1.

INTRODUCTION
There are three domains of life, which are archaea, eukaraya and bacteria. Archaea are further divided into four main groups among them Crenarchaeota and the Euryarchaeota are most extensively studied. The second domain, eukarya, consits of organism whose cells contain complex structures enclosed within membranes. They comprises of all animals, plants, fungi, and protists. The third domain, bacteria are a large group of unicellular microorganisms lacking the nuclear membrane. Important groups of organisms are displayed in Fig.1.

Fig.1. Three domains of life. This figure is adopted from http://www.biologie.uni-hamburg.de/bonline/library/micro229/terry/images/other/3domains.gif

Bacterial domain is divided into three major groups: 1) Gram-negative eubacteria that contain cell walls. They have a complex cell wall comprising of outer membrane and a thin inner membrane.

2) Gram-positive eubacteria that have a cell wall. They are able to retain the crystal violet stain because of the high amount of peptidoglycan in the cell wall. Their cell walls typically lack the outer membrane found in Gram-negative bacteria. 3) Eubacteria lacking cells walls. They are non motile but some have gliding movement. Cells stain gram negatively. The percentage of guanine plus cytosine of ribosomal RNA is less than gram-positive and gram-negative bacteria. These microorganisms are further divided on the basis of phenotypic grouping comprising of biochemical and morphological characteristics. These groups are also classified by DNA relatedness, molecular finger printing (Grimont and Grimont 1991) and on the basis of 16S rRNA relation (Clarridge 2004). There are various factors, which directly approach the DNA relatedness. They include: i) Genome size ii) Guanine + Cytosine content iii) DNA reassosiation under optimal condition iv) Related sequences having thermal stability v) DNA relation under supra optimal conditions The phylogenetic relation is usually based on 16S rRNA sequence because it is highly conserved in the process of evolution. Final identification of a microorganism is a polyphasic approach comprising of all the above factors (Clarridge 2004). E. coli and Bacillus subtilis are the most studied microorganisms among gram-negative and grampositive bacteria, respectively.

1.1. B. subtilis
Members of genus Bacillus are ubiquitous in nature. B. subtilis strains are aerobic, rod shaped and endospore formers. Endospores are resistant to chemical and physical

agents. Production of spores plays a vital role for dispersal of the genus in the environment and its ubiquity in the world (Nakano and Zuber 1988). The genome sequence of B. subtilis was completed in 1997 and was the first published sequence for a gram-positive bacterium. The genome is 4.2 Mega-base pairs along with 4,100 protein-coding regions (Kunst et al., 1997). B. subtilis is an organism which is easy to manipulate genetically and is considered as a model organism. The microorganism is heavily flagellated which enables it to move swiftly. Bacillus may contaminate the food but food poisoning is rarely caused. B. subtilis is not considered as the human pathogen and according to food and drug administration (FDA) B. subtilis is generally recognised as safe (GRAS).

1.1.1. B. subtilis: a protein secretory machine

Secretary proteins of B. subtilis are comprised of three parts including signal peptide, propeptide and the mature protein. Signal peptides are present at the N-terminal and have an importance in the export of the protein. Most of the amino acids in this region are hydrophobic in nature (Briggs and Gierasch 1986; Gierasch 1989). Positively charged amino acids in this region have an affinity towards the negatively charged cell membrane (Jones et al., 1990). Average length of this hydrophobic core region comprises of 17 amino acid residues, which is embedded in the membrane. The hydrophobic core region of the signal peptide is thought to span in the cell membrane from where the translocation starts. Distribution of amino acid in this hydrophobic core is as follows: L, 24%; S, 13%; F, I, A, V and T, 10% each; M and G, 4% each; C, W, Y, P, Q and N are collectively 5% (Perlman and Halvorson 1983). Position -1 and -3 of the C-terminal of signal peptides usually contains >90% Ala and 50% of the +1 mature protein contains alanine (Smith et al., 1988). The steps involved in the secretion of these proteins are:

i) ii) iii)

The secretary proteins first are inserted into the membrane. They are translocated across the membrane. They are then passed through the cell wall and secreted outside the cell.

1.1.2. Industrial potential B. subtilis

B. subtilis is of high importance in industry due to variety of reasons including high growth rate, short life cycle, secretion of extracellular proteins, generally regarded as safe and availability of complete genome sequence which has opened up a new horizon in this specie which comprises of 4100 protein coding genes (Kunst et al., 1997). The economic market of the industrial enzymes comprised of 1.6 billion US dollars at the start of the twenty first century (Outtrup and Jorgensen 2002). The industrial enzymes comprise of general technical enzymes, which are 56%, food enzymes are 29% and feed enzymes are 15% of the world market. Species of genus Bacillus in general and B. subtilis in particular are good source of industrially important enzymes. Enzymes from this genus make upto 50% of the total enzyme market. The enzymes produced by various species of genus Bacillus include cellulases, xylanases (Singh and Hayashi 1995; Kuhad et al., 1997), amylases (Pandey et al., 2000), chitinases (Thamthiankul et al., 2001), proteases (Rao et al., 1998), CGTases (Gawande et al., 1999), esterases (Kademi et al., 2000) and many other enzymes. Apart from production of industrial enzymes, the members of genus Bacillus are important because of their ability to produce antibiotics and biosurfactants (Eppelmann et al., 2001; Peypoux et al., 1999; Fox and Bala 2000; Moran et al., 2000). The antibiotics produced by B. subtilis include subtilin, bacilysin, subsporin A-C. lipooligopeptide and rhizocticins A-D, phosphooligopeptide (Priest 1992; Defuria and Claridge 1976). B. subtilis also produces a biosurfactant. Biosurfactants are chemically complex molecules

comprising of different structures, which include peptides, glycopeptides, glycolipids, phospholipids and fatty acids (Mercade and Manresa 1994). These are produced on the surface of the microorganisms or extracellularly secreted and contain both hydrophilic and hydrophobic moieties. Chemically produced surfactants are rapidly being replaced by the biosurfactants (Lin et al., 1998). Biosurfactants have a lot of applications in on going industrial processes involving emulsification, wetting, dispersing, foaming, detergency and solubulization, which are being utilized in household and different industrial processes (Makkar and Cameotra 1997). The biosurfactant produced by this organism is called surfactin which is a very powerful surfactant commonly used as an antibiotic. It is a cyclic lipopeptide, largely prominent for its exceptional surfactant power (Mor 2000). It is one of the 24 different types of antibiotics produced by B. subtilis (Peypoux et al., 1999). B. subtilis has also been utilized to produce high levels of riboflavin through recombinant DNA techniques and fermentation (Perkins et al., 1999). D-Ribose has been used as a flavor enhancer in food, pharmaceutical, cosmetics, animal food and health food. It has also been used for the treatment myocardial ischemic and muscular pain so the Dribose production is of immense importance. Several strains of B. subtilis are reported to produce D-ribose (De Wulf and Vandamme 1997). Bacillus spores are easy to manipulate genetically and are being used as an attractive vehicle for oral and intranasal vaccination. Spores are commercially produced and used as probiotics (Casula and Cutting 2002). Its sopres are also being used as a vehicle for tetnus toxin fragment C antigen for oral and intranasal immunization (Duc et al., 2003). All the potential applications described earlier make B. subtilis a potent organism for many industries. Therefore further exploration of novel strains of B. subtilis may open

the new horizons of industrial applications. This study comprises of isolation and characterization of a novel strain of B. subtilis.

1.2. Enzymes
Enzymes are biomolecules that speed up a chemical reactions. They act as catalysts for specific chemical reactions, converting specific substrates into specific products. Most of the known enzymes are proteins. Enzymes are classified into following 6 groups: i) Oxidoreductases (EC 1) Oxidoreductases catalyze oxidation-reduction reactions in which electron transfer takes place from one specie to another specie. A + B ii) Transferases (EC 2). Transferases are enzymes that transfer a functional group e.g. a methyl or phosphate from one compound to another compound. AX + B iii) Hydrolases (EC 3) Hydrolases catalyze the hydrolytic cleavage of C-O, C-N, C-C and some other bonds as well, including phosphoric anhydride bonds. They include lipases, amylases, cellulases, xylanases, chitosanases and proteases. AB + H2O iv) Lyases (EC 4) Lyases are enzymes which cleaves C-C, C-O, C-N, and other bonds by elimination, leaving double bonds or rings, or conversely adding groups to double bonds. ATP cAMP + PPi AOH + BH A + BX A + B

v) Isomerases (EC 5) Isomerases catalyze geometric or structural changes within one molecule. They are named owing to different types of isomerism; they may be called racemases, epimerases, cis-trans-isomerases, isomerases, tautomerases, mutases or cylcoisomerases. A vi) Ligases (EC 6) Ligases catalyze the joining together of two molecules usually coupled with the hydrolysis of a pyrophosphate bond in ATP or a similar triphosphate. The bonds formed are often high-energy bonds. Ab + C AC + b or Ab + cD AD + b + c B

1.3. Hydrolases or Hydrolytic Enzymes

Hydrolytic enzymes catalyze the hydrolysis of a chemical bond of a compound such as proteins, nucleic acids, starch, fats, phosphate esters, and other macromolecular substances. AB + H2O AOH + BH

Hydrolytic enzymes which cleave substrates with the incorporation of water, falls into the general categories of glycosidases (amylases, xylanases and cellulases), lipases, esterases (including nucleases, phosphatases, and kinases), polymerases, and proteases. Hydrolases are classified according to their action on different bonds. They are: EC 3.1 EC 3.2 EC 3.3 EC 3.4 EC 3.5 Acting on ester bonds Acting on glycosyl bond Acting on ether bonds Acting on peptide bonds (Peptidases or peptide hydrolases) Acting on carbon-nitrogen bonds, other than peptide bonds

EC 3.6 EC 3.7 EC 3.8 EC 3.9 EC 3.10 EC 3.11 EC 3.12

Acting on acid anhydrides Acting on carbon-carbon bonds Acting on halide bonds Acting on phosphorus-nitrogen bonds Acting on sulfur-nitrogen bonds Acting on carbon-phosphorus bonds Acting on sulfur-sulfur bonds

1.4. Chitosanase
Chitosanases (EC 3.2.1.132) are members of glycosyl hydrolase family 8 and 75. They act on chitosan, a polymer of -(1-4) linked glucosamine residues. Chitosan and Nacetylated analogue of chitin are the most abundant glycans in nature (Pantaleone et al., 1992) and they are substrates of chitosanases. Main sources of different chitosanases include bacteria, fungi and plants. Chitosanase from different species have different hydrolytic activity pattern which is dependent on degree of substrate acetylation (Kurita et al., 1977; Sakai et al., 1991; Seino et al., 1991). Most of the fungi and bacteria secrete chitosanases extracellularly (Monaghan et al., 1973; Yoshihara et al., 1990) whereas in plants they are intracellular (Grenier et al., 1991). Bacillus subtilis 168, one of the strains of genus Bacillus whose full genome have been sequenced, harbors an open reading frame that encodes a chitosanase. Infact the gene product have been partially purified and characterized (Rivas et al., 2000). The protein (accession no. CAB14630) consists of 277 amino acids and have a homology of 77% with a thermostable chitosanase from Bacillus sp. strain CK4 (Yoon et al., 2000).

1.4.1. Structure and domain

Chitosanases comprises of two helices and a three stranded beta sheet, which collectively form the substrate binding and catalytic cleft. This structural property is not only present in this enzyme but also in chitinase and lysozyme although they share very low significant amino acid similarities. Presence of cystein residues is considered important for the quarternary structure of a protein in order to make disulphide bridges. Enzymes from mesophilic sources usually contain fewer cystein residues compared to their thermophilic counterparts. A thermostable chitosanase from Bacillus sp. CK4 was reported to contain three cystein residues at poisiton 49, 72 and 211. Mutagenesis studies at position 49 and 72 showed a slight decrease in enzyme activity where as a mutation at 211 position showed a drastic effect on the enzyme activity (Yoon et al., 2000) Chitosnase originating from Streptomyces sp. N174 has been extensively characterized and its crystal structure is available. Circular dichorism studies on this enzyme have proved that Glu-22 and Asp-40 are directly involved in the catalytic centre (Monzingo et al., 1996). This enzyme has an inverting mechanism reaction in the binding cleft. Site directed mutagenesis in the binding cleft has further confirmed that Glu-22 and Asp-40 are the catalytic residues. Tryptophan present in the binding cleft is not directly involved in the catalysis but it stabilizes the chitosanases by interacting with other hydrophobic and carboxylic side chain of the amino acids (Fukamizo and Brzezinski 1997). Based on structural similarties a putative substrate recognition mode proposes that Glu-22 acts as an acid and Asp-40 as general base to activate a water molecule for an SN2 reaction, which act on the glycosidic bond (Marcotte et al., 1996).

1.4.2. Difference of chitosanase and chitinase

Chitinases closely resembles to chitosanases and they have a common substrate that is chitosan. The functional difference between chitosanase and chitinase is not much prominent because both have the ability to degrade various chitosans with different degree of acetylation (Ohtakara et al., 1988). There are two main features which distinguish them are: i) Chitinases prefer to attack the highly N-acetylated polymer as compared to chitosanase (Fenton and Eveleigh 1981). ii) Chitosanases have low molecular mass as compared to chitinases (Grenier et al., 1991).

1.4.3. Enzymatic action

Microbial chitosanases are classified on the basis of their specificity of the cleavage of partially acetylated chitosan into three subclasses. Chitosanases belonging to class I can split GlcNAc-GlcN and GlcN-GlcN linkages where as chitosanases grouped in class II can cleave GlcN-GlcN linkages only Chitosanase which are placed in class III can split both GlcN-GlcN and GlcN-GlcNAc linkages (Fukamizo et al., 1994). Enzymatic action of chitosanase cleaves a glycosidic linkage of polymer containing varying degree of acetylated GlcN. For example, chitosanase activity of Penicillium islandicum has been documented to be optimal for degrading a polymer containing equal amounts of GlcN and GlcNAc (Fenton and Eveleigh, 1981), whereas chitosanase of B. circulans possess highest activity on 80% deacetylated chitosan (Yabuki et al., 1988). Additionally there are examples of chitosanases, which can break a bond of GlcN-GlcN, or GlcN-GlcNAc but not a bond of GlcNAc-GlcNAc as reported by studies on Nocordia orientalis (Sakai et al., 1991). This varying degree of acetylation in chitosans (Araki and Ito, 1975) was proposed to exert an evolutionary pressure to the development

10

of chitosanases with differential specificities for different substrates (Fenton and Eveleigh, 1981). It has been reported that about 25-35% N-acetylated chitosan was digested by chitosanase from B. pumilus BN-262. As a result glucosamine disaccharides were abundantly produced by splitting the -1,4-glycosidic linkage of GlcN-GlcN. The analysis of the product demonstrated the production of hetero oligosaccharide containing glucosamine and N-acetylglucosamine. These results showed that chitosanase also cleaves at the site of GlcNAc-GlcN (Fukamizo et al., 1994).

1.4.4. Applications
There are profound applications of chitosanases in generating low molecular weight oligomers of varying sizes of chitosan. These size specific oligomers have pharmaceutical importance (Yabuki et al., 1988). Moreover, chitosanases have also been used for producing fungal protoplast that is being used in basic studies (Fenton et al., 1978). In plants the chitosanases are believed to be involved in defense mechanism against pathogenic fungi (Grenier and Asselin 1990).

1.5. Lipases
One of the basic components of the biomass on the earth is lipid. Lipases play an important role for its mobilization in the cell and their transfer from one organism to another organism (Beisson et al., 2000). Lipases hydrolyze lipids into fatty acid and glycerol. Both of the products have importance in soap industry (Hoq et al., 1985). Bacteria produce lipolytic enzyme carboxyl esterase [E.C.3.1.1.1], which hydrolyzes small water-soluble ester containing molecules while true lipases [3.1.1.3] shows maximum activity on long chain triglycerides (Titball 1998; Songer 1997).

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1.5.1. Classification of lipases

Classification of true lipases comprises of six sub families. Recently, knowledge about structure of liapses increased owing to gene sequences and several crystal structures (Cygler and Schrag 1997; Schrag and Cygler 1997). Based on these sequences and crystal structures lipases have further been divided into families and subfamilies (Anthonsen et al., 1995; Drablos and Peterson 1997). A lot of work has been done to identify different sequence motifs, which are conserved in lipolytic enzymes present in higher and lower vertebrates, fungi and bacteria. The super family / hydrolase (Ollis et al., 1992) consists of a catalytic triad consisting of Ser, His and Asp that is similar to trypsin and subtilisin but not structurally similar. They also have a consenses pentapeptide composed of Gly-Xaa-Ser-Xaa-Gly. True lipases have a catalytic activity, which shows a competency in the industry because its substrate is water insoluble (Derewenda 1994). There is only one amino acid difference between the conserved pentapeptide of lipases from gram-positive bacteria and other organisms. The first Glycine of pentapeptide has been replaced by alanine and conserved pentapeptide is Ala-Xaa-Ser-Xaa-Gly. Two mesophillic strains of Bacillus subtilis and B. pumilus have smallest true lipase that is 20 kDa and sequence similarties is approximately 15% with other Bacillus and Staphylococcus lipases but the thermophillic bacterial strains like B. thermocatenulatus and B. staerothermophillus have 45 kDa molecular weight (Schmidt et al., 1996; Kim et al., 1998). Staphylococcal lipases are approximately 75 kDa when they are precursor proteins. This enzyme is cleaved by special protease into 400 amino acids mature protein and a propeptide of 207-267 residues. This propeptide helps the enzyme to move away from the intracellular to extracellular environment (Gotz et al., 1998). Lipases have been divided into eight families, which are given below.

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1.5.1.1. Family I and II Most of the enzymes whose crystal structure has been resolved belong to family I and II. Subfamily 1.1 of lipases comprises of 30-32 kDa and subfamily 1.2 consists of 33 kDa proteins (Noble et al., 1993; Lang et al., 1996; Schrag et al., 1997). These proteins contain two aspartates other than the aspartic acid present in the catalytic triad. These two aspartic acid residues are involved in the Ca++ binding and it is present in all sequence. Two cystein residues form the disulfide bridge. These two aspartic acid residue and disulfide bridge is present in the vicinity of the catalytic triad, which is proposed to stabilize the catalytic triad (Kim et al., 1997). Subfamily 1.3 members have higher molecular weight than subfamily 1.1 and 1.2. Secretion of these enzymes is reported via ATP binding cassette transporter system (Duong et al., 1994; Li et al., 1995). The enzyme grouped in family II are also called GDSL family because they contain Gly-Asp-Ser-(Leu) [GDS(L)] instead of conventional pentapeptide. The important residues lie close to the N-terminus (Upton and Buckley 1995). 1.5.1.2. Family III and IV Family III has the canonical fold of / hydrolases and also has typical catalytic triad. Enzymes of family IV are also called hormone sensitive lipase (HSL) family because they have a sequence homology with the HSL family (Hemila et al., 1994). These enzymes display relatively high activity similar to HSL (Feller et al., 1991; Langin et al., 1993). 1.5.1.3. Family V These enzymes are present in psychrophiles, mesophiles as well as in thermophiles. They share 20-25% homology with bacterial non-lipolytic enzymes, which

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also posses / hydrolase fold and catalytic triad (Verschueren et al., 1993; Misawa et al., 1998). 1.5.1.4. Family VI Enzymes in this group are the smallest esterases of 23-26 kDa. Three dimensional structure of one of the member has been resolved (Kim et al., 1997). The active enzyme is in the dimeric form having / hydrolase fold and a classical Ser, Asp and His catalytic triad. They hydrolyze small chain substrate but not long chain triglycerides (Hong et al., 1991). 1.5.1.5. Family VII These are large bacterial esterases of 55 kDa having significance homology with eukaryotic acetylcholine esterases and intestine/liver carboxyl esterases (Pohlenz et al., 1992). 1.5.1.6. Family VIII Enzymes of this family show similarity with class C -lactamase. The members of this family show approximately 45% similarity with Enterobacter cloacae amp C gene product (Galleni et al., 1988). This reflects that active site in the enzymes of this family is more reminiscent than the classs C -lactamase category although Ser-Xaa-Xaa-Lys motif is conserved in the N-terminal of both the enzymes (Nishizawa et al., 1995; Lobkovsky et al., 1993).

1.5.2. Industrial importance of lipases

Lipases have been involved in various industrial reactions either in organic or aquous systems, depending on their specificity (John and Abraham 1990; Kotting and Eibl 1994; Villeneuve and Foglia 1997; Godfrey and Hawkins 1991). In the dairy industry, 14

lipases are extensively used for the hydrolysis of milk fat. Applications of lipase include acceleration of cheese ripening, flavour enhancement of cheese, manufacturing of cheeselike products, and lipolysis of cream butter and fat (Falch 1991). It is the enzymatic hydrolysis, which offers the hope for fat splitting without investing hugely in expensive equipment as well as in huge investment in the production of thermal energy. The use of lipases in washing powders still remains the single biggest market for industrial enzymes (Arbige and Pitcher 1989). The application of lipases in the oleochemical industry is enormous, reason is that it saves energy and reduces thermal degradation during glycerolysis, alcoholysis and hydrolysis (Arbige and Pitcher 1989; Hoq et al., 1985). The introduction of the newly generated lipase enzymes can change the economic balance in the direction of this enzyme (Macrae and Hammond 1985). Owing to these processes the result is in the form of increased productivity. Acidolysis, the reaction that is done by 1,3-specific lipases, is utilized for the preparation of nutritionally important products, which contain mediumchain fatty acids (Eibl and Unger 1990). Lipases are being extensively studied with regard to the modification of oils rich in high-value polyunsaturated fatty acids. Substantial enhancement in the polyunsaturated fatty acid content of mono-glyceride fraction has been achieved by alcoholysis or hydrolysis and these reactions are catalyzed by lipases (Fregapane et al., 1991). They are also useful in the synthesis of a variety of biodegradable surfactants (amphoteric) (Hills et al., 1990). Wax esters have the applications in personal care products like skin and bath oils, and suntan creams. These products are being manufactured enzymatically using lipases (Hoq 1985) Lipases as industrial catalysts have application for the resolution of racemic alcohols in the preparation of some steroids, prostaglandins, and analogues of carbocyclic

15

nucleosides. They also useful through regioselective hydrolysis of octa-acetylsucrose for the synthesis of the artificial sweetner sucralose. The stereoselectivity is the phenomenon useful for synthesis of optically active polymers by lipases (Margolin 1987). These are asymmetric reagents, which are being used as absorbents. Transesterification of alcohols catalyzed by lipases produces suitable monomers, which are used in the field of liquid crystals (Pavel and Ritter 1992). Furthermore, this enzyme has potential application in many fields, for example they have successfully been used in paper industry, pulp treatment with lipase leads to a higher quality product and huge reduction in the area of waste cleaning. Similarly, the enzyme used in association with a microbial mixture for the treatment of fat-based effluents from an ice-cream plant. This is also used in waste processing of many food industries (Godfrey and West, 1996). The global market of the industrial enzyme was 2 billion dollars in 2004 and it is expected 2.4 billion dollars in 2009. Industrial enzymes are divided into three parts: Animal feed enzyme, food enzymes and technical enzymes. It is expected that in the current year the demand of animal feed enzyme would be highest (Rajan 2004). In technical enzymes, hydrolases including carbohydrases, proteases and lipases have a 95% share (Godtfredsen, et al., 1986). Carbohydrases and proteases are the major enzyme in food and animal feed. In laundry detergents, dishwashing and other cleaners enzymes such as cellulases, proteases, lipases and amylases are being used. Lipases also used in detergent to remove the fat stains from fabrics (Andree et al., 1980). Laundry detergent market exceeds 60% of the total world enzyme production. Alkaline enzymes have additional properties. Lipases are the third largest group (total sale volume) other than proteases and carbohydrases and the business of the lipases is a 1 billion dollars in the global market (Jaeger et al., 1999).

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1.5.3. Use of lipases in waste treatment

One of the beauties of the enzymes like lipase is that they are biodegradable. Therefore, their selective use produces less accumulation of waste (Posorske 1984). In the leather industry the removal of the fats and protein debris was previously done by using chemicals (Seittz 1974) but now this is done with the help of proteases and lipases (Posorske 1984). Waste treatment is a big problem in all the times in leather; food processing and other industries (Godfrey and Reichelt 1983). Tschocke (Tschocke 1990) immobilized the lipase generating bacteria, which were capable of removal of 90% waste. The enzymes needed in waste treatment should be theromostable and microbial enzymes are more stable than the enzymes taken from animals and plants. Therefore they are being used in waste treatment. Another good reason is that it is easy to extract the enzymes from the microbial sources (Wiseman 1995) compared to other sources. It is reported that many lipases from mesophilic origin are stable at high temperature (Sugihara et al., 1991) so the thermostable enzymes can also retrieve not only from thermophilic but also from mesophilic origin of organisms. Thermophilic organisms have the property to produce thermostable enzymes. Highly thermostable enzymes have been isolated and characterized from hyperthermophilic archaea such as Pyrococcus furiosus and Thermococcus kodakaraensis and bacteria such as Thermotoga sp. (Adams et al., 1995; Adams and Kelly 1998; Fischer et al., 1996). Advent of the protein engineering has helped to harness the structure and function of the enzyme especially proteases and lipases (Cheetham 1995).

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1.5.4. Other uses of lipases

Lipases are active under mild conditions, stable in organic solvents, have high substrate specificity and usually show high stereo and/or region selectivity in catalysis (Snellman et al., 2002). Like other enzymes, lipases can be immobilized on the oxygen/pH electrode (in association with glucose oxidase). These are called as lipids biosensors (Karube and Sode 1988) and used in blood cholesterol (Imamura et al., 1989) and triglyceride determination (Iwai 1990). Lipases are increasingly produced via recombinant DNA technology. The enantio, chemo and regio-specific behavior of these enzymes have made them interesting for industrialist and scientists (Saxena et al., 2003). Lipases have a wide range of properties regarding positional specificity, thermostability, fatty acid specificity, pH optimum etc (Huang 1984). Lipolytic enzymes have strong biotechnological potential (Jaeger and Reetz 1998; Reetz and Jaeger 1998). Hence there is a dire need to explore new enzymes and their potential use in industry.

1.6. Glycosyl Hydrolases (EC 3.2)

Glycosyl hydrolases are enzymes that catalyze the hydrolysis of glycosides. They can also be classified according to the outcome of the stereospecifc hydrolytic reaction product. On this basis glycosyl hydrolases are classified as inverting or retaining enzymes (Sinnott 1990). These enzymes can also be classified on the basis of amino acid sequence. Classification based on sequencing may suggest the function of the newly sequenced enzymes whose function has not been experimently determined. There are more than 100 different families established on the basis of sequence similarity (Henrissat et al.,1995; Henrissat and Davies 1995; Bairoch 1999) and these are available on CAZy website (http://www.cazy.org) (Henrissat and Coutinho 1999). These families are converged into

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different clans on the basis of their 3D structure comparison of these families (Bourne and Henrissat 2001). Xylanases and chitosnases are the members of glycosyl hydrolase families and xylanase enzyme activity has been found in families 5, 7, 8, 10, 11, 16, 26, 43, 52 and 62 (Flint et al., 1993). Family 8, also includes chitosanases in addition to xylanases.

Table 1: Glycoside Hydrolase family having activity on xylan, fold, mechanism and catalytic site
residue

General GH Fold Family Residue 5 8 10 11 (/)8 (/)6 (/)8 -jelly role 5-fold-43 propeller GH-F Inverting Glutamate Aspartate GH-A GH-M GH-A GH-C Retaining Inverting Retaining Retaining Glutamate Glutamate Glutamate Glutamate Glutamate Aspartate Glutamate Glutamate Clan Mechanism Acid/Base General Base Nucleophilic/

1.6.1. Glycoside hydrolase family 5

This is the largest glycosyl hydrolase family having heptapeptide conserved amino acid region, which comprises of nucleophile and general acid/base residues. It comprises enzymes with several activities including endoglucanase (EC 3.2.1.4), -mannanase (EC 3.2.1.78), exo-1,3-glucanase (EC 3.2.1.58), endo-1,6-glucanase (EC 3.2.1.75), xylanase (EC 3.2.1.8) and endoglycoceramidase (EC 3.2.1.123). Structural alignment shows rms

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(root mean square) deviation of 1.25 0.12 between residues in its members and it has nine sub-families (Lo Leggio et al., 1999).

1.6.2. Glycoside hydrolase family 8

Family 8, previously named as family D, consists of cellulases (EC 3.2.1.4), chitosanases, (EC 3.2.1.132) lichenases, (EC 3.2.1.73) and endo-1,4--xylanases (EC 3.2.1.8) (Coutinho and Henrissat 1999). Most of the xylanases in this family are originated from species of genus Bacillus (Takami et al., 2000; Takami and Horikoshi 2000).

1.6.3. Glycoside hydrolase family 10

This family comprises of endo-1,4--xylanase (EC 3.2.1.8), endo-1,3--xylanase (EC 3.2.1.32) and cellobiohydrolase (EC 3.2.1.91) (Coutinho and Henrissat, 1999). The major enzyme in this family is endo-1,4--xylanase, which may not be specific for xylan and may also be active on low molecular mass cellulose substrate (Biely 2003; Gilkes et al., 1991).

1.6.4. Gycoside hydrolase family 11

Irrespective of all other families, this family is highly specific which comprises of xylanases and these xylanases are called as true xylanases. They are active exclusively on
D -xylose

containing substrate. They have less catalytic efficiency that is why their

products are further catalyzed by the family 10 enzymes (Biely et al., 1993; Biely et al., 1997). They have high pI, low molecular weight, a double displacement catalytic mechanism, catalytic residue comprises of two glutamates and a -jelly role fold structure.

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1.6.5. Glycoside hydrolase family 43

Glycoside hydrolase family 43 includes enzymes with beta-xylosidase (EC 3.2.1.37), alpha-L-arabinofuranosidase (EC 3.2.1.55), arabinanase (EC 3.2.1.99) and xylanase (EC 3.2.1.8).

1.7. Xylanases
These are the glycosidases, which catalyze the endo-hydrolysis of 1,4--Dxylosidic linkages in xylan. In 1961, the International Union of Biochemistry and Molecular Biology (IUBMB), recognized the enzyme as EC 3.2.1.8 and named it as endo1,4--xylanase. Xylanase is such a versatile enzyme that it is active under extreme conditions. It exhibits the enzyme activity over a wide range of temperature, pH and NaCl concentration. There are reports describing the enzyme activity at a temperature range of 5-105 C (Collins et al., 2005; Li et al., 2000; Kulkarni et al. 1999), pH from 2-11 (Kimura et al., 2000; Kulkarni et al., 1999; Puls et al., 1987) and NaCl concentration as high as 30% (Waino and Ingvorsen, 2003; Wejse et al., 2003). Xylan, the substrate for xylanase, is the most abundant hemi-cellulosic part of wood. It is present in the secondary wall and it connects inter phase of lignin and the polysaccharide. It is covalently as well as non-covalently attached to cellulose, lignin, pectin and other polysaccharide to maintain cell wall integrity (Coughan and Hazlewood 1993; Hori and Elbein 1985). Xylan is homopolymers of D-xylose, linked through -1,4 glycosyl bond. In nature they are partially substituted by acetyl 4-O-methyl-Dglucoronosyl and l-arabino-furanosyl residues.

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Fig.2. Xylan structure showing different groups which are being attacked by microbial xylanase.

Xylan is a polysaccharide comprising of xylose residues linked by -1,4-glycosidic bond as shown in Fig. 2. The main chain of xylan is composed of -xylopyranose residues (Whistler and Richards 1970). Xylan is the most common hemicellulosic part of cell wall of plants, comprising 3035% of the total dry weight (Joseleau et al., 1992). The xylan from hardwood is acetyl-4O-methylglucuronoxylan. This

polysaccharide consists of at least 70 -xylopyranose residues linked by -1,4-glycosidic bonds. A 4-O-methylglucuronic acid is attached to the 2 position of xylose after every tenth residue. Acetylation in hardwood xylans is more frequent at the C-3 than at the C-2 position. During alkali extraction these acetyl groups are readily removed from xylan (Sunna and Antranikian 1997). Softwood xylans are composed of arabino-4-Omethylglucuroxylans. They have a higher content of 4-O-methylglucuronic acid than xylans of hardwood. Softwood xylans are not acetylated and instead of an acetyl group they have -L-arabinofuranose units linked by -1,3-glycosidic bonds at the C-3 position of the xylose. The arabinosyl substituents are 12% of the xylosyl residues (Wong et al., 22

1988). Softwood xylans are shorter than hardwood xylans, and also they are less branched (Sunna and Antranikian 1997). The common substituents found on the backbone of xylan are arabinosyl, glucuronysyl and acetyl residues (Whistler and Richards 1970). The xylanolytic enzyme system which carries out the xylan hydrolysis is of following hydrolytic enzymes: -1,4-endoxylanase, -L-arabinofuranosidase, -xylosidase, acetyl xylan esterase, -glucuronidase, and phenolic acid (p-coumaric acid and ferulic) esterase as shown in Fig. 2. These all enzymes act collectively to convert xylan into its constituent sugars. The system in which all xylanolytic enzymes are functional is present in eukaryotes (Belancic et al., 1995; Biely et al., 1985) as well as in prokaryotes (Dey et al., 1992; Elegir et al., 1995).

1.7.1. Structure of xylanase

The family 10 of glycosyl hydrolases comprises of endo-1,4--xylanase, endo-1,3-xylanase and cellobiohydrolase (Coutinho and Henrissat 1999) and their catalytic domain is cylindrical / barrel resembling a salad bowl with a catalytic site at the narrower end near the C-terminal of the -barrel (Derewenda et al., 1994; Harris et al., 1994). The number of pyranose rings that the enzyme will bind effectively determines the nature of the oligoproduct formation. Xylanases belonging to family 11 of glycosyl hydrolases is specific for xylan and their catalytic domain consists of -pleated sheets forming a two-layered trough that covers the catalytic site (Miao et al., 1994; Withers and Aebersold 1995). Going down into the trough there is a long loop terminating at isoleucine. Xylanases belonging to family 11 have relatively low molecular weight compared to family 10. Family 11 xylanases are usually called true xylanases because they only exhibit xylanase enzyme activity but no cellulase enzyme activity.

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1.7.2. Multiple forms and domains of xylanase

Several microorganisms produce multiple xylanases (Gilbert and Hazlewood 1993; Yang, et al., 1989; Gilbert et al., 1988). Aspergillus niger produces as many as fifteen extra-cellular xylanases (Biely et al., 1985) and Trichoderma viride secretes thirteen xylanases out side the cell (Luthi et al., 1990). This multiple form of xylanases is the result of genetic redundancy (Wong et al., 1988) but these isozymes have different posttranslational processing (Biely 1985). Xylanases in majority are extracellularly secreted due to the reason that substrate of xylanase is quite large in size which cannot enter in the cell. Presence of xylan in the growth media induces the enhanced production of xylanase (Subramaniyan and Prema 2002; Defaye et al., 1992; Biely 1985). Sequencing of xylanase genes orignated from various sources have revealed that the encoded amnio acid residues contain multi-domains. In these xylanases catalytic domain is usually joined with one or more domains with different functions (Gilkes et al., 1991; Tomme et al., 1995). This additional domain may be a xylan binding domain or cellulose binding domain, suggested to promote the proximity of the active site to its substrate (Black et al., 1997; MillwardSadler et al., 1995; Irwin et al., 1994), dockerin domain in Clostridium thermocellum (Hayashi et al., 1997; Grepinet et al., 1988), thermo-stabilizing domain (Winterhalter et al., 1995) and other domains whose function is still to be elucidated (Black et al., 1997; Gilkes et al., 1991). Sequence analysis of xylanases from the thermophillic microorganism have proposed that there is a thermo-stabilizing domain (Fontes et al., 1995), removal of which decreases the optimum temperature and thermo-stabilty of the enzyme (Winterhalter et al., 1995; Zverlov et al.,1996). A few studies have shown that the proposed thermostable domains are not only increase the thermostability but also increase the stability of the enzyme against pH denaturation (Kulkarni 1999). However there are a few exceptions, although certain amino acid sequences of thermostable xylanases, which

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are considered to be responsible for the thermostability of the enzyme, are present in mesophilic bacterial enzymes but these enzymes dont show substantial thermal stability (Margolles-Clark et al., 1996).

1.7.3. Xylanases from Bacillus

Different xylanases are produced by the genus Bacillus. Several of which have been cloned and characterized. Although xylanases produced from different bacteria have multi domains in it but the sequencing of the xylanases from Bacillus strain have shown that they have a single domain only, except xylanase from Bacillus polymyxa which also contains a catalytic domain and cellulose binding domain (Gosalbes et al., 1991)

1.7.4. Industrial applications of xylanases

Xylanases have been utilized in various industries since long. The major industry where xylanases are being used extensively is the paper and pulp. Paper and pulp industry is one of the largest users of plant biomass. In United States, paper consumption exceeds 300 kg/capita annually (Anon 1996) and paper consumption increases with the increase of economic development. As xylanases play a vital role in this industry therefore these enzymes are indispensable for the economic growth of a country. In paper industry the first step is make pulp from wood and it is important to remove the lignin. It can be accomplished through combination of chemo-mechanical pretreatment, chemical or enzymatic hydrolysis and extraction procedures to get the maximum yield. Enzymatic fabrication is economical than traditional chemical methods (Jefferies and Viikari, 1996). Viikari and co-workers (1986) first reported the importance of endo-xylanase and decreasing chemical needs. After improvement it was commercialized (Paice et al., 1986; Clark et al., 1990). Hemicellulase and mannases (Viikari et al., 1990) have also been used 25

for pulp formation and they affect differently on hard wood and soft wood (Buchert et al., 1992), whereas an equally good role of xylanases have been reported in pulp formation from both types of wood (Saake et al., 1995). There are two models proposed to enhance pulp bleaching. First model proposes that action of xylanases and mannases increases access to pulp fiber by removing precipitated xylan, which is physically entraped within the lignin (Kantelinen et al., 1993). Second model proposes that hemicellulases release chromophore and lignin from cellulose by breaking their covalent linkage. This evidence has also been supported by various studies (Wong et al., 1996; Suurnakki et al., 1996). Xylanase showed ability to release chromophore and kappa reduction at the dose of 3 IU/g of pulp. It attains brightness 80, which is 12 points above from the control pulp, which was without enzyme treatmet (Elegir et al., 1995). Evidence also proves that chromophore release is a part of hemicellluose degradation only and not lignin (Zibrio 1990a, 1990b). Apart from xylanase use in the paper and pulp industry, they are also being used as additives to food in poultry (Bedford and Classen 1992). They are also being used in flour for making better quality of baked products (Maat et al., 1992). Their used in extraction of plant oils, coffee, and starch (Wong and Saddler 1992) as well as in the enhancement of nutritional value of grain feed and agricultural silage is well established (Kuhad and Singh 1993). A mixture of xylanases, cellulases and pectinases have also been used for clarification of fruit juices (Biely 1985) and degumming of plant fibers such as flax, ramie and jute (Kapoor et al., 2001; Puchart et al., 1999; Sharma 1987). These were some industrial applications of microbial xylanolytic enzymes, which are discussed here with the main emphasis on biobleaching.

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