is to compare the encapsulation and catalytic performance of lipase from Mucor miehei and trypsin from bovine pancreas, two hydrolases with rather dissimilar properties and structures. Mesoporous silica with hexagonal structure (SBA-15) is used as host for the enzymes and we demonstrate the importance of the pore dimensions and the pH for proper function of the encapsulated enzyme. The pore size of the silica particles was increased from 50 to 89 by increasing the synthesis temperature from 80 C to 140 C. For trypsin optimal encapsulation with the largest loading was obtained at pH 7.6 using particles with 60 pores while for lipase pH 6.0 and 89 pores was optimal. The encapsulation rate was faster and the loading was larger for trypsin than for lipase. The difference may be due to electrostatic attraction between trypsin and SBA-15, since trypsin is positively charged while lipase and SBA-15 are negatively charged in these buffers. However, the activity tests show that trypsin is inactivated rapidly and the product yield is much lower compared to free trypsin, while the product yield for encapsulated lipase is more than twice as large compared to free lipase. This may be due to the created interface between the silica pore walls and the water phase which is essential for lipase activation.
________________ [1] Daz, J.F. and K.J. Balkus, Journal of Molecular Catalysis B: Enzymatic, 1996. 2(2-3): p. 115-126.
Pyruvate
Acetyl CoA Isocitrate NH3 Oxaloacetate alapha-Ketoglutarate BcAT

NH2 O R COOH
Glutamate
Succinate
COOH
Figure 1. Production of unnatural amino acids from the corresponding -keto acids in C. glutamicum
October 2-6, 2011
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Biocatalytic routes for polyesters derived from biobased building blocks
David I. Habeych a,b, Benjamin P. Juhl c, Jrgen Pleissc, Gerrit Egginka,b, Carmen G. Boeriub,d a Food and Bioprocess Engineering, Wageningen University, Wageningen, The Netherlands; b Wageningen UR Food & Biobased Research, Wageningen, The Netherlands, c Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany; dDepartment of Chemical and Biological Sciences, University Aurel Vlaicu, Arad, Romania E-mail: carmen.boeriu@wur.nl The interest for polymers from renewable resources as alternative to commonly used petrochemical polymers has increased continuously in the past decade, in the context of the transition to a sustainable biobased economy. Three major routes to obtain biopolymers are konwn: (i) isolation of natural polymers from plant and animal materials; (ii) production by microorganisms; (iii) synthesis from bio-derived monomers by in vitro enzymatic catalysis. While the first two are based on biosynthetic pathways and are basically limited to the synthesis of naturally occurring polymers by in vivo enzymatic catalysis, the third is more versatile and offers many possibilities to design new synthetic routes for existing polymers, or to obtain new polymers that are difficult to be synthesized by conventional chemical catalysis. Here we present an enzymatic approach for the production of polyesters derived from biobased building blocks. The condensation polymerization of both flexible and rigid biobased monomers was explored using immobilized lipase B from C. antarctica as catalyst (CALB). Medium chain (C4 to C10) dicarboxylic acids, short chain (C3 and C4) diols and dianhydrohexitols (e.g. isosorbide, isomannide and isoidide) were among the monomers used. Linear and cyclic ester oligomers were obtained in all the condensation polymerisation reaction catalyzed by immobilized CALB. The type (cyclic vs. linear) and the length of the polymers produced depended on the chain length, the structure and the steric conformation of the monomers. The reactivity of the dianhydrohexitols, for example, decreased in the order isomannide > isosorbide >> isoidide [1]. Substrate-imprinted docking was used to model the formation of (poly)esters by the enzyme and to understand the molecular basis of the observed CALB specificity (Fig. 1).

Development of process technology for two-stage enzymatic FAEE-biodiesel production
Yuan Xua, Mathias Nordblada, Jesper Braskb and John M. Woodleya a Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800 Lyngby, Denmark b Novozymes A/S, Krogshjvej 36, DK-2880 Bagsvrd, Denmark Email of presenting author: xuy@kt.dtu.dk Fatty acid ethyl ester (FAEE) biodiesel production catalyzed by immobilized lipases is attracting considerable interest as an alternative to conventional base-catalysed biodiesel production, due to its potential of improved sustainability[1]. However, FAEE has thus far not been a well recognized biodiesel product and the high cost of the immobilized lipases has limited its commercial application[2]. Here, we present a two-stage biodiesel production, catalyzed by two immobilized lipases in two reactors, which aims to make FAEE-biodiesel qualified for the market in terms of specifications and to enhance the economic viability for large-scale industrial application by improving the process efficiency. For the first stage of the process (transesterification of glycerides), the activity and stability of the catalyst have been evaluated in a stirred tank reactor with respect to the effects of mechanical stirring and ethanol concentration. In the second stage (esterification of fatty acids), catalyst performance has been evaluated in a packed bed reactor, focusing on the effect of the by-product water.
_______________________ [1] Nielsen, P.M., Brask, J., Fjerbaek, L., 2008. Enzymatic biodiesel production: Technical and economical considerations. Eur J Lipid Sci Technol 110, 692700. [2] Fjerbaek, L., Christensen, K.V., Norddahl, B., 2009. A review of the current state of biodiesel production using enzymatic transesterification. Biotechnol Bioeng 102(5), 1298-1315.

Biocatalytic alternatives for green-synthesis of glycerol carbonate from renewable glycerol
Madalina Sandulescu, Loredana Protesescu, Florentina Neg, Georgiana Sanda, Simona Coman, Vasile I. Parvulescu University of Bucharest, Department of Chemical Technology and Catalysis, Bd. Regina Elisabeta 4-12, Bucharest 030016, Romania, Tel./Fax: 4021 4100241 E-mail: madalina.sandulescu@g.unibuc.ro Glycerol carbonate (4-hydroxymethyl-1,3-dioxolan-2-one) is a relatively new material in the chemical industry with a large potential as a novel component of gas-separation membranes, a non-volatile solvent for several types of materials (e.g. dyes, lacquers, pharmaceuticals, detergents, adhesives and cosmetics), a precursor in biomedical applications, and biolubricant owing to its adhesion to metallic surfaces and resistance to oxidation, hydrolysis, and pressure. The present industrial synthesis of glycerol carbonate from glycerol substrate involves harsh reaction conditions, moderate yield of targeted product was obtained and another purified step using distillation or extraction was required. Actually, the glycerol as principle by-product from biodiesel process seems to be an inspired alternative source for glycerol carbonate synthesis knowing the reality of biodiesel field. The biodiesel production of worldwide is increasing dramatically over the last decade. Consequently, a large volume of crude glycerol generated will far exceed the actual demanded leading to environmental problem with its storage. The immense amount of bio-glycerol stacking unsold gave a visual image of the huge loss of energy and material resources. On the other hand, the overcapacity of glycerol production drives its price down. Industry reacted to this situation by starting research to find new applications of bio-glycerol Such that, glycerol as versatile biofeedstock has good multiple functionality and a number of immediately valuable or promising products can be envisaged based on the exploitation of Green Chemistry and green chemical technologies. We propose a biocatalytic route for glycerol carbonate synthesis from renewable glycerol. This presentation is intended as overview on our research activity dedicated to glycerol carbonate green-synthesis. Glycerol is enzyme-converted to glycerol carbonate in solvent-free reaction system in which excess dimethyl carbonate is used as coupling reagent as well as reaction medium. The synthesis is performed in heterogeneous biocatalytic conditions. The bioreactor designed is varied from lipase-enzyme supported on the micro/macro porous silica to lipase immobilised on the magnetic nano-/micro-particles in order to emphasise the influence of the reactor structure on the biocatalytic system performance. Also, a screening of the enzyme sources was performed before choosing the optimum lipase used for further experiments. The enzyme was immobilized on the magnetic beads surface using different immobilization approach. Therefore, the enzyme was attached via its COOH, -NH2 or OH terminal group. The experimental parameters, such as reagents concentration, enzyme-beads concentration and configuration, temperature and incubation time were set up at optimum values, too. In this experimental context, the developed biocatalytic system shows as valuable way to use renewable glycerol (e.g. the glycerol conversion and the glycerol carbonate selectivity had maximum values of 64 and 95 %, respectively).
Acknowledgements: This work was financially supported by CNCSIS (PN II TE 90/2010 project). _______________ [1] M. Aresta, A. Dibenedetto, F. Nocito and C. Ferragina, Journal of Catalysis 2009, 268, 106-114. [2] M. J. Climent, A. Corma, P. De Frutos, S. Iborra, M. Noy, A. Velty and P. Concepcin, Journal of Catalysis 2010, 269, 140-149. [3] J. George, Y. Patel, S. M. Pillai and P. Munshi, Journal of Molecular Catalysis A: Chemical 2009, 304, 1-7.
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Feruloyl esterases immobilized into mesoporous materials leads to improved transesterification yield
Christian Thrna, D.B.R.K Gupta Udathaa, Nils Carlssonb, Evangelos Topakasc, Lisbeth Olssona a Industrial Biotechnology, Chalmers University of Technology, Sweden b Physical Chemistry, Chalmers University of Technology, Sweden c Biotechnology Laboratory, National Technical University of Athens, Greece E-mail: christian.thorn@chalmers.se Feruloyl esterases is a class of enzymes used in biocatalysis for refinement of hydroxycinnamic acids. These compounds have shown to have antioxidant and antibacterial properties, though modification of solubility is necessary for the compounds to be of interest in different commercial products. Immobilization of enzymes is often required for sufficient enzyme stability and to enable recovery in industrially feasible and efficient processes. Mesoporous silica have become popular as immobilization support for enzymes due to advantages such as high protein loading capacity and enhanced enzyme activity because of confinement into pores. Upon immobilization of feruloyl esterases (FoFAEC) into mesoporous silica (SBA-15), the selectivity was altered towards transesterification while the hydrolytic activity was decreased compared to free enzyme. It is remarkable how adsorption of the enzyme so drastically can change its catalytic properties. We have demonstrated that the immobilization efficiency is highly affected by the pH during the immobilization, which also had an effect on the specific activity when testing the immobilized enzymes for transesterification of methyl ferulate to butyl ferulate. Consequently there is a pH memory effect from the immobilization, which could be reverted by subsequent washing with a buffer of different pH. The aim of our research is to understand how mesoporous materials can be used to alter the enzymatic activity upon immobilization and in the end develop improved feruloyl esterase biocatalysts that allow customization of the antioxidant properties of hydroxycinnamic acids. We will approach this by testing a pH probe bound to the enzyme which will give information of the microenvironment pH close to the enzyme. Additionally, an in silico model of FoFAEC has been developed to simulate the enzyme structure at different pH and to predict orientation and adsorption behavior.
Figure 1. (a) Products of the enzymatic polymerization of succinic acid and isomannide and (b) productive placement of transition-state analogues in the CALB binding pocket.
______________ [1] Habeych et al., (2011), J. Mol. Cat. B. Enzymatic. In Press. Doi:10.1016/j.molcatb.2011.02.015
Figure 1. Changed product selectivity after immobilization of feruloyl esterase into mesoporous silica.
377
Improvement of immobilization of a GH 43 xylosidase
M-L Desrousseauxabc, N. Boonvitthyaabcd, C. Dumonabc, M. ODonohueabc Universit de Toulouse; INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France ; bINRA, UMR792, Ingnierie des Systmes Biologiques et des Procds, F-31400 Toulouse, France ; cCNRS, UMR5504, F-31400 Toulouse, France ; dBiological Science Program, Chulalongkorn University, Bangkok, Thailand E-mail: marie-laure.desrousseaux@insa-toulouse.fr
a
378
Engineering platforms for the directed evolution of ligninolytic laccases
Susana Camareroa, Isabel Pardoa, Diana Matb and Miguel Alcaldeb a Centro de Investigaciones Biolgicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain; b Instituto de Catlisis y Petroleoqumica, CSIC, Marie Curie 2, 28049 Madrid, Spain. E-mail: susanacam@cib.csic.es High-redox potential laccases (HRPLs) produced by white-rot fungi during lignin biodegradation display a huge potential applicability in the frame of industrial and environmental biocatalysis. Above all, Pycnoporus cinnabarinus laccase (PcL) comprises interesting biochemical characteristics in terms of redox potential, stability and turnover rates for a wide range of substrates. Indeed, PcL applicability in biobleaching of paper pulps, pitch control, dye decolorization or degradation of PAHs have been exhaustively investigated [1,2]. All these features make PcL a good target for laccase engineering to attain better catalytic turnovers or improve its stability under operational conditions by directed evolution. However, the poor functional expression levels in the evolutionary host and the lack of suitable screening assays are the main bottlenecks for the directed evolution of basidiomycete laccases, making necessary the development of new tools that overcome these hindrances. Here, we describe a directed evolution platform on which developing PcL and other HRPLs towards different fates can be achieved. We combined the construction of a PcL fusion gene with the optimization of conditions to induce functional expression in Saccharomyces cerevisiae, along with its directed evolution and semi-rational engineering. Moreover, we fine-tuned and validated a multi-screening assay system based upon the oxidation of synthetic laccase substrates and compounds of natural origin derived from lignocellulose. The PcL native signal peptide was replaced by the -factor prepro-leader and jointly subjected to six rounds of evolution coupled to a multi-screening assay based on the oxidation of natural and synthetic compounds at more neutral pHs. Whilst the laccase activity was enhanced up to 8000-fold over parent type and the pH profile was shifted onwards, both the thermostability and the broad substrate specificity of PcL were kept intact. The mutational exchange in hot positions with another ligninolytic laccase (PM1L) evolved in parallel [3] provided new insights about the transit and function of the evolved pre-proleader in yeast for a successful laccase secretion. The possible role of certain residues on the oxidation of phenolic and non-phenolic substrates by HRPLs were discussed on the basis of the mutations in the mature protein, which were mapped in the recently solved PcL crystal structure and compared with those selected in the in vitro evolution of PM1L. The directed evolution platforms presented here might be useful departure guide-templates for laccase chimeragenesis or for developing HRPLs with new features such as high specificity/activity onto natural compounds of biotechnological relevance [4], both topics are currently under study in our group.
________________ [1] D. Ibarra, et al., "Exploring the enzymatic parameters for optimal delignification of eucalypt pulp by laccase-mediator," Enzyme Microb. Technol. 39, 1319 (2006). [2] A. Caas, et al., "Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil," Environ. Sci. Technol. 41(8), 2964 (2007). [3] D. Mat, et al., "Laboratory evolution of high redox potential laccases," Chem. Biol. 17, 1030 (2010). [4] A. I. Caas and S. Camarero, "Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes," Biotechnol. Adv. 28, 694 (2010).
381
Cellulose Hydrolysis performed with enzymes extracted and not extracted from solid culture medium
Larissa Cardillo Afonso, Beatriz Vahan Kilikian Chemical Engineering Department, University of So Paulo (USP),P.O.Box:61548 CEP:05424-970 So Paulo, Brazil. E-mail: kilikian@usp.br The effectiveness of cellulase produced through solid state culture, was evaluated by means of yield on glucose released in two types of experiments of cellulose hydrolysis: (1) using enzymes extracted from solid culture medium; (2) using the whole culture medium, i.e., enzymes adhered to the medium. Cellulases were produced by Myceliophthora sp. M77, a recently isolated fungus through an environmental program for biodiversity preservation in So Paulo state (BIOTA FAPESP). The fungus was cultivated in a solid medium composed by sugar cane bagasse and soybean bran (9:1 w/w) with initial moisture of 80% in dry basis. The inoculated medium was incubated at 45C for five days, reaching a cellulase production of 10 FPU. gdm-1. The extraction method, widely applied, was made by addition of 100 mL of acetate buffer (0.1 M, pH 5.0) to each 4 g of culture medium and shaking the contents at 180 rpm for 60 min. Hydrolysis occurs as follows: to 1g of cellulosic substrate (Avicel), 10 FPU of cellulase activity was added using the (1) enzymatic extract or (2) the whole solid culture medium. In order to reach 1% of solids in the reaction medium, acetate buffer pH 5.0 was added. Besides, sodium azide (32mg.L-1) and cyclohexane (10mg.L-1) were added to prevent bacterial and fungal growth. The reaction medium was supplemented with -glucosidase (15 U.gdm-1) to enhance the complete hydrolysis. Flasks were put in a shaker at 180 rpm, 50C, and, samples were periodically withdrawn. The results are presented in the figure below.
382
Enzymatic hydrolysis of castor bean residue by an multienzyme complex produced from solid state fermentation
Olavo L. Moreiraa, Mariana M. P. Texeirab, Jimmy A. Lpezc, Aline M. de Castrod, Leda dos Reis Castilhob, Denise M. G. Freirec a School of Chemistry, Federal University of Rio de Janeiro (UFRJ), Brazil; bCOPPE, Chemical Engineering Program (UFRJ), Brazil; cInstitute of Chemistry (UFRJ), Brazil; d Biotechnology Division, Research and Development Center, PETROBRAS, Av. Horcio Macedo, 950. Ilha do Fundo. Rio de Janeiro, Brazil; Email: alinebio@petrobras.com.br The sustainability of bioprocesses and biorefineries is a factor totally depending on an efficient utilization of cheap renewable raw materials such as agricultural by-products and industrial residues [1]. Castor bean (CB) cake, a residue generated from the biodiesel production process based on the utilization of this agricultural source, contains high amounts of starch (48%) and protein (35%), besides cellulose and hemicellulose [2, 3]. Due to its valuable composition, the implementation of a hydrolysis process employing a combination of different enzymes would generate a hydrolysate rich in fermentable sugars and nutrients which could be the raw material for producing high-value metabolites according to a biorefinering strategy. This work evaluates the possibility of using a CB residue as: (1) sole substrate for crude enzymes production by solid state fermentation (SSF); (2) sole raw material for obtaining sugars and nutrients through hydrolysis using the produced crude enzyme complex. After 144 h of SSF using the filamentous fungus Aspergillus awamori IOC-3914, multienzyme complex solutions containing -amylases (11 U g-1), proteases (2.6 U g-1), xylanases (202 U g-1), and cellulases (3.9 U g-1) were produced. Two different preparations of these enzyme-rich solutions were used for transforming the main natural polymers present in castor bean residue into fermentable sugars (measured as total reducing sugars, TRS) and nutrients (measured as free amino nitrogen, FAN). Hydrolysis experiments were carried out at 45C and an initial CB concentration of 137 g L-1 (db). Results are shown in Figures 1 and 2.
Hemicelluloses are the second most abundant source of renewable carbon after cellulose and represent a sugar feedstock for biotechnological processes. Nevertheless the development of methodologies focused on pentose transformation are hampered both by the complexity of hemicellulose structures and their relative fragility with respect to some chemical pre-treatments [1-2]. To promote the use of pentose syrups as valuable platform intermediates for the production of fuels and chemicals, it is thus desirable to develop processes that will provide efficient recovery of high quality pentose sugars from lignocellulose biomass. In this context, enzymes are interesting tools that can be potentially incorporated into extraction processes that are characterized by mild temperatures and pH. Certain alkaline pretreatments, some organosolv technologies and enzyme-based hydrolysis of biomass can potentially produce pentose streams containing both monomers and xylo-oligomers. To complete the processing of such streams into monomeric syrups, we have previously devised an immobilized xylosidase-based packed bed reactor. This rudimentary prototype was produced using classical immobilization technology and a chitosan solid support. This system provided a proof of concept, but the overall performance was far from meeting industrial requirements [3]. Therefore, in our recent work we have strived to improve upon this concept, using state of the art immobilization technology and industrially compatible solid phases, In this report, we will communicate upon recent work that has focused on the use of a combinatory approach [4], which combines protein engineering and epoxy-activated Sepabeads to achieve orientated and optimized immobilization of the GH43 xylosidase from B. halodurans C-125. Our experimental strategy has involved the creation of several enzyme variants (which combine site-directed mutations and C or N-terminal His-tags) and the use of two different Sepabeads supports (EC-EP and EC-HFA). By testing all of the different enzyme-solid phase combinations, we have identified a best performing immobilized enzyme system, which will be described. In our presentation we will describe the experimental strategy and the key biochemical and operational features of the selected immobilized enzyme and will discuss the influence of polyhistidine tag location.
________________ [1] F.M. Girio, C. Fonseca et al., 2010, Bioresource Technology, 101, 47754800 [2] N. Mosier, C. Wyman et al., 2005, Bioresource Technology, 96, 673-686. [3] I. Smaali, C. Rmond, et al., 2009, Bioresource Technology, 100, 338-344. [4] C. Mateo, V. Grazu et al., 2007, Nature Protocols, 2, 1022-1033.
Figure 1. TRS production at two different enzyme concentrations. Figure 1. Cellulose hydrolysis yield as function of reaction time, applying (1) enzymatic extract and (2) whole solid culture medium. The hydrolysis using enzymes adhered to the solid culture medium, resulted a yield 35% higher than that made by the enzyme extract. These results indicated the feasibility of promoting the hydrolysis with enzymes in whole solid culture medium, thus, eliminating the need for extraction, as well as are a positive indicative on exploring the hydrolytic capability of the enzymes produced by solid state culture.
Figure 2. FAN production at two different enzyme concentrations.
The level of fermentable sugars and nutrients increased when higher amounts of the enzyme complex were used. Thus, the experimental results give evidence of the ability of the produced crude enzyme complex to catalyze the hydrolysis of the main natural macromolecules present in castor bean residue.
__________________ [1] Kamm, B; Kamm, M. Appl. Microbiol. Biotechnol. 64 (2004) 137. [2] Melo, W.C. et al. J. Braz. Chem. Soc. 19 (2008) 418. [3] Madeira Jr, J.V. et al. Bioresource Technol. In Press, doi: 10.1016/j.biotech.2011.04.099.
October 2-6, 2011
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Use of a lipase from Jatropha curcas L. in the enrichment of vegetable oils with polyunsaturated fatty acids
a

Synthesis of methyl-ethyl-ketone from levulinic acid via enzymatic catalysis using acetoacetate decarboxylase from Clostridium acetobutylicum
Kyoungseon Mina, Youngsoon Uma, Byoung-In Sangb a Clean Energy Research Center, Korea Institute of Science and Technology, Seoul 130-650 b Department of Chemical Engineering, Hanyang University, Seoul 133-791, Korea E-mail: presenting.author@affiliation.domain Lignocellulosic biomass is converted to 5- or 6-carbon sugars via a robust hydrolysis process such as acidic saccharification. Under the acidic conditions, 6-carbon sugars are dehydrated and consequently LA is formed. According to the DOE report, LA can be used as an important building block from biomass for the production of value-added chemicals including levulinate esters and -aminolevulinate. The goal of this study is the development of the biocatalytic process for conversion of LA to methyl-ethyl-ketone (MEK). The product MEK is a widely used commercial solvent in the field of plastic and textile industry. MEK also can be converted to 2-butanol via hydrogenation using chemical catalysis. Herein, acetoacetate decarboxylase from Clostridium acetobutylicum is employed as the biocatalyst and expressed in E.coli with 6xHis tag for the conversion of levulinic acid to MEK. Whole cells of E. coli expressing AADC converted 8.8% of LA into MEK, while no MEK was detected in control cells lacking AADC. AADC with a 6xHis tag was purified by nickel-affinity chromatography, and the purified enzyme converted 26.1% of LA into MEK. The kinetic parameters and stability of the enzyme were also determined. LA showed the substrate inhibition and played a role as a competitive inhibitor in the co-existence of acetoacetate. The enzyme maintained over 85% of its initial activity for 15 days. Several mediators were tested to improve the conversion rate by enhancing the electron transfer efficiency. The detailed results will be discussed.

Bioresponsive polymers based modified polysaccharides for enzymatic controlled release
Anton Gliedera, Konstantin Schneidera, Andrea Hasmanna, Eva Wehrschuetz-Sigla, Teresa Flocka, Ulrike Gewesslera, Doris Schifferb, G. M. Guebitza,b, a ACIB GmbH, Petersgasse 14, Graz, Austria; bDepartment of Environmental Biotechnology E-mail: k.schneider@tugraz.at; guebitz@tugraz.at The idea of a bioresponsive polymer (BRP) is focused to detect the presence of contaminating bacteria or fungi. Microorganisms release extracellular enzymes which can be used as triggers to permit a controlled release of dyes or antimicrobial molecules. Common systems of (bio) responsive polymers are designed to be sensible to pH or temperature changes in different environments [1-2]. A more selective delivery is obtained with devices responding to trigger enzymes. Natural polysaccharides e.g. polygalacturonic acid (PGA) or carboxymethylcellulose (CMC) can be hydrolyzed by different hydrolytically enzymes like pectinases or cellulases. In this work, in order to make a physical stable hydrogel out of these natural polysaccharides a cross-linking system was developed. The covalent integration of methacrylate substituents via esterification directly to the polysaccharide backbone led to an improved stability of the polymer. These methacrylate groups were polymerized by radical polymerization. Two major parameters of BRPs, namely stability and sensitivity were be optimized by changing the ratio between backbone and linker molecule. To test the controlled release alizarin as a model molecule was incorporated into the hydrogel matrix and tested against different activities of pectinases and cellulases. In addition to enzyme triggered release, B. subtilis and Y. entercolitica, were used as models for contaminating. Indeed, these organisms successfully triggered the release of Alizarin compared to a blank (figure 1). The amount of released test substance alizarin was detected in the supernatant [3] by spectrophotmetric measurements
169 388
A mathematical study of an enzymatic reaction with substrate and product inhibition in a continuous stirred-tank reactor with a dead zone
Guillaume Nogaroa, Ahlem Saddouda, Tewfik Sarib, Jrme Harmandc, Eric Dubreucqa, Alain Rapaportd, Robert Lortiee a Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier, France b Universit de Haute Alsace, LMIA, Muhouse and EPI INRA/INRIA MODEMIC, 2 Place Viala, F-34060 Montpellier, France c INRA, LBE, EPI INRA/INRIA MODEMIC, Avenue des Etangs, F-11100 Narbone, France d INRA, UMR MISTEA, EPI INRA/INRIA MODEMIC, 2 Place Viala, F-34060 Montpellier, France e IRB-CNRC, 6100 Royalmount Av., Montral, H4P 2R2, Qubec, Canada E-mail: Eric.Dubreucq@supagro.inra.fr In an effort to contribute to the understanding of enzyme reactors involving complex kinetics and space heterogeneity, we have decided to develop a new mechanistic model which takes into account the non homogeneity in medium composition. A mathematical model was constructed to describe an enzymatic reaction with both substrate and product inhibition in two Interconnected Continuous Stirred Tank Reactors (ICSTRs), where one of them represents a dead zone. The main purpose is to estimate the kinetic and operating parameters that can lead to steady state multiplicity, to investigate at steady state the biological performance of the enzymatic system, and to derive the experimental parameters which give the optimal performance in the domain of multiplicity. We demonstrate analytically that the existence of multiple steady states can be guaranteed by necessary and sufficient conditions. Based on literature data, we chose the hydrolysis of cellobiose by Novozyme 188 as a model reaction, and used published kinetic parameters (Vm, KM, cellobiose inhibition constant KiS and glucose inhibition constant KiP) with a Haldane-derivated model to demonstrate that in some conditions of initial substrate concentrations and flow rates, three steady states should exist, associated with different reaction and space-time yields. However, a significant transglycosylation activity of Novozyme 188 was observed in our experiments that did not fit the classically described behaviour of this enzyme and was not compatible with steady-state multiplicity evaluation. The modelling approach described here will be applied to other enzymatic reactions in the future.
Joab Sampaio de Sousaa, Alexandre Guedes Torresa, Denise Maria Guimares Freirea Universidade Federal do Rio de Janeiro - Instituto de Qumica, Rio de Janeiro - RJ, Brazil e-mail: joabsousa@me.com / freire@iq.ufrj.br Plant lipases are widely available in nature and are more readily accepted by the food and pharmaceuticals industries than microbial lipases. They normally have some kind of selectivity, mostly for a particular substrate, and as such can be used strategically for specific enrichment of oils or the isolation of a kind or class of lipid [1]. One interesting application for plant lipases in the food industry is the synthesis of structured lipids, which are triacylglycerols that have been restructured or modified to change their fatty acid composition and/or their distribution in the glycerol molecule [2]. They can be used for nutritional or therapeutic purposes, treating specific diseases or abnormal metabolic conditions. The present work demonstrates the potential of the lipase encountered in the Jatropha seed (Jatropha curcas L.)[3] to replace microbial lipases and classic chemical catalysts in the production of oils enriched in polyunsaturated fatty acids (PUFAs) for human consumption. After the interesterification reaction was catalyzed by this biocatalyst, total PUFAs, which account for just 10.5% of palm oil, reached 21.8%, i.e. their concentration was 107% higher. The final PUFA composition included 2.3% eicosapentaenoic acid (EPA) and 1.5% docosahexaenoic acid (DHA). When coconut oil was interesterified using this biocatalyst, the total PUCA concentration was around 880% higher (1.8% vis--vis 17.7%), of which 2.3% was EPA and 1.4% was DHA. These fatty acids are essential for the human organism and were not initially detected in the oils. The new composition effectively represents a new vegetable oil with specific health benefits derived from its enhanced nutritional value. This procedure resulted in the production of a vegetable oil suitable for human consumption with a high PUFA content, and with EPA and DHA levels as high as those recommended by the WHO [4]. Furthermore, this process assures the provision of essential fatty acids that permit the easy absorption and digestibility of the medium-chain fatty acids [5] present in coconut and palm oils.
________________ [1] Freire, D. M. G. & Castilho, L. C. (2008). Lipases em Biocatlise em: Enzimas em Biotecnologia: Produo, aplicao e mercado, 1 edio Rio de Janeiro, RJ, Editora intercincia, Cap 16, 369-385. [2] Lee, K. T. & Akoh, C. C. (1998). Characterization of Enzymatically Synthesized Structured Lipids Containing Eicosapentaenoic, Docosahexaenoic and Caprylic acids. Journal of American Oil Chemists Society, 75, 495-499. [3] Sousa, J. S., Cavalcanti-Oliveira, E. D., Aranda, D. A. G., & Freire, D. M. G. (2010). Application of lipase from the physic nut (Jatropha curcas L.) to a new hybrid (enzyme/chemical) hydroesterification process for biodiesel production. Journal of Molecular Catalysis B: Enzymatic, 65, 133-137. [4] Institute of Medicine (IOM). (2005). Dietary Reference Intakes for Energy and Macronutrients. Washington, DC: National Academy Press. [5] Osborn, H. T. & Akoh, C. C. (2002). Structured lipids novel fats with medical, nutraceutical, and food applications. Comprehensive Reviews in Food Science and Food Safety, 1, 93-103.
Figure 1. Bacterial triggered release of test substance alizarin from a modified polysaccharide based bioresponsive polymer.
________________ [1] E.S. Sil et al, (2004) Prog. Polym. Sci, 29:1173-1222 [2] L.S. Nair et al, (2007) Prog. Polym. Sci, 32:762-798 [3] K.P. Schneider et al, (2011) Enzym Microb. Technol, 48: 312-318
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Effect of high pressure on lipase-catalyzed production of structured lipids rich in -3 polyunsaturated fatty acids
Joana Rodriguesa, Natlia Osrioa,b, Pamela Torresc, Vronique Perrierd, Francisco Plouc, Maria H. Ribeiroe, Eric Dubreucqd, Suzana Ferreira-Diasa a Instituto Superior de Agronomia, CEER-Biosystems Engineering, Technical University of Lisbon, Tapada da Ajuda, P-1349-017 Lisbon, Portugal; bInstituto Piaget, Ncleo de Investigao em Eng Alimentar e Biotecnologia, ISEIT de Almada, Quinta da Arreinela de Cima, 2800-305 Almada, Portugal; cInstituto de Catlisis y Petroleoqumica, CSIC, Marie Curie 2, 28049 Madrid-Spain; dMontpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier, France; eFaculdade de Farmcia, Research Institute for Medicines and Pharmaceutical Sciences (i-Med.UL), University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal. E-mail: nosorio@almada.ipiaget.org The interesterification of natural fats can improve certain physical and nutraceutical properties by modification of their acylglycerol profile. The aim of this work was to study the effect of high pressure on the interesterification kinetics of a fat blend, carried out in solvent-free medium, in the presence of three commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM and Novozym 435) or of a lipase/acyltransferase from Candida parapsilosis immobilized on Accurel MP1000. The reaction medium was a ternary blend of palm stearin, palm kernel oil and a concentrate of triacylglycerols (TAG) rich in omega-3 polyunsaturated fatty acids (EPAX 4510TG). Reactions were carried out at 60C in 6 successive batches of 3h each, for each pressure tested: 0.1 MPa (atmospheric pressure), 25, 50 and 100 MPa. The interesterification was followed by the decrease in Solid Fat Content values of the blend at 35C (SFC35C) and by the changes in the acylglycerol profile. The observed modifications in acylglycerol profiles showed that the four biocatalysts were still active after 6 batches (18h) at up to 100 MPa. Moreover, the values of SFC35C reduction and the acylglycerol profiles varied differently depending on the enzyme.
386
Comparative evaluation of two commercial cellulase complexes in the hydrolysis of pretreated wood at high solids concentration for bioethanol production
Lorena Solera, Lorena Alvarezb, Nelly Zamudiob, Germn Arocaa, Andrs Illanesa a School of Biochemical Engineering, Pontificia Universidad Catlica de Valparaso, General Cruz 34, Valparaso, Chile. b Bioenercel S.A. Barrio Universitario s/n, Concepcin, Chile. www.bioenercel.com E-mail: lsoler@bioenercel.com Two commercial cellulase preparations: NS50013 supplemented with NS 50010 from Novozymes (Denmark) and Accelerase 1500 from Genencor (USA), were evaluated in the hydrolysis of Organosolv-pretreated wood from Eucalyptus globulus. The enzymes were first characterized in terms of: global cellulolytic activity (filter paper units, FPU), -glucosidase activity (p-nitrophenylglucosidase units, pNPGU), xylanase activity, protein content (Bradford) and thermal stability. 1 FPU was defined as the amount of enzyme that produces 2 mg of glucose from a 50 mg Whatman N 1 filter paper piece, in 1 hour at 50 C and pH 4.8. 1 pNPGU was defined as the amount of enzyme that hydrolyses 1 mol of p-nitrophenilglucopyranoside per minute at 50 C and pH 4.8. 1 unit of xylanase activity was defined as the amount of enzyme that hydrolyses 1 mol of birch xylan per minute at 50C and pH 4.8. The NS50013 cellulase complex was supplemented with -glucosidase activity (NS 50010, Novozymes) in order to avoid cellobiose inhibition during hydrolysis, while the Genencor preparation needed no -glucosidase supplementation. Both enzymes were evaluated on the hydrolysis of a 10% (w/w) suspension of pretreated wood at 50 C and pH 4.8, at 10 and 20 FPU/g glucan using a 1:3 FPU/pNPGU supplementation ratio for the Novozymes enzymes. The reactions of hydrolysis were conducted in stirred-tank reactors at 200 rpm and monitored for 72 hours. Samples were analyzed by HPLC with refractive index detector using Zorbax Carbohydrate Aminopropylsilane column. Conversions close to 100% were obtained with the supplemented NS 50013 complex for both FPU/g glucan ratio, while with Accelerase 1500 only 74 and 91% conversions were obtained with 10 and 20 FPU/g glucan, respectively. A possible explanation for these differences between both complexes is the amount of xylanase activity in each enzyme preparation, since the xylanolytic activity in NS50013 plus NS50010 was twice than in Accelerase 1500.
389
Enzyme-catalyzed modification of poly(ethersulfone) membranes
Norhan Nadya,b,c, Karin Schronb, Maurice C.R. Franssena, Remko M. Boomb, Mohammed S. Mohy Eldinc and Han Zuilhofa a Laboratory of Organic Chemistry, Wageningen University, Dreijenplein 8, 6703 HB Wageningen, the Netherlands; bFood Process Engineering group, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, the Netherlands; cPolymers Department, Advanced Technology and New Materials Research Institute (ATNMRI), Mubarak City for Scientific Research and Technology Applications (MuCSAT), New Boarg El-Arab City 21934, Alexandria, Egypt E-mail: maurice.franssen@wur.nl Poly(ethersulfone) (PES) is the thermoplastic material of choice for the manufacture of ultrafiltration and microfiltration membranes, due to its structural and chemical stability. Unfortunately, the separation performance of PES membranes often deteriorates because of membrane fouling, which is attributed to the intrinsic hydrophobic character of this material. Therefore, introduction of different polar functional groups to the PES membrane surfaces has been reported in literature by incorporation of hydrophilic polymer through blending, coating, and radiation induced-grafting [1]. Although successful to some extent, these methods only offer random control over the resulting surface structure and may be environmentally adverse. This study presents the first successful example of enzyme-catalyzed modification of PES membranes. Various phenolic acids were coupled to the membrane at room temperature using laccase in aqueous medium, and the resulting surfaces were investigated by XPS, FT-IR and 1H-NMR [2]. The extent of modification was monitored by coloration of the modified membranes and by weighing (grafting yield). The effect of modification on both membrane flux and protein repellence were determined. Based on our results, it is clear that it is not just hydrophilicity of the surface that influences protein adsorption.
390
Engineering an improved commercial glucoamylase for the production of fuel ethanol
Daniel Torres Pazmioa, Richard Bottb, Wolfgang Aehlea, Martijn Scheffersa, Don Wardc, Jackie Huitinkb, Piet van Solingena, Igor Nikolaeva, Lydia Dankmeijera, Pim van der Kleija, Veli Alkana, Marco van Brussela, Casper Vroemena a Genencor - a Danisco Division, Archimedesweg 30, 2333 CN Leiden, The Netherlands; b Genencor - a Danisco Division, 925 Page Mill Road, Palo Alto, California 94304, U.S.A. c Genencor - a Danisco Division, 1000 41st Avenue Drive SW, Cedar Rapids, Iowa 52404, U.S.A. E-mail: daniel.torres.pazmino@danisco.com Glucoamylases (1,4--D-glucan glucohydrolases) catalyze the removal of successive glucose units from the non-reducing ends of starch. These enzymes are applied in large scale fermentations to convert starch into fuel ethanol. In order to make this process more efficient, new glucoamylases with improved specific activities are needed. The glucoamylase from Trichoderma reesei is an excellent starting point for a protein engineering effort, as the three-dimensional structure of this enzyme with an intact starch binding domain was recently solved by X-ray crystallography (1). In addition, this glucoamylase has a specific activity that is twice higher than that of the glucoamylase from Aspergillus awamori. The three-dimensional structure revealed that the starch binding domain forms a stable complex with the catalytic domain to form an extended binding site. The variant, which comprised of substitutions at sites selected on the basis on the knowledge of this and related enzyme structures, having the best overall performance included a combination of sites from both the catalytic and starch binding domains.
___________________ 1. R. Bott et al. Biochemistry 47, 5746-5754 (2008)
Figure 1. Structures of poly(ethersulfone) (PES) and the phenolic acid substrates used.
Acknowledgment: this work is funded by ISPT Institute for Sustainable Process Technology ________________ [1] N. Nady, M.C.R. Franssen, H. Zuilhof, M.S. Mohyeldin, R. Boom and K. Schron. Surface modification methods for poly(arylsulfone) membranes: a mini-review focusing on surface modification. Desalination, in press. [2] N. Nady, K. Schron, M.C.R. Franssen, B. van Lagen, S. Murali, R.M. Boom, M.S. Mohyeldin and H. Zuilhof. Mild and highly flexible enzyme-catalyzed modification of poly(ethersulfone) membranes. ACS Appl. Mater. Interfaces 2011, 3, 801-810.
October 2-6, 2011
170 391
Chemical modification and stabilization of raw starch digesting amylase (RSDA) using acetic acid anhydride
Tochukwu N. Nwagua, Bartho N. Okoloa, Hideki Aoyagib, Shigeki Yoshida a Microbiology Department, Faculty of Biological Sciences, University of Nigeria, Nsukka. b Life Sciences and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan E-mail: tochukwu.nwagu@unn.edu.ng Raw starch digesting amylases (RSDAs) have enormous potentials in biotechnology limited by enzyme instability. RSDA from Aspergillus carbonarius was chemically modified with different concentrations (10, 15 and 20 mg) of acetic anhydride (AAA). Effect of AAA concentration on enzyme structure, activity, stability and kinetics were evaluated. Acetylation levels were determined using trinitrobenzene sulphonic acid (TNBS) assay. Circular dichroism and fluorescent intensity spectra were used to probe structural changes to the modified enzyme. Modification with 15 and 20 mg AAA led to 1% and 13% increase in RSDA activity respectively, while 10 mg AAA resulted in 9 % loss of native enzyme activity. pH optimum remained at 5 when modified with 15 and 20 mg AAA, same as the native enzyme but was altered to pH 4 when 10 mg AAA was used. Over 90 % residual activity was retained over the range of pH 3-9 by modification with 10 mg AAA, compared to average of 60 % by native enzyme. Optimum temperature shifted from 30oC to 50oC for 15 mg AAA and 20 mg AAA derivative while 10 mg AAA was most temperature stable retaining up to 90 % activity at 80oC. Modification led to a decrease in the Km of the enzyme. Activity of 10 mg AAA derivative was stimulated by Zn2+ (6.76%), SDS (47.88%), cholic acid (57.91%), Ca2+ (15.25%), Triton X-100 (9.14%) and Tween 80% (65.25%). Using 10 mg AAA to modify the lysine groups proved a simple and effective way of improving the stability profile of the RSDA.

Biogas production from cassava tubers in semi-continuous mode anaerobic digestion
Wipada Siri-anusornsak1,2, Suphang Chulalaksananukul 3 Chompunuch Virunanon2,4 Warawut Chulalukkananukul*2,4 1 Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 2 Biofuels by Biocatalysts Research Unit, Faculty of Science, Chulalongkorn University, Bangkok 10330 3 Department of Chemical Engineering, Faculty of Engineering, Mahidol University, Nakornpathom 73170 4 Department of Botany, Faculty of Science, Chulalongkorn University, Bangkok 10330 Biogas was generated from starch-rich of cassava tubers combined with seed culture prepared from cow dung in single stage bioreactor. The operation was conducted in 5 liters digester with 3 liters of working volume at ambient temperature. The system was developed for methane production effectively, consisted of a starch hydrolysis unit together with a combined acidogenesis and methanogenesis unit operation. After 10 days of incubation, the effects of different hydraulic retention time (HRT) to biogas production at HRTs of 5, 10, 15, 20, and 25 days in a semi-continuous mode system. The results show high stability condition with pH of 7.14- 7.21 during methane production. The maximum methane concentration of 43.23% was obtained at HRT of 20 days of fermentation, as well as, chemical oxygen demand (COD) removal efficiency was reach 64.50% at HRT of 20 days. The maximum amount of biogas production was 1.95 l of average of 1.50/day of gas production.
Keywords: Hydraulic Retention Time (HRT), Semi- continuous mode, Anaerobic digestion, Biogas, Methane, Cassava tuber

Kinetic analysis of enzymatic saccharification of lignocellulose
a
171 396
Lipase Catalyzed Esterfication and Extraction of Acetic Acid from Fermentation Broth
Anett Kstner, Doris Schieder, Martin Faulstich, Volker Sieber TU Mnchen, Germany The separation of organic acids, like acetic acid, from fermentation broth requires sophisticated methods due to the good solubility of the acids in aqueous solutions. Lipases are able to catalyze both hydrolysis and esterification. This offers the possibility to separate water soluble acid by transformation into water insoluble esters. In our work we studied the esterification of acetic acid with 1-octanol by various lipases. Tests were performed in model solutions as well as in a fermentation broth of homoacetate fermentation. Within the reaction, 1-octanol is supposed to activate the lipase and to act as reactant on the one hand and as an organic extractant for the resulting ester on the other. Our experiments showed that acetic acid could be esterified with 1-octanol and the ester could be completely extracted in 1-octanol without an additional organic solvent. A lipase from Aspergillus oryzae as well as the immobilized CaLB Novozym 435 offered the best results with respect to the degree of esterification. We tested a range of 0.1 to 1 M acetic acid. The esterification yields were only marginally affected by the enzyme load and the concentration of acetic acid while the conversion rates increased with both increasing enzyme load and acid concentration. At single stage esterification conversion degrees of about 50% could be achieved. Multi-stage esterification enabled enhanced yields. The best yields were achieved at pH below 4. The experiments with fermentation broth showed the same results as the model solutions. By our experiments we could demonstrate the potential of lipase catalyzed esterification for the recovery of acetic acid from fermentation broth which may be further improved.
Monschein Mareikea, Schwarz Alexandraa, Nidetzky Berndb Austrian Centre of Industrial Biotechnology, Petersgasse 14, A-8010 Graz b Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz E-mail: mareike.monschein@tugraz.at Bioethanol produced from lignocellulosic material is a promising candidate for meeting the growing demand for alternative renewable energy sources. However, large scale process commercialization is mainly hindered by high costs associated with enzymatic hydrolysis. Among others, the dramatic decrease of hydrolysis rate at high degrees of conversion results in long process times and a need for high enzyme concentrations. To overcome these limitations a more fundamental understanding of relevant enzyme and substrate variables as well as the identification of rate-limiting factors is necessary [1]. In a systematic approach, binding studies were performed on lignocellulosic (wheat straw) and microcrystalline (Avicel) model substrates using an enzyme mixture from Trichoderma reesei in varying loadings. Determination of protein binding, glucose release and release of reducing sugars, were used to monitor enzyme adsorption and progress of hydrolysis. Quantitative modelling represents a powerful tool for advanced understanding of enzymatic hydrolysis [2]. Based on our results an empiric model describing cellulose hydrolysis was developed. The definition of hydrolysis parameters enables the analysis and evaluation of experimental set-ups with regard to hydrolytic efficiency.
______________ [1] Bansal P., et al. Biotechnology Advances 27, 833-848 (2009) [2] Zhang Y.-H. P., et al. Biotechnology and Bioengineering 88, 797-819 (2004)
393
Functionalization of chitosan by laccase-catalyzed oxidation of ferulic acid and ethyl ferulate under heterogeneous reaction conditions
Abdulhadi Aljawisha, Isabelle Chevalotb, Bernadette Piffauta, Jordane Jasniewskia, Jol Schera, Lionel Munigliaa. a Laboratoire dIngnierie des Biomolcules (LIBio), Nancy-Universit. 2 avenue de la Fort de Haye, F-54500 Vanduvre-ls-Nancy. France b Laboratoire Ractions et Gnie des Procds (LRGP), Nancy-Universit. 2 avenue de la Fort de Haye, F-54500 Vanduvre-ls-Nancy. France E-mail: abdulhadi.aljawish@ensaia.inpl-nancy.fr Chitosan particles were functionalized with ferulic acid (FA) and ethyl ferulate (EF) as substrates using laccase from Myceliophtora thermophyla (Suberase) as biocatalyst. The reactions were performed in enzymatic reactor with chitosan particles under an ecofriendly procedure, in a heterogeneous system at room temperature, in aqueous medium (phosphate buffer) and at neutral pH (7.5). Results showed that laccase from Myceliophtora thermophyla could oxidize FA and EF to reactive radicals and that oxidation rate of EF was higher than of FA. Furthermore, the presence of chitosan did not interfere with the oxidation rate of FA or EF. Color analyses demonstrated that the FA-chitosan derivative presented an intense yelloworange color which was stable even after several procedures of washings with different solvents while the EF-chitosan derivative was colorless. The spectroscopic analyses by UV-Visible, FT-IR and 13C NMR of the derivatives indicated that the reaction products bound covalently to the free amino groups of chitosan at C-2 region via Schiff base bond (N=C). Additionally, antioxidant properties of chitosan derivatives, including DPPH, ABTS and reducing power, showed that the chitosan derivatives presented improved antioxidant properties, especially for FA-chitosan derivative. These results clearly emphasized the interests for using renewable resources and biochemical processing to create functional polymers. These innovative molecules present high value-added properties and maybe used as active food-packaging and antioxidant additives for foods as well as for cosmetics.
394
Immobilized in porous carrier whole-cell lipase preparations for polyols production
Lukasz Stanczyk, Miroslawa Szczesna-Antczak, Katarzyna Struszczyk, Magorzata Rzyska, Stanisaw Bielecki, Tadeusz Antczak Technical University of Lodz, Institute of Technical Biochemistry, Stefanowskiego 4/10 Str., 90-924 Lodz, Poland; E-mail: lukasz.stanczyk@p.lodz.pl Presented study is a part of task 2.2 of a project realized by a consortium formed by Technical University of Lodz, Institute of Biopolymers and Chemical Fibers in Lodz, Centre of Molecular and Macromolecular Studies PAS in Lodz, University of Agriculture in Krakow and Central Mining Institute in Katowice. The leading idea of the Project involves the transformation of various kinds of biomass by biotechnological methods into environmentally friendly polymer materials. Task 2.2 consists in the development of a method of inexpensive lipase preparations production tools in directed chemo-enzymatic conversion of lipid biomass into oligo-/polyols which will be used as an additive in manufacturing of biodegradable alkyl-aryl polymers. Immobilized whole-cell lipase preparations are obtained through binding of mycelia of the oleaginous filamentous fungi Mucor circinelloides and M. racemosus from ITB culture collection onto porous carrier during their cultivation. Both the fungal strains produce intracellular lipases that are predestined to catalyze reactions in non-aqueous media [1-2]. Preliminary investigations comprised among other: selection of induction conditions of the biosynthesis of lipases active in transesterification processes (including acidolysis) and stabilization of lipases contained in mycelial Mucor preparations. To obtain the immobilized biocatalyst, a porous carrier was chosen among commercial polyester and polyether foams with the open porosity and different pore size, and conditions of effective binding of the lipolytic strains to this carrier were optimized (including culture medium composition and % content of the carrier in the culture medium). It was found that efficient lipase synthesis (>18000 U/dm3) was achieved during relatively short, 3 day culture in the medium containing nitrogen (e.g. corn steep liquid) and carbon sources (triacylglycerols and/or fatty acids). Optimization of immobilized lipase preparations production in the large-laboratory scale included selection of shape and size of porous material, its location in typical bioreactors purchased from Infors (volumes of 1 dm3 and 24 dm3) and the method and rate of mixing and aeration.. Also alternative methods of de-fatting and de-hydration of the biocatalyst immobilized on the porous carrier and destined for catalysis in non-aqueous media (containing plant oils and fatty acids or alcohols) were developed. pH of lipase microenvironment and water content expressed as water activity (aw) were optimized. The obtained, Mucor lipase preparations immobilized in porous carriers display the high operation stability (above 100 days) in continuous transesterification processes including alcoholysis (aliphatic alcohols C2-C5 [3]) as well acidolysis (saturated fatty acids) of plant oils and waste fats. The study was realized within the scope of the project POIG 01.01.02-10-123/09: Application of biomass in production of environmentally friendly polymer materials task PZ 2.1., co-financed from the funds of European Fund of Regional Development within the frames of Operation Program Innovative Economy 2007-2013.
________________ [1] Antczak T., et al.: J. Mol. Cat. B, 19-20, 2002, 287-294 [2] Szczesna-Antczak M., et al.:: J. Mol. Cat. B, 29, 2004, 163-171 [3] Szczsna-Antczak M. et al.: Renewable Energy, 34, 2009, 1185-1194
October 2-6, 2011
172 397
Development of an enzymatic kit for ethanol determination
Camila A. O. Diasa, Flvia Danielia, Karina R. Lopesa, Ftima P. Souzab, Marcelo A. Fosseyb, Cleslei F. Zanellia, Sandro R. Valentinia, Edwil A. L. Gattsa a School of Pharmaceutical Sciences UNESP, Araraquara, Brazil b Institute of Biosciences, Language, and Science UNESP, So Jos do Rio Preto, Brazil Ethanol determination in alcoholic beverages, drugs and fuel is very important for increasing the yield of the production processes and product quality. Although there are several methods to determine ethanol concentration, the enzymatic methods have shown to be the most effective. The aim of this work was to develop an ethanol enzymatic quantification kit using recombinant alcohol dehydrogenase enzyme from S. cerevisiae. The yeast gene encoding the alcohol dehydrogenase (ADH1) was cloned into the pET28a vector for expression in E. coli (BL21) and several conditions for optimum production of the recombinant enzyme were screened. After production of insobuble alcohol dehydrogenase in E. coli, purification by affinity chelating chromatography was carried out under denaturing conditions using the 6xHis tag fused to the enzyme and followed by protein refolding. The activity and stability of the recombinant enzyme alcohol dehydrogenase was determined using standard protocols (UV) at different temperatures and times. The results obtained demonstrated in vitro activity of the recombinant enzyme similar to that of a commercial endogenous S. cerevisiae enzyme (Sigma) and showed a minimal detection limit of 2,3 x 104 g/L of ethanol in 2 minutes. In addition, our recombinant alcohol dehydrogenase was stable up to 120 days when stocked at -20C. All the features presented by the recombinant alcohol dehydrogenase produced using the methods determined herein enable the use of this enzyme in a kit to be low-cost, fast and accurate for assaying ethanol concentration.
Supported by FAPESP, CNPq.
Industrial processes research & development 398

Polymeric resins as strategy for removal of chiral amines, produced using -transaminase.
Watson Neto, Pr Tufvesson and John M. Woodley Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark (DTU), DK-2800 Kgs. Lyngby, Denmark E-mail: wan@kt.dtu.dk In situ product recovery brings several advantages to processes where equilibrium and product inhibition are an issue limiting the process to achieve economically significant yield and productivity. The production of chiral amines using -Transaminase (-TAm) is an excellent representative example, where the removal of the product drastically improves the process yield. For instance, in the production of (S)--methylbenzylamine (MBA) (Fig 1) which is significantly limited by equilibrium and product inhibition, the continuous removal of the product will allow the reaction to proceed faster towards the product (by shifting the equilibrium as well as alleviating product inhibition) [1].

High-scale expression of a recombinant 17-hydroxysteroid dehydrogenase
Fogal Stefanoa, Motterle Riccardob, Arvotti Giancarlob, Castellin Andreab, Bergantino Elisabettaa a University of Padova, Department of Biology via Ugo Bassi 58b-35121 Padova, Italy; b F.I.S.- Fabbrica Italiana Sintetici S.p.a, Alte di Montecchio Maggiore viale Milano 2636075 Vicenza, Italy E-mail: stefano.fogal@unipd.it The optimization of a new procedure to produce the recombinant 17-hydroxysteroid dehydrogenase (17-HSD) type 5 from mouse is presented. The enzyme has to be used in the bio-catalyzed synthesis of Testosterone (TS) from Androstendione (AD) (fig. 1). The coding sequence of the enzyme, cloned in a suitable plasmid in fusion with an His-tag, is expressed in E.coli. For its characterization, activity tests were performed with enzyme purified from small-scale cultures1. The enzyme catalyses the transformation with complete regio- and stereo-selectivity, since neither reduction of the 3-keto group nor the presence of the 17-hydroxyl stereoisomer have been detected. However for its industrial application, high enzyme quantities are needed. To support the optimization of the biocatalytic process, the production procedure has been furthermore investigated. Different components of the fermentation broth have been considered and tested to optimise the process and to reduce costs of scaling up. LB medium has been replaced with the less expensive M9, while in the induction IPTG was replaced by lactose. An auto-inducing procedure was also chosen in order to avoid the periodic optic density measurements[2]. These optimized conditions give enzymatic yields comparable with those obtained by the standard ones[1]. Eventually we have been able to scale the culture up to 500 litres and the subsequent IMAC purification has allowed the gain of enough enzyme to perform a Kilo-lab study of the bio-catalytic process.
173 402
Construction and modification of two human PAM gene domains separately and its expression in CHO dhfr-cells.
D. Mikiewicz, M. Zielinski, M. Ksik-Brodacka, A. Wjtowicz-Krawiec, P. Kiery, A. Jagieo, L. Chojnacka-Puchta, G. Pucienniczak, A. Pucienniczak. Institute of Biotechnology and Antibiotics, Staroscinska 5, 02-516 Warsaw, Poland mikiewiczd@iba.waw.pl Peptidylglycine -amidating monooxygenase (PAM) is a multifunctional protein containing two enzymes that act sequentially to catalyze the -amidation of neuroendocrine peptides. Peptidylglycine -hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-hydroxyglycine -amidating lyase (PAL) catalyzes the second step of the reaction. We obtained two enzymes PHM and PAL separately in the eukaryotic expression vector pCG. Both genes are derived from the recombinant human bifunctional PAM Satani gene obtained previously in pCG vector in our Department. The pCG/dhfr/PHM and pCG/dhfr/PAL plasmids contained CMV promoter, dhfr gene, MTX resistant variant, with promoter SV40 and the human PHM or PAL sequence deprived of transmembrane domain sequence, but with added different recombinant Tags on the C-end. The different Tags, FLAG or c-Myc Tag were used to recombinant PHM or PAL identification. The PHM and PAL genes have changed signal sequence which influenced the stability in transfected cells and secretion peptides into a medium.
Several techniques such as extraction using solvents or membranes, or cascade reactions can be applied to remove a specific product from a complex mixture [2]. This work will focus on the use of polymeric resins to selectively remove product MBA in the above mentioned reaction. Different resins have been studied and compared with respect to affinity and selectivity to the product, and the implications for process design will be discussed.
________________ [1] P. Tufvesson, J. L. Ramos, J. J. Skibsted, N. Al-Haque, W. Neto, J. M. Woodley; Biotechnology and Bioengineering 108 (2011) 1479-1493 [2] A. Freeman, J. M. Woodley and M. D. Lilly; Bio/Technology 11 (1993) 1007-1012
Figure 1. Enzymatic conversion of Androstendione (AD) to Testosterone (TS) catalysed by mouse 17-hydroxysteroid dehydrogenase type 5 (17-HSD).
________________ [1] Fogal, S.; Motterle, R.; Castellin, A.; Arvotti, G.; Bergantino, E. Chemical Engineering Transactions 2010, 20, 61-66. [2] Studier, F.W.; Protein Expression and Purification 2005, 41, 207-234.
399
Microuidic enzymatic reactors using -transaminases
Vijaya Krishna Bodla, Ulrich Krhne, John M. Woodley, Krist V. Gernaey Center for Process Engineering and Technology, Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK-2800, Denmark, E-mail: jw@kt.dtu.dk In recent years, miniaturizing reaction formats has gained interest since the productivities of some chemical reactions can be enhanced by orders of magnitude. Miniaturization helps diffusion-limited reactions to occur significantly faster than they would at the larger scale. Of particular interest is the potential for high throughput experimentation for rapid screening of reactions, determining kinetics, exploring hazardous chemistry and developing chemical reactions[1]. The aim of this study is to evaluate the effect of miniaturization, if we can improve or gain better insight, on biochemical enzymatic reactions using biocatalytic transamination as a model reaction.
400
Practical aspects of integrated operation of biotransformation and SMB separation for fine chemical synthesis
Nina Wagnera, Markus Fueredera, Andreas Bossharta, Sven Pankea, Matthias Bechtolda a Bioprocess Laboratory, ETH Zurich, Switzerland E-mail: nina.wagnerr@bsse.ethz.ch Integrated operation of biotransformation and simulated moving bed (SMB) separation constitutes an attractive option for high-yield manufacturing of commercially relevant compounds such as rare sugars from equilibrium- limited isomerase- and epimerase reactions. In principle, the established epimerases and isomerases allow the synthesis of numerous rare sugars from low cost substrates such as D- psicose and D- tagatose from D- fructose and D-galactose[1]. However, in order to overcome the intrinsic yield limitation of such biotransformations a selective removal of product from the reaction space is required. Due to the small differences in physico-chemical properties of product and substrate a very powerful separation method such as column chromatography is required[2]. In this work we present the first lab-scale implementation of such an integrated process consisting of an enzyme membrane reactor (EMR), continuous chromatography (SMB) and nanofiltration (NF) (Fig.). As test system the production of the rare, but commercially attractive sugar D- psicose [3] by D- tagatose epimerase catalyzed epimerization from D- fructose was selected. While a typical batchwise eprimerization of D- fructose would stop at 25%, a yield of 97% was obtained when operating the fully integrated process for a number of days with absolute product purities. Next to demonstrating the proof of principle, other practical aspects that equally determine the applicability of a process such as stability, start up and process robustness were evaluated. Long term operation of the respective units indicated a potential process time of 5-6 days that could be further extended in the future by engineering a more stable enzyme variant and by implementing a cleaning in place approach for SMB column regeneration. Further, reasonable start-up times of approximately 5 hours and robust system behaviour towards changes in the feed concentration could be demonstrated. It is worth noting that this is the first report on such an integration of biotransformation and SMB separation.
403
The new expression system with using deubiquitinating UBPD2C protease and fusion proteins for an efficient synthesis of proteins that would be useful in industrial processes.
Wjtowicz-Krawiec A., Chojnacka-Puchta L., Pucienniczak G., Mikiewicz D., Sokoowska I., ukasiewicz N., Marciniak-Rusek A., Mazurkiewicz A., Jagieo A., Plucienniczak A. The Institute of Biotechnology and Antibiotics, Staroscinska5, 02-516 Warsaw, Poland E-mail: wojtowicza@iba.waw.pl The aim of this work was to obtain an expression system for an efficient synthesis of the UBP1 protease variant that would be useful in industrial processes. Ubiquitin is composed of 76 amino-acid residues with a total molecular mass of 8.6 kDa. This protein is an element of the universal protein modification in eukaryots called ubiquitination, a phenomenon which does not occur in bacteria. In spite of that, it has been shown that proteins fused to ubiquitin undergo greater expression in Escherichia coli and are easier to purify and renaturate than nonhybrid foreign proteins [1]. However, to take advantage of these properties of hybrid proteins in technological processes, large amounts of proteases for cleaving specifically ubiquitin from those proteins are necessary. This is the reason why hybrid proteins containing ubiquitin at their N-end cannot be used in large scale biotechnological processes. We have obtained the truncated variant of the sequences encoding UBP1 without the transmebrane region. We have described the synthesis of the yeast deubiquination enzyme UBP1 mutein in E. coli. The obtained variant of the enzyme may be successfully used for processing large amounts of hybrid proteins comprising ubiquitin or tagged ubiquitin at their N-ends. We have shown that using UBPD2C protease analogue, tagged at the C-end by 6xHis, for cleaving fusion proteins containing N-end His-tagged ubiqutin of the type 6xHisUb::protein for example: Ub::hGH (hGH human grow hormone) and Ub::S (S the substrat protein) facilitates purification of the protein present in the hybrid to a great extent. The high expression level in E. coli of our UBP1 analogue and the expression purification system allows the use of ubiquitin::protein fusion in large scale production of recombinant proteins.
__________________ [1] Baker R: Protein expression using ubiquitin fusion and cleavage. Curr Opin Biotechnol. 1996, 7:5416.
404
Carbon-carbon bond hydrolases and lyases for industrial applications
Annika Frank* and Gideon Grogan York Structural Biology Laboratory, University of York, Department of Chemistry, Heslington, YO10 5DD, York, United Kingdom * E-mail: annika@ysbl.york.ac.uk The cleavage and formation of carbon-carbon bonds are key reactions in numerous biotransformations and catalysed by different classes of enzymes. Phenolic acid decarboxylases belong to the carbon-carbon bond lyases and catalyse the removal of carboxyl groups from various phenolic acids. The styrene derivatives produced are useful chemical backbones in the pharmaceutical industry or form the basis of food and fragrance compounds. In this project, a phenolic acid decarboxylase from Bacillus subtilis (BsPAD) (1) was analysed with respect to substrate turnover and structural aspects of ligand binding. A mutant library could be prepared which includes alterations in seven active site residues, each of them having potential catalytic functions. All proteins of the library were expressed, purified and analysed. For the first time it was possible to co-crystallise a mutant in complex with its substrate and obtain the structure of a BsPAD with ligand bound in its active site. Data collection and molecular replacement presented the ligand density surrounded by a number of potential catalytic acid/base and aromatic residues. This suggests a reaction mechanism similar to that proposed previously (2,3) and is supported by semi-quantitative kinetic data collected for all mutants using high-performance liquid chromatography. These results now provide an important insight into the degradation of phenolic acids and open possibilities for protein engineering and optimization.
Fig 1: Biocatalytic transamination The main challenges are: (1) an unfavourable thermodynamic equilibrium position, necessitating processes to shift the equilibrium; (2) substrate and product inhibition; (3) low substrate solubility, giving low volumetric productivities; (4) high biocatalyst cost2. Here we attempt to study the performance of this reaction in an aqueous/organic segmented flow capillary microreactor. A simple Y shaped micro-mixer has been fabricated where two inlet fluids, organic and aqueous phase, join at a junction creating alternate fluid segments. Different reaction conditions have been studied simultaneously due to a micro-milling based parallel fabrication of the flow system. The focus has been on the user friendliness of the system in order to perform rapid screenings. Some specific issues requiring careful consideration while developing such microsystems for biocatalytic reactions will be discussed: surface modifications, control of fluid behaviour in microchannels and detection limitations[1]. The micro-mixer is to be used for a) screening of various organic solvents; b) studying the reaction rate - mass transfer rate limitations (Damkhler number); c) conducting experiments with varying process conditions like flow velocity, enzyme concentration and phase ratio. Further, CFD (computational fluid dynamics) modelling has been used to study the different reactor formats. This system is expected to give better understanding of the rate limitations, rapid screening of organic solvents and process conditions.
______________________ [1] Matosevic S., Szita N., Baganz F.; J Chem Tech and Biotechnol., (2011) 86, 325471. [2] Tufvesson P.; Lima-Ramos J.; Jensen SJ.; Al-Haque N.; Neto W.; Woodley JM.; Biotechnol. Bioeng., (2011) 108, 1479-1493.
Figure: Flow scheme of the integrated process: D-fructose fed to the bioreactor is partially converted to D-psicose, the resulting reaction mixture is separated in the SMB. In order to account for the inherent dilution of the SMB the substrate SMB outlet is subjected to a nanofiltration concentration and subsequently recycled to the reactor feed.
____________________ [1] Izumori, K. J. Biotechnol. 2006, 124, 717 722. [2] Bechtold, M.; Panke, S. Chimia 2009, 63, 345. [3] Oh, D. K.; World J. Microbiol. Biotechnol. 2007, 23, 559 563.
Fig.1: BsPAD accepts various phenolic acids as substrates and decarboxylates them to give their styrene derivatives. The structure of a mutant protein with ligand bound in the active site shows the phenolic acid to be surrounded by potential catalytic residues.
_________________ 1. Cavin, J. F., Dartois, V., and Divies, C. (1998) Applied & Environmental Microbiology 64, 1466-1471 2.Hashidoko, Y., and Tahara, S. (1998) Archives of Biochemistry & Biophysics 359, 225-230 3. Rodrguez, H., Angulo, I., de las Rivas, B., Campillo, N., Pez, J. A., Muoz, R., and Mancheo, J. M. (2010) Proteins: Structure, Function,and Bioinformatics 78, 1662-1676
October 2-6, 2011
174 405
Integrated operation of racemization and SMB- enantioseparation for the high-yield production of amino acids
Markus Fuereder, Christian Femmer, Matthias Bechtold , Sven Panke Bioprocess Laboratory, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland; E-mail: markus.fuereder@bsse.ethz.ch SMB technology, the technical realization of continuous chromatography, has matured into a standard tool for large-scale enantioseparations over the last decade. Incorporating a mild enzymatic racemisation procedure into a chiral SMB process (Figure 1.) allows for obtaining the target enantiomer from the racemic mixture in theoretically 100% yieldcompared to 50% yield with the a stand-alone chiral SMB [1]. Next, such a process gives access to both enantiomers with one installation compared to the three enzymes required in dynamic kinetic resolution approaches for the same flexibility. Arguably, amino acids constitute one of the most intriguing areas of applications of such an integrated process due to the prominence of this molecule class in fine chemistry and the availability of an amino acid racemases with a broad substrate spectrum (P. putida DSM 3263). In this work we present the design and operation of a process integration of a chiral SMB equipped with Chirobiotic TAG (Sigma Aldrich, USA) columns and an enzymatic racemisation for the production of enantiopure D-methionine. The design and subsequent optimization of a fully integrated production system requires a detailed characterization of all units involved, specifically reaction kinetics and stability, adsorption isotherms and rejection of the nanofiltration membrane. Based on these parameters model-based design is applied in order to identify appropriate operating points for a set of specifications such as product purity. The usefulness of the obtained design is evaluated by experimental runs of the fully integrated process which constitutes the first report on the experimental implementation of such a biotransformation - chiral SMB integration.

New enzymes for industrial applications from non-conventional yeasts
Silvia Nicotra,a Chiara Dugoni,a Roberto Verga,b Silvia Rapacioli,b Benedetta Turchetti,c Pietro Buzzinic a Resindion srl (Italian Subsidiary of Mitsubishi Chemical Corporation), via Roma 55, Binasco (MI), Italy bBiCT srl, via Einstein snc, Lodi, Italy cDepartment of Applied Biology & Industrial Yeasts Collection DBVPG, University of Perugia, Borgo XX Giugno 74, Perugia, Italy E.mail: roberto.verga@bict.it Biotransformation processes are increasingly used to replace conventional chemical processes and to ease new products development. Accordingly, the use of whole cells or isolated enzymes (both in free or immobilized form) represents a valid and flexible tool for biotransformation. Screening of novel activities expressed by microbial germplasm is always strategic: in this framework, the so-called non-conventional yeasts (NCYs) could represent an interesting opportunity [1]. In this background, new NCYs (including psychrophilic ones) from natural environments have been recently isolated and studied [2, 3]. University and private companies are joined into a synergic project focused to the selection of enzymes from these NCYs and their applications in industrial processes. All NCYs cultures involved into the project are deposited in the Industrial Yeasts Collection DBVPG (www.agr.unipg.it/dbvpg). A practical example considering psychrophilic strains are esterases produced by some strains belonging to the facultative psychrophilic NCY Cryptococcus gilvescens, which are able to catalyze specific reactions in the range between 4C and 20C [2]. Another interesting example is represented by enoate reductases (ERs), enzymes belonging to the OYE family and able to catalyze asymmetric bioreduction of electron-poor alkenes. The ERs can undoubtedly be considered as emerging biocatalysts, since they catalyze a key step in the synthesis of increasingly complex drugs and fragrances. A number of NCYs were preliminarily tested, such as species of the genera Cryptococcus, Debaryomyces, Hanseniaspora, Kazachstania, Lindnera, Nakaseomyces, Vanderwaltozyma, and Wickerhamomyces. For the purification and the possible immobilization purposes are used Resindion resins and carriers, particularly the recent new product lines ReliSorbTM (ReliChromTM in pre-packed columns) and ReliZymeTM. More NCYs are at present under screening to develop further biocatalysts. The main target is the immobilization (where possible) onto solid carriers to permit the heterogeneous biocatalysis and enzyme recycling. This project represents a case of successful partnership among small Enterprise, big Industry and University, where all are focused to the same goal, joining different and synergic skills.
________________ [1] M. Goretti, C. Ponzoni, E. Caselli, E. Marchegiani, M.R. Cramarossa, B. Turchetti, L. Forti, P. Buzzini, Biores. Technol. 102 (2011) 3993-3998. [2] B. Turchetti, P. Buzzini, M. Goretti, E. Branda, G. Diolaiuti, C. DAgata, C. Smiraglia, A. VaughanMartini, FEMS Microbiol. Ecol. 63 (2008) 7383. [3] E. Branda, B. Turchetti, G. Diolaiuti, M. Pecci, C. Smiraglia, P. Buzzini, FEMS Microbiol. Ecol. 72 (2010) 354-36

Plug Flow reactors for bio manufacturing.
Edward Jonesa , Gilda Gasparinib, Kay McCleana, Ian Archerc C-Tech Innovation Ltd , Capenhurst Technology Park , Chester CH1 6EH , United Kingdom; bAMTechnology, The Heath Business & Technical Park, Runcorn,Cheshire, WA7 4QX, United Kingdom cIngenza Ltd. Wallace Building, Roslin BioCentre, Midlothian, EH25 9PP, United Kingdom. E-mail: Kay.McClean@ctechinnovation.com The adoption of more efficient development strategies and manufacturing techniques will be essential for future success in the bio manufacturing sectors. Continuous operation of biocatalytic processes has the potential to offer many advantages over established batch process methodologies. There exist opportunities for improved process control; ease of scale up; minimising of interruptions in production; reducing reactor size; and economic use of biocatalysts. The CofloreTM Agitated Cell Reactor (ACR) is a dynamically mixed plug flow reactor. The Coflore design employs a patented mixing technique where free moving agitators within each reaction stage promote mixing when the reactor body is subjected to lateral shaking. Multiple discrete (interlinked) reaction cells give good mixing and plug flow, and the design permits the use of slurries and handling of gas/liquid mixtures. The Coflore ATR is an industrial tube flow reactor for homogenous and two phase fluids. Employing the same mixing principle as the lab scale Coflore ACR, it uses lateral movement to generate mixing and stage separation to prevent back mixing. We describe the application of these continuous plug flow reactors for bioprocess development starting from simple lab scale batch processes; through benchtop plug flow reactors (ACR); and on to the multi-litre production scale agitated tube reactor (ATR). The presentation will compare the results of an oxidation reaction catalysed by D-amino acid oxidase (DAAO) operated under batch and continuous conditions, and will illustrate how application of the ACR and ATR reactors can facilitate process development.
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175 410
Dynamic cell based emulsions in biphasic biocatalytic systems and strategies for their downstream processing
a
J. Collinsa, C. Brandenbuschb, G. Sadowskib, A. Schmida, B. Bhlera Technische Universitt Dortmund, Department of Chemical and Biochemical Engineering, Laboratory of Chemical Biotechnology, Emil-Figge-Str.66, D-44227, Dortmund, Germany; bTechnische Universitt Dortmund, Department of Chemical and Biochemical Engineering Laboratory of Thermodynamics, Emil-Figge-Str.70, D-44227, Dortmund, Germany E-mail: jonathan.collins@bci.tu-dortmund.de The use of biphasic systems has emerged as a viable strategy for the implementation of a number of challenging whole-cell biotransformations. Often, the advantages of this technology are countered by the difficulty in handling the stable emulsions which are regularly produced in the process. In this work, the enantioselective epoxidation of styrene catalyzed by recombinant Escherichia coli is used as a model system to investigate the influence of changing reactor conditions on the formation of stable emulsions in an aqueous-organic bioreactor. Specifically, the emulsion stability was examined over the full course of the biotransformation. An abrupt decrease in stability was observed early on and was definitively linked to the accumulation of toxic oxidation products in the reactor. A method for the fractionation of the primarily cell-based emulsion was developed allowing for separation of the cells and measurement of their contact angles. The obtained results now enable effective downstream processing of these emulsions by means of a novel method utilizing supercritical carbon dioxide.[1]
Figure 1. Process scheme: By directing the SMB distomer outlet (L-Met) to an enzymatic racemization reactor(EMR) and subsequent recycling of the newly formed racemate to the SMB feed full conversion is achieved. In order to account for the inherent dilution of the SMB a nanofiltration (NF) is implemented for concentration of the distomer before the reactor.
________________ [1] M. Bechtold, S. Makart, M. Heinemann, S. Panke, Journal of Biotechnology 124 (2006) 146.
Figure 1. (a) Enantioselective epoxidation of styrene in a biphasic system; (b) Enhanced view of stable aqueous/bis(2-ethylhexyl)phthalate emulsion with stained cells (methyl violet); (c) View of stable emulsion after harvesting and prior to treatment with supercritical carbon dioxide Figure1 Coflore ACR Reactor Figure 2 ACR mounted in the shaking platform
[1] Brandenbusch B, Bhler B, Hoffmann P, Sadowski G, Schmid A. 2010. Efficient Phase Separation and Product Recovery in Organic-Aqueous Bioprocessing Using Supercritical Carbon Dioxide. Biotechnol Bioeng 107:642-641.
407
New application of lentikats biocatalyst for immobilisation of biologically active compounds
Radek Stloukal LentiKat's a.s., Evropska 423/178, 160 00 Prague 6, Czech Republic E-mail: stloukal@lentikats.eu Biocatalysis has been receiving an increasing interest over the last years in many different fields of industry. An important milestone was the advancement made in the use of immobilised forms of many types of biologically active compounds like free cells or enzymes. One of the recently developed immobilisation methods, Lentikats Biotechnology, uses polyvinyl alcohol (PVA) hydrogel entrapment, which has several advantages over the other currently available techniques. Application of this technology into existing or newly designed production processes leads to a substantial increase in process yields and a reduction in operational and investment costs. Lentikats Biotechnology has already found numerous applications in the pharmaceutical, food, chemical, environmental and biofuels industry. Significant achievement in the applications of this novel technology have been recently achieved in (I) the production 6-aminopenicillanic acid (6-APA) using immobilised penicillin G acylase activity, (II) the production of lactose free milk using immobilised -galactosidase, (III) the degradation of 1,2,3-tichloropropane (TCP) using multienzymatic Biocatalyst with haloalkane dehalogenase (DhaA), haloalcohol dehalogenase (HheC) and epoxide hydrolase (EchA), (IV) the increased stability of Lentikats Biocatalysts in organic solvents for the usage in non-agueous media and (V) in the improvement of enzymes properties by pre-immobilisation to magnetic nanoparticles and encapsulation of resulting conjugates into PVA hydrogel matrix. These projects clearly demonstrate the comprehensive nature and extremely high potential of this technology.
408
Continuous reduction of hardly water-soluble ketones in an enzyme membrane reactor
a
411
Encapsulation of lipase into biopolymer-based composites by using biocompatible ionic liquid
Min Hoo Kima, Keehoon Wonb, Hyung Joo Kima, Sang Hyun Leea Dept. of Microbial Engineeing, Konkuk University, Seoul 143-701, South Korea; bDept. of Chemical and Biochemical Engineering, Dongguk Univ-Seoul, Seoul 100-715, South Korea E-mail: sanghlee@konkuk.ac.kr
a
412
Production of -glucosidase, -arabinofuranosidase and feruloyl esterase at high substrate concentration and recovery
a
Susanne Leuchsa, Lasse Greinera,b DECHEMA e.V., Theodor-Heuss-Allee 25, 60486 Frankfurt am Main, Germany; bSeInstitut fr Technische und Makromolekulare Chemie, Worringerweg 1, 52074 Aachen, Germany E-mail:Leuchs@dechema.de The biocatalytic conversion of hardly water-soluble substrates is challenging due to the aqueous surrounding required for the biocatalyst. A two-phase approach is possible if the substrate shows at least a minimum solubility in aqueous media. In the case of nearly unsoluble substrates, this approach will lead to negligible reaction rates. With a biocompatible solubiliser, a monophasic approach is advisable. TEGO IL K5 (Evonik/Goldschmidt) was identified as good solubiliser for the biocatalytic reduction with an alcohol dehydrogenase from Lactobacillus brevis (LbADH) of 2-Octanone, 2-Nonanone, 2-Decanone, and 3-Octanone. LbADH is NADPH dependent, for the in situ cofactor-regeneration, a glucose dehydrogenase (GDH) is used. The continuous reduction is carried out in an enzyme membrane reactor (EMR) with an ultrafiltration membrane (MWCO: 10 kDa) to retain the enzymes in the reactor (see Fig. 1). For product separation, a stainless steel column filled with a solid phase extraction (SPE) material (HRP, Macherey-Nagel) is integrated in the product stream. The product adsorbs on the SPE material and is later eluted with an organic solvent. This setup allows for an easy recycling of the substrate-solution (including IL and glucose). For the reduction of 2-Octanone with 100 g/L TEGO IL K5 high turnover numbers (TON) of 4,100,000 for LbADH and 970,000 for GDH at a space-time yield (STY) of 374 mmol L-1 d-1 are reached. The recycling of 90 % of the substrate solution reduces the E-factor (kgwaste/kgproduct) by a factor of 10. LbADH is exclusively (R)-selective, so that the product, (R)-2-Octanol is obtained with an enantiomeric excess of > 99.9 %. Hence, by using TEGO IL K5 a stable process is possible with integrated downsream processing and recycling, resulting in high TON for both enzymes and good STY.
The demand for biopolymers-based materials has recently gained worldwide attention in the biomedical fields such as tissue engineering, drug delivery systems, dialysis membranes, and biosensors owing to their inherent biocompatibility and biodegradability. However, the insolubility of unmodified native biopolymers in most organic solvents has limited the applications of biopolymer-based materials and composites. Recently, ionic liquids have been reported to be good solvents to dissolve biopolymers and develop biopolymer-based materials, because of their synthetic flexibility by changing the combinations of cation and anion, and green solvent properties such as non-volatility, non-flammability and recyclability [1]. In this work, biocompatible ionic liquid, [Emim][acetate], was used to encapsulate lipase into various biopolymer-based composite beads. Cellulose, cellulose/chitosan, cellulose/ agarose, cellulose/agar, and cellulose-carrageenan beads containing lipase from Candida rugosa were prepared by co-dissolution of biopolymers and lipase in [Emim][acetate] followed by reconstitution with distilled water (Figure 1). Size and composition of biopolymer/lipase bead were controlled by changing flow rate of syringe pump and concentration of biopolymer. Average diameter of hydrogel bead was 2 mm and about 1 wt % of lipase per dried bead was entrapped. Specific activity of entrapped lipase was highly influenced by the types and concentration of biopolymer dissolved in [Emim][acetate]. Cellulose/ agarose was the most efficient composite to encapsulate and retain activity of lipase. Cellulose/agarose/lipase bead showed 2.5 times higher specific acitity than cellulose/carrageenan/lipase bead. Simple method to entrap lipase into various biopolymer composites was successfully developed by using biocompatible ionic liquid.
Riveros, R.a, Meza, M.b, Aguirre, C.b, Diaz A.a, Illanes, A.a School of Biochemical Engineering, Pontificia Universidad Catlica Valparaso, Av. Brasil 2241, Valparaso, Chile b Biotechnology Department, Faculty of Sciences, Universidad Catlica de la Santsima Concepcin, Alonso de Rivera 2850, Concepcin, Chile E-mail: rriveros@ucsc.cl Penicillium chrysogenum was cultivated in a designed medium supplemented with yeast extract (0,1%) and sugar beet pulp exhausted (SBPE) as carbon source at different concentrations in a liquid media in Erlenmeyer flask at 28 C, initial pH 4.0 and 200 rpm for 14 days. SBPE was previously selected as the carbon source that produced increased activity of the enzymes -glucosidase (GLU), -arabinofuranosidase (ARA) [1] and feruloylesterase (FE) in comparison with glucose, arabinose, glutamic acid, glycerol, ramnose and birch xylan. The production kinetic were folllowed for GLU, ARA and FE, which were measured with the artificial substrates p-nitrophenyl-glucopyranoside (PNPG), p-nitrophenyl arabinofuranoside (PNPA) and methyl ferulate (MF) at pH 5.0, 4.0 and 6.0, respectively. Activity GLU, ARA and FE for a design of 3 g/L of biomass are 952105 IU/L; 59068 IU/L and 22845 IU/L respectively, at 10 days of cultivation. Increasing the substrate concentration, increases activity, reaching 2722200 IU/L, 2700280 IU/L and 60771 UI / respectively, with a medium designed for 28 g/L of biomass. However, the highest activity per gram of substrate (IU/g) corresponds to 6.4 g/L of SBPE for the design of 3 g/L of biomass. For the recovery of enzymes was used distilled water, phosphate buffer 50 mM, pH 4.0 and 7.0, by varying the volume added by washing between 10 and 100 mL. The addition of the extracting agent is made subsequent to the separation of solids by centrifugation (20 min, 1C at 11,000 rpm) (method 1). In addition, the recovery of enzymes was studied but by adding the extracting agent prior to the separation of the solid (method 2). Each washing considers the agitation of the solid with the liquid during 30 min in a bath of ice and subsequent rest during 10 min [2]. The extraction of method 2 was selected because after the first centrifugation is obtained about 98% of all the enzymes in study on the filtrate when working with 20 g/L or lower concentrations of substrate. When the substrate concentration increases, a second washing is required for the achievement of equal percentage of recovery. With phosphate buffer pH 4.0 and 7.0, lower levels of extraction enzyme are obtained, being water the selected extracting agent and 30 mL the volume. Higher water volumes only dilute enzyme activity.
_____________ [1] Spagna, G. Barbagallo, R., Greco, E., Manenti, I., Pifferi, P. (2002). Enzyme and Microbial Technology. 30:80-89. [2] Rustiguel, C., De Oliveira, A., Terenzi, H., Jorge, J., Guimaraes, L. (2011). Electronic Journal of Biotechnology; 14(2). http://dx.doi.org/10.2225/vol14-issue2-fulltext-1
Figure 1. Procedure for the preparation of biopolymer-based bead containing lipase.

________________ [1] S. H. Lee, M. Miyauchi, J. S. Dordick, and R. J. Linhardt (2010) ACS symposium Series Ionic liquids application: Pharmaceutical, Therapeutics, and Biotechnology 1038: 115-134.
Figure 1. Reaction setup for the continuous reduction of 2-Octanone
October 2-6, 2011
176 413
A microuidic toolbox for the development of multi-step biocatalytic processes
a

Lipase catalyzed esterification of solketal with stearic acid under continuous ow conditions
Ivaldo I. Junior, a Marcela C. Flores, a Felipe K. Sutili, a Selma G. F. Leite, b Leandro S. de M. Miranda, a Ivana C. R. Leal, a Rodrigo O. M. A. de Souza a* a Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, Fax:+552125627001, CEP22941-909. bEscola de Qumica, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil, Fax:+552125627005, CEP22941-909. Partial acylglycerols, mono- and diacylglycerols (MAG and DAG), are well known biodegradable, biocompatible and non-toxic nonionic surfactants, widely used in food, pharmaceutical and industrial applications. They are commonly produced based on alkalinecatalyzed chemical glycerolysis of natural oil and fats at high temperatures (220-250C). Besides the energy costs, the high temperatures are the main responsible for the formation of dark-colored and burned-tasting products, which requires extensive and costly purification steps. In this work we have optimized a biocatalytic continuous flow process, with packed bed reactor, to the esterification reaction between solketal and stearic acid using response surface methodology (RSM) in a laboratory setting. The lipase-catalyzed esterefication have been investigated as a potential substitute to the traditional chemical glycerolysis since, lipases as biocatalysts demand milder reaction conditions which minimize the energy costs, allow a better reaction control and consequently provide higher-quality products.4 RSM is a statistical tool for developing and optimizing processes with one or more responses that are influenced by several variables. The advantage of RSM is that it allows the user to gather large amounts of information from a small number of experiments. The use of RSM is also possible to observe the effects of individual variables and their combinations of interactions on the response. The results obtained shows that we have developed with the assistance of response surface methodology (RSM) a continuous flow process to the esterification of solketal with stearic acid leading to the desired product in excellent conversions and short reaction time (yield > 90% and reaction time < 2 minutes). The optimized process also allows the use of palmitc acid with high conversions while the use of unsaturated fatty acids or short chain fatty acids leads to a significant reduction on conversion and are still been investigating by our group. The negative effects of the variables studied were easily observed in the response surface (Figure 1). When the flow decreases, ester conversion increases. This also happens with the substrate concentration in the studied range. There is an optimal range of work between 0.3 to 0.4 mL / min for the flow and from 65.9 to 71.9 mM for the substrate concentration reaching yields of up to 95.2%

Combining technologies: the large scale synthesis of (S)-allysine ethylene acetal utilising hydroformylation and biocatalysis
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177 418
Sustainable biocatalytic processes for the synthesis of pharmaceutical and cosmetic intermediates
Rob ter Halle, Daniel Auriol, Fabrice Lefvre, Renaud Nalin Libragen,3 rue des Satellites, 31400 Toulouse, France E-mail: r.terhalle@libragen.com Both the pharmaceutical and cosmetics industry face today the urgent need for producing intermediates in a green and sustainable manner. Libragen is a French Biotechnology company that has a strong competence in the discovery and development of new biocatalytic processes. Starting from a metagenomic platform which allows the discovery of new enzymes from a wide natural variety, diverse innovative bioprocesses have been developped. In this talk, different exemples of this approach will be discussed. A very diverse library of transaminases have been discovered using the metagenomic approach. Screening and subsequent process-development allowed cost-efficient production of enantiopure building blocks for the pharmaceutical industry.
O R1 R2 NH2
J. Lawrencea, B. OSullivana, H. Al-Bahrania, R. Wohlgemuthb, H.C. Hailesa, N. Szitaa University College London, London, WC1E 6BT, UK bSigma-Aldrich, Industriestrasse 25, 9470 Buchs, Switzerland E-mail: james.lawrence@ucl.ac.uk We have developed a modular continuousflow reactor and filter system as a first step towards a process development toolbox for multi-enzyme or chemo-enzymatic synthesis. Previous work has investigated biocatalytic processes on-chip, but has not taken the downstream purification into account, nor been capable of use with several enzymes in solution[1]. Furthermore, in our system both the reactor and filter devices (fig. 1A, B) were fabricated using rapid pro- Figure 1: Design of microfluidic reactor (A) totyping methods, allowing quick changes and filter unit (B) in poly(methylmethacrylate) of design if necessary, for example, to adapt (PMMA); setup for combined reaction and to the requirements of different enzyme sysfiltration experiments (C) tems. The transketolase (TK)-catalysed reaction of hydroxypyruvate (HPA) and glycolaldehyde (GA) to L-erythrulose was used as a model to evaluate the system, due to its relevance to organic synthesis as a catalyst for C-C bond formation. The reaction was performed as described previously [2]; the output was fed into the retentate inlet of the filter (fig. 1C). The filter was clamp-sealed, allowing integration of different membrane types, and was robust at pressures up to 100psi. Filtration pressure was regulated by attaching Figure 2: Volumetric output of combined capillaries of specific diameter and length reactor/filter over 8 hours; SDS-PAGE of filter to the outlet. input/output streams (inset) Under long-term operation the system was capable of achieving high yields, and the catalyst was retained in a concentrated form, suitable for reuse (fig. 2).
________________________ [1] Malic acid production within a microreactor with surface immobilised fumarase, G. Stojkovi et al., Microfluid. Nanofluid., 2011, 10, 627 [2] Continuous flow microfluidic reactor for biocatalytic synthesis, H. Al-Bahrani et al., Thirteenth International Conference on Miniaturized Systems for Chemistry and Life Sciences, Jeju, Korea, 2009, pp. 1940-1942
Michael Lloyda, Christopher Cobleya, Shaun Simmondsa and Christopher Hansonb Chirotech Technology Ltd., Dr Reddys Laboratories (EU) Ltd., Unit 162 Cambridge Science Park, Cambridge CB4 0GH UK b Dr Reddys Laboratories (EU) Ltd., Steanard Lane, Mirfield, West Yorkshire WF14 8HZ UK E-mail: mlloyd2@drreddys.com The compound, (S)-2-amino-5-[1,3]dioxolan-2-yl-pentanoic acid [(S)-allysine ethylene acetal, (S)-5], is a key intermediate in a number of Angiotension-I converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors currently in clinical trials. Through a combination of our hydroformylation and biocatalysis technologies[1] we have developed an efficient five step synthetic route to this material starting from crotonaldehyde.
amine-donor
Transaminase
R1
R2
(S)- or (R)-selectivity
Diverse naturally occuring polyphenolic compounds have been derivatized by glycosylation. A glycosylating enzyme effectively prepared different innovative compounds for the cosmetics industry in good yields and excellent purities from a simple reaction mixture of polyphenol and sugar. Special emphasis will be put on the development of an industrial process. OH Scheme 1. Synthetic route to (S)-allysine ethylene acetal from crotonaldehyde. Several enzymatic approaches to (S)-allysine ethylene acetal were evaluated and our rationale for utilising the route using a thermophilic L-acylase from archaeon Thermoccocus litoralis [2] will be discussed. The development of this process led to a large scale manufacturing campaign that produced multi hundred kilograms of this valuable chiral intermediate in >99% ee and >99% chemical purity. Key considerations for the scale up will be discussed.
_______________ [1] Christopher J. Cobley, Christopher H. Hanson, Michael C. Lloyd, and Shaun Simmonds, Wei Jun Peng, Org. Process Res. Dev., 2011, 15 (1), pp 284290 [2] Taylor, I.N.; Brown, R.C.; Bycroft, M.; King, G.; Littlechild, J.A.; Lloyd, M.C.; Praquin, C.; Toogood, H.S. and Taylor, S.J.C. Biochem. Soc. Trans. 2004, 32(2), 290-292
HO HO CO2H
OH O O OH O OH OH OH OH
HO
glucosylating enzyme
OH O O HO CO2H
HO HO
HO
water
Caffeyl Glucoside
Figure 1: Response surface for the esterification reaction catalysed by RM IM in continuous flow in function of flow rate and substrate concentration.
______________ [1] Frost, C. G., Mutton, L.; Green Chem. 2010, 12, 1687 [2] Mark D., Haeberle S., Roth G., von Stetten F., Zengerle R.; Chem Soc. Rev. 2010, 39, 1153.
Another process for a cosmetic application is the phosphorylation of naturally occuring sugars. For example, N-acetylglucosamine can be enzymatically phosphorylated to produce 6-phospho-N-acetyl glucosamine (a key intermediate in the biosynthesis of hyaluronic acid). In this case a phosphorylating enzyme, can be used to allow the reaction between N-acetyl glucosamine and pyrrophosphate in 96% yield and 90% purity. The phosphorylated carbohydrate is obtained in only one step in high yield without the need of protecting groups. O
OH HO HO O O OH NH
pyrrophosphate
phosphorylating enzyme
O HO HO
P OH OH
O OH NH O
415
Immobilization of Bacillus circullans b-galactosidase in hierachiral meso-macroporous silica
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416
Lipase-catalyzed esterifications within microuidic systems using free and immobilized enzyme
Andrej Pohar, Igor Plazl, Polona nidari-Plazl Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia E-mail: polona.znidarsic@fkkt.uni-lj.si Miniaturization, as a method for process intensification, allow for the harnessing of the small volumes, high surface to volume ratio obtained in an effort to achieve higher yields over shorter periods of time, higher product purity and better process control along with the reduction of cost and equipment associated with downstream processing.[1] Microprocess engineering allows for high levels of parallelization and reduced requirements of chemicals; intensive screening of biocatalysts and process variables has become more feasible, and reproducibility of bioconversion processes has been substantially improved.[2] Our studies in the field of the development of continuous microreactor biotransformation systems include lipase-catalyzed esterifications within two-phase (water/n-hexan3 and ionic liquid/n-heptan4) media with dissolved biocatalyst, as well as with immobilized lipase within a miniaturized packed-bed reactor. In the first case, the esterification of isoamyl alcohol and acetic acid occurred at the interface of a parallel laminar flow between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product (isoamyl acetate) remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. In order to forecast microreactor performance, a three-dimensional mathematical model was developed, considering the velocity profile developed and consisted of convection, diffusion, and enzyme reaction terms.[3] The same short-chain ester was further synthesized from acetic anhydride using free lipase B in the 1-butyl-3-methylpyridinium dicyanamide/n-heptane two-phase system within a continuously operated -shaped microreactor. A very high yield obtained was mainly a consequence of efficient reaction-diffusion dynamics in the microchannel system, where the developed flow pattern comprising of intense emulsification provided a large interfacial area for the reaction and simultaneous product extraction.[4] For the lipase-catalyzed transesterification of 1-butanol and vinyl butyrate to butyl butyrate in 1butyl1methylpyrrolidinium bis(trifluoromethanesulfonyl)imide with immobilized Candida antarctica lipase B, a miniaturized packed-bed reactor was developed and further integrated with a miniaturized continuous separator, which exploited the ionic liquids high boiling point compared to the products. The product was simultaneusly isolated and the ionic liquid cleaned for further use. The reaction diffusion dynamic inside the packed bed microbioreactor was described with a dispersion model, which incorporates axial dispersion or local backmixing of reactants and products in the small interstices of the packed bed and through the macropores. In all studied cases, continous flow microreactors have presented increased efficiency when compared to conventional reactors.
_______________ [1] L. L. Woodcock, C. Wiles, G. M. Greenway, P. Watts, A. Wells and S. Eyley, Biocatal. Biotransform., 2008, 26, 501-507 [2] P. Fernandes, Int. J. Mol. Sci., 11, 858-879. [3] P. nidari-Plazl, I. Plazl, Proc. Biochem, 2009, 44, 1115-1121. [4] A. Pohar, I. Plazl, P. nidari-Plazl, Lab Chip, 2009, 9, 33853390.
Claudia Bernala, Ligia Sierraa, Monica Mesaa Grupo Ciencia de los Materiales, Instituto de Qumica, Universidad de Antioquia. Medellin, Colombia E-mail: claberz@gmail.com The porous silica materials offer some special properties for using as enzyme supports due to their high surface area, thermal and mechanical stability, non toxicity, and high resistance against microbial attacks and organic solvents. Also they can have adjustable pore size for immobilization of large variety of biomolecules via hydrogen bonding and electrostatic interactions due to the existence of surface silanol groups. Some relatively small enzymes have been immobilized in these materials [1,2], but an enlargement of their mesopore size is desired for immobilization of bigger enzymes like b-gal from Bacillus circullans, with a diameter ~ 11 nm and MW of 212 kDa [3], in order to allow conformational changes during the catalytic process and at the same time to avoid lixiviation. b-Galactosidases (E.C. 3.2.1.23), catalyzes the hydrolysis of b-galactosidic linkages present on lactose or analogue molecules, producing high power reduction sugars such as glucose and galactose. b-galactosidases have been used for hydrolysis of lactose in dairy products, useful for people who suffer lactose intolerance. The bioconversion of lactose of the whey into products of higher value would help to solve the pollution problem caused by whey disposal. Implementing their repeated or continuous use at industrial level requires high operational enzymatic stability, which can be achieved by their immobilization onto a solid support. The aim of this work is to report the immobilization of Bacillus circullans b-galactosidase like big enzyme model onto hierachiral porous silicas by physical adsorption. These inorganic supports were synthesized under controlled conditions for tailoring the porous and chemical surface properties: The presence of macroporous (~ 0.07- 20 m) favors the difussion of enzyme molecules towards the mesopores (~ 10-50 nm), where they are encapsulated by electrostatic interactions with the ionized silanol groups of their surface. The enzyme loading capacity is higher than 80mg/g support and the lixiviation is less than 20% after 72 hours of continuos catalysis. The new immobilized b-gal biocatalysts exhibit optimum activity at and pH similar to the soluble enzyme (45 C and 6.0). The immobilization of b-gal onto silica improve its stability at 55C temperatures and at pH 4.0. This immobilized biocatalyst is applied in lactose hydrolisis of whey with good results
[1] Takahashi, H., et al., Catalytic Activity in Organic Solvents and Stability of Immobilized Enzymes Depend on the Pore Size and Surface Characteristics of Mesoporous Silica. Chem. Mater., 2000. 12(11): p. 3301-3305. [2] Yiu, H.H.P., P.A. Wright, and N.P. Botting, Enzyme immobilisation using siliceous mesoporous molecular sieves. Microporous and Mesoporous Materials, 2001. 44-45: p. 763-768. [3] Amedeo Vetere, Sergio Paoletti. Separation and characterization of three b-galactosidases from Bacillus circulans. Biochimica et Biophysica Acta 1380 1998 223231
October 2-6, 2011
178 419
Deciphering rationales determining the stability of a lipase in non-conventional solvents
Josiane Frauenkron-Machedjoua, Leilei Zhua, Ulrich Shwaneberga, Alexander Fultonb, Susanne Wilhelmb, Karl-Erich Jgerb a RWTH Aachen University, Lehrstuhl fr Biotechnologie, Worringerweg 1, 52074 Aachen; Germany b Institute of Molecular Enzyme Technology, University of Dsseldorf, 52426 Jlich, Germany E-mail: j.frauenkron-machedjou@biotec.rwth-aachen.de Understanding the general principles causing stabilization and destabilization of enzymes in non-conventional solvents is crucial to improve their biotechnological application. Due to the small size [1,2] (19.3 kDa and 181 amino acids) and its minimal / hydrolase [3]fold, lipase A from Bacillus subtilis (BSLA) is used here as a model enzyme. In this study we will systematically analyze how each amino acid position influences the stability of BSLA in various non-conventional solvents. For this purpose, a site saturation mutagenesis library targeting the complete BSLA sequence will be generated and screened in presence of different non-conventional solvents comprising three organic solvents with different polarity, four detergents with different functional groups and three ionic liquids with different carbon chain lengths. Analyzing the effect of each of these compounds on BSLA structure will reveal important insights into the contribution of H-bonds, salt-bridges as well as hydrophobic interactions to the enzyme stability. This project will be carried out by two PhD students (Josiane FrauenkronMachedjou and Alexander Fulton). In my part I will focus on the optimization of the screening conditions for three ionic liquids and two organic solvents and Alexander Fulton will optimize the screening conditions for four detergents and one organic solvent. The screening assay is based on the spectrophotometrical detection of the activity using para-nitrophenyl butyrate as a substrate [4]. All optimized screening conditions will be applied by each student to analyze half of the BSLA sequence. The obtained results will be combined to determine a stability pattern for the whole BSLA sequence based on chemical properties of the used non-conventional solvents. Hence, we will develop a predictive model and hypotheses for other / hydrolases.
BIONOCO & THDP dependent enzymes 420

Quantification of cellulose hydrolysis in ionic liquids by viscosity and chain length distribution measurements
Benjamin Bonhagea, Philip Engela, Antje C. Spiessa a BioNoCo, AVT Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany E-mail: benjamin.bonhage@avt.rwth-aachen.de Ionic liquids are a new generation of solvents known to effectively dissolve cellulose [1]. Until now the dissolution of cellulose in ionic liquids was mainly used as pretreatment with a subsequent precipitation of the cellulose in water. This results in suspended particles that are more accessible for cellulases, significantly increasing hydrolysis rates [2]. Even more attractive regarding accessibility is the possibility to hydrolyse the cellulose without precipitation, i.e. in a homogeneously dissolved system with ionic liquid. Recently, a cellulase has been discovered that shows activity in a system with dissolved cellulose and more than 80 % (v/v) ionic liquid [3]. To quantify the reaction kinetics of this unconventional reaction system, the change of the cellulose molecules over time was characterized based on their chain length distribution and viscosity. The chain length distribution was measured with size exclusion chromatography and the viscosity was measured with a rheometer at shear rates between 1e-3 and 3e3 s-1. To gain additional information the release of soluble sugars was monitored using colorimetric assays. The hydrolysis rates were then analyzed for two ionic liquids. Since large parts of the hydrolysis of cellulose take place on the long chains they cannot be easily measured with soluble sugar assays, such as DNS or PAHBAH. Therefore, the measurement of the chain length distributions and the viscosity provide complementary information about the reaction kinetics. It was identified that one ionic liquid hydrolysed cellulose also without the presence of a cellulase. Consequently, this effect has to be considered when analysing cellulose hydrolysis in ionic liquids. By adding the enzyme the hydrolysation rate could be increased. The results illustrate that the chain length distribution data provide a powerful technique to the understandingof the cellulase reaction kinetics in homogeneously dissolved polymer reactions. The knowledge of the kinetic behaviour can lead to activity optimized reaction systems.
________________ [1] M. Zavrel, D. Bross et al., Biores. Technol. 2008, 100, 2580. [2] P. Engel, R. Mladenov, et al., Green Chem., 2010, 12, 1959. [3] M. Girfoglio. PhD thesis, Universit di Napoli Federico II, 2008.

Solvent-free lipase-catalyzed kinetic resolution of alcohols.
Zaira Maugeri, Pablo Domnguez de Mara Institute of Technical and Macromolecular Chemistry (ITMC). RWTH Aachen University. Germany. E-mail: maugeri@itmc.rwth-aachen.de Enzyme-catalyzed reactions can be performed in aqueous-based environments, as well as in non-conventional media like organic solvents, gas, supercritical fluids (e.g. ScCO2) and ionic liquids or deep-eutectic-solvents. Addressing non-conventional media, working in solvent-free conditions could represent a promising, efficient and environmentally-friendly alternative. Despite these possible advantages, the set-up of solvent-free biocatalytic reactions has attracted very little attention, with only some examples for the synthesis of low-added-value bulk commodities (1,2), as well in asymmetric synthesis, yet with low substrate loadings (50 mM) (3,4). Herein lipase-catalyzed kinetic resolution of ()-1-phenylethanol was taken as model reaction to assess solvent-free conditions, using isopropenyl acetate as solvent and acyl donor (Figure). Several substrate:acyl donor ratios, temperatures, enzyme concentrations were investigated to find the best reaction conditions, together with successful catalyst recycling. Based on results, an economic assessment on enzyme contribution costs to product production was conducted.
a
179 424
Enzyme catalysis in emulsions
Susanne Wiesea,c, Antje C. Spieb,c, Walter Richteringa Institute of Physical Chemistry, RWTH Aachen, Landoltweg 2, 52056 Aachen, Germany; b AVT-Enzyme Process Technology, RWTH Aachen, Worringerweg 1, 52056 Aachen, Germany; cDFG Research Training Group BioNoCo Biocatalysis using non-conventional media, RWTH Aachen, Germany E-mail: wiese@pc.rwth-aachen.de Enzyme reactions are a good alternative for producing chiral compounds compared to conventional organic synthesis. One issue of the enzymatic approach is that many substrates are only poorly soluble in the aqueous environment needed by the enzyme. Until now, this reaction is performed in two phase systems [1], however, the transport of the substrate and product between organic and aqueous phase may limit it. An approach to enhance the productivity of the reaction is to increase the surface area by creating an emulsion using an emulsifier. After the reaction, the product needs to be separated, requiring emulsion breakage. Stimuli-sensitive microgels provide the opportunity to first stabilize and then break the emulsion; in other words: the microgels act as switchable emulsifier [2]. We report on temperature and / or pH-sensitive microgels that stabilize and break emulsions consisting of buffer and organic phase under conditions suitable for the enzymatic reaction. The model reaction system is the conversion of acetophenone to R-phenylethanol using the enzyme Lactobacillus brevis alcohol dehydrogenase (Lb-ADH). The experimental data provide clear a proof-of-concept that enzyme catalyzed reactions can be sucessfully performed in switchable (smart) emulsions.
________________ [1] M. F. Eckstein, M. Peters, J. Lembrecht, A. C. Spiess, L. Greiner, Advanced Synthesis & Catalysis 2006, 348, 1591. [2] B. Brugger, W. Richtering, Langmuir 2008, 24, 7769
Figure 1. Kinetic resolution of ()-1-phenylethanol under solvent-free conditions Acknowledgements. Financial support from DFG training group 1166 BioNoCo (Biocatalysis in Non-conventional Media) is gratefully acknowledged. We thank C-lecta for kindly providing a sample of immobilized CAL-B.
__________________ [1] C. Korupp, R. Weberskirsch, J.J. Mller, A. Liese, L. Hilterhaus, Org. Proc. Res. Dev. 2010, 14, 1118-1124. [2] P. Tufvesson, A. Annerling, R. Hatti-Kaul, D. Adlercreutz, Biotechnology Bioengineering, 2007, 97, 447-453. [3] M. Solymar, F. Flp, L.T. Kanerva, Tetrahedron: Asymmetry, 2002, 13, 2383-2388. [4] M. Solymar, L.T. Kanerva, F. Flp, Tetrahedron: Asymmetry, 2004, 15, 1893-1897
________________ [1] G van Pouderoyen u. a., The crystal structure of Bacillus subtilis lipase: a minimal alpha/beta hydrolase fold enzyme, Journal of Molecular Biology 309, Nr. 1 (Mai 25, 2001): 215-226. [2] Eerappa Rajakumara u. a., Structural basis for the remarkable stability of Bacillus subtilis lipase (Lip A) at low pH, Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics 1784, Nr. 2 (Februar 2008): 302-311. [3] David L. Ollis u. a., The / hydrolase fold, Protein Engineering 5, Nr. 3 (April 1, 1992): 197 -211. [4] U K Winkler und M Stuckmann, Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens., J. Bacteriol. 138, Nr. 3 (Juni 1, 1979): 663-670.
421
Reaction engineering for laccase-catalysed lignin degradation
Simon Rotha, Nora Chena, Antje C. Spiea a BioNoCo, AVT Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany E-mail: simon.roth@avt.rwth-aachen.de In the last decades biomass has gained rising importance as renewable resource for fuels and platform chemicals. Whereas plant derived molecules like starch, sugars and proteins are well accessible for biotransformations the main components of wooden biomass - cellulose, hemicelluloses and lignin - are difficult to transform into valuable products. Lignin is one of the most abundant renewable raw materials available on earth and pulp and paper industry still produces millions of tons as a widely unutilised side product [1,2]. Both, the high availability and the content of phenolic subunits, feature lignin as an attractive resource to obtain platform chemicals which nowadays are only supplied by petrochemical processes. The big challenge here is to break down the lignin polymer into its monolignolic subunits which than can be employed for further transformations. The following enzymatic approach to degrade lignin is pursued: Laccases are multinuclear copper-containing blue oxidases produced by certain white rot fungi [3]. They catalyse a four electron transfer from a substrate over a mediator to oxygen which is reduced to water [4] (Fig. 1).
422
Reaction kinetics identification of benzaldehyde-lyase wild type and a variant
a
425
Solvent-free asymmetric reduction of ketones by E. coli whole cells with quantitative yields
Andre Jakoblinnerta, Fabrizio Sibillaa, Radoslav Mladenova, Albert Paula, Ulrich Schwaneberga, Marion Ansorge-Schumacherb, Pablo Domnguez de Marac a Chair of Biotechnology, RWTH Aachen, Worringer Weg 1, 52074 Aachen, Germany b Institute of Chemistry, Department of Enzyme Technology TU Berlin, Strae des 17. Juni 124, 10623 Berlin, Germany c Institute of Technical and Macromoelcular Chemistry (ITMC), RWTH Aachen, Worringer Weg 1, 52074 Aachen, Germany E-mail: a.jakoblinnert@biotec.rwth-aachen.de The asymmetric reduction of carbonyl compounds by oxidoreductases is a cheap and effective route for the production of enantiopure alcohols, which are useful building blocks for pharmaceuticals, agrochemicals and food industry. Herein, whole cells are commonly employed to efficiently regenerate the expensive cofactor (NAD(P)H). Other limitations for a practical use of enzymes are derived by the limited solubility of the substrates in aqueous systems. Whole cell biocatalysis can be performed in non-conventional media (e.g. biphasic systems) to increase the productivity [1]. However, in many cases waste production and extensive product work-up are major drawbacks towards green large-scale processes. In this communication, we show for the first time that E. coli whole cells, overexpressing a carbonyl reductase, are able to perform enantioselective carbonyl reduction with substrate-coupled co-factor regeneration in a solvent-free system (Fig. 1).
426
Fluorescence reporters for analysis of whole cell biocatalysts in non-conventional media
Kathrin Kleina, Daniel Okrobb, Martina Pohlb, Karl-Erich Jaegera and Ulrich Kraussa a Institut fr Molekulare Enzymtechnologie der Heinrich-Heine-Universitt Dsseldorf im Forschungszentrum Jlich, 52426 Jlich, Germany b Institut fr Bio- und Geowissenschaften, Forschungszentrum Jlich, 52425 Jlich, Germany E-mail: k.klein@fz-juelich.de Biocatalysis in non-aqueous reaction media (e.g. pure organic solvents) has rapidly developed into an industrially attractive alternative to conventional biotransformations in aqueous systems. Due to their physico-chemical properties, non-aqueous media are highly demanding on the employed biocatalysts. Their use may eventually result in enzyme modification, denaturation and/ or aggregation [1,2]. Fluorescent reporter proteins may serve as a useful tool for tracking the fate of the biocatalysts during enzymatic conversions. Proteins of the green fluorescence protein (GFP) family are widely used for the study of gene expression[3], protein folding processes and in localization studies[4] in living organisms. Although, their properties have not yet been investigated thoroughly, the recently developed Light-Oxygen-Voltage (LOV)-based fluorescent reporters (LOV-FRPs) might serve as an attractive alternative to GFP. The smaller size of LOVFRPs compared to GFP might be particularly advantageous. To evaluate different fluorescent reporter proteins for biotechnological application, we generated a set of fusion proteins of either the yellow fluorescent protein (YFP) or the LOV-FRP derived from the Bacillus subtilis YtvA-protein[5] with the hydroxynitrile lyase (HNL) of Arabidopsis thaliana. HNLs catalyze the synthesis and cleavage of chiral cyanohydrins[6], which are important building blocks for e.g. fine chemicals. From an application perspective, the use of whole cells in biocatalytic transformations has several important advantages compared to purified, lyophilized or immobilized enzyme preparations. First of all they are easy to produce, handle and store. Moreover, no laborious and expensive protein purification steps are needed. Biocatalyst recycling of the cells expressing the biocatalyst is feasible. Using both the wild-type HNL and fluorescent HNL fusion proteins we carried out whole cell biotransformation using hydrogen cyanide and benzaldehyde as substrates to yield enantiopure (R)-mandelonitrile. All reactions were performed in pure methyl tert-butyl ether, using whole Escherichia coli cells expressing the respective biocatalyst. Even after three subsequent recycling steps, cells expressing wild-type HNL, YFP-HNL and LOV-HNL fusion proteins, still show almost complete conversion within 60 minutes with an excellent ee > 97%. Thus, in conclusion the presented whole cell biotransformation approach represents a suitable alternative for the synthesis of (R)-mandelonitrile. Further investigations regarding the integrity of the cells as well as of the fluorescent fusion proteins during the course of the synthesis reaction and the subsequent recycling steps will be conducted using confocal microscopy. Hereby, the fused fluorescent reporter will enable us to visualize and track cellular- and fusion protein integrity in this non-conventional reaction system.
Kai F. Mahnkena, Melanie Schwarzb, Martina Pohlb, Antje C. Spiessa BioNoCo, AVT Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany b Institute of Bio- and Geosciences IBG-1: Biotechnology, FZ Jlich, 52425 Jlich, Germany E-mail: Kai.Mahnken@avt.rwth-aachen.de Thiamine diphosphate (ThDP)-dependent enzymes are multifunctional biocatalysts taking part in numerous biosynthetic pathways and catalyzing a broad range of reactions. They are able to form and cleave bonds between carbons and other atoms like hydrogen, oxygen, sulphur and nitrogen [1]. In this group, benzaldehyde lyase (BAL) is very promising since BAL is an exceedingly active catalyst to perform carboligation reactions yielding chiral hydroxyketones, which are important building blocks of drugs and natural products [2]. In the reaction mechanism of BAL, the cofactor thiamine diphosphate (ThDP) plays a very crucial role. The reaction mechanism consists of three possible rate-limiting reaction steps. In the first reaction step, the activated cofactor binds to an aldehyde A and forms an enamine intermediate ThDP-A. Then, the enamine intermediate ThDP-A performs a nucleophilic attack on a further aldehyde B resulting in a ternary complex ThDP-AB. In a third step, the hydroxyketone AB is released. Characteristic for BAL is a long C-terminal -helix on the protein surface which partly covers the substrate channel entrance which hinders release of steric demanding products. Kokova et al. assumed in their work that deleting the -helix could lead to higher overall reaction rates [3]. In this project, reaction kinetic identification [4] is applied for quantification of the effect of deleting the -helix on each of the rate-determining reaction rates. For the reaction kinetic identification, progress curve experiments with different substrate starting concentrations are conducted. The concentration courses are differentiated and used for the estimation of macro kinetic parameters according to the incremental method [5]. The quality of the resulting parameters is subsequently validated statistically and their sensitivities are analyzed. The estimated macro-kinetic parameters are used to calculate micro-kinetic rate constants to quantify each of the reaction steps and compare the kinetic abilities of both BAL types on a mechanistic level. Additionally, this method of reaction kinetic identification allows suggestions for optimized experiments to enhance the parameter accuracy based on the performed sensitivity analysis.
________________ [1] M. Mller et al., Febs J. 2009, 276(11): 2894 [2] A.S. Demir et al., J Chem. Soc. Perk. T 1 2001, 7: 633 [3] M. Kokova et al., J Mol. Catal. B-Enzym. 2009, 61 (1-2): 73 [4] C. Michalik et al., Chem. Eng. Sci. 2007, 62 (18-20): 5592 [5] W. Marquardt, Chem. Eng. Res. Des., 2005, 83: 561
Figure 1. Schematic representation of laccase-catalyzed redox cycles for substrates oxidation in the presence of chemical mediators [4]. Within this project a suitable reaction system for laccase-catalysed lignin degradation shall be validated. Therefore, different laccase-mediator-systems (LMS) are evaluated under varied conditions. To follow the degradation process several analytical methods are established: Cyclovoltammetry provides information about laccase-mediator-interactions. Particle size distribution analysis should visualize the degradation progress on suspended lignin particles in aqueous solution. Gel permeation chromatography shows lignin break down on molecular level. Detection of several degradation products is achieved by HPLC analysis. The results obtained by these methods should lead to first conclusions concerning the kinetic parameters of this process and in long term to a suitable reactor concept.
Figure 1. Reaction scheme of ketone reduction by carbonyl reductase in E. coli with substrate-coupled cofactor regeneration. The bioconversion is conducted by only using three components: substrate, co-substrate and biomass, obtaining yields up to 97 %, corresponding to ~ 300 g/L enantiopure product in case of acetophenone as model substrate. The addition of NADH is not necessary since it is intracellularly recycled via coupled substrate (iso-propanol). The carbonyl reductase system can be attractive for industrial applications as the space time yield is dramatically increased compared to the existing conventional concepts for chiral alcohol production. Additionally, the process is very environmentally friendly, since no waste material is produced and the work-up involves cost-effective work-up steps (e.g. acetone evaporation).
Acknowledgement: Financial support from DFG training group 1166 BioNoCo (Biocatalysis in Nonconventional Media) is gratefully acknowledged. ________________ 1. Groger, H., et al., Angew Chem Int Ed Engl, 2006. 45(34): p. 5677-81.
________________ [1] R.J.A. Gosselink, E. de Jong, et al. Co-ordination network for ligninstandardisation, production and applications adapted to market requirements (EUROLIGNIN). Ind. Crops Prod. 2004, 20, 121. [2] H.P. Call, I. Mcke. History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym-process). J. Biotechnol. 1997, 53, 163. [3] A. I. Yaropolov, O.V. Skorobogatko, et al.: Laccase - properties, catalytic mechanism, and applicability. Appl. Biochem.. Biotechnol. 1994, 49, 257. [4] S. Riva: Laccases: blue enzymes for green chemistry. Trends Biotechnol. 2006, 24, 219.
_________________ [1] K. Griebenow, A. M. Klibanov J. Am. Chem. Soc. 47, 118 (1996) [2] H. K. Weber, J. Zuegg J. Mol. Catal. B-Enzymatic. 4-1, 3 (1997) [3] J. C. March et al. Appl. Microbiol. Biotechnol. 4, 62 (2003) [4] G. S. Waldo, B. M. Standish Nat. Biotechnol. 7, 17 (1999) [5] U. Krauss et al. Microb. Biotechnol. 15-23, 3 (2010) [6] J. Andexer et al. Angew. Chem. Int. Ed. 46, 8679 (2007) [7] U. Hanefeld et al. Biochim. Biophys. Acta 185-193 1432 (1999)
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Productivity optimized biocatalyst for oxidoreductions in organic solvents
Jens Begemanna, Jurek K. Failmezgera, Marion Ansorge-Schumacherb, Antje C. Spiessa a BioNoCo, Aachener Verfahrenstechnik - Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, 52056 Aachen, Germany b Institute of Chemistry, Enzyme Technology (TC4), Technical University of Berlin, Strasse des 17. Juni 124, 10623 Berlin, Germany E-Mail: jens.begemann@avt.rwth-aachen.de Chiral alcohols constitute important building blocks for the pharmaceutical industry [1]. Due to their enantioselectivity, alcohol dehydrogenases (ADH) are increasingly applied for the production of secondary alcohols, but the hydrophobicity of the substrates limits the productivity in aqueous medium. The use of a two-phase system of an organic phase that dissolves hydrophobic substrates and products and a hydrogel stabilized aqueous phase for the protection of the biocatalysts against the organic phase, has been proven to be applicable [2]. In a model process on 1 L scale carbonyl reductase from Candida parapsilosis (CPCR) is applied in a two phase system for the production of 1-(S)-phenylethanol. The unpolar substrate acetophenone is dissolved in heptane in high concentrations whereas CPCR as well as formate dehydrogenase from Candida boidinii (FDH) for cofactor regeneration are immobilized in polyvinylic alcohol (PVA) beads. For sufficient supply with the cosubstrate formic acid a fed batch system has been developed. To maintain a constant pH during biocatalysis, the formic acid feed is controlled via online measurement of the reaction progress via the emitted co-product CO2. The molar amount of CO2 is resupplied to the reactor as formic acid. This process concept has now to be optimized for improved productivity. The enzyme load in the PVA-hydrogel beads is currently the process bottleneck. It is limited by the amount and the residual activity of the biocatalysts and therefore, by the material properties and the formation procedure of the PVA-hydrogel beads. The first step to optimize the biocatalyst load is a sufficient characterization of the immobilization efficiency and the material properties. First results show that residual activity after immobilization is strongly dependent on the biocatalyst. Therefore, the ratio of activity of the value adding and the cofactor regenerating enzymes can be adjusted for optimal economic utilization of the biocatalyst phase.
__________________ [1] Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Sturmer R, Zelinski T. 2004. Industrial methods for the production of optically active intermediates. Angewandte Chemie International Edition. 43(7). 788 - 824. [2] Ansorge-Schumacher MB, Greiner L, Schroeper F, Mirtschin S, Hischer T. 2006. Operational concept for the improved synthesis of (R)-3,3-furoin and related hydrophobic compounds with benzaldehyde lyase. Biotechnology Journal. 1. 564 - 568

Systematical approach to decipher the rationales behind the detergent and solvent stability of a model lipase
Alexander Fultona, Josiane Frauenkron-Machedjoub, Ulrich Schwanebergb, Susanne Wilhelma, Karl-Erich Jaegera a Insitute for molecular Enzyme Technologie, Heinrich-Heine-University Dsseldorf, Research Center Jlich, 52426 Jlich, Germany; bLehrstuhl fr Biotechnologie, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany E-mail: a.fulton@fz-juelich.de The stability and activity of the biocatalysts is often compromised by the use of certain additives, e.g. detergents and organic solvents, to increase the solubility of certain reactants. This effect is not surprising and due to the evolutionary design of the enzyme. Enzymes have been evolved by nature to work efficiently in aqueous environments and thus require a water shell surrounding the protein surface to retain enzymatic activity. Solvents and detergents interfere with the surrounding water shell and protein electrostatics. This interference can lead to the unfolding and aggregation and a loss of activity. Despite these effects the influence of solvents on the enzyme structure and function has neither been studied systematically nor been understood theoretically so far. We aim to discover the potential of stabilizing a model enzyme in non-conventional media through a systematic mutagenesis study. We are interested in the development of a predictive stability model for the customized design of biocatalysts in respect to the intended application. The model enzyme for our purpose is BSLA (LipA from Bacillus subtilis), a minimal /hydrolase which can be easily expressed in Escherichia coli. BSLA has already been well characterized and is of known structure, the biotechnological potential has been demonstrated with the production of enantiopure cyclohexane-trans-1,2-diol[1]. In preparation of this screening we have performed a saturation mutagenesis along the whole sequence of BSLA. Degenerated codons were used to substitute the wild type amino acid by every other naturally occurring amino acid, resulting in a total of 3439 BSLA variants (181 amino acids x 19 possible substitutions). We are now developing a high throughput screening system to monitor the stability of every variant in different detergents and organic solvents. The selection of the solvents is justified through different interferences towards the intra protein interactions that will be weakened. The results will give us an insight into the contribution of every single amino acid towards the stability of the whole enzyme. The library construction and mutant screening is performed in cooperation with a project partner(b) which will focus on the stability in other non-conventional media. We will present the results from the screening of several exemplary mutants.
________________ [1] Jean Detry, Thorsten Rosenbaum, Stephan Ltz, Doris Hahn, Karl-Erich Jaeger, Michael Mller & Thorsten Eggert (2006) Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol using extracellular lipases from Bacillus subtilis. Appl Microbiol Biotechnol. 72:1107-16. PMID:16586103

Stabilization of the hydroxynitrile lyase from Arabidopsis thaliana by rational protein engineering
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432
Inuence of recombinant protein production on respiration activity of E. coli shake ask cultures in autoinduction medium
a
Daniel Okroba, Julia Metznera, Wolfgang Wiecherta, Karl Gruberb, Martina Pohla Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jlich GmbH, 52425 Jlich, Germany; bInstitute of Chemistry Structure biology, Karl Franzens University, A-8010 Graz E-mail: d.okrob@fz-juelich.de Hydroxynitrile lyases (HNLs) constitute an industrial important class of enzymes which is applied in stereoselective syntheses of chiral cyanohydrins from aldehydes or ketones and HCN [1]. The stereoselectivity of this enzymatic reaction is strongly compromised by a non-catalyzed side reaction resulting in racemic cyanohydrins. This side reaction is influenced by the pH, the temperature and the water content of the reaction medium [2]. In aqueous media it can be suppressed at low pH (4-5) and by lowering the temperature. However, both approaches are not possible with the wild type hydroxynitrile lyase from Arabidopsis thaliana (AtHNL), since the enzyme is inactivated already at pH 5.0 within 10 minutes, which prevents its use in aqueous media or two-phasic aqueous-organic reaction systems [3]. AtHNL is a very interesting biocatalyst as it is the first R-selective HNL with an /-hydrolase fold and thus easier to express in bacteria than its R-selective relatives from Prunus species [4]. The stabilization of the dimeric AtHNL toward acetic pH was inspired by the related and much more acid stable HNL from Manihot esculenta, which is a tetramer [3]. In order to allow tetramer assembly of the dimeric enzyme 11 amino acid residues on AtHNLs surface were exchanged. A detailed characterization of the variant revealed a significant increased activity and stability already at pH 4.5, although size-exclusion chromatography demonstrated that the variant is still dimeric.
Martin Kunzea, Natalie Rahmena, Jochen Bchsa BioNoCo, AVT Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringer Weg 1, 52074 Aachen, Germany E-mail: natalie.rahmen@avt.rwth-aachen.de As E. coli is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure because appropriate on-line monitoring systems are still lacking. In the presented study the Oxygen Transfer Rate (OTR), determined on-line in shake flasks by applying a RAMOS device (Respiration Activity MOnitoring System), was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in a commercial autoinduction medium, containing 0.5 g/L glucose, 2.0 g/L lactose and 12.6 g/L glycerol, to identify relationships between the respiration activity and the target protein production. All sixteen clones showed a remarkably different respiration activity, biomass and protein formation under induced conditions. However, the clones could be classified into three distinct types and correlations between OTR patterns and target protein production could be made. The acquired knowledge was used to modify the autoinduction medium for each of the three types in order to increase the target protein yield. Thereby it could be shown that by changing the induction strength the cultures could be shifted from one type into another. Within the project Influence of recombinant enzyme on host cell metabolic activity and expression of the Research Training Group BioNoCo the recent findings will be further investigated. As a first step the inhomogeneity of screening cultures of enzymes, explored in BioNoCo, will be assessed by various techniques. Subsequently the differences in expression between the different proteins shall be reduced by developing dedicated induction profiles and culture methods for the individual clones. A robotic procedure which will handle this individualized treatment shall be developed.
______________ [1] T. Purkarthofer, W. Skranc, C. Schuster, H. Griengl, Appl. Microbiol. Biotechnol. 2007, 76, 309. [2] J. von Langermann, J. Guterl, M. Pohl, H. Wajant, U. Kragl, Bioprocess Biosyst. Eng. 2008, 31, 155. [3] J. Guterl, J. Andexer, T. Sehl, J. von Langermann, I. Frindi-Wosch, T. Rosenkranz, J. Fitter, K. Gruber, U. Kragl, T. Eggert, J. Biotechnol. 2009, 141, 166. [4] J. Andexer, J. von Langermann, A. Mell, M. Bocola, U. Kragl, T. Eggert, M. Pohl, Angew. Chem., Int. Ed. 2007, 46, 8679.
429
Valorization of natures gifts: pretreatment & enzymatic hydrolysis of cellulose in seawater
Philipp M. Grandea, Walter Leitnera,b, Pablo Domnguez de Maraa a Institut fr Technische und Makromolekulare Chemie (ITMC), RWTH Aachen University, Worringerweg 1, D-52074, Aachen, Germany; bMax-Planck-Institut fr Kohlenforschung, 45470, Mlheim an derRuhr, Germany. E-mail: Grande@itmc.rwth-aachen.de In the scope of depleting sources the full valorization of renewable resources (e.g. lignocellulosic/green biomass) is a key problem to be addressed. The main component of biomass is cellulose, which can be hydrolyzed to fermentable sugars (e.g. glucose, xylose) via cellulases. One mayor issue to be tackled here is the inhibition/deactivation of the cellulases by lignin and crystallinity of the lignocellulose. Hence pretreatment/pulping procedures have a great importance in this field. In the presented work a pulping procedure for the full valorization of lignocellulosic biomass is introduced, leading to a significant improvement of activity with a commercial cellulase (Figure 1) [1,2]. It is shown that this improvement is versatile, independent of the type of biomass (e.g. lignocellulose or green biomass). As a second topic the utilization of seawater as a convenient, bio-based and green solvent for enzyme reactions is shown (Figure 2). Processing of amorphous cellulose as well as commercial microcrystalline cellulose AVICEL showed analogous good results in seawater as in distilled water. Even in higher saline concentrations, which occur in waste streams of the drinking water production from seawater, a considerable glucose formation was observed, showing up a huge potential for genetically evolved cellulases. [3]
430
Benzaldehyde lyase catalyze synthesis of (r)-3,3-Furoin in non-conventional media
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433
Stereochemical promiscuity of thiamine enzymes: engineering the stereoselectivity of MenD from Escherichia coli
Robert Westphala, Michael Widmannb, Jrgen Pleissb, Michael Mllerc, Drte Rothera, Martina Pohla a Institute of Bio- and Geosciences 1: Biotechnology, Forschungszentrum Jlich GmbH, D-52425 Jlich, Germany b Institute of Technical Biochemistry, University of Stuttgart, D-70569 Stuttgart, Germany c Institute of Pharmaceutical and Medicinal Chemistry, University of Freiburg, D-79104 Freiburg, Germany E-mail: r.westphal@fz-juelich.de Thiamine diphosphate (ThDP)-dependent enzymes exhibit the synthetic potential for asymmetric carboligations resulting in a vast number of valuable enantiomerically different products and building blocks (e.g. 2-hydroxy ketones) with high potential for pharmaceutical applications [1]. Nevertheless, the access to (S)-products is still very limited at the moment. The predominant (R)-selectivity of thiamine enzymes seems to be an intrinsic property, directed mainly by their structures. To understand stereoselectivity in ThDP-catalyzed reactions asymmetric carboligations have been already studied for many thiamine enzymes and appropriate models concerning stereoselectivity were developed in recent years [2, 3]. To evaluate existing models and expand the product range of 2-hydroxy ketones further, principles that control stereoselectivity of thiamine enzymes will be transferred to the (1 R, 6 R)-2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD) from Escherichia coli. MenD is a relatively new member of the thiamine enzyme family and displays (R)-selectivity in mixed carboligations [4] like most of the thiamine enzymes. However, the stereoselectivity is not understood yet. The elucidation of principles that control stereoselectivity in MenD is the main goal of this project including the attempt to invert the stereoselectivity by rational design to gain hardly accessible (S)-products. Therefore, steric properties of the MenD active site are compared with already known thiamine enzymes by modelling studies followed by preparation of appropriate variants. Those variants are currently characterized concerning their substrate spectrum and stereoselectivity.
________________________________ [1] Mller, M., Gocke, D. & Pohl, M., FEBS J. 2009, 276: 2894-2904. [2] Dnkelmann, P., Kolter-Jung, D., Nitsche, A., Demir, A.S., Siegert, P., Lingen, B., Baumann, M., Pohl, M. & Mller, M., J. Am. Chem. Soc. 2002, 124: 12084-12085. [3] Gocke, D., Dissertation 2007, Heinrich-Heine-University Dsseldorf. [4] Kurutsch, A., Richter, M., Brecht,V., Sprenger, G.A. & Mller, M., J. Mol. Catal. B: Enzym. 2009, 61: 56-66.
434
Inuencing the stereoselectivity of ThDP-dependent enzymes through the addition of water-miscible organic solvents
T. Gerhards, U. Mackfeld, E. von Lieres, W. Wiechert, M. Pohl, D. Rother Institute of Bio- and Geoscience (IBG-1: Biotechnology), Forschungszentrum Jlich, D-52426 Jlich, Germany E-mail: t.gerhards@fz-juelich.de Based on initial studies of thiamine diphosphate (ThDP)-dependent enzymes in non-conventional solvents, it became obvious that these enzymes have a reasonable stability in non-conventional media [1], although they require water for their activity. These studies have been expanded in a comprehensive study towards the bimolecular benzoin-like carboligase reaction with varying ThDP-dependent enzymes. As a consequence of the mixed reaction a total of four different ligation products can principally occur, each in both enantiomeric forms (Figure 1).
Dessy Nataliaa, Walter Leitnera, Marion Ansorge-Schumacherb, Lasse Greinera,c ITMC, RWTH Aachen University, Aachen, Germany; bTC 4, Technische Universitt Berlin, Berlin, Germany, cDECHEMA e. V. Karl-Winnacker-Institut,Frankfurt am Main, Germany; Email: Natalia@itmc.rwth-aachen.de Benzaldehyde lyase (BAL) is an extremely versatile catalyst for the organic synthesis of -hydroxyketones compounds [1]. BAL accepts a broad substrate range and has outstanding stereoselectivity. Biocatalysis reaction using BAL were normally done in aqueous system with addition of co-solvent, however, the solubility limit of the product and substrate is still the major drawbacks in this system [1]. Non-conventional media such as organic solvent, ionic liquid, gases, and supercritical fluids (SCF) could be an alternative for biocatalysis, especially using BAL. The use of this non-conventional media could enhance the solubility of both substrate and product. There were two approaches taken, one is using SCF and the other is by immobilisation in PVA beads. The first approach was to use SCF as a reaction medium with the BAL in the lyophilisate form. SCF deliver the substrate to the reactive site and carry the product out of the reactive site. For this approach, residual activity and batch experiment were tested. Surprisingly, preparation of the BAL has more impact on the residual activity and stability compared to the nature of SCF. Furthermore, water is needed for high enantio- and chemo-selectivity. The second approach was to use BAL immobilised in PVA beads. BAL has been known to be unstable at phase boundaries; therefore PVA can be used for stabilisation [2]. Continuous application with continuous organic solvent will be presented and analysed for productivity and stability.
Figure 1. Possible product range of a bimolecular carboligase reaction catalyzed by ThDP-dependent enzymes. The chemo- and stereoselectivity of the reaction are influenced by the enzyme, the reaction conditions and the substrates. A very good example to study the influence of water-miscible organic solvent addition on enzyme selectivity is e.g. the branched-chain 2-keto acid decarboxylase from Lactococcus lactis, an enzyme which allows in principle the access to both mixed ligation products of 2-hydroxy ketones (Fig. 1) starting from different aromatic and various aliphatic aldehydes [2]. Further the pyruvate decarboxylase from Acetobacter pasteurianus [3] and variants thereof exhibit interesting synthetic properties and represent suitable targets to study selectivity shifts. Here we proof that different water-miscible organic solvents have an influence on both, chemo- and stereoselectivity of ThDP-dependent enzymes. The influence on the stereoselectivity is most pronounced, if the enzymes selectivity in aqueous buffer is < 95 % ee.
________________ [1] Kokova M, Zavrel M, Tittman K, Spiess A, Pohl M. 2009. J. Mol. Cat. B, 61: 73-79 [2] Gocke D, Nguyen CL, Pohl M, Stillger T, Walter L, Mller M. 2007. Adv. Synth. Catal. 349:14251435. [3] Gocke D, Graf T, Brosi H, Frindi-Wosch I, Walter L, Mller M, Pohl M. 2008. J. Mol. Cat. B, 61:3035.
Figure 1. Process for the fractionation of lignocellulosic biomass.
Figure 2. Process for the enzymatic hydrolysis in seawater as solvent.

________________ [1] P. Domnguez de Mara, T. vom Stein, P. M. Grande, F. Sibilla, W. Leitner, EP 11154705.5, 2011. [2] T. vom Stein, P. M. Grande, H. Kayser, F. Sibilla, W. Leitner, P. Domnguez de Mara, Green Chem. 2011, accepted, DOI:10.1039/C1GC00002K. [3] P. M. Grande, P. Domnguez de Mara, 2011, submitted.
Figure 1. (a) The scheme for the batch application of BAL in SCF, (b) The scheme for the continuous application of immobilised BAL in organic solvent.
________________ [1] Marion B. Ansorge-Schumacher, et.al., Biotechnology Journal. 2006, 1, 564-568. [2] Anne van den Wittenboer, et.al., Journal of Molecular Catalysis B: Enzymatic. 2010, 67 (3-4), 208-213
October 2-6, 2011
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An E. coli tktA/tktB double mutant allows in vivo selection both for transketolase and phosphoketolase activity
Natalie Trachtmann, Bettina Buerle, Georg A. Sprenger* Institute of Microbiology, Universitt Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany. *e-mail: g.sprenger@imb.uni-stuttgart.de The thiamine diphosphate (ThDP)-dependent enzyme transketolase (TKT; EC 2.2.1.1) catalyzes C-C-bonding and cleaving reactions in the non-oxidative branch of the pentose phosphate cycle. TKT is being used as biocatalyst for a variety of non-physiological C-C bonding reactions in biotransformations [1]. In E. coli, two isoenzymes, TKTA and TKTB, exist. A tktA tktB double mutant is unable to grow on pentose sugars as sole carbon sources. It is furthermore auxotrophic for aromatic amino acids as the precursor, erythrose-4-phosphate, cannot be formed, and finally -due to the lack of sedoheptulose7-phosphate formation, lipopolysaccharides of the outer membrane cannot be attached. Therefore, E.coli tktA tktB mutants are unable to grow in the presence of detergents (0.1% of SDS) or bile salts (e.g. MacConkey agar). We made use of these three features and used tktA tktB mutant strains for the expression of transketolase genes from different organisms, e.g. of human, plant or microbial origin. The phenotypes (prototrophic growth, growth on pentoses and resistance to bile salts) were restored in the tktA tktB deletion strain by expression of recombinant TKT (E. coli, human and plant origin) on appropriate selective media (minerals salts agar without aromatic supplements, growth on xylose, growth on MacConkey agar plates, respectively). Thus, exposition of the E. coli tktA tktB mutant strain with recombinant genes under selection conditions provides easy and rapid in vivo test systems for the detection of heterologous TKT activities. Moreover, the gene for the ThDP-dependent enzyme phosphoketolase from Bifidobacterium (splitting fructose-6-phosphate into erythrose-4-phosphate and acetyl-phosphate) also was able to complement the growth defects (bile salt sensitivity, aromatic amino acid auxotrophy and metabolism of pentoses). Use of the tktA/B double mutant now allows for rapid characterisation of active TKT enzymes or of enzyme variants (fusion proteins, isoenzymes, muteins, etc.). Examples will be provided.
_______________________ [1] Sprenger, G.A., & Pohl, M. (1999) Synthetic potential of thiamin-dependent enzymes. Journal of Molecular Catalysis B: Enzymatic 6:145-159.

Novel routes to antiobiotics using synthetic biology approaches
David Steadmana, Panwajee Payongsrib, Paul A. Dalbyb and Helen C. Hailesa a Department of Chemistry, University College London, 20 Gordon St, London WC1H 0AJ, UK; bDepartment of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK E-mail: david.steadman.09@ucl.ac.uk The aminodiol moiety is present in many biologically active molecules, including the broad spectrum antibiotics chloramphenicol and thiamphenicol. Biocatalytic strategies are useful alternative routes to such molecules, compared to traditional synthetic approaches, as enzymes can offer high degrees of stereoselectivity, substrate tolerance and atom efficiency. Aminodiols can be synthesized using two enzymes, transketolase (TK) and transaminase (TAm) (Figure 1).
O HO O CO2 O O- Li+ TK, Mg2+, ThDP OH OH O Amine Ketone TAm, PLP NH2 OH OH
List of Authors A
Abad, S Abdoul-Zabar, J Abedi, V Abian, O Abreu, JC Acero, EH Acher, F Achour, O Adir, NL Adlercreutz, P Aehle, W Afonso, CAM Agui, V Aguieiras, ECG Aguirre, C Al-Bahrani, H Albertini, AM Albertyn, J Alcalde, M Alcntara, AR Al-Haque, N Alib, S Aljawish, A Alkan, V Allen, MJ Almeida, RV Almeida Vegele Sousa de, Y Althoff, E Altschuh, D Alvarez, HM Alvarez, L lvaro, G Amati, G Ambrogelly, A Amin, MA Andberg, M Andexer, JN Andrade, LH Andre, M Andr, I Ang, EL Ansorge-Schumacher, MB Antczak, T Aoyagi, H Araujo,Y Arajo, LS Araya, E Archelas, A Archer, I Arends, IWCE Arizpe, A Aroca, G Arroyo, PA Arvotti, G Arzhanik, V Asano, M Asano, Y Ashrafuzzaman, MD Aslam, R Atrauskait, M Augustin, P Auriol, D Aymes, A Azerad, R Azevedo, VH Aziz, M Aziz, S PC-358 PC-27, 28 PC-12 PC-146 PC-111 PC-363 PC-217 PC-124 PC-326, 340 PC-160 PC-390 PC-190, 371 PC-302 PC-113 PC412 PC-179, 413 OC-33; PC-365 PC-72, 85 PC-4, 216, 248, 252, 264, 378 PC-184 PC-127 PC-76 PC-393 PC-390 PC-76 PC-210, 313 PC-165 OC-35 PC-302 PC-207, 275, 357 PC-386 PC-51, 185 OC-33 IL-10 PC-328 PC-8 OC-13, PC-100, 111, 122, 208, 278 PC-52 PC-218, 280, 303 OC-4 PC-66, 144, 184, 425, 427, 430 PC-347, 348, 394 PC-391 PC-251 PC-278 PC-205 PC-159, 271 PC-409 OC-7; PC-94, 116, 161, 297 PC-156 PC-386 PC-352 OC-5; PC-401 OC-37 PC-164 PC-60, 290, 309 PC-364 PC-39 PC-67 PC-57, 64 PC-418 PC-291 PC-175, 217 PC-118 PC-157 PC-157 PC-284 PC-38, 53 PC-59 PC-109, 182, 312 PC-55, 179 PC-246 PC-25 PC-222 PC-4,186, 204, 216 PC-197 OC-35 PC-310 Baraibar, A Barao, CE Barbe, S Bariotaki, A Barletta, GL Bartmaska, A Bartsch, S Basso, A Buerle, B Baum, S Baxter, S Bayn, C Bechtold, M Becker, J Bedini, E Bednar, D Begemann, J Behrendt, D Beigi, M Belsare, KD Ben Rhouma, G Benaissi, K Benson, S Berenguer, J Bergantino, E Berglund, P Bernal, C Bernath-Levin, K Berriel, RF Bertolani, A Bertolesi, GM Besenmatter, W Besnard, M Bezouka, K Bezze, C Biagiotti, M Bidmanova, S Bidouil, C Biedermann, M Bielecki, S Biondi, D Bischop, M Bishnu, P Bisogno, FR Black, GW Blank, L Blumenrder, B Bobrov, KS Boer, H Boeriu, CG Bing, C Bojarov, P Bommarius, AS Bonhage, B Bonugli-Santos, RC Boom, RM Boomer, M Boonvitthya, N Borchert, S Bordeaux, M Bordes, F Bordi, F Bordier, F Borghese, G Borisova, AS Bornscheuer, UT Boros, Z Borowska, R Bosshart, A Bossius, B Bott, M Bott, R Bou Saab, H Boulanger, A Bour, A Bovicelli, P Bozov, P Bracco, P Braiuca, P Braa, AF Branco, RV Brandenbusch, C Branneby, S PC-117 PC-352, 372 PC-218, 303 PC-203 PC-169 PC-198 PC-240, 331 OC-40 PC-435 PC-21 PC-121 PC-192 OC-41; PC-214, 292, 400, 405 PC-212 PC-258 OC-28 PC-427 PC-120 OC-22 PC-293 PC-367 PC_26 PC-315 PC-192 OC-5; PC-401 PC-12 PC-415 PC-273 PC-161 PC-172 PC-145, 270 PC-316 PC-223 PC-7 OC-5 PC-365 OC-28 PC-437 PC-329 PC-394 PC-24 OC-12 IL-5 PC-48, 133 PC-23, 234, 285, 333 PC-61, 82 PC-70 PC-319 PC-8 PC-375 PC-71 PC-7 PC-310, 332 OC-26; PC-420 PC-299 PC-389 PC-311 PC-377 PC-70 OC-2; PC-68 PC-303 PC-337 PC-223 PC-365 PC-319 KL-1; PC-245, 259, 268, 286 OC-10 PC-348 PC-214, 400 PC-341 PC-242 PC-390 PC-105 PC-105 PC-130 PC-24 PC-369 PC-98 OC-40; PC-325, 341 PC-148 PC-210 PC-410 PC-160
183
Figure 1. A transketolase and transaminase strategy towards aminodiol synthesis TK is an enzyme responsible for asymmetric carbon-carbon bond formation.[1] The TK catalysed condensations of hydroxypyruvate with glycolaldehyde and non-hydroxylated aliphatic aldehydes have been reported in the literature;[2,3] however with a view to developing TK enzymes suitable for use with a wider range of substrates for general synthetic applications, cyclic and aromatic aldehydes were explored. We have recently reported the use of mutant TKs which accept aromatic aldehydes, giving access to aromatic and heteroaromatic dihydroxyketones with varying optical purities (53% - 97% ee).[4] TAms convert a ketone or aldehyde moiety into an amine functionality, in the presence of a pyridxoxal phosphate (PLP) co-factor and an amine donor. TAm from Chromobacterium violaceum can accept aromatic dihydroxyketones, highlighting its potential for use in a two-step route to aminodiols.[5] We are currently using novel combination mutant TKs to increase yields and optical purities of the resulting aromatic dihydroxyketones and aim to use new and existing TAms to access the corresponding aminodiols. Recent progress in this area will be presented.
________________________________________ [1] Schneider G., Lindqvist Y., Biochim. Biophys. Acta 1385 (1998) 387. [2] Hibbert E.G., Senussi T., Costelloe S.J., Lei W., Smith M.E.B., Ward J.M., Hailes H.C., Dalby P.A., J. Biotechnol. 131 (2007) 425. [3] Czares A., Galman J.L., Crago L.G., Smith M.E.B, Strafford J., Ros-Sols L., Lye G.J., Dalby P.A., Hailes H.C., Org. Biomol. Chem. 8 (2010) 1301. [4] Galman J.L., Steadman D., Bacon S., Morris P., Smith M.E.B., Ward J.M., Dalby P.A., Hailes H.C., Chem. Commun. 46 (2010) 7608. [5] Smithies K., Smith M.E.B., Kaulmann U., Galman J.L., Ward J.M., Hailes H.C., Tetrahedron: Asymmetry 20 (2009) 570.
437
Selectivity of lipase-catalyzed acetylation of quercetin: a molecular modelling study
Christelle Bidouila, Eduardo Basilio De Oliveirab, Latifa Chebila, Elaine-Rose Maiac, Bernard Maigretd, Evelyne Ronat-Heita Mohammed Ghoula, Jean-Marc Engassera and Catherine Humeaua a Laboratoire dIngnierie des Biomolcules, ENSAIA-INPL, 2, Avenue de la Fort de Haye 54500 Vandoeuvre-ls-Nancy, France; bDepartamento Tecnologia de Alimentos, Universidade Federal de Viosa, 3 6570. 000 Viosa - MG Brasil; cLaboratorio de Estudos Estruturais Moleculares (LEEM), Instituto de Quimica, Universidade de Brasilia, CP 4478, 70904-970, Braslia-DF, Brasil; dLaboratoire Lorrain de Recherche en Informatique et ses Applications, CNRS, BP 239, 54506 Vandoeuvre-ls-Nancy, France E-mail : christelle.bidouil@ensaia.inpl-nancy.fr Quercetin is an aglycon flavonoid abundant in human diet and known for its various beneficial health effects and especially its antioxidant activity [1]. However, the use of quercetin in several application fields is limited by its low solubility, stability and bioavailability. One solution to overcome these limitations consists in acylating regioselectively quercetin by a lipase-catalyzed process, in order to preserve the functionality of the hydroxyl groups responsible for its antioxidant activity. Among commercially available lipases, many enzymes as Candida antarctica lipase B (CALB) were not able to catalyze quercetin acylation. Only Pseudomonas cepacia lipase (PCL) allowed the synthesis of quercetin mono-, di- and triesters on the 4, 3 then 7 hydroxyl groups. So far, no explanation was given at a molecular scale neither for the absence of reaction with CALB, nor for the lack of regioselectivity with PCL. Recently, computer-based modelling methodologies have been increasingly used to understand the mechanism and the selectivity of enzyme-catalyzed reactions [2]. The aim of the present study is to investigate the selectivity of Candida antarctica lipase B and Pseudomonas cepacia lipase towards quercetin in acetylation reaction. A methodology combining docking and molecular dynamics (MD) simulations was applied to study the interactions between acyl enzyme targets (CALB or PCL with an acetate bound to the catalytic serine) and quercetin. Results showed that with CALB, the serine-bound acetate was not correctly positioned within the oxyanion hole whatever the orientation of quercetin, suggesting that no product could be obtained. With PCL, the acetate remained within the oxyanion hole during all MD trajectories. Depending on quercetin orientation, either the 7-OH group or the 3, 5, 3', 4'-OH groups came alternatively near the catalytic residues, suggesting that all of them should be acylated. However, previous experimental study reported the absence of acetylation of the 3-OH and 5-OH groups. This result was explained by their poor nucleophilic character which was not taken into account in the models.
________________ [1] Chen, C., J. Zhou, and C. Ji, Quercetin: a potential drug to reverse multidrug resistance. Life Sciences, 2010. 87(11-12): p. 333-338. [2] De Oliveira, E.B., et al., A molecular modelling study to rationalize the regioselectivity in acylation of flavonoid glycosides catalyzed by Candida antarctica lipase B. Journal of Molecular Catalysis B: Enzymatic, 2009. 59: p. 96-105.
438
Carba analogues of PAF- enzyme assisted syntheses and biological activities
Bernd Jakob, K. Lange and M.P. Schneider* FB C Bergische Universitt, D-42097 Wuppertal, Germany, schneid@uni-wuppertal.de Platelet activating factor (PAF), an ether lipid, is interacting with the G-protein coupled PAF receptor and thus activates multiple intracellular signalling pathways[1]. It is an important mediator of platelet aggregation, inflammation and anaphylaxis. Structural analogues of PAF such as 1-Octadecyl-2-O-Methyl-sn-glycero-3-phosphocholine (ET- 18-OCH3) display anticancer properties[2]. Since PAF is metabolically instable with a half-life of only a few minutes due to hydrolysis of the acetate function in the sn-2 position by acetyl hydrolases, numerous derivatives such as alkyl ethers, thio ethers and carbamoyl derivatives have been prepared and studied regarding their biological activities. All of them, however, change the stereo-electronic character of PAF and we thus decided to synthesize a series of carbaanalogues of these molecules in which the sp3 oxygens of the acetyl functions are replaced by sp3- carbons. This change results in isosteric mimics of the natural molecules with minimal deviations regarding bond angles and bond distances (Figure)[3].
Key steps are enantioselective, enzyme (lipase) catalyzed esterifications and hydrolyses, thereby differentiating between either the enantiotopic hydroxy groups in achiral 2-substituted 1,3 diol precursors or the enantiomers of the corresponding esters[4]. Both enantiomeric series of the title compounds were synthesized, carrying a variety of alkyl and acyl groups with the potential of also varying the polar head groups[5]. The thus obtained molecules are currently tested regarding their biological activities. While the originally expected antitumor and antiproliferative activities are only weak to moderate, first results indicate that the title compounds (somewhat surprisingly) display extremely high antimicrobial (antimycotic) activities towards micro organisms such Mycobacterium vaccae, Candida albicans and Pennicillium notatum comparable to commercial drugs such as ciprofloxacin and amphotericin B[6].
Acknowledgements: We are grateful to Dr. Christian Lehmann (Max Planck Institut fr Kohlenforschung, Mlheim/Ruhr, Germany) for the determination of absolute configurations via X-ray crystallography and to Corinna Lange (Hans Knll Institut, Jena, Germany) for biological testing. ________________ [1] S.M.Prescott,G.A.Zimmerman, D.M.Stafforini,T.M.McIntyre, Annu.Rev.Biochem. 69 (2000) 419-445; [2] J.D. Winkler, C.M. Sung, M. Chabot Fletcher, R.J. Mayer, M.E. Surette, F.H. Chilton; J. Pharm. Exp. Therapeutics; 179(2) (1996); 956 966; [3] For further examples see: P.Andersch, B.Jakob, R.Schiefer and M.P.Schneider in Ed. K.W.A Wirtz Molecular Mechanisms of Signalling and Membrane Transport (proceedings of the NATO ASI on Molecular Mechanisms of Signalling and Targeting 1996, Spetses Island), Springer Berlin Heidelberg 1997, 247 264; [4] K.Lange, M.Schneider, Tetrahedron:Asymmetry 15 (2004) 2811 2815, [5] K.Lange, Ph.D.thesis Wuppertal 2004, manuscript in preparation; [6] unpublished results.
B
Baas, B-J Babich, L Baboo, JZ Bckvall, JE Baganz, F Baganza, F Baier, S Baierl, A Ballesteros, AO Balloumi, A Ban, YA Bansal, P
October 2-6, 2011
184
Brask, J Brenna, E Brennan, T Breuer, M Brezovsky, J Bridiau, N Brondani, PB Brouk, M Bruce, NC Brussel, M Brzeziska-Rodak, M Buchhaupt, M Buchholz, S Bchs, J Buko, M Bhler, B Bujons, J Bulon, A Burda, E Burton, S Bury, A Bussamara, R Busto, E Buzzini, P PC-350, 376 PC-40 IL-10 OP-14; PC-78, 171 OC-28; PC-339 PC-124, 194 PC-122 OC-3 OC-30 PC-390 PC-90, 181,183 PC-99 PC-71 PC-432 PC-6 PC-61, 82, 96, 193,221, 410 PC-32 PC-280 PC-70 OC-38; PC-97 PC-53 PC-143 PC-131 PC-406 Claps, P Classen, T Clay, D Clemente-Jimnez, JM Cobley, C Cobucci-Ponzano, B Coelho, MAZ Cohelo Breda, G Collins, J Coman, S Comelles, F Commandeur, U Conti, P Cordeiro, Y Cornelissen, S Corsaro, MM Corts-Cabrera, A Costa, S Crestini, C Creus, M Cruz Neves, B Cuetos, A Cunha, AG Czarnotta, E IL-4; PC-32, 33, 49 OC-12 PC-129, 151 PC-17 PC-417 PC-258 PC-114, 165 PC-313 PC-410 PC-379 PC-186 OC-26; PC-212, 344 PC-172 PC-320 PC-193 PC-258 PC-184, 192 PC-128 IL-9 PC-65 PC-313 PC-133 PC-114 PC-96
List of Authors
List of Authors
Dumon, C Dupont, J Duquesne, S Durchschein, K Drrenberger, M Dutilleul, P Dvorak, P Dziworska, G PC-377 PC-143 PC-218 PC-187 PC-231, 247 PC-157 OC-28 PC-366 Fotheringham, I Fouchard, S Fraaije, MW Frank, A Franssen, MCR Frauenkron-Machedjou, J Freddi, G Freire, DMG Freitas, L Frick, O Fricks, AT Fryszkowska, A Fu, S Fuchs, CS Fuchs, M Fuciosa, P Fukuta, Y Fulton, A Freder, M Fusettir, F PC-121 PC-105 OP-15; PC-77, 279, 289, 332 PC-404 PC-389 PC-419, 428 PC-363 PC-114, 146, 210, 237, 382, 383 PC-170 PC-61 PC-275, 357, 372 PC-121 PC-56 PC-202 PC-50, 110 PC-276 PC-290 PC-419, 428 OC-41; PC-292, 400, 405 PC-300
185
E
Ebert, C Egger, S Eggert, T Eggink, G Eichenauer, N Eiteljoerg, I Elling, L Emmerstorfer, A Eneyskaya, EV Engasser, J-M Engel, P Engel, PC Engstrm, K Enrich, M Eras, J Escalante-Garca, J Escalettes, F Esquivel, R Estrine, B Ettrich, R EunOk, J PC-58, 324, 325 PC-22 PC-281 PC-375 OC-12 PC-363 OC-9 PC-57, 64 PC-319 PC-367, 437 OC-26; PC-212, 420 PC-224 PC-182, 312 PC-51 PC-368 PC-149 PC-229 PC-180 OC-25 PC-339 IL-5
G
Gaber, Y Galarneau, A Gallifuoco, A Gama, WS Gambera, G Gandolfi, S Gani, R Garca-Junceda, E Garcia-Ruiz, E Gardossi, L Gargiulo, S Garrabou, X Gasparini, G Gattas, EAL Gatti, FG Gatti-Lanfranconi, P Gavagan, JE Gavezzotti, P Gak, R Geertsema, EM Geinitz, C Gemeiner, P Gerhards, T Gernaey, KV Gershater, MC Gewessler, U Ghazi, I Ghisla, S Ghoul, M Gibka, J Giesecke, U Giordano, A Giordano, RC Giordano, RLC Giovannini, PP Giraldelli, MG Girfoglio, M Giulietti, AM Gadkowski, W Glieder, A Gloor, G Glueck, SM Gnach, A Goldfeder, MS Golovleva, L Golubev, AM Gonalves, C Gonalves, KM Gonalves Torres, FA Gonzles, ISC Gonzlez, R Gonzalez-Perez, D Gonzlez-Sabn, J Gonzlez-Siso, MI Gorseling, M Gotor, V Gotor-Fernndez, V Graber, M Grabs, D Grande , PM PC-328 OC-2; PC-68 PC-18 PC-275 PC-24 PC-5 PC-127 PC-54, 136, 283 PC-4, 264 OC-40; PC-58, 324, 325, 356 PC-94, 116 PC-32 PC-409 PC-397 PC-40 OC-18 PC-244 PC-9 PC-7 PC-284 PC-125 PC-6 PC-434 OC-11; PC-43, 399 PC-134 PC-387 PC-216 PC-327 PC-367, 437 PC-348 OC-39 PC-153 PC-143, 351 PC-143, 351 PC-128, 142, 189 PC-355 OC-26; PC-212, 344 PC-249 PC-132 PC-119, 235, 387 IL-10 PC-135 PC-90 PC-326 PC-58 PC-319 PC-299 PC-320 PC-313 PC-207, 275 PC-276 PC-4, 264 PC-148 PC-276 PC-277 PC-48, 69, 77, 131,133, 148, 156 PC-48, 69, 131,156 PC-124, 197 OC-35 PC-429
C
Cabrera, Z Caicedo, A Calvio, C Camarero, S Camattari, A Campopiano, D Camps-Bres, F Canduri, F Canela, R Cannio, R Cantarella, L Cantarella, M Carballeira, JD Crdenas-Fernndez, AM Cardillo, AB Cardillo Afonso, L Carlsson, N Cartwright, J Cassimjee, KE Castellin, A Castiglione, F Castilho, LR Castillo, E Castro, MF Castro, AM Castro, HF Cativiela, C Caufin, S Cavaco-Paulo, A elebi, B Cerdn, ME Cerioli, L Cesarini, S Chabbert, B Chadha, A Chaloupkova, R Chamantray, F Chaput, L Charles, TC Chebil, L Chekir-Ghedira, L Chen, C Chen, N Cheong, CS Chernykh, A Chevalot, I Chiappe, C Choi, S-L Choi, Y-H Chojnacka, A Chojnacka-Puchta, L Chong, S Chronopoulou, L Chulalaksananukul, S Chulalaksananukule, W Chulalukkananukul, W Churakova, E PC-178 PC-32 PC-365 PC-4, 252, 378 PC-235 PC-121 PC-54, 136 PC-251 PC-368 OC-26; PC-212 PC-18 PC-18 PC-184 PC-185 PC-249 PC-381 PC-380 PC-76 PC-12 OC-5; PC-401 PC-361 PC-382 PC-149 PC-118 PC-237, 382 PC-372 PC-156 PC-9 PC-363 PC-174 PC-276 PC-11, 16, 123, 349, 361 PC-359 PC-302 PC-155 PC-339 PC-26, 27, 28 PC-197 PC-362 PC-367, 437 PC-367 PC-330 PC-421 PC-364 PC-58 PC-291, 393 PC-123 PC-260, 322 PC-374 PC-132 PC-298, 402, 403 PC-262 PC-337 PC-392 PC-218 PC-392 OC-7; 116
D
DArrigo, P Da Ros, PCM da Silva, MAP Dadashipour, M Dalby, PA Damborsky, J Danieli, B Danieli, F Dankmeijer, L Dantas, JH D'Antona, N Daramwar, PP Darbre, T Dariva, C Darvas, F de Abreu, MA De Angelis, E de Beer, R de Beer, RJAC de Berardinis, V De Castro, HF de Gonzalo, G de Oliveira, JR De Oliveira, EB de Paris, LD Debard, A Decina, S Dejonghe, W Dekker, FJ Delaunay, S Delenne, M Demir, AS Dennig, A Desmet, T-K Desrousseaux, M-L Devamani, T di Giuro, C Dias, CAO Diaz, A Diaz, P DiCosimo, R Diels, L Diociaiuti, M Dirk, H Dolors Benaiges, M Dominguez de Maria, P Donova, MV Doo, EH Dordick, JS Dorel, G Dover, GL Driscoll, BT Drone, J Dubreucq, E Dudek, HM Duetz, W Dugoni, C PC-11, 16, 123, 267, 349, 361 PC-355 PC-113 PC-290 PC-436 OC-28; PC-339 PC-9 PC-397 PC-390 PC-352 PC-24, 108 PC-63 PC-34 PC-357 OC-10 PC-204 PC-356 PC-204 PC-196 PC-209, 211, 223 PC-170, 355 PC-77, 133, 279 PC-147 PC-437 PC-352 PC-223 IL-9 PC-15 PC-253 PC-291 OC-30 OC-20; PC-174 PC-242, 272 PC-330 PC-377 PC-27, 28 OC-29 PC-397 PC-412 PC-359 PC-244 PC-15 PC-337 PC-238 PC-51 PC-101, 236, 266, 423, 425, 429 PC-80, 81 PC-82 PL-3 PC-239 PC-333 PC-362 OC-2; PC-68 PC-305, 335, 345, 388, 385 PC-289, 332 OP-15 PC-406
F
Faber, K Fabian, WMF Failmezger, JK Fajula, F Falcioni, F Falus, P Famrov, V Faria de Moraes, F Farrs, A Fattor, D Faulstich, M Faur, N Federsel, HJ Femmer, C Feringa, BL Fernandes, C Fernandez-Arrojo, L Fernandez-Lafuente, R Fernandez-Lobato, M Feroz, S Ferrandi, EE Ferrari, F Ferrario, V Ferreira-Diaz, S Fessner, WD Fibriansah, G Filho, PSC Filipkowski, P Findrik, Z Fink, MJ Fiore, MF Fioroni, M Fisch, F Fischer, R Fischer, T Fisher, T Fishereder, EM Fishman, A Flake, C Flankenberg, M Flicker, K Flinn, T Flock, T Flores, MC Fogal, S Fokina, VV Forano, C Foscato, M Fossey, MA PC-107, 110, 129, 135, 150, 151, 187, 255, 334 PC-187 PC-427 OC-2; PC-68 PC-61 OC-10 PC-271 PC-352 PC-180 OC-40; PC-58, 324, 356 PC-396 PC-205 PC-12 PC-292, 405 PC-253 PC-54 PC-204 PC-159 PC-204 PC-332 PC-10, 145, 270 PC-291 PC-58, 324, 325 PC-385 PC-27, 28, 29, 30, 31, 33 PC-321 PC-206 PC-232 PC-36 PC-3, 329 PC-355 PC-92 OC-30 PC-212, 344 PC-329 OC-12 OP-16 OC-3; PC-273, 326, 340 PC-343 PC-343 PC-119 PC-333 PC-387 PC-414 OC-5; PC-401 PC-80 PC-26, PC-58, 324, 325 PC-397
October 2-6, 2011
186
Greimel, K Greiner, L Greinera, L Grey, C Grischek, B Groeneveld, M Grogan, G Grger, H Gruber, C Gruber, CC Gruber, K Gruber, S Gruber-Khadjawi, M Grukien, R Gschaedler, A Guchhait, K Gudiminchi, RK Guebitz, GM Gurard-Hlaine, C Guerrero, C Guisan, JM Gupta Udata, DBRK Guranda, DT Gureviciene, V Gustafsson, H Gutarra, MLE PC-363 PC-408 PC-430 PC-160 PC-48, 106 OP-15 OC-30; PC-75, 76, 79, 106, 229, 404 IL-3; PC-70, 71, 87 PC-315 PC-255, 388 PC-22, 255, 288, 363, 431 PC-225, 307 OC-31; PC-213 PC-67 PC-354 PC-317 PC-85 OC-23; PC-363, 387 PC-54, 136, 209 PC-188 PC-47, 146, 159, 210, 237 PC-380 PC-173 PC-1 PC-370 PC-146, 237 PC-346 PC-375 IL-8; PC-130, 134, 141, 179, 413, 436 PC-256, 288 PC-55 PC-129, 150, 151, 310 PC-418 OC-36 IL-1; PC-10, 204, 226 OC-32 PC-171 PC-360 PC-168, 259, 277, 297 PC-417 PC-250 PC-388 PC-38, 86 PC-53 PC-23 PC-387 PC-158, 191, 328 PC-78, 88, 125, 171 OP-14 PC-29, 30 PC-167 PC-240, 331 PC-26, 27, 28 PC-65, 247 PC-26, 27, 28 PC-20 PC-221 PC-192 PC-115 PC-259 OC-19 PC-46 OC-32; PC-104, 164 IL-2 PC-348 PC-309 PC-95 OC-32 PC-161 PC-343 PC-219 PC-245 PC-313 OC-18 OC-7; PC-94, 116, 137, 301 PC-370 PC-92, 293 PC-121 PC-78, 88 PC-306 OC-10 PC-343 Hoyos, P Huang, H Hub, S Huitink, J Hult, K Humeau, C Hummel, A Hummel, W Huszcza, E PC-184 PC-104 PC-25 PC-390 OC-27; PC-182 PC-291, 437 PC-268 PC-70, 87 PC-198
List of Authors
List of Authors
Khan, N Kiebasiski, P Kieryl, P Kim, B-G Kim, D-H Kim, HJ Kim, H-J Kim, HK Kim, JH Kim, J-Y Kim, KS Kim, MH Kim, P Kim, SJ Kim, YH Kim, S-J Kima, MH Kino, K Kirilin, E Kirsebom, H Kishino, S Kittelmann, M Kittl, R Klebensberger, J Kleija, P Klein, K Kleinait, E Klempier, N Klimek-Ochab, M Klose, H Knapic, L Knaus, T Knrr, L Kobayashi, R Kfinger, P Khler, V Kohlmann, C Koivula, A Kolb, M Koley, M Kollerov, VV Kondor, B Knst, P Konstantinovics, C Korpak, M Koschorreck, K Kotik, M Kourist, R Kozyra, K Kratzer, R Krauss, U Kreen, M Ken, V Kenek, K Krishna Bodla, V Kroutil, W Krne, U Kubac, D Kudanga, T Kudryashova, M Kuhn, D Kulig, J Kullartz, I Kulminskaya, AA Kulschewski, T Kumar, R Kumar Padhi, S Kunze, M Kurutsch, A Kuthan-Stycze, J Kwiatkowska, M Kwon-Young, C OC-38 OC-34; PC-112 PC-402 IL-5; PC-374 PC-374 PC-411 PC-263 PC-323, 360 PC-374 PC-373 PC-364 PC-411 PC-263 PC-261 PC-261 PC-322 PC-317 PC-250 OC-37 PC-227 PC-296 IL-2 IL-1 PC-171 PC-390 PC-426 PC-67 PC-22, 23 PC-90, 181, 183 PC-344 PC-324, 325 PC-107, 334 PC-65 OC-32; PC-104 PC-225, 307 PC-65 IL-2 PC-8 OC-24 PC-41 PC-80, 81 PC-200 PC-94 OC-29 OC-12 PC-93 PC-271 PC-259, 295 PC-181 PC-22 PC-426 PC-163 IL-6; PC-7, 271 PC-7 PC-399 OP-16; PC-48, 50, 69, 91, 106, 110, 133, 137, 202 PC-399 PC-24 OC-23 PC-163 PC-82 PC-137 OC-12; PC-274 PC-319 PC-338 PC-243 PC-286 PC-432 OC-22 PC-298 OC-34 IL-5 Las Heras-Vzquez, FJ Lavandera, I Lawrence, J Lazarevi Pati, T Lazos, O Le Joubioux, F Le Roes-Hill, M Leadlay, PF Leal, ICR Leal, IRC Lee, HJ Lee, JH Lee, N Lee, S-G Lee, SH Lee, SJ Leese, C Lefvre, F Leggewie, C Lehmann, C Lehmann, A Lehwald, P Leite, SGF Leitner, C Leitner, W Lejczak, B Lekhal, A Lemaire, M Li, T Li, XZ Li, Z Lie, A Lienhart, WD Lihammar, R Lillea, Lima, AS Lima-Ramos, J Litthauer, S Littlechild, J Lloyd, M Lopes, KR Lopez, JA Lpez, C Lpez-Gallego, F Lpez-Iglesias, M Lpez-Lpez, O Lpez-Santn, J Lorovere, A Lortie, R Louis, D Loureno, NMT Lozano, P Lucchese, AM uczak, J Ludkin, E Ludwig, R Lukasiewicz, N Luna, H Lupi, S Lutz, S Ly, A Lye, GJ Lyskowski, A ywa, P PC-17 PC-48, 69, 133 PC-179, 413 PC-14 OC-13 PC-124 OC-38; PC-97 OC-13 PC-320, 414, PC-177 PC-166 PC-310 PC-374 PC-260, 322 PC-411 PC-260, 322 PC-229 PC-418 PC-281 PC-266 PC-315 OC-22 PC-414 PC-83 PC-120, 429, 430 PC-183 IL-10 PC-54, 136, 209 IL-10 PC-74 OC-1; PC-201 PC-195 OP-16 PC-109, 182 PC-163 PC-357, 372 PC-37, 139 PC-301 PC-311 PC-75, 417 PC-397 PC-382 PC-51, 185 PC-47 PC-131 PC-276 PC-51, 185 PC-5 PC-388 PC-28 PC-190, 371 PC-192 PC-207, 275 OC-34 PC-333 IL-1; PC-10 PC-403 PC-115 PC-337 IL-2 PC-362 PC-59 PC-22, 288, 255 OC-34
187
I
Iannuzzi, MC Ihssen, J Iliev, I Illanes, A In Pyo, J Inoue, T Inthanavong, L Irague, R Iribarren, M Irina, S Isaschar, S Issaoui, N Isupov, M Itabaiana , JI PC-172 PC-89 PC-369 PC-188, 205, 386, 412 PC-364 PC-25 PC-102 PC-280 PC-138 PC-239 PC-273 PC-194 PC-311 PC-177, 414
Haas, T Habeych, DI Hailes, HC Hajnal, I Halin, AA Hall, M Halle, R Halling, PJ Haltrich, D Hamada, M Hammer, S Han, JY Hanefeld, U Hanson, C Hara, R Harmand, J Hartog, AF Hartog, L Hasenhrl, C Hasmann, A Hatti-Kaul, R Hauer, B Hauer, D He, N Heath, R Heberling, MM Hecquet, L Heinisch, T Helaine, V Hellriegel, C Hermann, I Hernaiz, J Hernndez-Vzquez, L Herter, S Hibi, M Hietanen, A Higashi, T Hildebrand, F Hiler, D Himi, M Hirakawa, H Hiraoka, C Hiseni, A Hoffmann, M Hofzumahaus, S Hhne, M Holanda Moura, MV Hollfelder, F Hollmann, F Holmberg, K Holtmann, D Holt-Tiffin, K Honda Malca, S Hong, S-H Hornyanszky, G Hospital, J
J
Jackson, C Jger, G Jger, K-E Jagieo, A Jakob, B Jakoblinnert, A Jane, K Janssen, DB Jasniewski, J Jendrossek, D Jennewein, S Jensen, CN Jensen, S Jimnez, M Jimenez-Barbero, J Jin, J Jochems, P Jochens, H Joglar, J Jones, E Joosten, H-J Juhl, BP Julsing, MK Jung, J Junker, SA PC-304 OC-26 PC-419, 426, 428 PC-298, 402, 403 PC-438 PC-425 OC-11; PC-43 PC-98, 219, 240, 253, 300, 331 PC-393 OP-14 IL-7 PC-76 PC-139 PC-185 PC-186, 204 PC-277 PC-15 PC-245 PC-49 PC-409 PC-342 PC-375 PC-96, 193 PC-323 PC-33
K
Kaczmarczyk, S Kaczmarek, A Kafarski, P Kagohara, E Khne, F Kalaitzakis, D Kaluzna, I Kamelb, G Kameya, M Kamon, T Kanerva, LT Kang, SY Kang, Y Kanteev, R Kapel, R Karboune, S Karmee, SK Kstner, A Kato, D Kawakami, K Kazlauskas, R Kdziora, K Kendrew, SG Kermasha, S Kesik-Brodacka, M Khabiri, M Khaliullin, I PC-112 PC-347 PC-90, 183 PC-278 PC-99 OC-8; PC-203 PC-57, 64 PC-337 PC-60 PC-140 PC-44, 46, 103 PC-263 PC-310 PC-326, 340 PC-291 PC-102 PC-168, 277 PC-396 PC-140 PC-154 PL-4 PC-48 OC-13 PC-157 PC-402 PC-339 PC-309
M
Maaheimo, H Machado, SS Macheroux, P Mackfeld, U Magomedova, Z Magrone, P Mahlala, MKA Mahnken, KF Maia, E-R Maietti, S Maigret, B Majewska, P Malik, V Malone, KJ Mangas-Snchez, J Mangold, KM Manitto, P PC-8 PC-207 OP-16; PC-57, 64, 107, 187, 334 PC-434 PC-225, 307 PC-9 PC-72 PC-422 PC-437 PC-142 PC-437 PC-183 PC-285, 333 PC-39, 167 PC-131 PC-92 OC-33; PC-126
L
Labuschagne, M Lack, I Ladkau, N Lambusta, D Langanke, J Lange, Kerstin Lange, Karsten Langone, MAP PC-72, 85 PC-6 PC-96, 221 PC-108 PC-120 PC-96 PC-438 PC-113
October 2-6, 2011
188
Mannazzu, I Manoel, EA Manrich, A Mao, J Maraite, A Marciniak-Rusek, A Marcos Nascimento, V Mariage, A Marienhagen, J Marlire, P Marquardt, W Marsaioli, AJ Martinelle, M Martinez, M Martnez, AT Martnez-Gmez, AJ Martnez-Rodrguez, S Martinez-Torres, J Martinkov, L Martins, E Marton, Z. Marty, A Marvalin, C Maseme, MJ Massou, S Mastronardi, F Mate, D Mateo, C Matijoyt, I Matsumi, R Mattiasson, B Maugard, T Maugeri, Z Maurs, M Mautner, B May, O Mazurkiewicz, A McClean, K Medici, A Mdici, R Meeuwissen, SA Meglei, G Mele, A Mellitzer, A Melone, F Mendez, JJ Mndez, C Mercalli, E Merkens, H Mes, M Meskys, R Metsala, A Metzner, J Meyer, AS Meyer, D Meza, M Michalak, M Micheletti, M Mifsud, M Mifsud Grau, M Mihovilovi, MD Mikhailopulo, IA Mikiewicz, D Mikkelsen, JD Milesi, T Milleta, R Milon, A Min, K Mink, D Miranda, LS Mitrovic, A Mitsukura, K Miyamoto, K Miyauchi, Y Mladenov, R Mohyeldin, MS Molina, P Molinari, F Molla, G Mollaahmad, A Mondragn, ME Monsan, P PC-358 PC-114 PC-351 PC-65 PC-184 PC-403 PC-351 PC-211, 223 PC-71, 220, 242, 272, 294 PC-28, 211 PC-120 PC-228, 299 OC-27 PC-359 PC-264 PC-17 PC-17 PC-130 PC-18, 19, 24, 269 PC-291 PC-197 PC-218, 303 PC-175 PC-72 PC-280 PC-172 PC-4, 248, 252, 378 PC-159, 237 PC-67 PC-20 PC-227 PC-124, 194 PC-101, 266, 423 PC-217 PC-133 PC-35, 341 PC-403 PC-409 PC-189 PC-236 PC-77 PC-362 PC-123, 349, 361 PC-119 IL-9 PC-368 PC-148 OC-39 PC-282 PC-415 PC-1, 2 PC-163 PC-431 PC-45, 162 OC-21 PC412 PC-162 PC-59 PC-49 PC-116 OC-6; PC-6, 41, 329, 332 PC-241 PC-298, 402, 403 PC-45, 162 PC-20 PC-109 PC-280 PC-384 IL-7 PC-177, 414 PC-119 PC-254 PC-295 PC-295 PC-425 PC-389 PC-4 PC-172 PC-265, 267, 327 PC-191 PC-180 PC-280 Monschein, M Monteiro, CM Monti, D Montserrat, FH Moracci, M Moreau, B Moreira, OL Morelli, CF Moreno, IS Morge, X Morin, F Mors, F Morknas, M Morrone, R Moss, S Motterle, R Mouad, AM Moulis, C Mourey, L Moussa, R Mousty, C Mller, CA Mller, Mi Mller, Mo Mundhada, H Muniglia, L Mutschler, A Mutti, FG Muzard, M PC-395 PC-190, 371 PC-9, 10, 40, 145, 270 PC-357 PC-258 PC-345 PC-382 OC-33; PC-126, 365 PC-209 PC-175 PC-102 PC-148 PC-2 PC-24, 108 OC-13 OC-5; PC-401 PC-251 PC-280 PC-303 PC-222 PC_26 PC-71, 87 OC-21, 22; PC-433 PC-35, 57, 64 PC-62, 220, 294 PC-393 PC-247 PC-91, 106, 202 OC-25
List of Authors
List of Authors
Nyhln, J Nynhongo, GS PC-312 OC-23 Perry, JJ Peruze, A Perzborn, M Pei, M Pesnot, T Peterbauer, CK Petit, JL Petkov, A Petschacher, B Pfeffer, J Pham, LTM Piamtongkam, R Pichler, H Pick, A Pietrow, O Pietruszka, J Piffaut, B Pinet, A Pisanelli, I Plantier-Royon, R Plazl, I Pleiss, J Plou, FJ Plucienniczak, A Plucienniczak, G Poelarends, GJ Pohar, A Pohl, M Pollegioni, L Poposki, J Poppe, L Pordea, A Portais, J-C Porto, ALM Porto, C Potocki-Vronese, G Poveda, A Pratter, S Pressnitz, D Prevot, V Prokop, Z Prost, JC Protesescu, L Puls, M Puthan Veetil, V Pyc, R Pyo, JI Pyo, S-H
189
PC-23, 234, 285, 333 OC-20 PC-13 PC-51 PC-134 PC-226 PC-209, 211, 223 PC-19 PC-22 PC-50, 110, 346 PC-261 PC-218 PC-57, 64 PC-215 PC-232, 233 OC-12; PC-84, 274 PC-393 PC-209 PC-267 OC-25 PC-416 OC-38; PC-314, 315, 338, 375, 433 PC-4, 186, 188, 204, 216, 385 PC-298, 402, 403 PC-298, 402, 403 PC-253, 284, 321 PC-416 OC-21; PC-117, 137, 141, 222, 422, 426, 431, 433, 434 PL-1; PC-265, 267, 327, 361, 349 PC-198 OC-10 PC-65 PC-280 PC-147, 206, 251 PC-228, 299 PC-280 PC-186, 204 OC-29 PC-202 PC_26 OC-28; PC-339 PC-65 PC-379 PC-281 PC-321 PC-347 PC-364 PC-158, 191 PC-196 PC-199 PC-253, 284 PC-357 PC-257
O
OSullivan, B Oakeshott, J Ochs, M Oda, Y O'Donohue, M Ogawa, J Ogino, H Ogolong, A Oh, D-K Oh, T-K Okolo, BN Okrob, D Okruszek, A Olejnik, S Olivarius, C Oliveira, PC Olsson, L Onaka, H Opperman, DJ Orfanopoulos, M Oroz-Guinea, I Osorio, N O'Sullivan, B Otten, LG Ottolina, G Oubrechtov, P Ouwehand, AC PC-179 PC-304 OC-25 PC-154 PC-377 OC-19; PC-296 OC-17 PC-343 PC-306 PC-317 PC-391 PC-426, 431 PC-366 PC-120 PC-175 PC-170 PC-380 PC-60 PC-301 PC-203 PC-136, 283 PC-385 PC-413 PC-161, 236 PC-5 PC-271 PC-162
N
Nabuurs, B Nady, N Nagamune, T Nagasawa, T Nagy, J Nahum, L Nakagawara, K Nakamura, M Nalin, R Nars, G Natalia, D Naudot, M Navarini, L Navarra, C Navarrete, S Navarro, A Navarro-Ocaa, A Neang, PM Neg, F Negoro, S Nestl, B Neto, ENR Neto, W Netto, CGCM Neubauer, P Neufeld, K Neunlist, S Ngo, TPN Nguyen, G-S Nicolosi, G Nicotra, S Nidetzky, B Nie, Y Nieguth, R Niitsu, Y Nikolaev, I Nikolay, P Nishimura, E Nogaro, G Nogueira, ES Noor, S Nordblad, M Nordschild, A Northen, J Nov, Y Nozac'h, H Nugroho Prasetyo, E Nuijens, T Nez, LE Nur-e-Alam, M Nwagu, TN PC-196 PC-389 PC-95 PC-254 OC-10 IL-5 OC-32 PC-100 PC-418 PC-303 PC-430 PC-291 PC-356 PC-9 PC-192 PC-180 PC-115 PC-335 PC-379 PC-140 PC-78, 88, 125, 171 PC-207 PC-160, 398 PC-100 PC-241 OC-12; PC-84 PC-105 PC-201 PC-259 PC-24, 42, 108 PC-406 PC-22, 395 PC-336 PC-144 OC-32 PC-390 PC-239 PC-140 PC-388 PC-231 PC-304 PC-349, 353, 376 OC-12 PC-333 OC-3 PC-305 OC-23 PC-196 PC-148 OC-13 PC-391
P
Pais, KC Pivi, M Palazzolo, MA Palmeira, DJ Palmer, Z Palocci, C Palomo, JM Pampn, B-J Panek, A Panke, S Panzeri, W Paradisi, F Paravidino, M Pardo, I Parini, G Park, CB Park, I-H Park, JB Park, JH Parkkinen, T Parmeggiani, F Parra, JL Parve, O Parvulescu, VI Pascholati Gusmo, LF Pasini, D Pastor, J Patel, IA Pau, A Paukner, R Paul, CE Pavkov-Keller, T Payne, MS Payongsri, P Pedersen, LH Pedotti, M Pedrini, P Pedrocchi Fantoni, G Pehk, T Pelayo, C Pellouin, V Pen, N Pennacchio, A Penttil, M Pereira, SC Perki, P Perret, A Perrier, V PC-114 PC-103 PC-138 PC-111, 122 PC-97 PC-337 PC-146, 178, 210, 237, 287 PC-276 PC-232, 233 OC-41; PC-214, 292, 400, 405 PC-123 PC-199 PC-259 PC-252, 378 PC-169 PC-166 PC-374 PC-82, 230, 373 PC-374 PC-8 PC-40 PC-186 PC-163 PC-379 PC-207 PC-365 PC-359 PC-10 PC-425 PC-83 PC-69 PC-255 PC-244 PC-436 PC-195 PC-265 PC-128, 142, 189 PC-11, 16 PC-163 PC-354 PC-223 OC-2; PC-345 PC-152, 153 PC-8 PC-143 PC-103 PC-211 PC-305, 335, 345, 385
Q
Quaedflieg, JLM Quaglia, D Quax, WJ Queiroz, MLB Quinto, T
R
Raemakers, E Raemekers-Franken, E Rahmen, N Raia, CA Rainer, F Raj, H Rajagopalan, A Rakotoarivonina, H Rale, MV Ramesh, H Ramiega, T Ramosa, TC Rancke-Madsen, A Ranoux, A Rapacioli, S Rapaport, A Razumas, R Razumiene, J Realff, MJ Rech, C Rehn, G Reis, JS Reisinger, C Reiss, R PC-35 PC-341 PC-432 PC-152, 153 PC-238 PC-253 PC-69, 91 PC-302 PC-33 PC-353 PC-347 PC-275 PC-353 PC-297 PC-406 PC-388 PC-1 PC-1 PC-310 OC-9 PC-160 PC-208 PC-119 PC-89
October 2-6, 2011
190
Reiter, J Remaud-Simon, M Remler, P Rmond, C Ren, J Renau-Ferrer, S Resch, V Resende, WCS Reymond, JL Reyo, A Rha, E Ribeiro, BD Ribeiro, IA Ribeiro, MHL Ribitsch, D Richard, P Richter, M Richtering, W Ringelov, A Ros-Lombarda, N Rioz-Martnez, A Ritthitham, R Riva, S Rivera, D Riveros, R Robins, K Roccatano, D Rocha, LC Rocha-Martn, J Roda, G Rder, J Rodrigues, J Rodrguez, C Rodrguez, R Rodriguez Talou, J Rodrguez-Alonso, MJ Rodriguez-Colinas, B Rodrguez-Hinestroza, RA Rodrguez-Mata, M Rodrguez-Vico, F Roman, A Romano, D Romero, O Ronat-Heit, E Rongjina, NL Roper, L Rosas, DO Rosche, B Rosencrantz, P Rosini, E Rossel, T Rossi, M Roth, S Rother, D Rouvinen, J Roytio, H Ra, ML Rudat, J Rudroff, FA Ruff, AJ Rhmann, B Ruiz-Dueas, FJ Russell, R Rutjes, FPJT Rychkov, GN Rzyska, M Saam, J Sabat, A Sacchetti, A Sacchetti, G Saddoud, A Sadowski, G Saini, P Saladino, R Salanoubat, M Salas, JA Salentin, S Samland, AK Sanchez, OC Snchez-Moreno, I Sanda, G Sandalova, T OC-24 PC-280 PC-213 OC-25; PC-302 PC-176 PC-302 OP-16 PC-372 PC-34 PC-115 PC-260 PC-165 PC-118 PC-118, 385 PC-363 PC-8 OC-22 PC-424 PC-19, 269 PC-131 PC-77, 133 PC-195 PC-7, 9, 10, 145, 270 PC-149 PC412 PC-245 PC-308, 318 PC-206 PC-47 PC-169 PC-344 PC-385 PC-133 PC-276 PC-249 PC-17 PC-204 PC-51 PC-131, 156 PC-17 PC-4 PC-172 PC-146 PC-437 PC-319 PC-79 PC-113 PC-74 OC-9 PC-265, 327 PC-247 PC-152, 153, 258 PC-421 PC-117, 137, 141, 433, 434 PC-8 PC-162 PC-276 PC-13, 52 PC-329 PC-220 PC-215 PC-264 PC-304 PC-53, 196 PC-319 PC-348, 394 PC-327 PC-280 PC-40 PC-142 PC-388 PC-410 PC-243 IL-9 PC-211 PC-148 PC-219 PC-25 PC-207 PC-54, 136, 283 PC-379 PC-25 Sandkuhl, D Sandoval, G Sandoval, M Sandstrm, AG Sandulescu, M Sanfilippo, C Sang, B-I Santacoloma, PA Santos, OAA Saravanan, T Sardo, A Sareen, D Sari, T Sattler, JH Satyawali, Y Saurel, O Sayago, FJ Sayer, C Schaffer, S Schallmey, A Schallmey, M Schatz, J Schtzle, S Scheffers, M Schenkmayerov, A Scheps, D Schera, J Schieder, D Schiffer, D Schilling, A Schirmer, T Schmid, A Schmid, H Schmid, J Schmidt, T Schneider, G Schneider, K Schneider, MP Schnellbcher, M Schnuerch, M Schober, M Schoenenberger, B Scholz, A Schlzel, M Schrader, J Schrewe, M Schrittwiser, JH Schron, K Schroer, K Schrmann, Martin Schrmann, Melanie Schwab, H Schwamberger, O Schwaneberg, U Schwarz, A Schwendenwein, D Scoffone, V Scott, C Scwarz, M Secundo, F Sehl, T Seifert, A Seitz, M Seki, T Seleghim, HR Sendovski, M Serra, CD Serra, I Servi, S Sette, LD Shabalin, KA Shafee, T Shafioul, ASM Shainsky, J Shalaeva, D Sheldon, RA Shimizu, S Shivange, AV Shleev, S Shoji, M Shuster, V Shutov, AA
List of Authors
OC-12 PC-354 PC-192 PC-312 PC-379 PC-42 PC-384 OC-11 PC-372 PC-155 PC-65 PC-243 PC-388 OP-16; PC-50, 110, 202 PC-15 PC-280 PC-156 PC-311 PC-346 PC-98, 219 PC-362 PC-70 PC-245 PC-390 PC-6 PC-78, 88 PC-393 OC-24; PC-396 PC-387 PC-120 PC-65 PC-82, 96, 193, 221, 410 PC-61 PC-215 PC-212 PC-25 PC-387 PC-438 PC-31 PC-41 PC-91, 107, 334 PC-20 PC-66 OC-12 PC-92, 99, 293 PC-96, 193 OP-16 PC-389 IL-2 IL-7; PC-35 PC-25 PC-57, 64, 213, 225, 255, 256, 288, 307, 363 PC-255 PC-62, 71, 87, 92, 120, 220, 242, 266, 272, 293, 294, 308, 318, 419, 425, 428 PC-395 PC-225, 307 PC-365 PC-304 PC-422 PC-169 PC-141 PC-314 PC-171 OC-32 PC-147 PC-340 OC-33 OC-33 PC-11, 16, 123, 267 PC-299 PC-319 OC-18 PC-364 PC-273 OC-37; PC-309, 316 PC-200, 234 PL-2; OC-19; PC-296 PC-87, 272, 293, 294, 308 PC-248 OC-32; PC-104, 164, PC-326, 340 PC-80, 81
List of Authors
Sibilla, F Sieber, V Siedenburg, G Sierra, L Sign, C Siirola, E Silva, CSP Silva, GS Simas, ABC Simmonds, S Simon, R imek, Sinb, G Sinigoi, L Sinisterra, JV Siri-anusornsak, W Sirim, D Sjstrand, Ulf Slmov, K Smit, MS Smonou, I Soares, CMF Soetaert, W Sokolowska, I Soler, L Solingen, P Song, BK Song, J-W Sorel, I Sorgedrager, M Soriano-Maldonado, P Sousa, JS Souza, FP Souza, RL Souza, ROMA Souza Santos, R Spera, A Speranza, G Spiess, AC Spizzo, P Sprenger, GA Srivastava, P Stanczyk, L Staudt, S Steadman, D Stecher, H Steffen-Munsberg, F Steiner, K Steinkellner, G Stepankova, V Stepczynska, M St-Louis, R Stloukal, R Stolz, A Straathof, AJJ Straganz, G Stratmann, A Strazzulli, A Streit, WR Strittmatter, H Strhle, F Struszczyk, K Stueckler, C Subileau, M Sudar, M Sugai, T Sukhodolskaya, GV Sulzenbacher, G Summers, BD Sundell, R Suplatov, D Sutili, FK Suzuki, M vedas, V Svedendahl Humble, M Svendsen, A Sygmund, C Syldatk, C Synowiecki, J Syrnb, P-O Szczesna-Antczak, M Szeker, K Szekrnyi, A PC-238, 266, 425 OC-24; PC-73, 215, 396 OP-14 PC-415 PC-123, 349 PC-106 PC-355 PC-170 PC-114 PC-417 PC-137 PC-174 OC-11 OC-40; PC-58 PC-184, 192 PC-392 OC-38 PC-160 PC-7 PC-72, 85 OC-8; PC-203 PC-357, 372 PC-330 PC-403 PC-386 PC-390 PC-261 PC-230 PC-28 PC-200 PC-17 PC-383 PC-397 PC-372 PC-177, 320, 414 PC-165 PC-18 OC-33; PC-126, 365 OC-26; PC-212, 343, 420, 421, 422, 424, 427 OC-40; PC-356 OC-22; PC-25, 435 PC-63 PC-394 PC-71, 87 PC-436 OC-31; PC-213 PC-245, 259 PC-255, 256, 288 PC-22, 255, 288, 363, PC-339 PC-347 PC-157 PC-407 PC-21 PC-236, 277 OC-29 OC-39 PC-258 PC-266, 362 OC-24 PC-13 PC-394 PC-150 PC-305, 335 PC-36 OC-32; PC-104, 164, PC-80 PC-258 PC-75 PC-44 OC-37; PC-309, 316 PC-177, 414 OC-32 OC-37; PC-173, 239, 309, 316, PC-12 PC-316 IL-1 PC-13, 52 PC-232, 233 PC-182 PC-347, 348, 394 PC-241 PC-32, 33, 49 Szita, N Szymanski, WIquam dolupti PC-179, 413 PC-253 PC-154 PC-140 PC-164 OC-27; PC-328 PC-224 PC-172 PC-349, 361 PC-163 PC-280 PC-129, 151 PC-50, 110 PC-2 PC-130 OC-31; PC-213 OC-33 PC-207 PC-11, 16, 123, 349, 361 PC-315 PC-219 PC-382 PC-85 PC-162 PC-259 PC-245 PC-89 PC-370, 380 PC-63 PC-321 PC-102 OC-21 PC-309 PC-100 PC-380 PC-300 PC-383 PC-237 PC-368 PC-4, 186, 216, 385 PC-390 PC_26 PC-435 PC-303 PC-35 PC-211 PC-343 PC-198 PC-363 OC-11; PC-37, 43, 127, 139, 398 PC-174 PC-406 PC-44 PC-35 PC-39, 167 PC-358
191
T
Takahashi, R Takeo, M Taketomi, S Takwa, M Tamar, AA Tamborini, L Tamborini, S Tamp, S Tarquis, L Tasndi, G Tauber, K Taurait, D Taylor, I Tengg, M Terreni, M Teshima, E Tessaro, D Tessaro, P Teune, IPG Texeir, MMP Theron, CW Thomassen, LV Thompson, ML Thontowi, A Thny-Meyer, L Thrn, C Thulasiram, HV Thunnissen, A-M Tian, F Tittmann, K Tokunan, K Toma, HF Topakas, E Toplak, A Torres, AG Torres, FAG Torres, M Torres, P Torres Pazmino, D Touisni, N Trachtmann, N Tranier, S Trefzer, A Tricot, S Trieu, T Tronina, T Trotscha, E Tufvesson, P Tural, B Turchetti, B Turcu, MC Turk, S Turner, NJ Turon, X
U
Ubiali, D Uemura, D Ueno, M Ulrich, C Um, Y-H Umezawa, K Urge, L Urlacher, VB Urrutia, P Ushakov, GA OC-33; PC-365 PC-295 PC-154 PC-238 PC-384 OC-32 OC-10 PC-93 PC-205 PC-173
V
Vahan Kilikian, B Vaidya, B Valentini, SR Vallin, M van Beek, HL van de Horst, MA van der Oost, J van Heerden, E van Hemert, L van Hest, JCM PC-381 PC-159 PC-397 PC-182 PC-279 PC-86 PC-20 PC-301 PC-53 PC-77
October 2-6, 2011
192
van Oosten, R van Pelt, S van Rantwijk, F van Rooyen, N Van Roy, S Vasi, V Vasi-Raki, D Vasileva, T Veetil, VP Veloso, CO Venturi, V Vera, C Verga, R Vergne-Vaxelaire, C Verma, R Vermote, L Vesel, AB Veteikyt, A Viani, F Viell, J Vikartovsk, A Villegas-Torres, MF Villo, L Vioget, S Virunanon, C Volont, F von Lieres, E Vowell, BJ Vroemen, C Vrsalovi Preseki, A PC-168 PC-234 PC-21, 234 PC-72 PC-15 PC-14 PC-36 PC-369 PC-253 PC-113 PC-142, 189 PC-188 PC-406 PC-223 PC-318 PC-341 PC-19, 269 PC-67 PC-11, 16, 267 PC-343 PC-6 PC-246 PC-163 PC-362 PC-392 PC-267 PC-117, 434 PC-310 PC-390 PC-36 Wu, Q Wuensch, C Whrer, P Wulfhorst, H PC-176 PC-135 PC-83, 226 OC-26; PC-343
List of Authors
List of Partecipants
ABE, Ikuro ADLERCREUTZ, Patrick AL-BAHRANI, Homam The University of Tokyo Tokyo, JAPAN Lund University Lund, SWEDEN University College London London, UNITED KINGDOM University of the Free State Bloemfontein, SOUTH AFRICA Institute of Catalysis, CSIC, Madrid, SPAIN Complutense University Madrid, SPAIN University College Dublin Dublin, IRELAND Technical University of Denmark Kgs. Lyngby, DENMARK ENSAIA-INPL Vanduevre les nancy, FRANCE Universitat Autonoma De Barcelona Bellaterra, SPAIN University College London London, UNITED KINGDOM VTT Espoo, FINLAND University of Freiburg Pharmaceutical and Medicinal Chemistry Freiburg, GERMANY Engineering School of Lorena University of So Paulo, Brazil Lorena, BRAZIL University of So Paulo SAO PAULO, BRAZIL KIT - Institute of Process Engineering in Life Sciences, Section II: Technical Biology Karlsruhe, GERMANY Codexis Laboratories Singapore Pte Ltd Singapore, SINGAPORE Technical University of Lodz Lodz, POLAND Norwegian University of Science and Technology Trondheim, NORWAY ISM2-Universit Paul Cezanne Facult des Sciences et Techniques de St Jrome Marseille, FRANCE Delft University of Technology Delft, NETHERLANDS NCSU Raleigh, USA Toyama Prefectural University Imizu, JAPAN UMR 8601 University Paris Descartes Paris, FRANCE Amsterdam University Amsterdam, NETHERLANDS University College London St.Albans, UNITED KINGDOM CSIC Instituto de Catalisis y Petroleoquimica Madrid, SPAIN Forschungszentrum Jlich Gmbh Jlich, GERMANY University of Groningen Groningen, NETHERLANDS University of Nottingham NottiNgham, UK ETH Zurich, Department of Biosystems Science Basel, SWITZERLAND Aachener Verfahrenstechnik Enzyme Process Technology RWTH Aachen University, BioNoCO Aachen, GERMANY RWTH Aachen University, CMT Aachen, GERMANY abei@mol.f.u-tokyo.ac.jp Patrick.Adlercreutz@biotek.lu.se albahrani98@gmail.com Albertynj@ufs.ac.za malcalde@icp.csic.es andresr@farm.ucm.es asma.alhaj@ucd.ie nah@kt.dtu.dk ah-jawish@hotmail.com gregorio.alvaro@uab.cat a.halim@ucl.ac.uk martina.andberg@vtt.fi jennifer.andexer@pharmazie.uni-freiburg.de
193
X
Xiao, R Xie, J Xochitl, N Xu, G Xu, Yan Xu, Yuan Xua, Y Xue, R PC-336 PC-262 PC-354 PC-56 PC-262, 336 PC-350, 376 PC-353 PC-45
ALBERTYN, Jacobus ALCALDE, Miguel ALCANTARA, Andres R ALHAJ TAMAR, Asma AL-HAQUE, Naweed ALJAWISH, Abdulhadi ALVARO, Gregorio AMANATUZZAKIAH, Abdul Halim ANDBERG, Martina ANDEXER, Jennifer
Y
Yalnkl, Yamashita, Y Yamazaki, M Yang, K-M Yang, L Yara, E Yi, D Yokozeki, K Yoshid, S Yoshida, T Yu, B Yu, X Yun, JW Yun, JY PC-174 OC-32 PC-290 PC-373 PC-56 PC-368 PC-27, 28, 30 OC-19; PC-296 PC-391 PC-254 PC-176 PC-262 PC-230 PC-82
W
Wachala, R Wagner, H Wagner, N Wagner, U Wallner, S Wang, C Wang, L-L Wang, R Wang, W Wani, SI Ward, D Ward, JM Ward, TR Watanabe, H Wawrzeczyk, C Wechsler, C Wehrschuetz-Sigl, E Weinhandl, K Weis, R Weissenbach, J Westphal, R Wetzl, D Wever, R Weyrauch, P Widmann, M Wiechert, W Wiemann, LO Wiese, S Wikmark, Y Wilding, B Wilhelm, S Wilkinson, B Willetts, A Willies, S Wilson, L Wilson, Y Winkler, CK Winkler, M Winkler, T Wittrup Larsen, M Wohlgemuth, R Wjtowicz-Krawiec, A Wolberg, M Won, K Wong, G Woodley, JM Wriessnegger, T Wu, B Wu, J PC-347 PC-78 OC-41; PC-214, 400 PC-107, 334 OP-16 PC-362 PC-262 PC-262 PC-201 PC-243 PC-390 PC-59, 130, 134, 141, 246, 311, PC-65, 231, 247, 257 PC-95 PC-132 OC-21 PC-387 PC-235 PC-119 PC-211 PC-433 PC-125 PC-38, 53, 86 PC-19 PC-433 PC-117, 137, 141, 222, 431, 434 OC-24; PC-73 PC-424 PC-182, 312 PC-22, 23 PC-419, 428 OC-13 PC-76 PC-39 PC-205 PC-65, 231, 247 PC-129, 150, 151, 255 PC-22, 23 PC-87 OC-27 PC-20, 179, 413 PC-298, 402, 403 IL-7 PC-166, 411 IL-10 OC-11; PC-37, 43, 45, 127, 139, 350, 353, 376, 398, 399 PC-57, 64 PC-240, 331 PC-56
ANDRADE, Grazielle Santos Silva
grazielle@debiq.eel.usp.br
Z
Zaks, A Zampieri, D Zamudio, N Zandvoort, E Zanelli, CF Zangger, K Zanghellini, A Zanin, GM Zaparucha, A Zaushitsyna, O Zeder-Lutz, G Zeithammel, E Zengin eki, S Zhang, M Zhang, W Zhou, X Zhu, D Zhu, L Zielinski, M Zimmermann, A Zimmermann, WR nidari-Plazl, P Zolandz, RR Zorzetti, C Zuilhof, H Zumarraga, M ymaczyk-Duda, E IL-10 PC-299 PC-386 PC-284 PC-397 PC-187 OC-35 PC-352, 372 PC-211, 223 PC-227 PC-302 PC-62 PC-92 PC-234, 285 PC-201 PC-241 PC-176 PC-419 PC-402 OC-13 PC-363 PC-416 PC-244 PC-258 PC-389 PC-4 PC-90, 181, 183
ANDRADE, Leandro ANDRE, Markus
LEANDROH@IQ.USP.BR markus.andre@kit.edu
ANG, Ee Lui ANTCZAK, Tadeusz ANTHONSEN, Thorleif ARCHELAS, Alain
eelui.ang@codexis.com tadeusz.antczak@p.lodz.pl Thorleif.Anthonsen@chem.ntnu.no a.archelas@univ-cezanne.fr
ARENDS, Isabel ARGYROPOULOS, Dimitris ASANO, Yasuhisa AZERAD, Robert
i.w.c.e.arends@tudelft.nl dsargyro@ncsu.edu asano@pu-toyama.ac.jp robert.azerad@parisdescartes.fr
BABICH, Lara BABOO, Jasmin BALLESTEROS, Antonio O. BARAIBAR, lvaro Gmez BARTSCH, Sebastian BATTAGLIA, Ugo BECHTOLD, Matthias BEGEMANN, Jens
l.babich@uva.nl j.baboo@ucl.ac.uk a.ballesteros@icp.csic.es a.baraibar@fz-juelich.de s.bartsch@rug.nl pcxub1@nottingham.ac.uk administration@bsse.ethz.ch jens.begemann@avt.rwth-aachen.de
BEHRENDT, Dominik
behrendt@cmt.rwth-aachen.de
October 2-6, 2011
194
BEL-RHLID, Rachid BELSARE, Ketaki Nestl Lausanne, SWITZERLAND Lehrstuhl fr Biotechnologie RWTH Aachen University Aachen, GERMANY University of Padova Padova, ITALY ITB - Institute of Technical Biochemistry University Stuttgart Stuttgart, GERMANY Royal Institute of Technology Stockholm, SWEDEN Universidad de Antioquia Medellin, COLOMBIA Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche Milano, ITALY Biocon Limited Bangalore, INDIA LIBIO INPL Vanduvre-ls-Nancy, FRANCE ICB- Consiglio Nazionale delle Ricerche Catania, ITALY Northumbria University Newcastle upon Tyne, UNITED KINGDOM Wageningen UR Food & Biobased Research Wageningen, NETHERLANDS Georgia Institute of Technology Atlanta, USA RWTH Aachen Enzyme Process Technology Aachen, GERMANY University of Erlangen-Nrnberg Erlangen, GERMANY Institut Charles Gerhardt Montpellier Montpellier, FRANCE INSA - LISBP Toulouse, FRANCE CEA-Genoscope Evry, FRANCE Greifswald University Institute of Biochemistry Greifswald, GERMANY ETH Zurich D-BSSE Basel, SWITZERLAND Universite De Haute-Alsace/ENSCMU Mulhouse, FRANCE University College London London, UNITED KINGDOM RWTH Aachen University Aachen, GERMANY Johnson Matthey Catalysts Dusseldorf, GERMANY Universidade Federal do Rio de Janeiro Departamento de Bioqumica Rio de Janeiro, BRAZIL Sanofi-Aventis Chilly-Mazarin, FRANCE BASF SE Fine Chemicals and Biocatalysis Researchv Ludwigshafen, GERMANY University of La Rochelle La Rochelle, FRANCE L'Oral Aulnay sous Bois,FRANCE Wroclaw University of Technology Wroclaw, POLAND DECHEMA e.V., Karl-Winnacker-Institut Biochemical Engineering Frankfurt/Main, GERMANY Institute of Chemistry Slovak Academy of Sciences Bratislava, SLOVAKIA Sanofi-Aventis Deutschland GmbH Frankfurt am Main, GERMANY
rachid.bel-rhlid@rdls.nestle.com k.belsare@biotec.rwth-aachen.de
BHLER, Bruno BULEGON BRONDANI, Patrcia BURTON, Stephanie CABRERA, Zaida CAIMI , Paolo TU Dortmund University Dortmund, GERMANY University of So Paulo So Paulo,BRAZIL University of Pretoria Pretoria, SOUTH AFRICA Pontificia Universidad Catolica de Valparaiso Valparaiso, CHILE Resindion Srl Binasco, ITALY Centro de Investigaciones Biolgicas, CSIC Madrid, SPAIN Technische Universitt Graz Graz University of technology Graz, AUSTRIA University of L'Aquila L'Aquila, ITALY Ctedra de Microbiologa Industrial y Biotecnologa. Facultad de Farmacia y Bioquimica. Universidad Buenos Aires, ARGENTINA Resindion Srl Binasco, ITALY Dipartimento CMIC Giulio Natta Politecnico di Milano Milano,ITALY University of Barcelona. Department of Microbiology Barcelona, SPAIN Libio INPL Vandoeuvre les nancy, FRANCE Ghent University Gent, BELGIUM Korea Institute of Science and Technology Seoul, SOUTH KOREA Seoul national university Seoul, SOUTH KOREA Institute Biotechnology and Antibiotics Warsaw, POLAND Universit degli Studi di Roma "La Sapienza" Rome, ITALY Department of Botany Faculty of Science Chulalongkorn University Pathumwan, THAILAND Delft University of Technology Delft, NETHERLANDS Instituto de Qumica Avanzada de Catalunya CSIC Barcelona, SPAIN TU Dortmund Faculty of Biochemical and Chemical Engineering Chair of Biotechnology Dortmund, GERMANY GlaxoSmithKline Ulverston, UNITED KINGDOM Technische Universitaet Darmstadt Darmstadt, GERMANY Universit di Ferrara Ferrara, ITALY Universit Picardie Jules Verne IUT Amiens - LPMV Amiens, FRANCE Universit Picardie Jules Verne IUT Amiens - LPMV Amiens, FRANCE Tor Vergata University Dipartimento di Scienze e Tecnologie Chimiche Rome, ITALY Engineering School of Lorena University of So Paulo, Brazil Lorena, BRAZIL University of So Paulo So Paulo, BRAZIL Toyama Prefectural University (TPU) Imizushi, JAPAN bruno.buehler@bci.tu-dortmund.de patyqmc@gmail.com stephanie.burton@up.ac.za zaida.cabrera@ucv.cl s.bonecchi@resindion.com susanacam@cib.csic.es
195
BENEVENTI, Elisa BENSON, Sven
elisa.beneventi@gmail.com sven.benson@itb.uni-stuttgart.de
BERGLUND, Per BERNAL ZULUAGA, Claudia patricia BERTOLESI, Giulia
perbe@kth.se claberz@gmail.com giuliberto@gmail.com
CAMARERO, Susana
CAMATTARI, Andrea
andrea.camattari@tugraz.at
BHAMIDIPATI, Satya vara prasad BIDOUIL, Christelle BIONDI, Daniela BLACK, Gary BOERIU, Carmen BOMMARIUS, Andreas BONHAGE, Benjamin
prasad.bsv@biocon.com christelle.bidouil@ensaia.inpl-nancy.fr daniela.biondi@icb.cnr.it gary.black@northumbria.ac.uk carmen.boeriu@wur.nl andreas.bommarius@chbe.gatech.edu benjamin.bonhage@avt.rwth-aachen.de
CANTARELLA, Maria CARDILLO, Alejandra Beatriz
maria.cantarella@univaq.it acardillo@ffyb.uba.ar
CARMINATI, Alessandra CERIOLI, Lorenzo
s.bonecchi@resindion.com lorenzo.cerioli@yahoo.it
CESARINI, Silvia
silviacesarini@ub.edu
CHEBIL, Latifa CHEN, Chao
latifa.chebil@ensaia.inpl-nancy.fr Chao.Chen@UGent.be c2496@kist.re.kr yun31707@snu.ac.kr chojnackal@iba.waw.pl laura.chronopoulou@uniroma1.it warawut.c@chula.ac.th
BORCHERT, Sonja BORDEAUX, Melanie BORDES, Florence BORDIER, Franck BORNSCHEUER, Uwe
sonja.borchert@chemie.uni-erlangen.de melanie.bordeaux@enscm.fr florence.bordes@insa-toulouse.fr fbordier@genoscope.cns.fr uwe.bornscheuer@uni-greifswald.de
CHEONG, Chan Seong CHOI, Yun-hee CHOJNACKA-PUCHTA, Luiza CHRONOPOULOU, Laura CHULALAKSANANUKUL, Warawut
BOSSHART, Andreas BOULANGER, Anna BOUR, Alex BRACCO, Paula BRAIUCA, Paolo BRANCO, Roberta Vieira
andreas.bosshart@bsse.ethz.ch anna.boulanger@uha.fr uccaadb@live.ucl.ac.uk p.bracco@biotec.rwth-aachen.de Paolo.Braiuca@matthey.com robertavbranco@yahoo.com.br
CHURAKOVA, Ekaterina CLAPS, Pere
e.churakova@tudelft.nl pere.clapes@iqac.csic.es
COLLINS, Jonathan
jonathan.collins@bci.tu-dortmund.de
COLLIS, Andrew J. CORDERO DI MONTEZEMOLO, Benedetta COSTA, Stefania COURTOIS, Bernard
andrew.j.collis@gsk.com benedetta.montezemolo@icrm.cnr.it stefania.costa@unife.it bernard.courtois@u-picardie.fr
BRASSEUR, Denis
denis.brasseur@sanofi-aventis.com michael.breuer@basf.com
BREUER, Michael BRIDIAU, Nicolas BROSSAT, Maude BRZEZINSKA-RODAK, Malgorzata
nicolas.bridiau@univ-lr.fr mbrossat@rd.loreal.com malgorzata.brzezinska-rodak@pwr.wroc.pl buchhaupt@dechema.de
COURTOIS, Josiane
josiane.courtois@u-picardie.fr
CRESTINI, Claudia
crestini@uniroma2.it
DA RS, Patrcia Caroline Molgero
patriciadaros@dequi.eel.usp.br
BUCHHAUPT, Markus BUCKO, Marek
Marek.Bucko@savba.sk
DA SILVA ARAJO, Lidiane
lidianepiaui@yahoo.com.br
BUDDE, Michael
michael.budde@sanofi-aventis.com
DADASHIPOUR, Mohammad
mdadashi@gmail.com
October 2-6, 2011
196
DAMINATI, Moreno Resindion Srl Binasco, ITALY Univerdade Estadual Paulista "Julio de Mesquita Filho" UNESP. Araraquara, BRAZIL CNR National Research Council of Italy Institute of Biomolecular Chemistry Catania, ITALY National Chemical Laboratory Pune, INDIA Dipartimento CMIC Giulio Natta Politecnico di Milano Milano,ITALY Radboud University Nijmegen Nijmegen, NETHERLANDS CEA-genoscope Evry, FRANCE University of Groningen Groningen, NETHERLANDS University of Groningen Groningen, NETHERLANDS VITO NV Mol, BELGIUM Middle East Technical University Department of Chemistry Ankara, TURKEY RWTH Aachen University Instute of Biotechnology Aachen, GERMANY INSA - LISBP Toulouse, FRANCE Technische Universitt Darmstadt Darmstadt, GERMANY FEDERAL UNIVERSITY OF RIO DE JANEIRO Rio de Janeiro, BRAZIL University of Barcelona. Spain Barcelona, SPAIN DuPont Wilmington, USA RWTH Aachen University Aachen, GERMANY Institute of Biochemistry & Physiology of Microorganisms, Russian Academy of Sciences Pushchino, RUSSIA Rensselaer Polytechnic Institute Troy, USA Institut Charles Gerhardt Montpellier UMR 5253 CNRS/ENSCM/UM2/UM1 Montpellier, FRANCE Montpellier SupAgro Montpellier Cedex 1, FRANCE University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute Groningen, NETHERLANDS Resindion Srl Binasco, ITALY INSA - LISBP Toulouse, FRANCE University of Graz Graz, AUSTRIA University of Basel Basel, SWITZERLAND Masaryk University, Faculty of Science Department of Experimental Biology Brno,CZECH REPUBLIC Technical Univrsity of Lodz Faculty of Biotechnology and Food Sciences Lodz, POLAND Inst. for Biotechnology and Helmholtz-Institute for Biomedical Engineering RWTH Aachen University Aachen,GERMANY ACIB GmbH Graz, AUSTRIA LIBIO INPL Vanduvre-ls-Nancy, FRANCE
s.bonecchi@resindion.com
ENGSTRM, Karin Stockholm University Stockholm, SWEDEN University of Lleida Lleida, SPAIN Universidad Nacional Autonoma De Mexico Mexico, MEXICO Wageningen University Department of Microbiology Wageningen, NETHERLANDS TU Dortmund, Chemical and biochemical engineering Dept., Chair of Biotechnology Dortmund, GERMANY Universit degli Studi di Trieste Trieste, ITALY ETH Zurich D-BSSE Basel, SWITZERLAND Vienna University Of Technology Institute Of Applied And Synthetic Chemistry Vienna, AUSTRIA Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche Milan, ITALY Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt Darmstadt, GERMANY Vienna University of Technology Vienna, AUSTRIA Technion - Israel Institute of Technology Haifa, ISRAEL University of Padova Padova, ITALY Sekisui Diagnostics (UK) Ltd West Malling, UNITED KINGDOM University of Trieste Trieste,ITALY University of Groningen Groningen, NETHERLANDS York Structural Biology Laboratory University of York, Department of Chemistry York, UNITED KINGDOM Wageningen University, Lab. of Organic Chemistry Wageningen, NETHERLANDS RWTH Aachen Aachen, GERMANY Israel state employee Rehovot, ISRAEL Dr Reddy's Laboratories Cambridge, UNITED KINGDOM University of Graz Graz, AUSTRIA RWTH Aachen / BioNoCo Aachen, GERMANY Lund University, Biotechnology Department. Lund, SWEDEN Prodotti Chimici E Alimentari Spa Basaluzzo, ITALY Istituto di Chimica Biomolecolare Consiglio Nazionale delle Ricerche Catania, ITALY Institute of Catalysis and Petroleochemistry CSIC Madrid, SPAIN Instituto de Qumica Orgnica General. CSIC Madrid, SPAIN University of Trieste Trieste, ITALY Delft University of Technology Delft,NETHERLANDS Sanofi-Aventis Warren,USA Dept. of Biochemistry University of Cambridge Cambridge,UNITED KINGDOM karin@organ.su.se
197
DANIELI, Flavia
flavia.danieli@gmail.com
ERAS, Jordi ESQUIVEL, Ricardo FALCICCHIO, Pierpaolo
eras@quimica.udl.cat farres@unam.mx pierpaolo.falcicchio@wur.nl
D'ANTONA, Nicola
nicola.dantona@icb.cnr.it
DARAMWAR, Pankaj D'ARRIGO, Paola
pp.daramwar@ncl.res.in paola.darrigo@chem.polimi.it FALCIONI, Francesco
francesco.falcioni@bci.tu-dortmund.de
DE BEER, Roseri DE BERARDINIS, Veronique DE VILLIERS, Marianne DE VILLIERS, Jandre DEJONGHE, Winnie DEMIR, Ayhan s.
r.debeer@science.ru.nl vberard@genoscope.cns.fr m.de.villiers@rug.nl j.de.villiers@rug.nl WINNIE.DEJONGHE@VITO.BE asdemir@metu.edu.tr
FATTOR, Diana FEMMER, Christian FEROZ, Saima
diana.fattor@phd.units.it christian.femmer@bsse.ethz.ch saima.feroz@ias.tuwien.ac.at
FERRANDI, Erica elisa
erica.ferrandi@icrm.cnr.it
FESSNER, Wolf-dieter
fessner@tu-darmstadt.de
FINK, Michael a.dennig@biotec.rwth-aachen.de FISHMAN, Ayelet FOGAL, Stefano FOLEY, Adrian FOSCATO, Marco FRAAIJE, Marco FRANK, Annika
mfink@ioc.tuwien.ac.at afishman@tx.technion.ac.il stefano.fogal@unipd.it adrian.foley@sekisuidiagnostics.com marco.foscato@phd.units.it m.w.fraaije@rug.nl annika@ysbl.york.ac.uk maurice.franssen@wur.nl
DENNIG, Alexander
DESROUSSEAUX, Marie-laure DEVAMANI, Titu DIAS RIBEIRO, Bernardo DIAZ, Pilar DI COSIMO, Robert DOMNGUEZ DE MARA, Pablo DONOVA, Marina
marie-laure.desrousseaux@insa-toulouse.fr tituiitm@gmail.com dias.bernardo@gmail.com pdiaz@ub.edu Robert.DiCosimo@usa.dupont.com dominguez@itmc.rwth-aachen.de donova-marina@rambler.ru
FRANSSEN, Maurice FRAUENKRON-MACHEDJOU, Josiane FRIDKIN, Gil FRYSZKOWSKA, Anna FUCHS, Michael FULTON, Alexander GABER HASAN, Yasser
j.frauenkron-machedjou@biotec.rwth-aachen.de tgfridkin@yahoo.com afryszkowska@drreddys.com mihi_fuchs@yahoo.de a.fulton@fz-juelich.de Yasser.gaber@biotek.lu.se administration@pcaitaly.it giovanni.gambera@icb.cnr.it
DORDICK, Jonathan DRONE, Jullien
dordick@rpi.edu jullien.drone@enscm.fr
DUBREUCQ, Eric DUDEK, Hanna
Eric.Dubreucq@supagro.inra.fr h.dudek@rug.nl
DUGONI, Chiara DUQUESNE, Sophie DURCHSCHEIN, Katharina DRRENBERGER, Marc DVORAK, Pavel
s.bonecchi@resindion.com sophie.duquesne@insa-toulouse.fr durchsck@stud.uni-graz.at marc.duerrenberger@unibas.ch dvorak.paul@seznam.cz
GALDI, Gianluca GAMBERA, Giovanni
GARCIA, Eva
evagarcia@icp.csic.es
GARCA-JUNCEDA, Eduardo
eduardo.junceda@iqog.csic.es
DZIWORSKA, Gabriela
gabriela.dziworska@p.lodz.pl
GARDOSSI, Lucia GARGIULO, Serena GARYANTES, Tina GATTI-LAFRANCONI, Pietro
gardossi@units.it s.gargiulo@tudelft.nl tina.garyantes@sanofi-aventis.com pg356@cam.ac.uk
ELLING, Lothar EMMERSTORFER, Anita ENGASSER, Jean-Marc
l.elling@biotec.rwth-aachen.de emmersto@sbox.tugraz.at jean-marc.engasser@ensaia.inpl-nancy.fr
October 2-6, 2011
198
GAVEZZOTTI, Paolo Istituto di Chimica del Riconoscimento Molecolare Centro Nazionale delle Ricerche Milano,ITALY Forschungszentrum Jlich Institute of bio- and geosciences (IBG-1) Jlich,GERMANY LIBIO INPL Vanduvre-ls-Nancy,FRANCE W42 Consulting GmbH Penzberg,GERMANY Federal University of Sao Carlos Department of Chemical Engineering (DEQ/UFSCar) Sao Carlos,BRAZIL Federal University of Sao Carlos Department of Chemical Engineering (DEQ/UFSCar) Sao Carlos,BRAZIL Universit di Ferrara Dipartimento di Chimica Ferrara,ITALY Ctedra de Microbiologa Industrial y Biotecnologa. Facultad de Farmacia y Bioquimica Buenos Aires,ARGENTINA Wroclaw University of Environmental and Life Sciences Wroclaw,POLAND austrian centre of industrial biotechnology Graz,AUSTRIA Roche Diagnostics GmbH Mannheim,GERMANY Technion-Israel Institute of Technology Haifa,ISRAEL Chemistry Institute UNICAMP Campinas,BRAZIL Federal University of Rio de Janeiro Chemistry Institute Rio de Janeiro,BRAZIL Entrechem S.L. Oviedo,SPAIN Oviedo University Oviedo,SPAIN Oviedo University Oviedo,SPAIN University of La Rochelle, UMR 6250 CNRS La Rochelle, FRANCE RWTH Aachen University Aachen,GERMANY DECHEMA e. V. Karl-Winnacker-Institut Frankfurt am Main,GERMANY Institute of Organic Chemistry Graz University of Technology Graz,AUSTRIA University of Groningen Groningen,NETHERLANDS University of York York,UNITED KINGDOM Bielefeld University Faculty of Chemistry Bielefeld,GERMANY ACIB/University of Graz Graz,AUSTRIA ACIB GmbH Graz,AUSTRIA Universidade de So Paulo Itapevi,BRAZIL Adisseo France SAS Toulouse,FRANCE Technische Universitt Graz Graz,AUSTRIA Universidade Federal do Rio de Janeiro Rio de Janeiro,BRAZIL Belozersky Institute of Physicochemical Biology Moscow,RUSSIA Chalmers University of Technology Gothenburg, SWEDEN paologave@yahoo.it
GUTARRA, Melissa HADI, Masood Federal University of Rio de Janeiro Rio de Janeiro, BRAZIL Sandia National Labs Joint BioEnergy Institute Livermore, USA Department of Chemistry University College London London, UNITED KINGDOM ACIB GmbH Graz, AUSTRIA Department of Chemistry University of Graz Graz, AUSTRIA WestCHEM, Dept P & A Chemistry University of Strathclyde Edinburgh, UNITED KINGDOM BOKU University of Natural Resources and Life Sciences Food Biotechnology Laboratory Vienna, AUSTRIA Institute of Technical Biochemistry Stuttgart, GERMANY The Catholic University of Korea Bucheon-si, SOUTH KOREA Technische Unversiteit Delft Delft, NETHERLANDS F. Hoffmann-La Roche Ltd Basel, SWITZERLAND Waseda University Shinjuku-ku, JAPAN INRA Reims, FRANCE University of Amsterdam Amsterdam, NETHERLANDS Lund University Lund, SWEDEN TU Darmstadt Darmstadt, GERMANY University of Manchester Manchester, UNITED KINGDOM University of Groningen Groningen Biomolecular Sciences and Biotechnology Institute Groningen, NETHERLANDS CNRS S.E.E.S.I.B. - UMR 6504 Aubire, FRANCE RWTH-Aachen/ Institute for Molecular Biotechnology Aachen, GERMANY CNRS S.E.E.S.I.B. - UMR 6504 Aubire, FRANCE CNRS S.E.E.S.I.B. - UMR 6504 Aubire, FRANCE KTH Royal Institute of Technology Stockholm, SWEDEN Richter Gedeon Nyrt., Biokmia II. zem Budapest, HUNGARY Complutense University Madrid, SPAIN Industrial Microbiology Graduate School of Agriculture, Kyoto University Kyoto, JAPAN University of Turku Turku, FINLAND The University of Tokyo Bunkyo-ku, JAPAN Dept. of Biotechnology & Enzyme Catalysis Institute of Biochemistry, University of Greifswald Greifswald, GERMANY RWTH Aachen University Institute of Biotechnology Aachen, GERMANY Department of Natural Science Engineering and Mathematics Sundsvall, SWEDEN Delft University of Technology Delft, NETHERLANDS gutarra@gmail.com mzhadi@sandia.gov
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GERHARDS, Tina
t.gerhards@fz-juelich.de HAILES, Helen mohamed.ghoul@ensaia.inpl-nancy.fr s.bonecchi@resindion.com raquel@ufscar.br HAJNAL, Ivan HALL, Mlanie
h.c.hailes@ucl.ac.uk
GHOUL, Mohamed GIESECKE, Ulrich Eberhard GIORDANO, Raquel
ivan.hajnal@acib.at melanie.hall@uni-graz.at
GIORDANO, Roberto
roberto@ufscar.br
HALLING, Peter j
P.J.Halling@strath.ac.uk
GIOVANNINI, Pier paolo
gvnppl@unife.it
HALTRICH, Dietmar
dietmar.haltrich@boku.ac.at
GIULIETTI, Ana mara
agiule@ffyb.uba.ar
HAMMER, Stephan HAN, Jin Yee
itbsch@itb.uni-stuttgart.de yni8708@naver.com u.hanefeld@tudelft.nl steven_paul.hanlon@roche.com ryo_h@aoni.waseda.jp harivony.rakotoarivonina@univ-reims.fr A.F.Hartog@uva.nl Rajni.Hatti-Kaul@biotek.lu.se he@mail.oc.chemie.tu-darmstadt.de rachel.heath@manchester.ac.uk m.m.heberling@rug.nl
GLADKOWSKI, Witold
glado@poczta.fm
HANEFELD, Ulf HANLON, Steven HARA, Ryotaro HARIVONY, Rakotoarivonina HARTOG, Aloysius Franciscus HATTI-KAUL, Rajni HE, Ning
GLIEDER, Anton GOEBEL, Jurgen GOLDFEDER, Mor GONALVES, Caroline
anton.glieder@acib.at juergen.goebel@roche.com mors@tx.technion.ac.il csgoncalves@iqm.unicamp.br
GONALVES AGUIEIRAS, Erika cristina
erikaaguieiras@gmail.com
GONZLEZ SABN, Javier GOTOR, Vicente GOTOR-FERNNDEZ, Vicente GRABER, Marianne GRANDE, Philipp GREINER, Lasse GRIENGL, Herfried
jgsabin@entrechem.com vgs@uniovi.es VICGOTFER@UNIOVI.ES mgraber@univ-lr.fr Grande@itmc.rwth-aachen.de greiner@dechema.de griengl@tugraz.at
HEATH, Rachel HEBERLING, Matthew
HECQUET, Laurence
laurence.hecquet@univ-bpclermont.fr
HEESEL, Dirk
dirk.heesel@rwth-aachen.de
HELAINE, Virgil HELAINE, Christine HENDIL-FORSSELL, Peter HRIN BERTA, Mnika HERNAIZ, Maria Jose HIBI, Makoto
Virgil.Helaine@univ-bpclermont.fr christine.helaine@univ-bpclermont.fr peterhf@kth.se berta.monika@gmail.com mjhernai@farm.ucm.es mhibi@kais.kyoto-u.ac.jp armahi@utu.fi hirakawa@bio.t.u-tokyo.ac.jp Matthias.Hoehne@gmx.net
GROENEVELD, Maarten GROGAN, Gideon GRGER, Harald
m.groeneveld@rug.nl gg12@york.ac.uk harald.groeger@uni-bielefeld.de
GRUBER, Christian GRUBER-KHADJAWI, Mandana GRUENWALDT, Caterina GUAIS, Olivier GUEBITZ, Georg GUIMARES FREIRE, Denise maria GURANDA, Dorel GUSTAFSSON, Hanna
christian.gruber@uni-graz.at mandana.gruber@acib.at caterinagcmn@gmail.com Olivier.guais@adisseo.com georg.guebitz@acib.at freire@iq.ufrj.br dorel@belozersky.msu.ru hanna.gustafsson@chalmers.se
HIETANEN, Ari HIRAKAWA, Hidehiko HOEHNE, Matthias
HOFZUMAHAUS, Sebastian
s.hofzumahaus@biotec.rwth-aachen.de
HGBERG, Hans-erik HOLLMANN, Frank
hans-erik.hogberg@miun.se f.hollmann@tudelft.nl
October 2-6, 2011
200
HLSCH, Kathrin Technische Universitt Mnchen Institute of Biochemical Engineering Garching, GERMANY DECHEMA e.V. Frankfurt am Main, GERMANY Institute of Technical Biochemistry University of Stuttgart Stuttgart, GERMANY Pfizer Inc Kalamazoo, USA Konkuk University Seoul, SOUTH KOREA Pfizer Kalamazoo, USA GlaxoSmithKline Worthing, UNITED KINGDOM Biochemistry, School of Biotechnology, Royal Institute of Technology (KTH) Stockholm, SWEDEN LIBio - INPL vandoeuvre-ls-Nancy, FRANCE Greifswald University Greifswald, GERMANY F. Hoffmann-La Roche Basel, SWITZERLAND Department of Biochemistry & Microbiology Plovdiv University Plovdiv, BULGARIA Pontificia Universidad Catlica de Valparaso Valparaso, CHILE Roche Diagnostics GmbH Penzberg, GERMANY Resindion Srl Binasco, ITALY RWTH Aachen University Aachen, GERMANY Danish Technical University Kgs. Lyngby, DENMARK University Stuttgart Stuttgart, GERMANY (YSBL) The University of York York, UNITED KINGDOM Instituto De Quimica Avanzada De Catalua Consejo Superior De Investigaciones Cientififcas Barcelona, SPAIN konkuk university Seoul, SOUTH KOREA Bio-Prodict Wageningen, NETHERLANDS Roche Diagnostics Deutschland GmbH Mannheim, GERMANY TU Dortmund University Dortmund, GERMANY The Catholic University of Korea Bucheon-si, SOUTH KOREA Federal University of Rio de Janeiro Rio de Janeiro, BRAZIL TU-Darmstadt Darmstadt, GERMANY Forschungszentrum Juelich Universitaet Duesseldorf Juelich, GERMANY Centre of Molecular and Macromolecular Studies Polish Academy of Sciences Lodz, POLAND Technical University of Lodz Lodz, POLAND Bio Springer Maisons-Alfort, FRANCE Toyama Prefectural University Imizu, JAPAN University of Turku Turku, FINLAND k.hoelsch@lrz.tum.de kwi@dechema.de
KARBOUNE, Salwa KARMEE, Sanjib kumar McGill University St-Anne de Belle Vue, CANADA Delft University of Technology Department of Biotechnology Delft, NETHERLANDS University of Hyogo Himeji, JAPAN Kyushu University Fukuoka, JAPAN University of Minnesota Saint Paul, USA University of Oviedo Oviedo, SPAIN McGill University Department of Food Science Ste. Anne-de-Bellevue, CANADA Lomonosov Moscow State University Moscow, RUSSIA CBMiM PAN Lodz, POLAND University of So Paulo So Paulo, BRAZIL Korea Research Institute of Bioscience and Biotechnology (KRIBB) Daejeon, SOUTH KOREA Korea Research Institute of Bioscience and Biotechnology (KRIBB) Daejeon, SOUTH KOREA The Catholic University of Korea Bucheon, SOUTH KOREA The Catholic University of Korea Bucheon-si, SOUTH KOREA Seoul National University Seoul, SOUTH KOREA Kwangwoon University Seoul, SOUTH KOREA Ewha Womans University Seodaemun-Gu, SOUTH KOREA Waseda University Shinjuku-ku, JAPAN Kyoto University Kyoto, JAPAN Novartis Institutes of Biomedical Research, Bioreactions Basel,S WITZERLAND Institut fr Molekulare Enzymtechnologie (IMET) im Forschungszentrum Jlich Jlich, GERMANY Institut of Organic Chemistry Graz University of Technology Graz, AUSTRIA Wroclaw University of Technology Wroclaw, POLAND Vertex Pharmaceuticals Cambridge, MA,USA RWTH/Molekulare Biotechnologie Aachen, GERMANY Universit degli studi di Trieste Trieste, ITALY Graz University of Technology Graz, AUSTRIA Keio University Department of Pharmaceutical Science Tokyo, JAPAN Graz University of Technology Institute of Molecular Biotechnology, Graz, AUSTRIA University of Basel Basel, SWITZERLAND Kyowa Hakko Bio CO., LTD. Tsukuba-shi, JAPAN Vienna University of Technology Vienna, AUSTRIA Institute of biochemistry and physiology of microorganisms (IBPM RAS) Pushchino, RUSSIA salwa.karboune@mcgill.ca S.K.Karmee@tudelft.nl
201
HOLTMANN, Dirk HONDA, Sumire
sumire.honda@itb.uni-stuttgart.de
KATO, Dai-ichiro KAWAKAMI, Koei KAZLAUSKAS, Romas KEDZIORA, Kinga KERMASHA, Selim
kato@eng.u-hyogo.ac.jp kawakami@chem-eng.kyushu-u.ac.jp rjk@umn.edu kedziorakinga@uniovi.es selim.kermasha@mcgill.ca
HONG, Kitae HONG, Seung hye HORNG, Jyh-song HUCKLE, Benjamin D. HULT, Karl
kitae.hong@pfizer.com hshplusyou@hanmail.net JYH-SONG.HORNG@PFIZER.COM benjamin.d.huckle@gsk.com kalle@biotech.kth.se
KHALIULLIN, Ilyas KIELBASINSKI, Piotr KILIKIAN, Beatriz KIM, Jihyun F. KIM, Myung Hee KIM, Hyung kwoun
ilyas@belozersky.msu.ru piokiel@cbmm.lodz.pl kilikian@usp.br jfk@kribb.re.kr mhk8n@kribb.re.kr hkkim@catholic.ac.kr rain320@catholic.ac.kr byungkim@snu.ac.kr metalkim@kw.ac.kr hwtj58@hanmail.net kkino@waseda.jp kishino@kais.kyoto-u.ac.jp matthias.kittelmann@novartis.com
HUMEAU, Catherine HUMMEL, Anke IDING, Hans ILIEV, Ilia
catherine.humeau@ensaia.inpl-nancy.fr anke.hummel@uni-greifswald.de hans.iding@roche.com iliailiev@uni-plovdiv.bg
ILLANES, Andrs INGO, Alexy ISOBE, Eiji JAKOBLINNERT, Andre JANES, Kresimir JENDROSSEK, Dieter JENSEN, Chantel JOGLAR, Jesus
aillanes@ucv.cl ingo.alexy@roche.com s.bonecchi@resindion.com a.jakoblinnert@biotec.rwth-aachen.de kreja@kt.dtu.dk imbdj@imb.uni-stuttgart.de chantel@ysbl.york.ac.uk joglar@iqac.csic.es
KIM, Hye-Jung KIM, Byung-Gee KIM, Yong Hwan KIM, Ju Yeon KINO, Kuniki KISHINO, Shigenobu KITTELMANN, Matthias
KLEIN, Kathrin ycjoo17@hanmail.net joosten@bio-prodict.nl uwe-michael.juhl@roche.com mattijs.julsing@bci.tu-dortmund.de airgreen13@gmail.com ivaldoufrj@gmail.com Sebastianjunker1@web.de d.jussen@fz-juelich.de KLEMPIER, Norbert KLIMEK-OCHAB, Magdalena KLINE, Billie j. kline KLOSE, Holger KNAPIC, Lorena KNAUS, Tanja KOBAYASHI, Ryohei
k.klein@fz-juelich.de
JOO, Youngchul JOOSTEN, Henk-jan JUHL, Uwe-michael JULSING, Mattijs JUNG, Jihye JUNIOR, Ivaldo JUNKER, Sebastian JUSSEN, Daniel
Klempier@tugraz.at magdalena.klimek-ochab@pwr.wroc.pl billie_kline@vrtx.com holger.klose@rwth-aachen.de lorena.knapic@phd.units.it tanja.knaus@tugraz.at ryo-kobayashi@z6.keio.jp
KACZMARCZYK, Sylwia
sylwiak@cbmm.lodz.pl
KFINGER, Petra
petra.koefinger@tugraz.at
KACZMAREK, Alicja KAD, Nadia KAMEYA, Masafumi KANERVA, Liisa
alicja.kaczmarek@p.lodz.pl nadia.kaid@biospringer.com mkameya777@st.pu-toyama.ac.jp lkanerva@utu.fi
KHLER, Valentin KOKETSU, Kento KOLEY, Moumita
valentin.koehler@unibas.ch kento.koketsu@kyowa-kirin.co.jp mkoley@ioc.tuwien.ac.at
KOLLEROV, Vyacheslav
svkollerov@rambler.ru
October 2-6, 2011
202
KOSCHORRECK, Katja Institute of Biochemistry II Heinrich-Heine University Dsseldorf Dsseldorf, GERMANY Institute of Microbiology Academy of Sciences Laboratory of Biotransformation Prague, CZECH REPUBLIC TU Mnchen Institute of Chemistry of Biogenic Resources Straubing, GERMANY Wroclaw University of Technology Wroclaw, POLAND FRSC Institute of Microbiology Academy of Sciences of the Czech Republic Laboratory of Biotransformations Czech Republic, CZECH REPUBLIC Forschungszentrum Jlich Jlich, GERMANY Heinrich-Heine-Universitt Dsseldorf im Forschungszentrum Jlich Jlich, GERMANY TU Dortmund University Dortmund, GERMANY F.I.S. - Fabbrica Italiana Sintetici S.p.A. Montecchio Maggiore, ITALY National Research Council (CNR) Institute of biomolecular chemistry (ICB) Catania, ITALY Biochemistry and Molecular Biology Almeria University Almeria, SPAIN Codexis Redwood City, USA University of Oviedo Oviedo, SPAIN CEA - Genoscope Evry, FRANCE UMR 6250 CNRS-ULR, LIENS Universit de La Rochelle La Rochelle, FRANCE Cape Peninsula University of Technology Bellville,SOUTH AFRICA Korea Research Institute of Bioscience and Biotechnology (KRIBB) Deajeon, SOUTH KOREA Korea Research Institute of Bioscience and Biotechnology (KRIBB) Deajeon, SOUTH KOREA Dept. of Microbial Engineering, Konkuk University Kwangjin-Gu, SOUTH KOREA University of York York, UNITED KINGDOM RWTH Aachen Aachen, GERMANY Clermont Universit Universit Blaise Pascal Aubire, FRANCE Istituto di Chimica Biomolecolare Consiglio nazionale delle Ricerche Catania, ITALY DECHEMA e.V. Frankfurt am Main, GERMANY Merck Co. Rahway, USA National University of Singapore Singapore, SINGAPORE Aalborg University. Department of Biotechnology, Chemistry and Environmental Engineering Aalborg, DENMARK Stockholm University Stockholm, SWEDEN Technical University of Denmark Kgs. Lyngby, DENMARK Cambrex Karlskoga AB Karlskoga, SWEDEN University of the Free State Bloemfontein, SOUTH AFRICA
Katja.Koschorreck@uni-duesseldorf.de
LITTLECHILD, Jennifer LLOYD, Michael University of Exeter Exeter, UNITED KINGDOM Dr. Reddy's Laboratories Ltd Cambridge, UNITED KINGDOM Universitat Autonoma de Barcelona Bellaterra, SPAIN Danisco A/S Brabrand, DENMARK Universidade Estadual de Feira de Santana Feira de Santana, BRAZIL AS Cambrex Tallinn Tallinn, ESTONIA Novartis Institutes for BioMedical Research Basel, SWITZERLAND Petrobras Rio de Janeiro, BRAZIL Institute of Molecular Biotechnology Technical University of Graz Graz, AUSTRIA RWTH Aachen University Aachen, GERMANY Northumbria University Newcastle, UNITED KINGDOM University of Mancehster Manchester, UNITED KINGDOM Universidade Federal do Rio de Janeiro (Federal University of Rio de Janeiro)/UFRJ/ Chemical Enginee Rio de Janeiro, BRAZIL Forschungszentrum Jlich GmbH Institute of Bio- and Geosciences, IBG-1: Biotechnology Juelich, GERMANY Universidade Estadual De Feira De Santana Feira de Santana, BRAZIL Chemistry Institute/ UNICAMP Campinas, BRAZIL KTH Stockholm, SWEDEN Resindion Srl Binasco, ITALY CSIC - Institute Of Catalysis And Petrochemistry Madrid, SPAIN Vilnius University Vilnius, LITHUANIA University of La Rochelle La Rochelle, FRANCE Institute of Technical and Macromolecular Chemistry RWTH Aachen University. Germany Aachen, GERMANY C-Tech Innovation Ltd Chester, UNITED KINGDOM Federal University of Rio de Janeiro Rio de Janeiro, BRAZIL Universidade de So Paulo Instituto de Qumica de So Carlos So Carlos, BRAZIL Resindion Srl Binasco, ITALY c-LEcta GmbH Leipzig, GERMANY Technical University of Denmark Kgs. Lyngby, DENMARK Instituto De Quimica Avanzada De Catalua Consejo Superior De Investigaciones Cientififcas Barcelona,SPAIN Vienna University of Technology Institute of Applied Synthetic Chemistry Vienna, AUSTRIA Institute Biotechnology and Antibiotics Warsaw, POLAND Technical University of Denmark Lyngby, DENMARK UNESP Rio Claro, BRAZIL ACIB GmbH Graz,AUSTRIA J.A.Littlechild@exeter.ac.uk mlloyd2@drreddys.com josep.lopez@uab.cat rikke.lorentsen@danisco.com angelica.lucchese@gmail.com mai.lyll@cambrex.com Stephan.luetz@novartis.com ALINEBIO@PETROBRAS.COM.BR zalina.magomedova@tugraz.at
203
KOTIK, Michael
kotik@biomed.cas.cz
LOPEZ-SANTIN, Josep LORENTSEN, Rikke LUCCHESE, Angelica LLL, Mai LTZ, Stephan
KOURIST, Robert
kourist@tum.de
KOZYRA, Kinga KREN, Vladimir
kinga.basalyga@pwr.wroc.pl kren@biomed.cas.cz
KULIG, Justyna KULLARTZ, Irene LADKAU, Nadine LAFORCE, Roger max LAMBUSTA, Daniela
j.kulig@fz-juelich.de i.kullartz@fz-juelich.de nadine.ladkau@bci.tu-dortmund.de roger.laforce@fisvi.com daniela.lambusta@icb.cnr.it
MACHADO DE CASTRO, Aline MAGOMEDOVA, Zalina
MAHNKEN, Kai MALIK, Vatsala MALONE, Kirk MANOEL, Evelin
Kai.Mahnken@avt.rwth-aachen.de vatsala.malik@northumbria.ac.uk Kirk.Malone@manchester.ac.uk evelin@ufrj.br
LAS HERAS VAZQUEZ, Francisco Javier
fjheras@ual.es
LATO, Susan LAVANDERA, Ivan LE BER, Pierre LE JOUBIOUX, Florian
susan.lato@codexis.com lavanderaivan@uniovi.es pleber@genoscope.cns.fr florian.le_joubioux@univ-lr.fr
MARIENHAGEN, Jan
j.marienhagen@fz-juelich.de
MARQUEZ, Heiddy MARSAIOLI, Anita Jocelyne MARTINELLE, Mats MASUDA, Satoru MAT, Diana MATIJOSYTE, Inga MAUGARD, Thierry MAUGERI, Zaira
marquezheiddy@gamil.com anita@iqm.unicamp.br matsm@biotech.kth.se s.bonecchi@resindion.com diana.mate@icp.csic.es inga.matijosyte@bti.vu.lt tmaugard@univ-lr.fr Maugeri@itmc.rwth-aachen.de
LE ROES-HILL, Marilize LEE, Seung-goo LEE, Sang jun LEE, Sang hyun LEESE, Charlotte LEHMANN, Christian LEMAIRE, Marielle
leroesm@cput.ac.za sglee@kribb.re.kr leesj@kribb.re.kr sanghlee@konkuk.ac.kr cl583@york.ac.uk c.lehmann@biotec.rwth-aachen.de marielle.lemaire@univ-bpclermont.fr
MCLEAN, Kay MEDEIROS GONALVES, Karen MELEIRO PORTO, Andr Luiz
dawn.barnes@ctechinnovation.com karen.goncalves@yahoo.com.br almporto@iqsc.usp.br
LEONE, Luigi
luigi.leone@icb.cnr.it
LEUCHS, Susanne LI, Tao LI, Zhi LIE, Aleksander
Leuchs@dechema.de tao.li@merck.com chelz@nus.edu.sg alie@bio.aau.dk
MERCALLI, Enrico MERKENS, Hedda MICHALAK, Malwina MIFSUD, Maria MIHOVILOVIC, Marko
s.bonecchi@resindion.com kontakt@c-lecta.de mmi@kt.dtu.dk maria.mifsud@iqac.csic.es mmihovil@pop.tuwien.ac.at
LIHAMMAR, Richard LIMA RAMOS, Joana LINDBERG, Lina LITTHAUER, Suzanne
richard@organ.su.se jlr@kt.dtu.dk lina.lindberg@cambrex.com litthauers@ufs.ac.za
MIKIEWICZ-SYGULA, Diana MIKKELSEN, Jorn Dalgaard MILAGRE, Cintia MITROVIC, Aleksandra
mikiewiczd@iba.waw.pl jdm@kt.dtu.dk cmilagre@rc.unesp.br aleksandra.mitrovic@acib.at
October 2-6, 2011
204
MITSUKURA, Koichi MOERKHOLT, Camilla kaer Gifu University Gifu,JAPAN Aalborg University Department of Biotechnology, Chemistry and Environmental Engineering Aalborg, DENMARK University of Milan DISTAM Industrial Microbiology Section Milano, ITALY Universit degli Studi dellInsubria Varese, ITALY IFP Energies Nouvelles Rueil-Malmaison, FRANCE Austrian Centre of Industrial Biotechnology Graz, AUSTRIA Faculdade de Farmcia/Universidade de Lisboa Lisboa, PORTUGAL Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche Milano, ITALY Institute of Protein Biochemistry National Research Council Naples, ITALY TECHNOLOGIE SERVIER Orleans, FRANCE Universit di Milano Dipartimento di Chimica Organica e Industriale Milano, ITALY Entrechem S.L. Oviedo, SPAIN Consiglio Nazionale delle Ricerche Catania, ITALY F.I.S. - Fabbrica Italiana Sintetici S.p.A. Montecchio Maggiore, ITALY INSA - LISBP Toulouse, FRANCE Universidade Federal do Rio de Janeiro Rio de Janeiro, BRAZIL Forschungszentrum Juelich GmbH IBG-1 Juelich, GERMANY Institute of Pharmaceutical Sciences University of Freiburg Freiburg, GERMANY DSM Innovative Synthesis Geleen, NETHERLANDS RWTH Aachen University Lehrstuhl fr Biotechnologie Aachen, GERMANY RWTH Aachen University Aachen, GERMANY Department of Chemistry, Organic and Bioorganic Chemistry University of Graz Graz, AUSTRIA Johnson Matthey Catalysts Dusseldorf, GERMANY Istituto di Chimica Biomolecolare Consiglio Nazionale delle Ricerche Catania, ITALY Austrian Centre of Industrial Biotechnology Graz,AUSTRIA Institute for Technical and Macromolecular Chemistry RWTH Aachen University Aachen, GERMANY Universidad Nacional Autonoma de Mxico Mexico, D.F.,MEXICO Montpellier SupAgro Montpellier cedex 1,FRANCE Universitaet Stuttgart Institute of Technical Biochemistry Stuttgart, GERMANY Department of Chemical and Biochemical Engineering Technical University of Denmark (DTU) Kgs. Lyngby, DENMARK Institute of Bioorganic Chemistry Heinrich Heine University Dsseldorf Jlich, GERMANY mitukura@gifu-u.ac.jp ckm@bio.aau.dk
NEUNLIST, Serge NGO, Thao nguyen phuong NGUYEN, Giang Son UHA ENSCMU Mulhouse, FRANCE National University of Singapore Singapore, SINGAPORE Delft University of Technology Delft, NETHERLANDS Istituto di Chimica Biomolecolare Consiglio Nazionale delle Ricerche Catania, ITALY Resindion Srl Binasco, ITALY Jiangnan University Wuxi, CHINA TU Berlin Enzyme Technology Berlin, GERMANY Centro de Investigacion y Asistencia en Tecnologia y Diseo del Estado de Jalisco Guadalajara, MEXICO University of Basel Basel, SWITZERLAND Technical University of Denmark (DTU) Kgs. Lyngby, DENMARK University of Nigeria, NSukka Nsukka, NIGERIA INRA Reims, FRANCE Osaka Prefecture University Sakai, JAPAN CJ CheilJedang BIO) R&D Institute Seoul, SOUTH KOREA Korea Research Institute of Bioscience and Biotechnology Yuseong, SOUTH KOREA Konkuk University Seoul, SOUTH KOREA Novacta Biosystems Limited Welwyn Garden City, UNITED KINGDOM Forschungszentrum Jlich GmbH Jlich, GERMANY BERTIN PHARMA Montigny-le-Bretonneux, FRANCE University of the Free State Bloemfontein, SOUTH AFRICA University of Crete, Department of Chemistry Heraklion, GREECE Instituto de Qumica Orgnica General CSIC Madrid, SPAIN Lonza AG Visp, SWITZERLAND Novozymes A/S Bagsvaerd, DENMARK University College London London,UNITED KINGDOM Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche Milano, ITALY TEVA Pharmaceutical Works Pltd Debrecen,HUNGARY Dept. of Biotechnology & Enzyme Catalysis Institute of Biochemistry, University of Greifswald Greifswald, GERMANY UFRJ - NPPN Rio de Janeiro, BRAZIL University of Turku Turku, FINLAND Johnson Matthey Catalysts Royston, UNITED KINGDOM Laboratorio de Biocatlisis y Biotransformaciones DCyT, Universidad Nacional de Quilmes Bernal, ARGENTINA University of So Paulo So Paulo, BRAZIL erge.neunlist@uha.fr g0800030@nus.edu.sg G.S.Nguyen@tudelft.nl giovanni.nicolosi@cnr.it
205
MOLINARI, Francesco
francesco.molinari@unimi.it
NICOLOSI, Giovanni
MOLLA, Gianluca MONOT, Frederic MONSCHEIN, Mareike MONTEIRO, Carlos MONTI, Daniela
gianluca.molla@uninsubria.it frederic.monot@ifpen.fr mareike.monschein@tugraz.at monteiro.cmft@gmail.com daniela.monti@icrm.cnr.it
NICOTRA, Silvia NIE, Yao NIEGUTH, Rene NIEHUS CHARLES, Xochitl del Rocio
s.bonecchi@resindion.com nieybird@hotmail.com Rene.Nieguth@chem.tu-berlin.de xrniehus@gmail.com
NOGUEIRA, Elisa m.moracci@ibp.cnr.it NORDBLAD, Mathias NWAGU, Tochukwu Nwamaka OCHS, Marjorie OGINO, Hiroyasu OH, Inseok OH, Tae-wang OH, Deok-kun OKRASA, Krzysztof OKROB, Daniel OLIVARIUS, Ccile OPPERMAN, Dirk monika.muller@dsm.com c.mueller@biotec.rwth-aachen.de ORFANOPOULOS, Michael OROZ-GUINEA, Isabel OSORIO - LOZADA, Antonio STERGAARD, Lars O'SULLIVAN, Brian OTTOLINA, Gianluca
elisa.nogueira@unibas.ch man@kt.dtu.dk tochuchukwu.nwagu@yahoo.com Marjorie.Ochs@reims.inra.fr ogino@chemeng.osakafu-u.ac.jp ohis80@cj.com otk@kribb.re.kr deokkun@konkuk.ac.kr Krzysztof.Okrasa@novactabio.com d.okrob@fz-juelich.de marketing@bertinpharma.com opperdj@ufs.ac.za orfanop@chemistry.uoc.gr isabel.oroz@iqog.csic.es
MORACCI, Marco
MOREAU-PEDRAGOSA, Sandrine MORELLI, Carlo
sandrine.moreau@netgrs.com carlo.morelli@unimi.it
MORIS, Francisco MORRONE, Raffaele MOTTERLE, Riccardo MOULIS, Claire MOURA, Marcelo MOUSSA, Roland MLLER, Michael
fmv@entrechem.com raffaele.morrone@icb.cnr.it lucia.ciatti@fisvi.com claire.moulis@insa-toulouse.fr marcelovhm21@gmail.com r.moussa@fz-juelich.de Michael.Mueller@pharmazie.uni-freiburg.de
MLLER, Monika MLLER, Christina andrea
MUNDHADA, Hemanshu MUTTI, Francesco g.
h.mundhada@biotec.rwth-aachen.de francesco.mutti@uni-graz.at
antonio.osorio@lonza.com laq@novozymes.com b.osullivan@ucl.ac.uk gianluca.ottolina@icrm.cnr.it
NAAMNIEH, Shukry NAPOLI, Edoardo
Shukry.Naamnieh@matthey.com edoardo.napoli@icb.cnr.it
NAPORA, Kamila NATALIA, Dessy
kamila.napora@acib.at natalia@itmc.rwth-aachen.de
PAL, Tihamr PADHI, Santosh Kumar
edina.biro@teva.hu skp_77@yahoo.com
NAVARRO-OCAA, Arturo NEANG, Pisey NESTL, Bettina
arturono@servidor.unam.mx Pisey.Neang@supagro.inra.fr bettina.nestl@itb.uni-stuttgart.de
PAIS, Karla PIVI, Mari
kceodaro@gmail.com mapaiv@utu.fi
PALACIOS-ALCOLADO, Maria Luisa
Maria_Luisa.Palacios-Alcolado@matthey.com
NETO, Watson
wan@kt.dtu.dk
PALAZZOLO, Martin Alejandro
mpalazzolo@unq.edu.ar
NEUFELD, Katharina
k.neufeld@fz-juelich.de
PALMEIRA, Dayvson J.
dayvson@usp.br \
October 2-6, 2011
206
PALOMO, Jose m CSIC - Institute of catalysis Department of Biocatalysis Madrid, SPAIN Gdansk University of Technology Gdansk, POLAND Faculty of Bioengineering and Bioinformatics Lomonosov Moscow State University Moscow, RUSSIA ETH Zurich Basel, SWITZERLAND Centro de Investigaciones Biolgicas CSIC Madrid, SPAIN Ewha Womans University Seoul, SOUTH KOREA Dipartimento CMIC Giulio Natta Politecnico di Milano Milano,ITALY University of Natural Resouces and Life Sciences Vienna, AUSTRIA University of Oviedo Oviedo, SPAIN Aalborg University Section of Biotechnology Aalborg, DENMARK Universit di Ferrara Dipartimento di Biologia ed Evoluzione Ferrara, ITALY Istituto di Biochimica delle Proteine Consiglio Nazionale delle Ricerche Napoli, ITALY Montpellier Supagro Montpellier Cdex 1, FRANCE Northumbria University Newcastle upon Tyne, UNITED KINGDOM Karlsruhe Institute of Technology Karlsruhe, GERMANY Universitat Autonoma de Barcelona Bellaterra,SPAIN University College London London, UNITED KINGDOM Evonik Degussa GmbH Marl, GERMANY Technical University Munich Chair of Chemistry of Biogenic Resources Straubing, GERMANY Gdansk Univerity of Technology Chemical Department, Department of Food Chemistry, Technology and Bi Gdansk, POLAND Heinrich-Heine-Universitt Dsseldorf Jlich, GERMANY University of Stuttgart Stuttgart, GERMANY CSIC - Instituto de Catalisis Madrid, SPAIN Forschungszentrum Jlich GmbH Institute of Bio- and Geosciences, IBG-1: Biotechnology Jlich, GERMANY Universit degli Studi dellInsubria Varese, ITALY Budapest University of Technology and Economics Department of Organic Chemistry and Technology Budapest, HUNGARY Natura Cosmetics Cajamar, BRAZIL Biocon Limited Bangalore, INDIA evocatal GmbH Duesseldorf , GERMANY Academy of Sciences of the Czech Republic Institute of Microbiology, Laboratory of Biotransformation Prague, CZECH REPUBLIC VTU Technology GmbH Grambach, AUSTRIA
josempalomo@icp.csic.es
PUTHAN VEETIL, Vinod Department of Pharmaceutical Biology University of Groningen, Groningen, NETHERLANDS Lund University, Department of Biotechnology Lund, SWEDEN DSM Innovative Synthesis B.V. Geleen, NETHERLANDS CSCB, UCD School of Chemistry and Chemical Biology Dublin, IRELAND University of Basel Basel, SWITZERLAND DSM Innovative Synthesis B.V. Geleen, NETHERLANDS BioNoCo - AVT.BioVT RWTH Aachen University Aachen, GERMANY Istituto di Biochimica delle Proteine Consiglio Nazionale delle Ricerche Napoli, ITALY University of Groningen Groningen, NETHERLANDS University of Graz Department of organic and bioorganic chemistry Graz, AUSTRIA Denmark Technical University (DTU) Lyngby, INDIA Universidade de So Paulo So Carlos, BRAZIL TU Delft Delft, NETHERLANDS Institute of Biochemistry Vilnius University Vilnius, LITHUANIA Dipharma Francis Srl Baranzate, ITALY Lund university Department of biotechnology Lund, SWEDEN University of So Paulo So Paulo, BRAZIL Empa St. Gallen, SWITZERLAND INRA Reims, FRANCE University of Bern Department of Chemistry and Biochemistry Berne, SWITZERLAND Faculdade Farmcia, Universidade Lisboa Lisboa, PORTUGAL Institute of Microbiology Academy of Sciences of the Czech Republic Prague, CZECH REPUBLIC Universidad de Oviedo Oviedo,SPAIN Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche Milano, ITALY Universidad Autonoma del Estado de Morelos Cuernavaca, MEXICO Pontificia universidad catlica de valparaso Valparaso, CHILE CSIC - Institute of catalysis Department of Biocatalysis Madrid, SPAIN CSIC - Institute of catalysis Department of Biocatalysis Madrid, SPAIN University of Oviedo Oviedo, SPAIN DiSTAM Universit degli Studi di Milano Milano, ITALY CSIC - Institute of catalysis Department of Biocatalysis Madrid, SPAIN v.puthan.veetil@rug.nl
207
PANEK, Anna PANIN, Nikolay
panus85@wp.pl panin@belozersky.msu.ru
PYO, Sang-hyun QUAEDFLIEG, Peter j.l.m. QUAGLIA, Daniela
Sang-Hyun.Pyo@biotek.lu.se peter.quaedflieg@dsm.com daniela.quaglia@ucd.ie
PANKE, Sven PARDO, Isabel
sven.panke@bsse.ethz.ch iparmen@cib.csic.es
QUINTO, Tommaso RAEMAKERS-FRANKEN, Elly RAHMEN, Natalie
tommaso.quinto@unibas.ch elly.raemakers@dsm.com Natalie.Rahmen@avt.rwth-aachen.de
PARK, Jin byung PARMEGGIANI, Fabio
jbpark06@ewha.ac.kr fparmeggiani@tiscali.it
PAUKNER, Regina PAUL, Caroline PEDERSEN, Lars haastrup
regina.paukner@boku.ac.at paulcaroline@hotmail.com lhp@bio.aau.dk
RAIA, Carlo A.
ca.raia@ibp.cnr.it
RAJ, Hans RAJAGOPALAN, Aashrita
h.raj@rug.nl aashrita.rajagopalan@uni-graz.at
PEDRINI, Paola
pdp@unife.it
RAMESH, Hemalata RANGEL, Julieta RANOUX, Adeline
hemalatarsarma@gmail.com julietarangel@iqsc.usp.br a.c.m.ranoux@tudelft.nl julija.razumiene@bchi.vu.lt
PENNACCHIO, Angela
a.pennacchio@ibp.cnr.it
PERRIER, Vronique PERRY, Justin PERZBORN, Mareike PESIC, Milja PESNOT, Thomas PFEFFER, Jan PICK, Andr
perrier@supagro.inra.fr justin.perry@northumbria.ac.uk mareike.perzborn@kit.edu milja.pesic@uab.cat t.pesnot@ucl.ac.uk jan.pfeffer@evonik.com a.pick@tum.de
RAZUMIENE, Julija
RAZZETTI, Gabriele REHN, Gustav
gabriele.razzetti@dipharma.it gustav.rehn@biotek.lu.se
REIS, Joel S. REISS, Renate REMOND, Caroline REYMOND, Jean-louis
jsreis@usp.br renate.reiss@empa.ch caroline.remond@univ-reims.fr jean-louis.reymond@ioc.unibe.ch
PIETROW, Olga
1olga@poczta.fm
RIBEIRO, Maria H. j.pietruszka@fz-juelich.de Juergen.Pleiss@itb.uni-stuttgart.de fplou@icp.csic.es ma.pohl@fz-juelich.de RINGELOV, Anna
mhribeiro@ff.ul.pt
PIETRUSZKA, Joerg PLEISS, Juergen PLOU, Francisco POHL, Martina
rinagelova.a@seznam.cz
RIOZ, Ana RIVA, Sergio
ANA.RIOZ@GMAIL.COM sergio.riva@icrm.cnr.it
POLLEGIONI, Loredano POPPE, Lszl
loredano.pollegioni@uninsubria.it poppe@mail.bme.hu
RIVERA RAMIREZ, Jose Domingo RIVEROS, Ruby ROCHA MARTN, Javier
aghomes315@hotmail.com rriveros@ucsc.cl javirocha@icp.csic.es
PORTO DA SILVA, Carla PULLELA, Venkata srinivas PULS, Michael PURCHARTOVA, Katerina
carlaporto@natura.net srinivas.pv@biocon.com m.puls@evocatal.com k.a.m.i.k@centrum.cz
RODRGUEZ, Barbara
brcolinas@icp.csic.es
RODRGUEZ MATA, Mara ROMANO, Diego
maria_rm_84@hotmail.com diego.romano@unimi.it
PURKARTHOFER, Thomas
thomas.purkarthofer@vtu.com
ROMERO, Oscar
oscar.romero@icp.csic.es
October 2-6, 2011
208
ROPER, Laila ROSCHE, Bettina ROSINI, Elena ROSSI, Mos ROTH, Simon ROTHER, Doerte RUA RODRIGUEZ, Maria luisa RUDROFF, Florian RUFF, Anna joelle RYCHKOV, Georgy SAMLAND, Anne SANDOVAL, Georgina The University of York York, UNITED KINGDOM The University of New South Wales Sydney, AUSTRALIA Universit degli Studi dellInsubria Varese, ITALY IBP-Consiglio Nazionale delle Ricerche Napoli, ITALY RWTH Aachen University Aachen, GERMANY Research Center Juelich Juelich, GERMANY University of Vigo Ourense, SPAIN Vienna University of Technology Vienna, AUSTRIA RWTH Aachen University Aachen, GERMANY Petersburg Nuclear Physics Institute Gatchina, RUSSIA Universitt Stuttgart Stuttgart, GERMANY CSIC - Institute of catalysis Department of Biocatalysis Madrid, SPAIN University of Bucharest Bucharest, ROMANIA Istituto di Chimica Biomolecolare Consiglio Nazionale delle Ricerche Catania, ITALY Technical University of Denmark Kgs. Lyngby, DENMARK Department of Bio-Technology IIT Madras,Chennai-600036 Chennai, INDIA Dept. of Biochemistry Panjab University Chandigarh, INDIA RWTH Aachen University - Institute of Biotechnology Junior Professorship for Biocatalysis Aachen, GERMANY RWTH Aachen University Lehrstuhl fr Biotechnologie Aachen, GERMANY RWTH Aachen, Dep of Molecular Biotechnology Aachen, GERMANY FB C - Bergische Universitt Wuppertal, GERMANY Technische Universitt Darmstadt Darmstadt, GERMANY University of Graz Insitute of Chemistry Graz, AUSTRIA Technical University of Berlin Berlin, GERMANY TU Dortmund / Chemical Biotechnology Dortmund, GERMANY University of Graz Graz, AUSTRIA DSM Innovative Synthesis Geleen, NETHERLANDS CSIRO Ecosystem Sciences Canberra, AUSTRALIA E.R.C.A. S.p.A. Bergamo, ITALY Istituto di Chimica del Riconoscimento Molecolare Consiglio Nazionale delle Ricerche Milano, ITALY Forschungszentrum Jlich GmbH Jlich, GERMANY University of Stuttgart Stuttgart, GERMANY University of Stuttgart Stuttgart, GERMANY lcf501@york.ac.uk b.rosche@unsw.edu.au
SERVI, Stefano Dipartimento CMIC Giulio Natta Politecnico di Milano Milano, ITALY Technion Israel Institute of Technology Haifa, ISRAEL TUDelft / CLEA Technologies B.V. Delft, NETHERLANDS Toray Industries New Prontiers Research Laboratories Kamakura, JAPAN Sanofi Pharmaceuticals Bridgewater, USA RWTH Aachen University Aachen, GERMANY Pfizer BioProcess Develoment Group Kalamazoo, USA TU Mnchen & Fraunhofer IGB BioCat Straubing, GERMANY University Stuttgart Institute for Microbiology Stuttgart, GERMANY Department of Chemistry, Organic and Bioorganic Chemistry University of Graz Graz, AUSTRIA SPRIN S.p.A. Trieste, ITALY Institute of Microbiology Academy of Sciences of the Czech Republic Praha 4, CZECH REPUBLIC Department of Biotechnology University of the Free State Bloemfontein, SOUTH AFRICA Sheffield Hallam University Sheffield, UNITED KINGDOM University of Crete Department of Chemistry Heraklio, GREECE Universidade Tiradentes Aracaju, BRAZIL Pontificia Universidad Catlica de Valparaso Consorcio Tecnolgico Bioenercel S.A. Valparaso, CHILE Ewha Womans University Seoul, SOUTH KOREA Federal University of Rio de Janeiro Rio de Janeiro,BRAZIL Istituto di Chimica Biomolecolare Consiglio Nazionale delle Ricerche Catania, ITALY Universit degli Studi di Milano Dipartimento di Chimica Organica e Industriale Milano, ITALY RWTH Aachen University AVT - Enzyme Process Technology Aachen, GERMANY University of Stuttgart, Institute of Microbiology Stuttgart, GERMANY Technical University of Lodz Institute of Technical Biochemistry Lodz, POLAND University of Erlangen-Nrnberg Erlangen, GERMANY University College London London, UNITED KINGDOM ACIB GmbH Graz, AUSTRIA ACIB GmBH Institute of Molecular Biosciences, KFU Graz Graz, AUSTRIA SANDOZ Kundl GmbH Kundl,AUSTRIA Masaryk University, Faculty of Science Department of Experimental Biology Brno,CZECH REPUBLIC Wageningen University Wageningen, NETHERLANDS stefano.servi@polimi.it
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SHAINSKY, Janna
shainsky.janna@gmail.com
elena.rosini@uninsubria.it m.rossi@ibp.cnr.it simon.roth@avt.rwth-aachen.de do.rother@fz-juelich.de mlrua@uvigo.es frudroff@ioc.tuwien.ac.at aj.ruff@biotec.rwth-aachen.de georgy-rychkov@yandex.ru anne.samland@imb.uni-stuttgart.de georgina@confluencia.net SINIGOI, Loris madalina.sandulescu@g.unibuc.ro claudia.sanfilippo@icb.cnr.it SMIT, Martie psa@kt.dtu.dk tsaravananiitm@gmail.com SMITH, Thomas SMONOU, Ioulia diptsare@pu.ac.in SOARES, Cleide Mara a.schallmey@biotec.rwth-aachen.de SOLER, Lorena SLAMOVA, Kristyna SHELDON, Roger SHIMIZU, Sakayu
r.sheldon@clea.nl Sakayu_Shimizu@nts.toray.co.jp
SHIMSHOCK, Stephen SHIVANGE, Amol Vaijanathappa SHORT, Kevin SIEBER, Volker SIEDENBURG, Gabriele
Stephen.Shimshock@sanofi-aventis.com a.shivange@biotec.rwth-aachen.de kevin.a.short@pfizer.com sieber@tum.de imbgsi@imb.uni-stuttgart.de
SIIROLA, Elina
elina.siirola@uni-graz.at
sinigoi@sprintechnologies.com slamova.kristyna@gmail.com
SANDULESCU-TUDORACHE, Madalina SANFILIPPO, Claudia
theroncw@ufs.ac.za
SANTACOLOMA, Paloma A. SARAVANAN, Thangavelu
t.j.smith@shu.ac.uk smonou@chemistry.uoc.gr
SAREEN, Dipti
cleide.soares@pq.cnpq.br lsoler@bioenercel.com
SCHALLMEY, Anett
SCHALLMEY, Marcus
m.schallmey@biotec.rwth-aachen.de
SONG, Ji-won SOUZA, Rodrigo SPAMPINATO, Marcella
izmsn@naver.com souzarod21@gmail.com spampinatom@hotmail.it
SCHMIDT, Thomas SCHNEIDER, Manfred SCHNELLBCHER, Mark SCHOBER, Markus
thomas.schmidt@molbiotech.rwth-aachen.de schneid@uni-wuppertal.de mschnellbaecher@googlemail.com markusschober@hotmail.com
SPERANZA, Giovanna
giovanna.speranza@unimi.it
SPEISS, Antje alexander.scholz@chem.tu-berlin.de manfred.schrewe@bci.tu-dortmund.de joerg.schrittwieser@edu.uni-graz.at martin.schurmann@dsm.com colin.scott@csiro.au labanalisi@gruppoerca.com SPRENGER, Georg STANCZYK, Lukasz
antje.spiess@avt.rwth-aachen.de
SCHOLZ, Alexander SCHREWE, Manfred SCHRITTWIESER, Joerg SCHRMANN, Martin SCOTT, Colin SECCOMANDI, Silvia
g.sprenger@imb.uni-stuttgart.de lukasz.stanczyk@p.lodz.pl
STAUDT, Svenja STEADMAN, David STEINER, Kerstin STEINKELLNER, Georg
svenja.staudt@chemie.uni-erlangen.de david.steadman.09@ucl.ac.uk kerstin.steiner@acib.at georg.steinkellner@uni-graz.at
SECUNDO, Francesco
francesco.secundo@icrm.cnr.it
SEHL, Torsten SEIFERT, Alexander SEITZ, Miriam
t.sehl@fz-juelich.de alexander.seifert@itb.uni-stuttgart.de miriam.seitz@itb.uni-stuttgart.de
STEINREIBER, Andreas STEPANKOVA, Veronika
andreas.steinreiber@sandoz.com stepankova.veronika@gmail.com
STEUNENBERG, Peter
peter.steunenberg@wur.nl
October 2-6, 2011
210
STEVENSON, David STLOUKAL, Radek STOLZ, Andreas STRAATHOF, Adrie National Starch & Corn Products International Bridgewater, USA LentiKat's a.s. Prague 6, CZECH REPUBLIC Institut fr Mikrobiologie Universitt Stuttgart Stuttgart, GERMANY Delft University of Technology Department of Biotechnology Delft, NETHERLANDS Graz University of Technology Graz, AUSTRIA Montpellier SupAgro Montpellier Cedex 1, FRANCE Keio University Department of Pharmaceutical Science Tokyo, JAPAN University of York Heslington, UNITED KINGDOM University of Turku - Institute of Biomedicine Department of Pharmacology, Drug Development and Therapeutics/L Turku, FINLAND Lomonosov Moscow State University Moscow, RUSSIA GlaxoSmithKline Stevenage, UNITED KINGDOM Belozersky Institute of Physicochemical Biology Lomonosov Moscow State University Moscow, RUSSIA Ranbaxy Laboratories Limited Gurgaon, INDIA Technical University of Lodz Lodz, POLAND TU Berlin Department of Biotechnology Berlin, GERMANY Instituto de Quimica Avanzada de Catalua Consejo Superior de Investigaciones Cientififcas Barcelona, SPAIN Keio University Department of Pharmaceutical Science Tokyo, JAPAN Metabomics, Inc. Zhangjiagang, CHINA ACIB GmbH Graz, AUSTRIA University of Graz Graz, AUSTRIA Vilnius University Institute of Biochemistry Vilnius, LITHUANIA ACIB GmbH Graz University of Technology Graz, AUSTRIA LIBRAGEN Toulouse, FRANCE University of Pavia Dipartimento di Scienze del Farmaco Pavia, ITALY Dipartimento CMIC Giulio Natta Politecnico di Milano Milano,ITALY Department of Biotechnology University of the Free State Bloemfontein, SOUTH AFRICA Chalmers University of Technology Chemical and Biological Engineering Industrial Biotechnology Gothenburg, SWEDEN Universidade Tiradentes Aracaju, BRAZIL Goettingen University Goettingen, GERMANY University of Groningen Groningen Biomolecular Sciences and Biotechnology Institute Groningen, NETHERLANDS
david.stevenson@nstarch.com hofova@lentikats.eu Andreas.Stolz@imb.uni-stuttgart.de a.j.j.straathof@tudelft.nl
TORRES, Pamela CSIC - Institute of Catalysis Department of Biocatalysis Madrid, SPAIN University of Lleida Lleida, SPAIN IBB - Bioengineering Department IST Lisbon, PORTUGAL Genencor International B.V. Leiden, NETHERLANDS Wroclaw University of Environmental and Life Sciences Wroclaw, POLAND Center for Process Engineering and Technology DTU Lyngby, DENMARK Dicle University Diyarbakir, TURKEY University of Manchester Manchester, UNITED KINGDOM Institut Quimic Sarria (IQS) Ramon Llull University Barcelona, SPAIN University Of Pavia Pavia, ITALY Korea Institute of Science and Technology Seoul, SOUTH KOREA School of Pharmaceutical Sciences UNESP Araraquara, BRAZIL University of Groningen Groningen, NETHERLANDS University of Amsterdam HIMS - Biocatalysis Amsterdam, NETHERLANDS Vinca Institute of Nuclear Sciences Belgrade, SERBIA University of Zagreb Faculty of Chemical Engineering and Technology Zagreb, CROATIA University of Ferrara Ferrara, ITALY Cargill Vilvoorde, BELGIUM Resindion Srl Binasco, ITALY Jacobs University Bremen Bremen, GERMANY Institute of Microbiology Academy of Sciences of the Czech Republic Prague, CZECH REPUBLIC University College London London, UNITED KINGDOM Tallinn University of Technology Tallinn, ESTONIA Codexis, Inc. Redwood City, USA Universidade Federal do Rio de Janeiro Instituto De Qumica Rio de Janeiro, BRAZIL ETH Zurich D-BSSE Basel, SWITZERLAND PROTEUS SA Nimes Cedex 1,FRANCE Forschungszentrum Jlich GmbH Jlich, GERMANY University of Stuttgart / Institute of technical biochemistry Stuttgart, GERMANY University of Amsterdam , Faculty of Science Amsterdam, NETHERLANDS University of Manchester Manchester, UNITED KINGDOM Fraunhofer IGB Straubing, GERMANY RWTH Aachen University Aachen, GERMANY pamela@icp.csic.es
211
TORRES, Merce TORRES LOURENO, Nuno
mtorres@tecal.udl.cat nmtl@ist.utl.pt
TORRES PAZMINO, Daniel grit.straganz@tugraz.at maeva.subileau@supagro.inra.fr TRONINA, Tomasz TUFVESSON, Pr
daniel.torres.pazmino@danisco.com tomasz.tronina@gmail.com pt@kt.dtu.dk
STRAGANZ, Grit SUBILEAU, Maeva
SUGAI, Takeshi
sugai-tk@pha.keio.ac.jp
TURAL, Bilsen TURNER, Nick TURON, Xavier
bilsentural@gmail.com nicholas.turner@manchester.ac.uk xavier.turon@iqs.edu
SUMMERS, Benjamin SUNDELL, Riku
summers@ysbl.york.ac.uk rhjsun@utu.fi
SUPLATOV, Dmitry SUTTON, Peter SVEDAS, Vytas
d.a.suplatov@belozersky.msu.ru peter.w.sutton@gsk.com vytas@belozersky.msu.ru
UBIALI, Daniela UM, Youngsoon VALENTINI, Sandro Roberto
daniela.ubiali@unipv.it yum@kist.re.kr valentsr@fcfar.unesp.br
SWARGAM, Sathyanarayana SZCZESNA-ANTCZAK, Miroslawa SZEKER, Kathleen
swargam.sathyanarayana@ranbaxy.com miroslawa.szczesna-antczak@p.lodz.pl k.szeker@mailbox.tu-berlin.de
VAN BEEK, Hugo VAN DER HORST, Michael
h.l.van.beek@rug.nl m.a.vanderhorst@uva.nl
VASIC, Vesna VASIC-RACKI, Durda
evasic@vinca.rs dvracki@fkit.hr
SZEKRNYI, Anna
anna.szekrenyi@iqac.csic.es
TAKETOMI, Shohei
taketomi@a8.keio.jp
VENTURI, Valentina VERCAUTEREN, Ronny VERGA, Roberto VERMA, Rajni VESEL, Alicja barbara
vntvnt@unife.it Ronny_Vercauteren@cargill.com s.bonecchi@resindion.com ra.verma@jacobs-university.de vesela@biomed.cas.cz
TAO, Junhua (alex) TASNADI, Gabor TAUBER, Katharina TAURAITE, Daiva
junhua_tao@yahoo.com gabor.tasnadi@acib.at tauberk@edu.uni-graz.at daiva.tauraite@bchi.vu.lt
TENGG, Martin
martin.tengg@tugraz.at
VILLEGAS TORRES, Maria VILLO, Ly
maria.torres.09@ucl.ac.uk lee@chemnet.ee mandy.vink@codexis.com volcan@iq.ufrj.br
TER HALLE, Robert TERRENI, Marco
am.crozet@libragen.com markt@ibiocat.eu
VINK, Mandy VOLCAN ALMEIDA, Rodrigo
TESSARO, Davide
davide.tessaro@polimi.it
WAGNER, Nina WAHLER, Denis WESTPHAL, Robert WETZL, Dennis
nina.wagner@bsse.ethz.ch dwahler@proteus.fr r.westphal@fz-juelich.de dennis.wetzl@itb.uni-stuttgart.de r.wever@uva.nl john.whittall@manchester.ac.uk lars.wiemann@igb.fraunhofer.de wiese@pc.rwth-aachen.de
THERON, Chris
theroncw@ufs.ac.za
THRN, Christian
christian.thorn@chalmers.se
TINOCO FRICKS, Alini TITTMANN, Kai TOPLAK, Ana
alinitf@yahoo.com.br ktittma@gwdg.de a.toplak@rug.nl
WEVER, Ronald WHITTALL, John WIEMANN, Lars WIESE, Susanne
October 2-6, 2011
212
WIKMARK, Ylva WILDING, Birgit WILLIES, Simon WILSON, Lorena WILSON, Yvonne WINKLER, Christoph WINKLER, Margit WOHLGEMUTH, Roland WOJTOWICZ-KRAWIEC, Anna WON, Keehoon Stockholm University Stockholm, SWEDEN Austrian Centre of Industrial Biotechnology Graz, AUSTRIA University of Manchester Manchester, UNITED KINGDOM Pontificia Universidad Catlica de Valparaso Valparaso, CHILE University of Basel Basel, SWITZERLAND Karl-Franzens University Graz Graz, AUSTRIA ACIB GmbH Graz, AUSTRIA Sigma-Aldrich Buchs, SWITZERLAND Institute Biotechnology and Antibiotcs Warsaw, POLAND Department of Chemical and Biochemical Engineering Dongguk University Seoul, SOUTH KOREA Technical University of Denmark Lyngby, DENMARK ACIB GmbH Graz, AUSTRIA Institute of Chemical and Engineering Sciences Singapore, SINGAPORE University of Natural Resources and Life Sciences Vienna, AUSTRIA University of Graz Department of Organic and Bioorganic Chemistry Graz, AUSTRIA RWTH, AVT-Enzyme Process Technology Aachen, GERMANY Technical University of Denmark lyngby, DENMARK Jiangnan University Wuxi,CHINA Technical University of Denmark Kgs. Lyngby,DENMARK University of Lleida Lleida, SPAIN Technische Universitt Darmstadt Darmstadt, GERMANY Jiangnan University Wuxi, CHINA Ewha Womans University Seoul, SOUTH KOREA Chemistry Institute/UNICAMP Campinas,BRAZIL University of Groningen Groningen, NETHERLANDS ARZEDA Corporation Seattle, USA State University of Maringa Maringa, BRAZIL CEA-Genoscope Evry, FRANCE Lund University Biotechnology Department Lund, SWEDEN RWTH Aachen Aachen, GERMANY SANDOZ Kundl GmbH Kundl, AUSTRIA Chinese Academy of Sciences Tianjin, CHINA University of Ljubljana Faculty of Chemistry and Chemical Technology Ljubljana, SLOVENIA Wroclaw University of Technology Wroclaw, POLAND ylva@organ.su.se birgit.wilding@acib.at
Contacts
213
Simon.willies@manchester.ac.uk lwilson@ucv.cl yvonne.wilson@unibas.ch christoph.winkler@edu.uni-graz.at margit.winkler@acib.at roland.wohlgemuth@sial.com wojtowicza@iba.waw.pl keehoon@dongguk.edu
Sponsors
BioNoCo
RWTH Aachen Templegraben 55 D-52056 Aachen GERMANY www.bionoco.rwth-aachen.de
CLEA
Delftechpark 34 2628XH Delft THE NETHERLANDS www.cleatechnologies.com
Codexis
200 Penonscot Drive Redwood City, CA 94063 USA www.codexis.com
COST
Action CM0701-CASCAT
WOODLEY, John WRIESSNEGGER, Tamara WU, Wu jinchuan WUEHRER, Petra WUENSCH, Christiane
jw@kt.dtu.dk tamara.wriessnegger@acib.at wu_jinchuan@ices.a-star.edu.sg petra.wuehrer@boku.ac.at christiane.wuensch@acib.at
www.cost-cascat.polimi.it www.cost.esf.org
Dipharma Francis S.r.l

Via Bissone, 5 20021 Baranzate , Milano ITALY www.dipharma.it
DSM Innovative Synthesis

a unit of Pharma Chemicals 6160 MD Geleen THE NETHERLANDS www.dsm.com
Evocatal GmbH
Merowingerplatz 1a D-40225 Dsseldorf GERMANY www.evocatal.com
E.R.C.A. S.p.A.
Via Padergnone 5/7 24050 Grassobbio, Bergamo ITALY www.gruppoerca.com
WULFHORST, Helene XU, Yuan XU, Yan XUE, Rui YARA, Edinson YI, Dong YU, Xiaowei YUN, Ji-yeong ZAMPIERI, Davila ZANDVOORT, Ellen ZANGHELLINI, Alexandre ZANIN, Gisella Maria ZAPARUCHA, Anne ZAUSHITSYNA, Oksana
Helene.Wulfhorst@avt.rwth-aachen.de xuy@kt.dtu.dk bioyuxw@yahoo.com.cn rxue@kt.dtu.dk yara@quimica.udl.cat charlesdyi@yahoo.com bioyuxw@yahoo.com.cn wnldud44@hanmail.net davila@iqm.unicamp.br e.zandvoort@rug.nl alexandre.zanghellini@arzeda.com gisella@deq.uem.br azaparuc@genoscope.cns.fr oksana.zaushitsyna@biotek.lu.se
F.I.S.
Fabbrica Italiana Sintetici S.p.A. Viale Milano 26 36075 Montecchio Maggiore, Vicenza ITALY www.fisvi.com
Flamma S.p.A.
Via Bedeschi, 22 24040 Chignolo d'Isola, Bergamo ITALY www.flamma.it
Gnosis
Gnosis S.p.A. Via Lavoratori Autobianchi,1 20033 Desio, Monza e Brianza ITALY www.gnosis-bio.com
Indena S.p.A.
Viale Ortles 12 20139 Milano ITALY www.indena.it
Jasco Europe Srl

Via Cadorna,1 23894 Cremella, Lecco ITALY serverone.jasco-europe.com
Lonza AG
Lonzastrasse CH-3930 Visp/VS Switzerland www.lonza.com
Novartis
Novartis Vaccines and Diagnostics Research Center Via Fiorentina 1 53100 Siena ITALY www.novartisvaccines.it
Prodotti Chimici e Alimentari S.p.A.

Via Novi 78, 15060 Basaluzzo, Alessandria ITALY www.pcaitaly.it
Resindion
subsidiary of Mitsubishi Chemical Corporation Via Roma, 55 20082 Binasco, Milano ITALY www.resindion.com
Sprin S.p.A.
Technology for Sustainable Chemistry c/o BIC Incubatori FVG via Flavia 23/1 34148 Trieste ITALY www.sprintechnologies.com
ZEITHAMMEL, Erik ZEPECK, Ferdinand ZHU, Dunming ZNIDARSIC-PLAZL, Polona
e.zeithammel@biotec.rwth-aachen.de ferdinand.zepeck@sandoz.com zhu_dm@tib.cas.cn polona.znidarsic@fkkt.uni-lj.si
ZYMANCZYK-DUDA, Ewa
ewa.zymanczyk-duda@pwr.wroc.pl
October 2-6, 2011
214
www.acib.at
Petersgasse 14 8010 Graz AUSTRIA
Exhibitors
Resindion
ESAB
ACIB GmbH
www.esabweb.org
www.resindion.com
Bertinpharma
Via Roma, 55 20082 Binasco, Milano ITALY
www.bertinpharma.com
Parc d'activits du Pas du Lac 10 bis av. Ampre 78180, Montigny-le-Bretonneux FRANCE
subsidiary of Mitsubishi Chemical Corporation
Austrian Centre of Industrial Biotechnology
Under the patronage of
Europeans Section on Appled Biocatalysis

00198 Roma ITALY www.buchi.it Viale Liegi 48
www.soc.chic.it
Nonnenwald 2 82377 Penzberg GERMANY
www.roche.com
www.assobiotec.it
AssoBiotech
Pal. A4 - Strada 4 20090 Assago , Milano ITALY
Bchi Labortechnik AG
Via Giovanni da Procida, 11 20149 Milano ITALY
BCHI Italia S.r.l.
Societ chimica italiana CLEA

Industriestrasse 25 9470 Buchs SWITZERLAND www.cleatechnologies.com www.sigma-aldrich.com
Roche Diagnostics GmbH
Sigma - Aldrich
Via Mancinelli, 7 20131 Milano ITALY www.dpm.cnr.it via dei Taurini, 19 00185 Roma ITALY c/o BIC Incubatori FVG via Flavia 23/1 34148 Trieste ITALY www.sprintechnologies.com
Delftechpark 34 2628XH Delft THE NETHERLANDS
Jasco Europe Srl
Sprin S.p.A.
Via Cadorna,1 23894 Cremella, Lecco ITALY
www.jasco-europe.com
Technology for Sustainable Chemistry
Dipartimento di Associazione Italiana Biocatalisi e Bioseparazioni Progettazione Molecolare
Consiglio Nazionale delle Ricerche
Contacts
Testo
Sunday, Oct 2
Monday, Oct 3
Tuesday, Oct 4
Wednesday, Oct 5
Thursday, Oct 6
ENZYMES STRUCTURES 09:00 - 09:45 09:45 - 10:15 Loredano POLLEGIONI Dietmar HALTRICH 09:00 - 09:45 09:45 - 10:15 Sakayu SHMIZU Byung Gee KIM 09:00 - 09:45 09:45 - 10:15 Jonathan DORDICK Claudia CRESTINI 09:00 - 09:45 09:50 - 10:10 10:10 - 10:30 Romas KAZSLAUSKAS Grit STRAGANZ Giedeon GROGAN
October 2-6, 2011

09:00 - 12:30 Registration 14:00 - 18:00 Registration 18:00 - 18:15 Opening Ceremony Welcome Speech Sergio RIVA 18:15 - 19:15 19:30 Welcome Reception
10:15 - 10:45
Coffee break
10:15 - 10:45
Coffe break
10:15 - 10:45
Coffe break
10:30 - 11:00
Coffe break
OXIDATIVE BIOCATALYSTS
BIOCATALYSIS AND NATURAL COMPOUNDS 10:45 - 11:05 11:05 - 11:25 11:25 - 11:45 10:45 - 11:05 11:05 - 11:25 11:25 - 11:45 11:45 - 12:05 12:05 - 12:25 12:25 - 12:55 Zhi LI Jullien DRONE Ayelet FISHMAN Ee Lui ANG Riccardo MOTTERLE Stephan LTZ 10:45 - 11:05 11:05 - 11:25 11:25 - 11:45 11:45 - 12:05 12:05 - 12:25 12:25 - 12:55 Jrg PIETRUZKA Jennifer ANDEXER Gabriele SIEDENBURG Maarten GROENEVELD Jorg SCHRITTWIESER Valdimir KREN 11:45 - 12:05 12:05 - 12:25 12:25 - 12:45
NEW APPLICATIONS FOR BIOCATALYSIS Georg GUEBITZ Volker SIEBER Marjorie OCHS Antje SPIESS Mats MARTINELLE Pavel DVORAK
BIOTRANSFORMATIONS
Testo
11:00 - 11:20 11:20 - 11:40 11:40 - 12:00 12:00 - 12:20 12:20 - 12:40
Mandana GRUBER-KHADJAWI Takeshi SUGAI Daniela UBIALI Piotr KIELBASINSKI Alexandre ZANGHELLINI
13:00 - 14:00
Lunch
13:00 - 14:00
Lunch
13:00 - 14:00
Lunch
13:00 - 14:00
Lunch
14:00 - 16:00
Poster Session I (1-150)
14:00 - 16:00
Poster Session II (151-300)
14:00
Excursions
14:00 - 16:00
Poster Session III (301-438)
CASCADE BIOCATALYSIS 16:00 - 16:30 16:30 - 16:50 16:50 - 17:10 17:10 - 17:30 Harald GRGER Marko MIHOVILOVIC Ekaterina CHURAKOVA Ioulia SMONOU 16:00 - 16:30 16:30 - 16:50 16:50 - 17:10 17:10 - 17:30
NEW ENZYMES FOR BIOTRANSFORMATIONS Martin SCHRMANN Hiroyasu OGINO Pietro GATTI-LANFRANCONI Makoto HIBI 16:00 - 16:20 16:20 - 16:40 16:40 - 17:00
STANDARDS AND BIOINFORMATICS Peter HALLING Dmitry SUPLATOV Marilize LE ROES-HILL
Key-note Lecture Uwe BORNSCHEUER
17:30 - 18:00
Coffee break
17:30 - 18:00
Coffee break
17:00 - 17:30
Coffee break
18:00 - 18:30 18:30 - 18:50 18:50 - 19:10 19:10 - 19:30
Pere CLAPES Lothar ELLING Lzl POPPE Paloma SANTACOLOMA
THDP-DEPENDENT ENZYMES 18:00 - 18:30 18:30 - 18:50 18:50 - 19:10 19:10 - 19:30 Helen HAILES Ayhan Sitki DEMIR Kai TITTMAN Micheal MLLER 17:30 - 17:50 17:50 - 18:10 18:10 - 18:30 18:30 - 19:00
SCALING-UP BIOTRANSFORMATIONS Ulrich GIESECKE Loris SINIGOI Mattias BECHTOLD Tao LI
Testo
19:00 - 19:15
Conclusions
20:30
Conference Dinner Poster Awards
215

Abstract Book Biotrans 2011

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Biotrans 2011 Italy

Permanent Steering Committee

Under the patronage of

Biotrans 2011 - Italy

October 2-6, 2011

Sunday, October 2nd

Monday, October 3rd

10:15 - 10:45 18:00 - 18:15

11:05 - 11:25 11:25 - 11:45

11:45 - 12:05 19:30

OC-4 OC-5 IL-2

12:05 - 12:25 12:25 - 12:55

Poster Session 1 (1-150)

Biotrans 2011 - Italy

October 2-6, 2011

Monday, October 3rd

Tuesday, October 4th

Session 2: Cascade Biocatalysis Chairs:

16:50 - 17:10 17:10 - 17:30

Session 3: Biocatalysis and Natural Compounds Chairs:

Jrg PIETRUSZKA, Dsseldorf Universitt and Forschungszentrum, Jlich, German

Jennifer ANDEXER, University of Cambridge, Cambridge, UK

11:25 - 11:45 11:45 - 12:05 12:05 - 12:25

OC-14 Biocatalytic cyclization of citronellal

Gabriele SIEDENBURG, University of Stuttgart, Stuttgart, Germany

Maarten GROENEVELD, University of Groningen, Groningen, The Netherlands

and beyond Joerg SCHRITTWIESER, University of Graz, Graz, Austria

Paloma SANTACOLOMA, Technical University of Denmark, Lyngby, Denmark

Poster Session 2 (151 - 300)

Biotrans 2011 - Italy

October 2-6, 2011

Tuesday, October 4th

Wednesday, October 5th

Session 4: New Enzymes for Biotransformations Chair:

OC-17 Development of organic solvent-tolerant enzymes

Pietro GATTI-LAFRANCONI, University of Cambridge, Cambridge, UK

Makoto HIBI, Kyoto University, Kyoto, Japan

Session 6: New Applications for Biocatalysis Chairs:

11:45 - 12:05 12:05 - 12:25

Session 5: ThDP-Dependent Enzymes (sponsored by Thiamine consortium) Chair:

synthesis Mats MARTINELLE, Royal Institute of Technology, Stockholm, Sweden

OC-28 In vitro protein and metabolic engineering of a biodegradation pathway

Pavel DVORAK, Institute of Microbiology, AVCR, Prague, Czech Republic

Kai TITTMAN, Georg-August-Universitt Gttingen, Gttingen, Germany

Michael MLLER, University of Freiburg, Freiburg, Germany

Biotrans 2011 - Italy

October 2-6, 2011

Thursday, October 6th

Thursday, October 6th

work in biocatalysis Peter HALLING, University of Strathclyde, Glasgow, UK

Jennifer LITTLECHILD, University of Exeter, Exeter, UK

OC-30 Structure and mechanism of bacterial maleate isomerase

Coffee Break Session 8: Biotransformations (sponsored by Evocatal GmbH)

Session 10: Scaling-up Biotransformations Chair:

Mandana GRUBER-KHADJAWI, Austrian Centre of Industrial Biotechnology, Graz, Austria

(DHMEQ) Takeshi SUGAI, Keio University, Tokyo, Japan

OC-33 Production, characterization and synthetic application of a purine nucleoside phosphorylase

from Aeromonas hydrophila Daniela UBIALI, Pavia University, Pavia, Italy

OC-34 Chemoenzymatic syntheses of selected biologically active chiral heteroorganic compounds

chemicals Mattias BECHTOLD, ETH, Zurich, Switzerland

Alexandre ZANGHELLINI, Arzeda Corporation, Seattle (WA), USA

13:00 - 14:00 14:00 - 16:00

Lunch Poster Session 3 (301 - 438)