**Abstract**

Lignocellulosic biomass is a renewable raw material. Industrial interest with new technology has grown to take advantage of this raw material. Different microbial enzymes are treated with biomass to produce the desired products under ideal industrial conditions. Xylanases are the key enzymes that degrade the xylosidic linkages in the xylan backbone of the biomass, and commercial enzymes are categorized into different glycoside hydrolase families. Thermophilic microorganisms are an excellent source of thermostable enzymes that can tolerate the extreme conditions of industrial processing. Thermostability of xylanases from thermophilic microorganisms has given the importance for a specific activity at elevated temperatures and distinction due to biochemical properties, structure, and mode of action. Optimized xylanases can be produced through genetic engineering: a novel xylanase is isolated from an extreme environment and then genetically modified to improve suitability for industrial contexts. Recombinant protein techniques have made it possible to engineer and express thermostable xylanases in bacteria, yeasts, and filamentous fungi. We will discuss the biotechnological potential of xylanases from thermophilic microorganism and the ways they are being optimized and expressed for industrial applications.

**Keywords:** xylanases, thermophilic xylanases, genetic engineering, applications

### **1. Introduction**

Increasing energy costs and environmental concerns have pushed the global demand for sustainable renewable fuels. The impacts of plant biomass gain particular interest due to their availability as the most abundant raw material worldwide and certainly play an important role because of its significant role as a renewable source of energy [1]. Plant biomass refers to lignocellulosic biomass and its growing demand for the more effective utilization appears to evolve as one of the most important area with great industrial interest. Lignocellulose is composed of the structural polymers of cellulose, hemicellulose, pectin, and lignin. Cellulose is the most abundant polysaccharide in nature followed by hemicelluloses. Xylan is a major structural component of hemicullose and makes up 20–40% of total plant biomass [1].

Various hydrolyzing enzymes are required to degrade lignocellulosic biomass. Carbohydrate-active enzymes, the second largest group of industrial enzymes are sourced from animals, plants, and microorganisms. The hydrolytic enzymes make up nearly 75% of the total extent of industrial enzymes worldwide [2]. Efficient

degradation of plant biomass remains a major challenge that requires the availability of enzymes for the hydrolysis of lignocellulosic biomass. Biomass can be efficiently degraded by combining multiple enzymes that hydrolyze complex polysaccharides into fermentable sugars under ideal industrial conditions. These enzymes must be active during a wide range of conditions, such as high temperature and pH [3]. Xylanases are a crucial group of depolymerizing enzymes used for the hydrolysis of the xylan that is a major component of hemicellulose. There is a critical need for thermophilic xylanases operating more efficiently at higher temperatures than current commercially available ones. Xylanase producing thermophilic microorganisms are of prime importance and well-suited for industrial applications. Therefore, there is growing interest in exploring thermophilic microorganisms from extreme environment for biotechnological applications toward biomass degradation. Because of their biotechnological characteristics, xylanases are most often produced from microorganisms for commercial applications. Recently, there has been much industrial interest in xylanases for biofuel production, chemical and pharmaceutical industries, wood pulp bioleaching, papermaking, the manufacture of food and beverages, and animal nutrition. Let us look briefly at the genesis and some promising industrial uses of xylanases.

β-xylosidase (xylan-1,4-β-xylosidase), α-glucuronidase (α-glucosiduronase), α-arabinofuranosidase (α-L-arabinofuranosidase) and acetylxylan esterase. Among them endo-xylanases and β-xylosidases are the two key enzymes responsible for the hydrolysis of xylan. Endo-xylanases randomly cleaves the xylan backbone into

oligosaccharides from non-reducing end into xylose monomers. The removal of side groups is catalyzed by α-D-glucuronidases, α-L-arabinofuranosidases, ferulic acid

Initially xylanases were classified into two groups, those with low molecular weight (less than 30 kDa) and basic isoelectric points (pI), secondly those with high molecular weight (greater than 30 kDa) and acidic pI. However, this classification system was unable to classify most of the recently discovered xylanases [11]. Afterward, another classification system was introduced that were based on the comparisons of primary structure of the catalytic domains and these enzymes were grouped into families based on related sequences. This classification system now considered the standard means for the classification of enzymes including xylanases. In addition, this classification system gave an extra edge that classifies the glycosidases in general [11]. The most extensive group of enzymes is "Glycoside hydrolases" that refers to catalyze the glycosidic bond cleavage between carbohydrates or between carbohydrate and non-carbohydrate moiety. In glycoside hydrolases (GH) families, some family protein folds are more conserved than their amino acid sequences, and these families are further grouped into clans. Presently, 14 different clans have been proposed (GH-A to GH-N), with most clans encompassing two or more than two

According to the information provided in the Carbohydrate-Active Enzymes Database (CAZy), xylanases have been classified into 13 families, however only the GH10 (formerly F) and GH11 families (formerly G) with exclusive activities for endo-β-xylanase in them. The difference between these two families based on sequence, different catalytic properties, substrate specificity, three-dimensional structure and mechanism of action [11, 12]. Besides the GH family 10 and 11, xylanases activity are also found in families of GH5, GH7, GH8, GH16, GH26, GH43, GH52 and GH62 [11, 13]. For the reason that some bifunctional enzymes are containing two catalytic domains, for example xylanases having domain of family GH10 or GH11 and it contains a domain of glycosidase as well. Among the other families, GH8 xylanases act solely on xylan whereas GH5, GH7, and GH43 xylanases also show activities as endo-glucanases, licheninases or arabino-furanosidases. Therefore, the enzymes with xylanase activity are solely not only confined to families GH10 and GH11 but also expanded to include other families like GH5, GH7,

Xylanases belongs to GH families 10 and 11, which hydrolyze glycosidic bonds by acid base-assisted catalysis through a double displacement mechanism leading to retention of anomeric configuration at the cleavage site [14]. The xylanases from GH family 10 belongs to clan GH-A and the crystal structures display an (α/β)8 barrel fold or "salad bowl" shape with extended loops creating a catalytic cleft that contains at least four to seven xylose-binding subsites [15]. The catalytic site contains two glutamate residues, one acting as a nucleophile and the other as an acid/base catalyst. Catalytic amino acids and enzymatic mechanism are conserved, presenting a domain for catalysis of 250–450 amino acids. From the biochemical point of view, most of them have high molecular weight though there are reports of

xylo-oligosaccharides, while β-xylosidases further hydrolyze the xylo-

esterases, acetylxylan esterases and p-coumaric acid esterases [9, 10].

**3. Classification of xylanases**

*Xylanase and Its Industrial Applications DOI: http://dx.doi.org/10.5772/intechopen.92156*

GH8, GH16, GH43, GH52, GH62 [11].

families [11].

**295**

### **2. Structure of xylan and xylanases**

Lignocellulosic biomass is a potential raw material and its growing demand for the more effective utilization appears to evolve as one of the most important technologies with great industrial interest. Hemicellulose is a complex of polymeric carbohydrates including xylan, xyloglucan (heteropolymer of D-xylose and D-glucose), glucomannan (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) [4]. Xylan is a major structural polysaccharide found in the cell walls of plants, in which they may constitute more than 30% of the dry weight [5].

The structure of xylan holds a backbone of β-1,4-linked xylopyranosyl residues with an equatorial configuration and a diversity of substituted groups such as arabinose, acetyl, glucuronic acids, ferulic acid, and p-coumaric acid [6]. However, the structure of xylan is variable, ranging from linear 1,4-β-linked polyxylose sugars other than D-xylose, but the main chain of xylan is analogous to that of cellulose, composed of D-xylose instead of D-glucose. Moreover, xylan interacts with lignin and polysaccharides through covalent and non-covalent linkages, respectively. Different types of covalent linkages are responsible to interconnect xylan and lignin in the secondary cell wall [7]. These covalent interactions include glycosidic linkages between xylopyranosyl and p-coumaric acid and ester linkages between arabinofuranosyl residues and p-coumaric acid or ferulic acid. Feruloyl residues in arabinoxylans are known to be bridging units between xylan and lignin. The distribution pattern of the substituents on xylan affects a series of functional properties, including their solubility, interactions with other polymeric cell wall substances, degradability by enzymes, and solution behavior [8]. In addition, a relationship between the chemical structure of xylan results in a certain degree of complexity of xylan-containing materials, that may have several different xylan polymers of related structures but differ by more or less important features [8].

Xylanases are a crucial group of depolymerizing enzymes used for the hydrolysis of the xylan that is a major component of hemicellulose. Enzymatic depolymerization of hemicellulose to monomer sugars needs the synergistic action of multiple enzymes. These enzymes include endo-xylanase (endo-1,4-β-xylanase),

#### *Xylanase and Its Industrial Applications DOI: http://dx.doi.org/10.5772/intechopen.92156*

degradation of plant biomass remains a major challenge that requires the availability of enzymes for the hydrolysis of lignocellulosic biomass. Biomass can be efficiently degraded by combining multiple enzymes that hydrolyze complex polysaccharides into fermentable sugars under ideal industrial conditions. These enzymes must be active during a wide range of conditions, such as high temperature and pH [3]. Xylanases are a crucial group of depolymerizing enzymes used for the hydrolysis of the xylan that is a major component of hemicellulose. There is a critical need for thermophilic xylanases operating more efficiently at higher temperatures than current commercially available ones. Xylanase producing thermophilic microorganisms are of prime importance and well-suited for industrial applications. Therefore, there is growing interest in exploring thermophilic microorganisms from extreme environment for biotechnological applications toward biomass degradation. Because of their biotechnological characteristics, xylanases are most often produced from microorganisms for commercial applications. Recently, there has been much industrial interest in xylanases for biofuel production, chemical and pharmaceutical industries, wood pulp bioleaching, papermaking, the manufacture of food and beverages, and animal nutrition. Let us look briefly at the genesis and some

Lignocellulosic biomass is a potential raw material and its growing demand for

The structure of xylan holds a backbone of β-1,4-linked xylopyranosyl residues

with an equatorial configuration and a diversity of substituted groups such as arabinose, acetyl, glucuronic acids, ferulic acid, and p-coumaric acid [6]. However, the structure of xylan is variable, ranging from linear 1,4-β-linked polyxylose sugars other than D-xylose, but the main chain of xylan is analogous to that of cellulose, composed of D-xylose instead of D-glucose. Moreover, xylan interacts with lignin and polysaccharides through covalent and non-covalent linkages, respectively. Different types of covalent linkages are responsible to interconnect xylan and lignin in the secondary cell wall [7]. These covalent interactions include glycosidic linkages between xylopyranosyl and p-coumaric acid and ester linkages between arabinofuranosyl residues and p-coumaric acid or ferulic acid. Feruloyl residues in arabinoxylans are known to be bridging units between xylan and lignin. The distribution pattern of the substituents on xylan affects a series of functional properties, including their solubility, interactions with other polymeric cell wall substances, degradability by enzymes, and solution behavior [8]. In addition, a relationship between the chemical structure of xylan results in a certain degree of complexity of xylan-containing materials, that may have several different xylan polymers of

related structures but differ by more or less important features [8].

**294**

enzymes. These enzymes include endo-xylanase (endo-1,4-β-xylanase),

Xylanases are a crucial group of depolymerizing enzymes used for the hydrolysis of the xylan that is a major component of hemicellulose. Enzymatic depolymerization of hemicellulose to monomer sugars needs the synergistic action of multiple

the more effective utilization appears to evolve as one of the most important technologies with great industrial interest. Hemicellulose is a complex of polymeric

carbohydrates including xylan, xyloglucan (heteropolymer of D-xylose and D-glucose), glucomannan (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) [4]. Xylan is a major structural polysaccharide found in the cell walls of plants, in which they may

promising industrial uses of xylanases.

*Biotechnological Applications of Biomass*

**2. Structure of xylan and xylanases**

constitute more than 30% of the dry weight [5].

β-xylosidase (xylan-1,4-β-xylosidase), α-glucuronidase (α-glucosiduronase), α-arabinofuranosidase (α-L-arabinofuranosidase) and acetylxylan esterase. Among them endo-xylanases and β-xylosidases are the two key enzymes responsible for the hydrolysis of xylan. Endo-xylanases randomly cleaves the xylan backbone into xylo-oligosaccharides, while β-xylosidases further hydrolyze the xylooligosaccharides from non-reducing end into xylose monomers. The removal of side groups is catalyzed by α-D-glucuronidases, α-L-arabinofuranosidases, ferulic acid esterases, acetylxylan esterases and p-coumaric acid esterases [9, 10].
