MICROBIAL ENZYMES

Johnry S. Maloles
MCB 260 Advanced Industrial Microbiology November 28, 2012

What are Enzymes?

Enzymes are active complex protein molecules (natural chemicals) that are produced and used by living organisms, catalyzing the rate of different biochemical reactions. Enzymes are very specific, fully biodegradable, and works under mild conditions (pH/temperature) Can be: intracellular (ENDOZYME) or extracellular (EXOZYME)

Although enzymes are formed within living cells, they can continue to function in vitro

What are Enzymes?

In Biochemistry, it can be classified based on the type of reaction catalysed: - Oxidoreductases - Transferases - Hydrolases - Lyases - Isomerases - Synthetases Can be obtained from:

PLANTS
Papain Ficin

ANIMALS

MICROBES
Amylase Protease Cellulase Xylanase

Rennet

Advantages of microbes as source of enzymes

1. Production of microbial enzymes is economical. 2. They can be easily extracted and purified.

3. Microbes can grow in a wide range of environmental conditions.

4. Microbes are capable of producing a wide variety of enzymes. 5. They can be genetically manipulated to improve their product productivity. Plants & Animals.
grow slowly in comparison with microorganisms; content often up to 1% enzyme of tissue weight; problems with antibodies and side reactions inhibiting their growth; and less competitive compared to fermentation of microorganisms.

ENZYME
PRODUCTION

Plant cell Animal organs

Microbial Fermentations
Extracellular enzymes

Plant Latex

Intracellular enzymes

liquid/solid separation solids Cell Disintegration

liquids

ADVANTAGES OF EXTRACELLULAR PRODUCTION OF ENZYMES

-Already outside the cell

liquid/solid separation solids liquids Nucleic acid Removal

WASTE

-Limited # of type of enzyme secreted (easier to isolate)

-More robust (highly resistant)

Purification
Concentration Finishing

PRODUCT
Source: Aehle, 2008 Enzymes in Industry

Extremozymes Enzymes that function at high temperatures or extreme environmental (cold, very high salt, very acid or very alkaline pH) - Used as biocatalysts - e.g. Taq polymerase, thermostable proteases, amylases,and cellulases

Immobilized Enzyme In industrial processes, it is desirable to attach enzymes to a solid surface Isolated enzymes can be immobilised so that they do not contaminate the end product and can be used again and again
Source: Madigan et al, 2011 Biology of Microorganisms

STRAIN SELECTION / DEVELOPMENT

Does is do what is required? Is it safe? Is it cost effective?

STRAIN SELECTION / DEVELOPMENT

Yeast

Each single strain of organism produces a large number of enzymes

Fungi

Produced various individual enzymes vary in amounts Commercial quantities can be obtained by isolating microbial strains

Bacteria
>Saccharomyces cerevisiae, Aspergillus sp., and Bacillus sp.
(http://etc.usf.edu/clipart/15400/15447/grwyeastcell_15447.htm) (http://www.bioquicknews.com/archive/story/2009?page=6)

STRAIN SELECTION / DEVELOPMENT

Yeast

PROPERTIES

Fungi

Bacteria
>Saccharomyces cerevisiae, Aspergillus sp., and Bacillus sp.
(http://etc.usf.edu/clipart/15400/15447/grwyeastcell_15447.htm) (http://www.bioquicknews.com/archive/story/2009?page=6)

1. Capable of producing the enzyme of interest 2. Grows fast and produces enzyme in large scale production 3. Compatible with substrates 4. Easily manipulated genetically 5. Genetically stable 6. Safe

STRAIN SELECTION / DEVELOPMENT

Suitable enzyme source available? Class I organism? YES Enzyme production sufficient? Cell strain stable? New enzyme source screening Gene transfer to class I organism success NO Improve production by gene tech Improve fermentation conditions

NO

NO

YES
Downstream processing cost minimized? Purity stable? YES

success NO
Improve downstream processing lower cost and waste, higher purity success NO

NO

STRAIN SELECTION / DEVELOPMENT

Development of Microbial Strains for Enzyme Production
involves microbiology, biochemistry, and genetics usually done when yield in wild type organism is low or desired enzyme is not in class I organism strategies/techniques for strain improvement - Mutation - Recombination - Recombinant DNA Technology

STRAIN SELECTION / DEVELOPMENT

Mutation
Spontaneous
Occur spontaneously at the rate of 10-10 and 10-15 per generation and per gene. Occur at low frequency and hence not used much in industrial strain improvement. any change in the base sequence of DNA deletion, insertion, inversion, substitution

Induced
The rate of mutation can be increased by various factors and agents called mutagens. ionizing radiations X-rays, gamma rays non-ionizing radiations ultraviolet radiations various chemicals mustard gas, benzene, ethidium bromide, Nitrosoguanidine-NTG

Site-Directed
Change in the base sequence of DNA

(http://users.wmin.ac.uk/~redwayk/lectures/sdm.htm)

STRAIN SELECTION / DEVELOPMENT

Recombination Protoplast Fusion

- cell fusion followed by nuclear fusion occur between protoplast

- efficient way to induce heterokaryon formation and recombination with high frequency
- the organism selected for fusion should be genetically related - Types: Intraspecific hybridization Interspecific hybtidization Intergeneric hybridization

STRAIN SELECTION / DEVELOPMENT

Recombination
A B C

Other Types

A. Transformation B. Transduction C. Conjugation

Source: Madigan et al, 2011 Biology of Microorganisms

STRAIN SELECTION / DEVELOPMENT

Recombinant DNA Technology
- to increase the yields and consistencies of enzymes - increases the production of heterologous proteins by:
(1) increasing the gene expression using strong promoters
(2) deletion of unwanted genes from the genome (3) manipulation of metabolic pathways

- Specific Goals:
(1) to get multiple copies of specific gene

(2) to get high amounts of specific protein (enzyme)

STRAIN SELECTION / DEVELOPMENT

Recombinant DNA Technology

Source: Lehninger The Foundations of Biocehmistry

STRAIN SELECTION / DEVELOPMENT

Source: Biocatalysis: Enzyme Production and Purification

STRAIN SELECTION / DEVELOPMENT

PROTEASES
proteolytic enzymes representing one of the three largest groups of industrial enzymes (accounting for 60% of total worldwide sale of enzymes Serine proteases Threonine proteases Cysteine proteases Aspartate proteases Metalloproteases

Source: Barredo, 2005 Microbial Enzymes and Biotransformations

STRAIN SELECTION / DEVELOPMENT

Isolation of Proteolytic Microorganisms (Barredo, 2005)
Suspend 1g of soil sample in 5mL sterile dH2O Streak aliquots of the clear suspension onto *nutrient agar medium containing 1% casein Incubate for 48h at 37 C Observe protease production as a clearing zone around the colondy in protease positive colonies
*nutrient agar medium (per 1L): 5 g peptic digest, 1.5 g beef extract,
1.5 g yeast extract, 5 g NaCl, and 15 g agar (pH of 7.4 at 25 C)

STRAIN SELECTION / DEVELOPMENT

Cloning of Gene for Alkaline Proteases (Sadeghi et al, 2009)
Genomic DNA of Bacillus subtilis with gene coding for alkaline protease is extracted - High Pure PCR Template Preparation Kit

Gene encoding for alkaline protease is amplified by PCR - template DNA - F primer (5- CATATGTTTGGGTACTCTATGG-3) - R primer (5-GGATCCTTATTGGCCGGGAACGGAA-3) - taq polymerase, dNTPs - optimal thermocycling profile
Product is analyzed using gel electrophoresis and digestion with restriction enzymes

STRAIN SELECTION / DEVELOPMENT

Cloning of Gene for Alkaline Proteases (Sadeghi et al, 2009)
Amplified gene inserted to a cloning vector - plasmid vector (pTZ57R) - PCR product - DNA ligase - buffer - 16 C, overnight E. coli HB101 competent cells are prepared by CaCl2 method for 39 C for 1min, and transformation is executed Screening of transformed colonies on LB agarampicillin culture medium (confirmed with appropriate restriction enzymes

FERMENTATION PROCESS
Most enzyme production is carried out in deep submerged fermentation; a few are best produced in semi-solid media.

FERMENTATION PROCESS

(http://www.anilbioplus.com/infrastructure/manufacturing.htm)

FERMENTATION PROCESS
Most industrial enzymes are products of batch processes; few are currently produced via continuous fermentation Fermenters for bulk enzyme production are up to 100m3 capacity, but fine enzymes may be produced on smaller scales of a few hundred litres or less The medium must be chosen to stimulate the microbe into synthesizing the correct enzyme What type of medium would you use to stimulate a microbe to synthesize a specific enzyme?

FERMENTATION PROCESS
Factors to consider prior to fermentation:
1) use of GRAS-listed (generally regarded as safe) organisms - incubation period 2) knowledge of the properties of the enzyme of interest - optimum pH and heat resistance

- ability to secrete the target enzyme

3) source of inexpensive carbon and nitrogen feedstock 4) knowledge of enzyme stability for downstream processing

FERMENTATION PROCESS
Semi-solid Medium
aka Koji or moldy bran method of solid state fermn
medium consists of moist sterile wheat/rice bran acidified with HCl; mineral salts including trace minerals are added; inducer is usually added organism used are fungi amenable to high enzyme production condition

labor intensive and expensive, prone to contamination

FERMENTATION PROCESS
Submerged Production
generally greater ease of controlling temperature, pH and other environmental factors in a fermentor
medium must also contain all the requirements for growth (C, N, various metals, trace elements)
*medium adequate for growth may not be satisfactory for enzyme production

inducers are also added that act as substrates rennet prodn soy bean proteins are added to induce protease prodn by fungi

FERMENTATION PROCESS
Submerged Production
inducer is not always the substrate for enzyme production but sometimes, the end product is the substrate (cellobiose stimulate cellulose prodn) medium components may repress enzyme production by catabolite repression
*glucose -amylase in B. licheniformis and B. subtilis *fructose enzymes in B. stearothermophilus *isoleucine and proline protease in B. megaterium *sulfur-containing aa protease in Aspergillus niger

temperature, pH, and oxygen requirements should be optimized (most mcgs used are aerobic)

FERMENTATION PROCESS
Production of Protease
proteases of all species of Bacillus are produced in a submerged culture fermentation system carbon source: starch, ground barley, or lactose ...nitrogen source: soybean meal or casein carbohydrate is added continuously in small amounts to keep concentration low usually supplemented with 10-15% dry substance and high protein content repressors: high [carbohydrate] and free amino acids ... inducers: peptides and proteins

FERMENTATION PROCESS
Production of Protease
Materials - culture medium casamino acids (variable), glucose (variable), 1 g/L KH2PO4, 3 g/L K2HPO4, 2 g/L Na2SO4 and 0.1 g/L MgSO47H2O - microorganism - orbital shaker - cooling centrifuge - agroindustrial residues (substrate) - salt solution - incubation chamber in Aspergillus oryzae - 4 proteases are formed in submerged fermentation - 2 proteases are formed in semi-solid medium and are less heat resistant

PRODUCT RECOVERY
- Whether enzyme is intracellular or extracellular - How pure the final product needs to be

PRODUCT RECOVERY
Generally account more than 50% of total enzyme production costs Dependent on degree of purity

Involves Separation, Purification, Stabilization, and Preservation

Source: Waites et al, 2009 Industrial Microbiology

PRODUCT RECOVERY
Stabilizers may be added: calcium salts proteins sugars starch hydrolysates Destabilizing metals are removed using EDTA Antimicrobials (optional) benzoates sorbate
Source: Waites et al, 2009 Industrial Microbiology

PRODUCT RECOVERY
Intracellular Enzyme
cells are needed to be disrupted mechanically or enzymatically

MECHANICAL
ball mills use of small abrasive particles ultrasonic disruption use of high sound frequency blenders blades rotate at speeds of 6,000 - 50,000 rpm freeze fracturing water crystals as abrasives

NON-MECHANICAL
chemical permeabilization (e.g., organic solvents, surfactants) enzymatic permeabilization (e.g., glycanases, proteases) osmotic shock (e.g., high sucrose medium)

PRODUCT RECOVERY
Intracellular and Extracellular Enzyme
separation of cellular debris from medium
A. Centrifugation
tubular bowl scroll

http://www.sgconsulting.co.za/flottweg-sedicanter.php

multichamber disc-stack

http://www.jvcgyroscreen.com/tab_cent.html

http://www.sciencedirect.com/scienc e/article/pii/S0015188205705875

PRODUCT RECOVERY
Intracellular and Extracellular Enzyme
separation of cellular debris from medium
B. Microporous Membrane Filtration
microfiltration (0.1 to 1 m) suspended solids, cells ultrafiltration (0.01 to 0.1 m) protein fractionation nanofiltration (0.001 to 0.01 m) low-molecular weight organics, color removal, desalination reverse osmosis (< 0.001m) demineralization
(http://www.winesecrets.com/wine-ultra-filtration/wine-ultra-filtration.asp)

PRODUCT RECOVERY
Further Purification by CHROMATOGRAPHY impurities (e.g., proteins, DNA and others) further purification when safety (e.g., recombinant DNA, viruses) or functional reasons (impurities disturbing catalytic function) basic knowledge of protein property necesary molecular weight (MW) isoelectric point (pI) cofactors pH range greater than 10% of enzyme is lost due to multiple-step manipulation

PRODUCT RECOVERY
Further Purification by CHROMATOGRAPHY
Enzyme Properties Size-exclusion chromatography Anion-exchange chromatography Cation-exchange chromatography Hydrophobic chromatography Metal-Affinity chromatography Bifunctional/Multifunctional chromatography Biospecific chromatography Molecular weight Surface charge (at pH > pI) Surface charge (at pH < pI) Hydrophobicity of enzyme surface Histidines at the surface or His-tags Surface charge and hydrophobicity

Specific hydrophobic and electrostatic binding interaction with inhibitors, antibody or biospecific tags

PRODUCT RECOVERY
Final treatment of Enzymes enzymes distributed as solutions, powders or immobilized Solution preservation and stabilization agents sterilization by filtration (e.g., therapeutical enzymes) Powder freeze-dried (small particles can be inhaled) spray-dried and coating/cover with protecting layer (particle size can not be inhaled)

Quality Control:

(1) Purity (2) Activity (3) Identity of enzyme produced

PRODUCT RECOVERY
Recovery and Purification of Protease
first step is separation of cell biomass and insoluble nutrient ingredients from the supernatant purification is by precipitation and chromatographic procedures recovery of protease: A. Ammonium Sulfate Fractionation - protein fractions are precipitated by successively increasing the concentration of salt (sulfate/sulfite) - proteins tend to aggregate and precipitate out of the solution B. Gel Filtration Chromatography (1) Purity Quality - separates protease using proper filtration matrix (2) Activity Control: (3) Identity of enzyme produced

PRODUCT RECOVERY
Recovery and Purification of Protease
Materials - 1% NaCl - 80% (NH4)2SO4 - 0.2M citrate phosphate buffer, pH 6.5 - magnetic stir plate and stir bar - centrifuge - dialysis tubing - chromatography equipment - appropriate gel matrix Quality check: measurement of protease activity - a colorimetric assay using azocasein (chemically modified (1) Purity protein) as a substrate can be used to determined the Quality (2) protease activity inActivity crude and purified samples

Control:

(3) Identity of enzyme produced

APPLICATIONS
Detergents Starch processing and related carbohydrases Cheese and plant juice production Textile and leather manufacturing Treatment of wood pulps Catalysts in organic synthesis

APPLICATIONS
Detergents Lower Temperature & No Phosphate Clothes Washing
- introduction of proteases that are active at alkaline pH - a well-known example is subtilisin, a bacterial alkaline serine protease from Bacillus licheniformis and B. subtilis, which is used extensively in laundry detergents - enzyme has now been engineered to improve pH and temperature characteristics, and reduce sensitivity to peroxide - protease (degrades protein stains) cutinase (degrades mixutre of fatty acids) amylases and lipases

APPLICATIONS
Starch processing and related carbohydrases Sugar syrups from Starch
- manufacturing of sugar syrups containing specific mixtures of sugars that could not be produced by conventional acid hydrolysis of starch

- -amylase generates glucose, maltose, and maltotriose industrially employed in brewing and baking thermostable types from Bacillus species - amyloglucosidase breaks down starch and dextrin into glucose from Aspergillus niger and Rhizopus species - glucose isomerase converts glucose into fructose (sweetener) from Bacillus and Streptomyces species - invertase converts sucrose into glucose and fructose (syrup and confectionery) from A. niger and A. oryzae

APPLICATIONS
Dairy Applications Cheeses
- rennet from animals were replaced with proteases from Rhizomucor miehei and R. pusillus (vegetarian cheese)

- calf chymosin gene has been introduced to several microorganisms like E. coli and Aspergillus species

Cheese Flavors
- microbial lipases for the hydrolysis of fatty acid esters to accelerate flavour development (wide variety of high quality cheeses)

Lactose-Free Dairy Products

- lactase converts milk sugar (lactose) into glucose and galactose *approximately 20-30% of adults are lactose intolerant

APPLICATIONS
Textile Manufacturing
- removing the adhesive components (may be composed of starch, starch derivatives, vegetable gum, and water-soluble cellulose derivatives) before dyeing, bleaching, and printing

- use of amylases and cellulases as they are non-corrosive and produce no harmful effluent wastes
- cellulases are used for biopolishing cotton and other cellulose fibers *for smoother and glossier appearance - cellulases for stonewashing *accelerates abrasion and aid the loosening of the indigo dye

APPLICATIONS
Plant Juice Production Leather Processing Treatment of Wood Pulps

Catalysts in Organic Synthesis

Contact Lens Cleanser Reduced Phosphorus Animal Feed Ethanol Fuel from Renewable Resources Paper Manufacturing Beverage Production

APPLICATIONS

(Waites, 2001)

(http://www.anilbioplus.com/aboutus/industrial-enzymes.htm)

THANK YOU

References:
Aehle W. 2008. In Enzymes in Industry.Hoboken, NJ: Wiley-VCH. Barredo, J. 2005. Microbial enzymes and biotransformations. Totowa, N.J.: Humana Press. Lehninger. The Foundations of Biochemistry Madigan M, J Martinko, D Stahl and D Clark. 2011. Brock: Biology of Microorganisms. 13th edition. San Francisco, CA. Benjamin Cummings Okafor, Nduka. 2007. Modern Industrial Microbiology And Biotechnology. Enfield, (NH): Science Publishers. Polaina, J and MacCabe, AP. 2007. Industrial enzymes: Structure, function and applications. Dordrecht: Springer. Sadeghi HM, Rabbani M, Naghitorabi M. 2009. Cloning of alkaline protease gene from Bacillus subtilis 168. Research in Pharmaceutical Sciences: Vol 4(1): p. 43-46. Vermelho AB, Supuran CT, and Guisan JM. 2012. Microbial Enzyme: Applications in Industry and in Bioremediation, Enzyme Research, vol. 2012, Article ID 980681, 2 pages, 2012. doi:10.1155/2012/980681 Waites MJ, Morgan NL, Rockey JS, and Higton G. 2009. Industrial Microbiology: An Introduction. In Industrial Microbiology pp19). Hoboken, NJ: Wiley-Blackwell.
http://www.iogen.ca/company/enzymes_technology/index.html http://www.bioquicknews.com/archive/story/2009?page=6 http://etc.usf.edu/clipart/15400/15447/grwyeastcell_15447.htm http://www.accessscience.com/search.aspx?rootID=792645 http://users.wmin.ac.uk/~redwayk/lectures/sdm.htm http://www.anilbioplus.com/infrastructure/manufacturing.htm http://www.sgconsulting.co.za/flottweg-sedicanter.php http://www.jvcgyroscreen.com/tab_cent.html http://www.winesecrets.com/wine-ultra-filtration/wine-ultra-filtration.asp http://www.sciencedirect.com/science/article/pii/S0015188205705875 http://loschmidt.chemi.muni.cz/peg/lecture/biocat_lecture08.pdf