*3.1.1. GH families 10 and 11*

Xylanase from the GH10 family (or family G) have a low molecular mass with a pI between 8–9.5, while those from the GH11 family (or family F) have a high molecular mass and lower pI values [35,36].

Glycoside hydrolase family 10 is composed of endo-1,4-β-xylanases and endo-1,3-β-xylanases (EC 3.2.1.32) [34]. Members of this family are also capable of hydrolyzing the aryl β-glycosides of xylobiose and xylotriose at the aglyconic bond. Furthermore, these enzymes are highly active on short xylooligosaccharides, thereby indicating small substrate-binding sites. Crystal structure analyses, kinetic analyses of the activity on xylooligosaccharides of various sizes and end product analyses have indicated that family 10 xylanases typically have four to five substrate-binding sites [37]. Members of this family also typically have a high molecular mass, a low pI and display an (α/β)8-barrel fold [10,34,38].

Compared to other xylanases, GH11 members display several interesting properties, such as high substrate selectivity and high catalytic efficiency, a small size, and a variety of optimum pH and temperature values, making them suitable in various conditions and in many appli‐ cations [39]. Family 11 is composed only of xylanases (EC3.2.1.8), leading to their consideration as "true xylanases," as they are exclusively active on D-xylose-containing substrates. GH11 enzymes are generally characterized by a high pI, a low molecular weight, a double-displace‐ ment catalytic mechanism, two glutamates that act as the catalytic residues and a β-jelly roll fold structure. Additionally, the products of their action can be further hydrolyzed by the family 10 enzymes [37]. Similar to family 10 xylanases, these enzymes can hydrolyze the aryl β-glycosides of xylobiose and xylotriose at the aglyconic bond, but they are inactive on aryl cellobiosides. Furthermore, in contrast to the family 10 xylanases, but similar to the family 8 cold-adapted xylanases, these enzymes are most active on long-chain xylooligosaccharides, and it has been found that they have larger substrate-binding clefts, containing at least seven subsites [10].

Xylanases belonging to GH10 exhibit greater catalytic versatility and lower substrate spe‐ cificity than those belonging to GH11 [37,40]. According to Davies *et al*. [41], the binding sites for xylose residues in xylanases are termed subsites, with bond cleavage occurring between the sugar residues at the -1 (non-reducing) and the +1 (reducing) ends of the polysaccharide substrate. As observed in assays using arabinoxylan as the substrate, GH10 products have arabinose residues substituted on xylose at the +1 subsite, whereas GH 11 products have arabinose residues substituted at the +2 subsite [42]. These results suggest that GH 10 enzymes are able to hydrolyze xylose linkages closer to the sidechain residues [43]. Therefore, xylanases from family 11 preferentially cleave the unsub‐ stituted regions of the arabinoxylan backbone, whereas GH10 enzymes cleave the decorated regions, being less hampered by the presence of substituents along the xylan backbone [37]. The xylan side-chain decorations are recognized by xylanases, and the de‐ gree of substitution in xylan will influence the hydrolytic products; this difference in substrate specificity has important implications in the deconstruction of xylan [43].

## *3.1.2. GH families 5, 7, 8 and 43*

catalytic residues. Inverting enzymes work via a single displacement reaction, in which one carboxylate provides for a general acid-catalyzed leaving group departure. The sec‐ ond function of these enzymes, acting as general base, activates a nucleophilic water molecule to attack the anomeric carbon, thereby cleaving the glycosidic bond and lead‐ ing to an inversion of the configuration at the anomeric carbon. Generally, the distance between the two residues allows for the accommodation of the water molecule between

256 Sustainable Degradation of Lignocellulosic Biomass - Techniques, Applications and Commercialization

Xylanase from the GH10 family (or family G) have a low molecular mass with a pI between 8–9.5, while those from the GH11 family (or family F) have a high molecular mass and lower

Glycoside hydrolase family 10 is composed of endo-1,4-β-xylanases and endo-1,3-β-xylanases (EC 3.2.1.32) [34]. Members of this family are also capable of hydrolyzing the aryl β-glycosides of xylobiose and xylotriose at the aglyconic bond. Furthermore, these enzymes are highly active on short xylooligosaccharides, thereby indicating small substrate-binding sites. Crystal structure analyses, kinetic analyses of the activity on xylooligosaccharides of various sizes and end product analyses have indicated that family 10 xylanases typically have four to five substrate-binding sites [37]. Members of this family also typically have a high molecular mass,

Compared to other xylanases, GH11 members display several interesting properties, such as high substrate selectivity and high catalytic efficiency, a small size, and a variety of optimum pH and temperature values, making them suitable in various conditions and in many appli‐ cations [39]. Family 11 is composed only of xylanases (EC3.2.1.8), leading to their consideration as "true xylanases," as they are exclusively active on D-xylose-containing substrates. GH11 enzymes are generally characterized by a high pI, a low molecular weight, a double-displace‐ ment catalytic mechanism, two glutamates that act as the catalytic residues and a β-jelly roll fold structure. Additionally, the products of their action can be further hydrolyzed by the family 10 enzymes [37]. Similar to family 10 xylanases, these enzymes can hydrolyze the aryl β-glycosides of xylobiose and xylotriose at the aglyconic bond, but they are inactive on aryl cellobiosides. Furthermore, in contrast to the family 10 xylanases, but similar to the family 8 cold-adapted xylanases, these enzymes are most active on long-chain xylooligosaccharides, and it has been found that they have larger substrate-binding clefts, containing at least seven

Xylanases belonging to GH10 exhibit greater catalytic versatility and lower substrate spe‐ cificity than those belonging to GH11 [37,40]. According to Davies *et al*. [41], the binding sites for xylose residues in xylanases are termed subsites, with bond cleavage occurring between the sugar residues at the -1 (non-reducing) and the +1 (reducing) ends of the polysaccharide substrate. As observed in assays using arabinoxylan as the substrate, GH10 products have arabinose residues substituted on xylose at the +1 subsite, whereas GH 11 products have arabinose residues substituted at the +2 subsite [42]. These results suggest that GH 10 enzymes are able to hydrolyze xylose linkages closer to the side-

the anomeric carbon and the general base [10,34].

a low pI and display an (α/β)8-barrel fold [10,34,38].

*3.1.1. GH families 10 and 11*

pI values [35,36].

subsites [10].

GH family 5 (or family A) is the largest glycoside hydrolase family, and only seven amino acid residues, including the nucleophile and the general acid/base residue, are strictly conserved among all members [10]. Structural alignment among the members of family 5 and 10 showed that these enzymes are as structurally different within family 5 as they are to the family 10 enzymes, therefore both families are classified into clan GH-A. The concept of clan or superfamily demonstrates a broader relationship between GH families, suggesting a more distant common evolutionary ancestor [44,45]. Further‐ more, the activity of these enzymes is affected by substituents on the xylan main chain, and it is unable to cleave linkages adjacent to substituted residues. Hydrolysis studies have shown that the shortest substituted fragments formed from glucuronoxylan and arabinoxylan are substituted xylotrioses, with the substitution being found on the inter‐ nal xylose residue. Therefore, the products produced by family 5 are shorter than those produced by family 7 [10].

GH family 8 (or family D) is composed of cellulases (EC 3.2.1.4), and also contains chitosanases (EC 3.2.1.132), lichenases (EC 3.2.1.73) and endo-1,4-β-xylanases (EC 3.2.1.8). This family of cold-adapted xylanases was found to hydrolyze xylan to xylotriose and xylotetraose and was most active on long-chain xylooligsaccharides. Similar to family 11 xylanases, a large substrate binding cleft containing at least six xylose-binding residues, with the catalytic site in the middle, was proposed [6]. However, unlike family 10 and 11 xylanases, enzymes from family 8 were found to catalyze hydrolysis with the inversion of the anomeric configuration and, under the conditions used, were found to be inactive on aryl β-glycosides of xylose, xylobiose and xylotriose [10,31].

GH families 7 and 43 contain only a few enzymes exhibiting xylanase activity that have been identified and studied. Family 7 has characteristics in common with both family 10 and 11 xylanases. Similar to the former family, those in family 7 have a high molecular weight and low pI, as well as a small substrate-binding site, containing approximately four subsites, with the catalytic site in the middle [10]. The members of family 43 have not been as thoroughly studied, and the structure of only one member has been determined, indicating that members of this family may display a five-blade β-propeller fold. Furthermore, a glutamate and aspartate in the center of a long V-shaped surface groove formed across the face of the propeller have been suggested as the catalytic residues. Family 43 is grouped with family 62 in clan GH-F, and, as also demonstrated in the family 8 enzymes, its members are believed to catalyze hydrolysis via a single displacement mechanism [10,31].
