**3. Xylanases**

#### **3.1. Classification and mode of action**

Xylanases, as glycoside hydrolase members, are able to catalyze the hydrolysis of the glycosidic linkage (β-1,4) of xylosides, leading to the formation of a sugar hemiacetal and the corre‐ sponding free aglycone (nonsugar compound remaining after replacement of the glycoside by a hydrogen atom [27]). Xylanases have been classified in at least three ways: based on the molecular weight and isoelectric point (pI) [28], the crystal structure [29] and kinetic properties, or the substrate specificity and product profile. As the first classification is not sufficient to describe all xylanases, several exceptions have been identified [10] because not all xylanases have a high molecular mass (above 30 kDa) and low pI or a low molecular mass (less than 30 kDa) and high pI [6]. Therefore, a more complete system, based on the primary structure and comparison of the catalytic domains, was introduced [10,30], analyzing both the structural and mechanistic features [10].

**iv.** Galactoglucuronoarabinoxylans, which are characterized by the presence of terminal

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

In each category there exists microheterogeneity with respect to the degree and nature of branching. The side chains determine the solubility, physical conformation and reactivity of the xylan molecule with the other hemicellulosic components and hence greatly influence the mode and extent of enzymatic cleavage [12]. Endospermic arabinoxylans of annual plants, also called pentosans, are more soluble in water and alkaline solutions than xylans of lignocellulosic

Due to the heterogeneity and complex chemical nature of plant xylan, its complete breakdown requires the action of a complex of several hydrolytic enzymes with diverse specificities and modes of action. Thus, it is not surprising for xylan-degrading cells to produce an arsenal of polymer-degrading proteins [1]. The xylanolytic enzyme system that carries out the xylan hydrolysis is normally composed of a repertoire of hydrolytic enzymes, including endoxyla‐ nase (endo-1,4-β-xylanase, E.C.3.2.1.8), β-xylosidase (xylan-1,4-β-xylosidase, E.C.3.2.1.37), αglucuronidase (α-glucosiduronase, E.C.3.2.1.139), α-arabinofuranosidase (α-Larabinofuranosidase, E.C.3.2.1.55) and acetylxylan esterase (E.C.3.1.1.72) [23]. All of these enzymes act cooperatively to convert xylan into its constituent sugars [24]. Among all xylanases, endoxylanases are the most important due to their direct involvement in cleaving

Xylan, being a high molecular mass polymer, cannot penetrate the cell wall. The low molecular mass fragments of xylan play a key role in the regulation of xylanase biosynthesis. These fragments include xylose, xylobiose, xylooligosaccharides, heterodisaccharides of xylose and glucose and their positional isomers. These molecules are liberated from xylan through the action of small amounts of constitutively produced enzymes [12]. Xylanase catalyzes the random hydrolysis of xylan to xylooligosaccharides, while β-xylosidase releases xylose residues from the nonreducing ends of xylooligosaccharides. However, a complete degrada‐ tion requires the synergistic action of acetyl esterase to remove the acetyl substituents from

Xylanases, as glycoside hydrolase members, are able to catalyze the hydrolysis of the glycosidic linkage (β-1,4) of xylosides, leading to the formation of a sugar hemiacetal and the corre‐ sponding free aglycone (nonsugar compound remaining after replacement of the glycoside by a hydrogen atom [27]). Xylanases have been classified in at least three ways: based on the molecular weight and isoelectric point (pI) [28], the crystal structure [29] and kinetic properties,

are typically found in perennial plants.

materials because of their branched structures [22].

the glycosidic bonds and in liberating short xylooligosaccharides [8].

the β-1,4-linked D-xylose backbone of xylan [25,26].

**3.1. Classification and mode of action**

**3. Xylanases**

**2.2. Enzymatic hydrolysis of xylan**

β-D-galactopyranosyl residues on complex oligosaccharide side chains of xylans and

Updated information on the characteristics and classification of enzymes may be found in the Carbohydrate-Active Enzyme (CAZy) database. This is a knowledge-based resource special‐ izing on enzymes that build and breakdown complex carbohydrates and glycoconjugates. This database contains information from sequence annotations found in publicly available sources (such as the National Center for Biotechnology Information, NCBI), family classifications and known functional information [31]. According to the CAZy database (http://www.cazy.org), xylanases (EC3.2.1.8) are related to glycoside hydrolase (GH) families 5, 7, 8, 9, 10, 11, 12, 16, 26, 30, 43, 44, 51 and 62. However, the sequences classified in families 16, 51 and 62 appear to be bifunctional enzymes containing two catalytic domains, unlike families 5, 7, 8, 10, 11 and 43, which have a truly distinct catalytic domain with endo-1,4-β-xylanase activity [10]. Using the same analysis, families 9, 12, 26, 30 and 44 may have residual or secondary xylanase activity.

Xylanases have been primarily classified as GH 10 and 11 based on the hydrophobic cluster analysis of the catalytic domains and similarities in the amino acid sequences [8]. Although members of these two families have been thoroughly studied, the catalytic properties of the members of the remaining families (5, 7, 8 and 43) are recent and remain very limited [32].

Members of GH families 5, 7, 8, 10, 11 and 43 differ in their physicochemical properties, structure, mode of action and substrate specificities [10]. Several models have been proposed to explain the mechanism of xylanase action. Xylanase activity leads to the hydrolysis of xylan. Generally, this hydrolysis may result either in the retention (GH families 5, 7, 10 and 11) or the inversion (GH families 8 and 43) of the anomeric center of the reducing sugar monomer of the carbohydrate [33,34].

Families 5, 7, 10 and 11 contain enzymes that catalyze the hydrolysis with the retention of the anomeric configuration, with two glutamate residues being implicated in the catalytic mechanism. This indicates a double-displacement mechanism, in which a covalent glycosylenzyme intermediate is formed and subsequently hydrolyzed, and two carboxylic acid residues, suitably located in the active site, are involved in the formation of the intermediate; one acts as a general acid catalyst by protonating the substrate, while the second performs a nucleophilic attack, which results in the departure of the leaving group and the formation of the α-glycosyl enzyme intermediate (β to α inversion). In the second step, the first carboxylate group instead functions as a general base, abstracting a proton from a nucleophilic water molecule, which attacks the anomeric carbon. This leads to a second substitution, in which the anomeric carbon again passes via a transition state to give rise to a product with the β configuration (α to β inversion) [10,34].

In contrast to the mechanism mentioned above, the enzymes in families 8 and 43 gener‐ ally act via an inversion of the anomeric center, and glutamate and aspartate may be the 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 the anomeric carbon and the general base [10,34].

chain 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

A Review of Xylanase Production by the Fermentation of Xylan: Classification, Characterization and Applications

http://dx.doi.org/10.5772/53544

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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

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

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

substrate specificity has important implications in the deconstruction of xylan [43].

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

produced by family 7 [10].

and xylotriose [10,31].

hydrolysis via a single displacement mechanism [10,31].
