**3. Classification of xylanases**

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 families [11].

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

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 low molecular weight enzymes [16]. The values of their pI are generally alkaline (8.0–9.5), however, some also have acid values and all of them sustain the same three-dimensional structure. Most of the substrate binding subsites are highly conserved in xylanases, but the affinity differences between these subsites significantly affect their mode of action, as well as substrate and product preferences [17]. As heat stability has great concern in commercial usages of xylanases. For this purpose, a number of studies analyzed the crystal structures of thermostable xylanases. Intraand intermolecular interactions in structural topography such as disulfide bond and hydrogen bond, compact the overall fold and stabilized N and C terminal end, fusion with CBM (carbohydrate-binding motif) and lower B-factor have been proposed to bestow the enzyme for increased heat stability [18].

GH 5 is the largest glycoside hydrolase family with varying activities including endo-1,4-β-xylanase. It hydrolyzes the β-1,4 xylan chain at a specific site directed by the position of an α-1,2-linked glucuronate moiety. The structural analysis XynA (of the family 5 xylanase) showed that, the catalytic domain displayed a common (β/α)8 barrel fold [21]; whereas, the β-barrels aligned well with those of another family 5 enzyme. The α-helices and loops were different, showing variances in the positioning, length and orientation. The xylanases belongs to family GH8 are classified in clan CH-M also contains endo-1,4-β-xylanase along with other glycoside hydrolase enzymes. It has also the aptitude to hydrolyze the β-1,4 xylan chain and exhibits the (α/α)6 barrel structure formed by six inner and six outer α helices [22]. Similarly, the GH26 are the member of the clan CH-A and exhibits the (β/α)8 structure. This family contains different glycoside hydrolase enzymes including β-1,3-xylanase, capable of hydrolyzing β-1,3-xylan. Activity, mechanisms and the structure of other member of glycoside hydrolase enzymes are listed in **Table 1**.

Demystifying definitional issue for common understanding, the xylanases are enzymes commonly found in microorganisms, marine algae, protozoans, snails, crustaceans, insects, seeds, plants, and other natural sources [23]. Recently, there has been much industrial interest in xylanases for wood pulp bioleaching, papermaking, the manufacture of food and beverages, animal nutrition, and bioethanol production. Because of their biotechnological characteristics, xylanases are most

Nature is replete with myriad microorganisms producing enzymatic complexes that degrade cellulose and hemicellulose releasing sugars, used for attainment of products with high economical value [24]. Microbial xylanases are of prime importance in industrial application. Most of commercial enzymes are accrued from mesophilic microorganisms. The thermostable enzymes from thermophilic micro-organisms can better meet the need of high temperatures in the industrial processes for preparing end products. Of course there is a growing interest for multiple studies in exploring the importance of enzymes producing thermophilic microorganisms in relationship with biotechnological application. The microorganisms being extremophilic in nature can survive and thrive in extreme environments on account of which thermo-stability is provided to industrial processes. Biological sources including bacteria, fungi and yeasts have been reported as xylanase

Xylanase producing thermophilic bacteria are found in variegated environments

*algeriensis* TH7C1(T), isolated from the hydrothermal hot spring has been reported as extracellular thermostable xylanase (XYN35) producing organism [26]. The isolation of thermophilic gram positive strain Rxl, a member of genus *Thermoanaerobacterium* with xylan degrading ability was reported from hot springs in Baoshan of Yunnan Province, China. The successful cultivation of the bacterium was made through utilization of xylan, starch and wide range of monosaccharide and

and the recent one was isolated in Tunisian hot springs. Various thermophilic *Bacillus* strains isolated and the identification of *Bacillus* strains was based on the phenotypic characteristics of *Bacillus* genus and phylogenetic analysis of the 16S rDNA sequence. Activity tests of these *Bacillus* strains confirmed the xylanase producing strains [25]. A thermophilic anaerobic bacteria *Caldicoprobacter*

often produced from microorganisms for commercial applications.

**4. Sources/genesis of xylanases**

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

producing organisms in a natural process.

**4.1 Bacterial source**

**297**

The xylanases from the GH11 family belongs to clan GHC. It displays exclusive substrate specificity toward xylose containing substrates and a preference for insoluble polymeric substrates. The structure of GH11 is highly homologous and contains a single major α-helix and two extended pleated β-sheets which form a jelly-roll fold [19]. The structural features include a compact globular structure and a thumb-like structure as an 11-residue long loop that connects β-strands β8 and β7, and a long cleft that spans the entire molecule and contains the active site [20]. The catalytic machinery is composed of two glutamate residues, acting as a nucleophile and an acid/base catalyst, located in the middle of the long cleft [19]. Moreover, catalytic amino acids and enzymatic mechanism of GH11 are conserved and presenting domains for catalysis of 180–200 amino acids that fold into β-sheet conformation curved on itself.


#### **Table 1.**

*Characteristics of different glycoside hydrolase family containing enzymes with a demonstrated xylanase activity.*

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

low molecular weight enzymes [16]. The values of their pI are generally alkaline (8.0–9.5), however, some also have acid values and all of them sustain the same three-dimensional structure. Most of the substrate binding subsites are highly conserved in xylanases, but the affinity differences between these subsites significantly affect their mode of action, as well as substrate and product preferences [17]. As heat stability has great concern in commercial usages of xylanases. For this purpose, a number of studies analyzed the crystal structures of thermostable xylanases. Intraand intermolecular interactions in structural topography such as disulfide bond and hydrogen bond, compact the overall fold and stabilized N and C terminal end, fusion with CBM (carbohydrate-binding motif) and lower B-factor have been pro-

The xylanases from the GH11 family belongs to clan GHC. It displays exclusive substrate specificity toward xylose containing substrates and a preference for insoluble polymeric substrates. The structure of GH11 is highly homologous and contains a single major α-helix and two extended pleated β-sheets which form a jelly-roll fold [19]. The structural features include a compact globular structure and a thumb-like structure as an 11-residue long loop that connects β-strands β8 and β7, and a long cleft that spans the entire molecule and contains the active site [20]. The catalytic machinery is composed of two glutamate residues, acting as a nucleophile and an acid/base catalyst, located in the middle of the long cleft [19]. Moreover, catalytic amino acids and enzymatic mechanism of GH11 are conserved and presenting domains for catalysis of 180–200 amino acids that fold into β-sheet conformation

posed to bestow the enzyme for increased heat stability [18].

*Biotechnological Applications of Biomass*

**Fold Clan Mechanism Nucleophile/**

**proton donor**

GH5 (β/α)8 CH-B Retaining Glu/Glu Endo-β-1,4-xylanase (EC 3.2.1.8),

GH8 (α/α)8 CH-M Inverting Asp/Glu Endo-1,4-β-xylanase (EC 3.2.1.8) 1H13

GH10 (β/α)8 CH-A Retaining Glu/Glu Endo-1,4-β-xylanase (EC 3.2.1.8),

GH30 (β /α) 8 GH-A Retaining Glu/Glu Endo-β-1,4-xylanase (EC 3.2.1.8),

*Characteristics of different glycoside hydrolase family containing enzymes with a demonstrated xylanase*

CH-B Retaining Glu/Glu Endo-β-1,4-glucanase (EC 3.2.1.4),

CH-A Retaining Glu/Glu Endo-1,4-β-xylanase (EC 3.2.1.8),

GH-F Inverting Asp/Glu Xylanase (EC 3.2.1.8), β-xylosidase

3.2.1.73)

3.2.1.37)

(EC 3.2.1.37)

**Xylanase Activity PDB**

Arabinoxylan-specific endo-β-1,4-

Endo-β-1,3–1,4-glucanase (EC

Endo-1,3-β-xylanase (EC 3.2.1.32)

Endo-1,3-β-xylanase (EC 3.2.1.32)

Endo-β-1,4-xylanase (EC 3.2.1.136), β-xylosidase (EC

xylanase (EC 3.2.1.-)

**No.\***

2Y8K 5G56 4U3A

1EG1 3OVW

1XW2

4QCE 1NQ6 1 W32

3WP3 1YNA 1XNK

4FMV 4FMV

5GLN 2EXJ

curved on itself.

GH7 β-jelly roll

GH11 β-jelly roll

GH43 5-fold βpropeller

*PDB, Protein data base number.*

*\**

**Table 1.**

*activity.*

**296**

**GH family**

GH 5 is the largest glycoside hydrolase family with varying activities including endo-1,4-β-xylanase. It hydrolyzes the β-1,4 xylan chain at a specific site directed by the position of an α-1,2-linked glucuronate moiety. The structural analysis XynA (of the family 5 xylanase) showed that, the catalytic domain displayed a common (β/α)8 barrel fold [21]; whereas, the β-barrels aligned well with those of another family 5 enzyme. The α-helices and loops were different, showing variances in the positioning, length and orientation. The xylanases belongs to family GH8 are classified in clan CH-M also contains endo-1,4-β-xylanase along with other glycoside hydrolase enzymes. It has also the aptitude to hydrolyze the β-1,4 xylan chain and exhibits the (α/α)6 barrel structure formed by six inner and six outer α helices [22]. Similarly, the GH26 are the member of the clan CH-A and exhibits the (β/α)8 structure. This family contains different glycoside hydrolase enzymes including β-1,3-xylanase, capable of hydrolyzing β-1,3-xylan. Activity, mechanisms and the structure of other member of glycoside hydrolase enzymes are listed in **Table 1**.

## **4. Sources/genesis of xylanases**

Demystifying definitional issue for common understanding, the xylanases are enzymes commonly found in microorganisms, marine algae, protozoans, snails, crustaceans, insects, seeds, plants, and other natural sources [23]. Recently, there has been much industrial interest in xylanases for wood pulp bioleaching, papermaking, the manufacture of food and beverages, animal nutrition, and bioethanol production. Because of their biotechnological characteristics, xylanases are most often produced from microorganisms for commercial applications.

Nature is replete with myriad microorganisms producing enzymatic complexes that degrade cellulose and hemicellulose releasing sugars, used for attainment of products with high economical value [24]. Microbial xylanases are of prime importance in industrial application. Most of commercial enzymes are accrued from mesophilic microorganisms. The thermostable enzymes from thermophilic micro-organisms can better meet the need of high temperatures in the industrial processes for preparing end products. Of course there is a growing interest for multiple studies in exploring the importance of enzymes producing thermophilic microorganisms in relationship with biotechnological application. The microorganisms being extremophilic in nature can survive and thrive in extreme environments on account of which thermo-stability is provided to industrial processes. Biological sources including bacteria, fungi and yeasts have been reported as xylanase producing organisms in a natural process.

#### **4.1 Bacterial source**

Xylanase producing thermophilic bacteria are found in variegated environments and the recent one was isolated in Tunisian hot springs. Various thermophilic *Bacillus* strains isolated and the identification of *Bacillus* strains was based on the phenotypic characteristics of *Bacillus* genus and phylogenetic analysis of the 16S rDNA sequence. Activity tests of these *Bacillus* strains confirmed the xylanase producing strains [25]. A thermophilic anaerobic bacteria *Caldicoprobacter algeriensis* TH7C1(T), isolated from the hydrothermal hot spring has been reported as extracellular thermostable xylanase (XYN35) producing organism [26]. The isolation of thermophilic gram positive strain Rxl, a member of genus *Thermoanaerobacterium* with xylan degrading ability was reported from hot springs in Baoshan of Yunnan Province, China. The successful cultivation of the bacterium was made through utilization of xylan, starch and wide range of monosaccharide and

polysaccharides [27]. Others like xylanase genes, was cloned from bacterial strain *Planococcus* sp., SL4, was isolated from the sediment of Soda Lake Dabusu of high alkalinity nature [28].

#### **4.2 Fungal source**

In comparison with the bacteria, the filamentous fungi have been in use as most potent industrial enzyme producers for the last five decades. Filamentous fungi are exuberant producers of xylanolytic enzymes in medium being used for the purpose. The genomes of lignocellulolytic fungi like for example *Trichoderma reesei*, *Aspergillus niger*, and *Myceliophthora thermophila* are producing diversity of enzymes that breakdown the complex cell wall components [29**–**31]. *M. thermophila* known as a powerful cellulolytic organism was used in new advanced technologies for industrial enzyme production, like the biomass-derived fuels. Due to its peculiarities *M. thermophila* distinguishes from rest of xylanase producers such *A. niger* and *T. reesei*. It is best source of gene encoding extracellular thermophilic xylanases. It has presence of a relatively high number of (glucurono) arabinoxylan degrading enzymes. It has lignocellulolytic enzymes that synthesize a complete set of enzymes necessary for the breakdown of cellulose. On the sequence analysis and the genome of the *M. thermophilia* has revealed a large repertoire of genes responsible for the production of thermostable lignocellulolytic enzymes such as carbohydrate-active enzymes, proteases, oxidoreductases, lipases and xylanase [30, 32, 33]. *M. thermophila* has 9110 genes, organized in 7 chromosomes, sequenced and annotated and also consists of 250 genes encode carbohydrate-active enzymes of which 180 are potential glycoside hydrolases. Thirteen out of 180 genes has ability to encode xylanases [34, 35].

Other thermophilic fungus like genus *Humicola* a nonpathogenic and nontoxic fungus also produces a wide range of hemicellulases and cellulases. Thermophilic *Humicola insolens* Y1 is an excellent producer of xylanolytic enzymes, including the thermophilic xylanases from family GH10 and GH11 [36]. More to the list is the *Thermoascus aurantiacus* another potential fungus with the ability to produce thermostable cellulases and xylanase, reported from Aravali forest area of University of New Delhi [37]. The fungus was able to produce antioxidant compounds as byproduct of its inoculum preparation process, which could be used for exploiting crop residues for biofuel production.

**5. Expression systems for xylanases**

*Sources of microbial xylanases with demonstrated activity.*

*RBB-Xylan, Remazol brilliant blue-Xylan.*

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

applications [9].

**299**

*\**

**Table 2.**

**5.1 Bacterial expression system**

To acquire a pure form of a particular enzyme from a given source is challenging. Also it is inconvenient to have cultivation of bacteria or fungi for large scale protein production that often leads to many interfering enzymes. It might need multiple purification steps to get the intended enzymes purified from a pool of proteins which in turn will increase the cost. Therefore, recombinant DNA technology is recommendable for application with success prospects for desired object [50]. Recombinant DNA technology allows large scale expression of enzymes in both homologous and heterologous protein expression. The genes of enzymes with industrial importance were reportedly cloned and expressed in expression hosts in order to enhance specific enzymes production plus improvement in substrate utilization, and other commercially useful properties. Likewise, genes encoding thermophilic xylanases from different sources have been cloned with the objectives of overproduction of the xylanases and changing its properties to suit commercial

**Sources Gene Substrate Xylanase activity References** *Humicola insolens* Xyn11B Beechwood Xylan 382.0 U/mg [36] *Streptomyces* sp*.* XynA Beechwood Xylan 250.69 U/mg [41] *Streptomyces* sp*.* — Birchwood Xylan 5098.28 U/mg [42] *Schizophyllum commune* XynA Beechwood Xylan 5768 U/mg [43] *Streptomyces* sp. XynBS27 Oat spelt Xylan 3272.0 U/mg [44] *Aspergillus niger* XAn11 Birchwood Xylan 909.4 U/mg [45] *Aspergillus. niger* XAn11 Birchwood Xylan 415.1 U/mg [45] *Planococcus* sp*.* XynSL4 Birchwood Xylan 244.7 U/mg [28] *Acrophialophora nainiana* Xyn6 Oat spelt xylan 172 mg/L [46] *Trichoderma reesei* Xyn2 Birchwood Xylan 1600 U/mg [47] *Myceliophthora thermophila* MYCTH\_56237 RBB-Xylan\* 1533.7 U/mg [48] *Myceliophthora thermophila* MYCTH\_49824 RBB-Xylan\* 1412.5 U/mg [48] *Thermothelomyces thermophila* MYCTH\_39555 Birchwood Xylan 105.42 U/mg [49]

*Escherichia coli* are the most promising host for cloning and expression of heterologous recombinant proteins. Success of this platform as a recombinant expression host mainly due to the ease of is attributed toward some factors such as wide choice of cloning vectors, rapid growth, inexpensive media and simple techniques required for transformation, secretion of heterologous proteins into the culture medium and avoid the difficulties associated with purification of the recombinant protein [9]. *E. coli* expression systems been used for recombinant proteins production both intracellularly and extracellularly. In spite of the many advantages of using *E. coli* as expression host, there are certain limitations such as upon gene over expression, recombinant protein aggregates to form inclusion bodies in the cytoplasm. In order

#### **4.3 Xylanases from archaeal domain**

In the field of biotechnology, the thermophilic micro-organisms from archeal domain have been reported/isolated with ability to express enzymes that can tolerate high temperatures (80–115°C), extreme pH, and high salt concentration [38]. These thermophilic enzymes with attribute of hydrolyzing lignocellulosic biomass were characterized, cloned and expressed in various hosts. *Sulfolobus solfataricus* is thermoacidophile that can live in acidic volcanic hot springs and grows optimally up to 87°C and pH 2–4. It produces enzymes with carbohydrate depolymerizing activities, such as endoglucanases and xylanases, as well as β-glucosidases/xylosidases involved in the degradation of plant-derived complex polysaccharides [39]. The genome of *S. solfataricus* has been sequenced, and three open reading frames (sso1354, sso1949, and sso2534) coding for putative extracellular endo-glucanases have been identified. These enzymes belong to a GH12 of glycoside hydrolases family and member of clan C [40]. Sources of microbial xylanases with demonstrated xylanase activity are listed in **Table 2**.

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


#### **Table 2.**

polysaccharides [27]. Others like xylanase genes, was cloned from bacterial strain *Planococcus* sp., SL4, was isolated from the sediment of Soda Lake Dabusu of high

In comparison with the bacteria, the filamentous fungi have been in use as most potent industrial enzyme producers for the last five decades. Filamentous fungi are exuberant producers of xylanolytic enzymes in medium being used for the purpose. The genomes of lignocellulolytic fungi like for example *Trichoderma reesei*, *Aspergillus niger*, and *Myceliophthora thermophila* are producing diversity of enzymes that breakdown the complex cell wall components [29**–**31]. *M. thermophila* known as a powerful cellulolytic organism was used in new advanced technologies for industrial enzyme production, like the biomass-derived fuels. Due to its peculiarities *M. thermophila* distinguishes from rest of xylanase producers such *A. niger* and *T. reesei*. It is best source of gene encoding extracellular thermophilic xylanases. It has presence of a relatively high number of (glucurono) arabinoxylan degrading enzymes. It has lignocellulolytic enzymes that synthesize a complete set of enzymes necessary for the breakdown of cellulose. On the sequence analysis and the genome of the *M. thermophilia* has revealed a large repertoire of genes responsible for the production of thermostable lignocellulolytic enzymes such as carbohydrate-active

enzymes, proteases, oxidoreductases, lipases and xylanase [30, 32, 33]. *M.*

New Delhi [37]. The fungus was able to produce antioxidant compounds as byproduct of its inoculum preparation process, which could be used for exploiting

*thermophila* has 9110 genes, organized in 7 chromosomes, sequenced and annotated and also consists of 250 genes encode carbohydrate-active enzymes of which 180 are potential glycoside hydrolases. Thirteen out of 180 genes has ability to encode

Other thermophilic fungus like genus *Humicola* a nonpathogenic and nontoxic fungus also produces a wide range of hemicellulases and cellulases. Thermophilic *Humicola insolens* Y1 is an excellent producer of xylanolytic enzymes, including the thermophilic xylanases from family GH10 and GH11 [36]. More to the list is the *Thermoascus aurantiacus* another potential fungus with the ability to produce thermostable cellulases and xylanase, reported from Aravali forest area of University of

In the field of biotechnology, the thermophilic micro-organisms from archeal domain have been reported/isolated with ability to express enzymes that can tolerate high temperatures (80–115°C), extreme pH, and high salt concentration [38]. These thermophilic enzymes with attribute of hydrolyzing lignocellulosic biomass were characterized, cloned and expressed in various hosts. *Sulfolobus solfataricus* is thermoacidophile that can live in acidic volcanic hot springs and grows optimally up to 87°C and pH 2–4. It produces enzymes with carbohydrate depolymerizing activities, such as endoglucanases and xylanases, as well as β-glucosidases/xylosidases involved in the degradation of plant-derived complex polysaccharides [39]. The genome of *S. solfataricus* has been sequenced, and three open reading frames (sso1354, sso1949, and sso2534) coding for putative extracellular endo-glucanases have been identified. These enzymes belong to a GH12 of glycoside hydrolases family and member of clan C [40]. Sources of microbial xylanases with demon-

alkalinity nature [28].

*Biotechnological Applications of Biomass*

**4.2 Fungal source**

xylanases [34, 35].

crop residues for biofuel production.

**4.3 Xylanases from archaeal domain**

strated xylanase activity are listed in **Table 2**.

**298**

*Sources of microbial xylanases with demonstrated activity.*
