**2.1. Structure and distribution**

A complex, highly branched heteropolysaccharide, xylan varies in structure between different plant species, and the homopolymeric backbone chain of 1,4-linked β-D-xylopyranosyl units can be substituted to varying degrees with glucuronopyranosyl, 4-O-methyl-D-glucurono‐ pyranosyl, α-L-arabinofuranosyl, acetyl, feruloyl or p-coumaroyl side-chain groups [12,13] (Figure 1).

Xylan is distributed in several types of tissues and cells and is present in a variety of plant species [12], being found in large quantities in hardwoods from angiosperms (15–30% of the cell wall content) and softwoods from gymnosperms (7–10%), as well as in annual plants (<30%) [14]. Wood xylan exists as O-acetyl-4-O-methylglucuronoxylan in hardwoods and as arabino-4-O-methylglucuronoxylan in softwoods, while xylans in grasses and annual plants are typically arabinoxylans [12]. Linear unsubstituted xylan has also been reported in esparto grass [15], tobacco [16] and certain marine algae [17,18], with the latter containing xylopyra‐ nosyl residues linked by both 1,3-β and 1,4-β linkages [17,19].

the process; therefore, genetic engineering and recombinant DNA technology have an important role in the large-scale expression of xylanases in homologous or heterologous

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

Considering the future prospects of xylanases in biotechnological applications, the goal of this review chapter is to present an overview of xylanase production via fermentation and to describe some of the characteristics of these enzymes and their primary substrate, xylan. Moreover, this review will discuss the fermentation processes as well as the genetic techniques

The three main components that constitute lignocellulosic substrates are cellulose, hemicellu‐ lose and lignin [8]. Schulze [9] first introduced the term 'hemicellulose' to represent the fractions isolated or extracted from plant materials using a dilute alkali. Hemicelluloses are composed of complex mixtures of xylan, xyloglucan, glucomannan, galactoglucomannan,

The substrate of xylanase, xylan, is the second most-abundant polysaccharide in nature, accounting for approximately one-third of the renewable organic carbon on Earth [10], and it constitutes the major component of hemicellulose, a complex of polymeric carbo‐ hydrates, including xylan, xyloglucan (heteropolymer of D-xylose and D-glucose), gluco‐ mannan (heteropolymer of D-glucose and D-mannose), galactoglucomannan (heteropolymer of D-galactose, D-glucose and D-mannose) and arabinogalactan (hetero‐ polymer of D-galactose and arabinose) [11]. Xylan is primarily present in the secondary cell wall and together with cellulose (1,4-β-glucan) and lignin (a complex polyphenolic compound) make up the major polymeric constituents of plant cell walls [12]. Within the cell wall structure, all three constituents interact via covalent and non-covalent linkages, with xylan being found at the interface between lignin and cellulose, where it is believed

A complex, highly branched heteropolysaccharide, xylan varies in structure between different plant species, and the homopolymeric backbone chain of 1,4-linked β-D-xylopyranosyl units can be substituted to varying degrees with glucuronopyranosyl, 4-O-methyl-D-glucurono‐ pyranosyl, α-L-arabinofuranosyl, acetyl, feruloyl or p-coumaroyl side-chain groups [12,13]

Xylan is distributed in several types of tissues and cells and is present in a variety of plant species [12], being found in large quantities in hardwoods from angiosperms (15–30% of the cell wall content) and softwoods from gymnosperms (7–10%), as well as in annual plants (<30%) [14]. Wood xylan exists as O-acetyl-4-O-methylglucuronoxylan in hardwoods and as arabino-4-O-methylglucuronoxylan in softwoods, while xylans in grasses and annual plants

to be important for fiber cohesion and plant cell wall integrity [1].

protein-expression hosts.

**2. Xylan**

applied to improve xylanase yields.

arabinogalactan or other heteropolymers [8].

**2.1. Structure and distribution**

(Figure 1).

Similar to other polysaccharides of plant origin, xylan has a large polydiversity and polymo‐ lecularity [20]. The degree of polymerization in xylans is also variable, with, for example, hardwood and softwood xylans generally consisting of 150-200 and 70-130 β-xylopyranose residues, respectively [12].

**Figure 1.** Structure of xylan and the xylanolytic enzymes involved in its degradation. Ac: Acetyl group; α-Araf: α-arabi‐ nofuranose; α-4-O-Me-GlcA: α-4-O-methylglucuronic acid. Source: Sunna and Antranikian [20].

Based on the common substituents found on the backbone, xylans are categorized as linear homoxylan, arabinoxylan, glucuronoxylan or glucuronoarabinoxylan. Homoxylans consisting exclusively of xylosyl residues are not widespread in nature; they have been isolated from limited sources, such as esparto grass, tobacco stalks and guar seed husks [20]. However, based on the nature of its substituents, a broad distinction may therefore be made among xylans, in which the complexity increases from linear to highly substituted xylans. Four main families of xylans can be considered [21]:


**iv.** Galactoglucuronoarabinoxylans, which are characterized by the presence of terminal β-D-galactopyranosyl residues on complex oligosaccharide side chains of xylans and are typically found in perennial plants.

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

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

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

255

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

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

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

mechanistic features [10].

carbohydrate [33,34].

configuration (α to β inversion) [10,34].

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 materials because of their branched structures [22].
