Chapter 1. General Introduction 1
1.3. Enzymatic degradation of lignocellulosic biomass…
(September 2019), the CAZy database contains a total 341 families of enzymes (regularly updated), which includes 165 families of GHs, 107 families of GTs, 37 families of PLs, 16 families of CEs and 16 families of AA. Among these five enzyme classes, glycosyltransferases are the enzymes involved in the synthesis of oligosaccharides and polysaccharides, while the rest four-class, i.e., Polysaccharide lyases, carbohydrate esterases, auxiliary activity enzymes and glycoside hydrolases are involved in either the breakdown of polysaccharides or the modification of the polysaccharides.
Glycosyltransferase (EC 2.4.x.y) is the only class, which catalyze the transfer of sugar moieties of activated donor molecules to specific acceptor molecules, forming glycosidic bonds. GTs contributed approx. 42.35% of total CAZymes at present (September 2019) and is virtually present in every single organism.
Polysaccharide lyase (EC: 4.2.2.x) enzymes catalyze non-hydrolytic cleavage of glycosidic bonds of uronic acid-containing polysaccharides using a β-elimination catalytic mechanism. Only 1.5% of total CAZymes are reported as PLs in the CAZy database, but these enzymes have various biotechnological and biomedical applications (Lombard et al., 2010).
The carbohydrate esterase enzymes catalyze the de-O-acylation or de-N- acylation of substituted saccharides by removing the ester-based modifications present in mono-, oligo- and polysaccharides to make it accessible for other hydrolytic enzymes such as PLs and GHs. CEs accounts for 5.10% of total CAZymes.
The auxiliary activities redox enzymes family contains lignin-degrading and lytic polysaccharide mono-oxygenase enzymes. These enzymes catalyze the degradation of lignin and cleavage of mono-oxygen bond from a crystalline
polysaccharide. AAs can be used in combination with other CAZymes for the complete degradation of lignocellulosic biomass. These AAs constitute only 0.92% of total CAZymes, but in the last one decade, the utilization of these enzymes in different applications have gained interest.
1.3.1. Glycoside hydrolase
Glycoside hydrolase (GHs), (EC: 3.2.1.x) represent the highest number of enzymes classified and contributed approximately 50.15% of total CAZymes. GHs includes glycosidases which can hydrolyze the glycosidic bond as well as transglycosidases which can transglycosylate the glycosidic bond between two or more monosaccharide moieties or between a monosaccharide and a non-carbohydrate moiety (Henrissat, 1991; Henrissat and Bairoch, 1993, 1996). Till date (September 2019) 664285 enzyme entries are classified into 165 glycoside hydrolase families in the CAZy database (http://www.cazy.org/Glycoside-Hydrolases.html). Some of the families out of 165 families based on conservation of protein structure folds were further categorized into 14 ‘clans or superfamilies.’ GH acts on polysaccharides or oligosaccharides substrates and hydrolyze the glycosidic bonds either within the substrate randomly or at reducing end, thus being termed as endo- or exo-acting enzymes, respectively. The shape or groove of active site of the endo-acting enzymes is present in the more open conformation, which allows random binding of non- crystalline polysaccharide resulted in the production of oligosaccharides (Davies and Henrissat, 1995). While the active site of exo-acting enzymes most often has a pocket or tunnel conformation to bind the reducing end of substrate for the release of disaccharides or monosaccharides (Rouvinen et al., 1990; Divne et al., 1994). Over the years, different types of GHs have been identified, biochemically and structurally
characterized. The common feature of several GHs is the modular nature, i.e. they also contain one or more ancillary modules that are often but not always carbohydrate- binding modules (CBMs) (Henrissat and Davies, 2000). Consideration of different modules of particular GHs is the importance of correct open reading frame (ORF) annotation and functional prediction (Bourne and Henrissat, 2001). The relative abundance of the polysaccharides, a group of enzymes, based on their substrate specificities involved in the degradation of these polysaccharides are classified into cellulolytic and hemicellulolytic enzymes.
1.3.1.1. Cellulolytic enzymes
Glycoside hydrolases that specifically target cellulosic polysaccharide present in the lignocellulosic biomass are known as cellulolytic enzymes. These cellulolytic enzymes hydrolyze the β-(1,4)-glycosidic linkages in cellulose and are generally named as endoglucanases (EGs), cellobiohydrolases (CBHs) or β-glucosidases.
Endoglucanases (EC 3.2.1.4) randomly catalyze the β-(1,4)-glycosidic linkages (internal bonds) in the amorphous regions of the polysaccharides and produces oligosaccharides. Currently, endoglucanases are found in 15 GH families viz. 5, 6, 7, 8, 9, 10, 12, 26, 44, 45, 48, 51, 74, 124 and 148 as single catalytic domain or as modular enzymes. Cellulose hydrolysis by endoglucanases produces one reducing and one non- reducing end, that may be utilized by cellobiohydrolases for further hydrolysis (Rouvinen et al., 1990; Divne et al., 1994). These CBHs are mainly found in family GH5, GH6, GH7, GH9 and GH48. The cellobiose released from CBH mediated hydrolysis acts as a substrate for β-Glucosidases (EC 3.2.1.21) for the conversion of cellobiose into glucose sugar. β-Glucosidases are mainly present in family GH1, GH2, GH3, GH5, GH9, GH16, GH30, GH39 and GH116 (Henrissat et al., 1985).
1.3.1.2. Hemicellulolytic enzymes
Diverse and complex hemicellulosic component interacts with the cellulosic component and forms a tight association. Inefficient removal of hemicellulose from the lignocellulosic biomass may limit the efficient and complete degradation of cellulose (Saha et al., 2013). Therefore, the enzymatic saccharification of hemicellulosic components enhances the overall product yield and now attracting more attention.
(Hemsworth et al., 2016). Hemicellulolytic enzymes differ in their mode of action on distinct substrates based on their substrate specificity are maybe endo- or exo-acting enzymes. These hemicellulolytic enzymes are classified in different GH families. Endo- acting hemicellulolytic enzymes include endo-β-xylanase and endo-β-mannanases.
However, for complete conversion of hemicellulose into monosaccharide and other valuable products, several exo-acting enzymes and some of which primarily act on substitutions as well as on oligosaccharides are required. This susbtitutuon removing enzymes are arabinofuranosidases, arabinanases, galactosidases, glucuronidases, mannosidases and xylosidases. The hemicellulolytic enzymes involved in the complete conversion of heteroxylans are termed as xylanolytic enzymes.