**5. Expression systems for xylanases**

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 applications [9].

#### **5.1 Bacterial expression system**

*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

to reduce the inclusion bodies, several strategies could be used such as regulation of the protein synthesis rate, co-expression of chaperone genes and empowering the secretion of proteins into the periplasm. However, the control rate of protein synthesis can be achieved by altering the promoter to regulate the level of expression, fusing the target gene to another gene, and adjusting the growth conditions, such as pH and temperature of the medium [9].

produced XynB under the egl2 promoter [58]. Expression of the *xynB* gene under all three different promoters resulted in improvements of the enzyme [58]. Two novel genes of family GH11 xylanases *xyn5* and *xyn6*, isolated from the thermophilic filamentous fungus *Acrophialophora nainiana,* were successfully expressed in an industrially-exploited fungal host *T.* reesei [46]. Moreover, the beneficial aspect of this fungus use for recombinant gene expression is the secretion of proteins into the growth medium and consequent gene products achieved comparatively straightforward. However, degradation of recombinant gene products also occurs due to

Yeasts considered as excellent and attractive host for the expression of heterologous proteins and offer many advantages over the other established expression systems especially in protein maturation [59]. The methylotrophic yeast *Pichia pastoris* is an established protein expression host for the production of industrial enzymes. It can be grown to very high cell densities, produces high titer of recombinant proteins and ability to secrete proteins into fermentation media thus it is a very useful expression

, respectively) under this pro-

host, especially when scaling up to industrial process [60]. *P. pastoris* can be expressed intercellularly and provides extra benefits over the other expression systems such as ability to perform eukaryotic post-translational modifications, glycosylation, proper folding of the proteins [61]. Moreover, the most significant feature the of this expression host are due to the availability of strong and regulatory promoter of alcohol oxidases AOX1, involved in the methanol utilization pathway which provided exceptionally high levels of heterologous recombinant protein [62]. Because of all such features, the expression of xylanase genes in *P. pastoris* preferred mostly and provides high yield of recombinant xylanases expression under methanol induction. The enzyme activity of xylanase was reported 3676 U mL<sup>1</sup> for the gene product of xylB from *A. niger*, when expressed under AOX1 in *P. pastoris*. In fact, this is one of the highest expressions of recombinant xylanase expressed from *P. pastoris* reported [63]. Similarly, Cheng et al. [61] and Chantasingh et al. [64] also attained high

moter (67-fold and 4-fold) higher recombinant xylanase activity, compared to the native fungal xylanases. The gene coding xylanase (*xynS14*) from a thermophilic xylan degrading actinomycetes *Actinomadura* S14, were expressed in both *E. coli* and *P. pastoris* [65]. The specific activity of purified recombinant xylanase from *P. pastoris* transformants was approximately 2.4-fold higher than that of purified recombinant xylanase from *E. coli* transformants*,* suggesting that *P. pastoris* is a better host for expression of recombinant XynS14*.* Although both recombinant XynS14 showed approximately the same basic properties, such as substrate specificity, optimal pH and temperature, stability for pH and temperature, and effects of EDTA and metal ions, whereas XynS14 (*P. pastoris*) showed higher specific activity and kinetic values (*V*max and *K*cat) than XynS14 (*E. coli*) [65]. These finding suggested the glycosyl chains present in XynS14 (*P. pastoris*) stabilized the enzyme and the enzymes were folded properly in *P. pastoris*. Cloning and expression of another xylanase gene belongs to family GH11 from *T. fusca* NTU22, also reported higher yield and thermo-

In most of the cases xylanases need to undergo some genetic modifications in order to enhance expression level, enzymes activity and that might have some

the secreted acidic proteases into the cultivation medium [58].

xylanase activity (342.2 U mL<sup>1</sup> and 238.5 mg mL<sup>1</sup>

stability than the original strain [61].

**301**

**6. Xylanases: genetic engineering and optimization**

**5.3 Yeast expression system**

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

Although previously it has been reported that the expression of the xylanase genes usually cannot be functionally expressed in *E. coli* due to some factors including the repetitive appearance of rare codons and the requirement for specific post translational modifications such as disulfide bond formation and glycosylation [51] and also it require N-glycosylation whereas *E. coli* can only perform simple O-glycosylation [52]. However, recently recombinant xylanase of family GH10 (XYN) from *Thermoanaerobacterium thermosaccharolyticum* (DSM 571) was successfully overexpressed in *E. coli* (strain BL21) [53]. Similarly, another xylanase gene (xyn10B) encoding the endo-xylanase from *Thermotoga thermarum*, was successfully cloned and expressed in *E. coli* (strain BL21) and exhibits the thermostability at high temperature [54]. These finding indicated that *E. coli* might be an effective and suitable host for the expression of xylanases. Furthermore, xylanase (gene xynA) from thermophilic fungus *Thermomyces lanuginosus* exhibits the activity endo-xylanase of GF11 and the expression of optimized sequence of xynA in *E. coli* was found to be a high level. However, the recombinant XynA was mainly found in inclusion bodies, and only a small proportion was soluble and active [55]. For this purpose, a strategy was exposed to overcome inclusion-body formation, an expression plasmid named pHsh exhibit a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ32). pHsh derivative was constructed by fusing a signal peptide to xynA2 gene, eases to export the recombinant protein to periplasm and xylanase was successfully produced in a soluble form [56].

## **5.2 Fungal expression system**

Filamentous fungus is the promising organism for protein expression and its production by fermentation has a long history in industrial area. Even developed other expression systems for recombinant protein expression, fungal expression system also considered an appropriate candidate for the expression [9]. Natural capability of fungal expression system to secrete large amounts of proteins into the medium gave an advantage to this expression system. Furthermore, it has feasibility for functional expression of other xylanases from remote sources by using of native xylanase expressing machinery [7, 9]. Most of the xylanase genes have been expressed in fungi under homologous expression system and frequently used fungus as expression hosts are *T. reesei*, *A. niger* and *Aspergillus oryzae* [57]. *T. reesei* system relies on the integration of the transforming DNA into the fungal genome, which results in excellent stability of transformants. The vectors construct provided a variety of N and C-terminal modifications that facilitate gene product processing and purification. In *T. reesei*, the most frequent choice of a promoter used for recombinant gene expression is the *cbh1* (cellobiohydrolase 1) gene encoding the cellulose. For high level expression of recombinant proteins in *T. reesei* is to use a variety of strong promoters that simultaneously transcribe the target gene, instead of one promoter with multiple copies of genes, which might be leads to the depletion of specific transcription factors for the promoter. Recently *T. reesei* strain expressed the recombinant bacterial xylanase XynB under the promoters of *egl2* (endoglucanase 2), *xyn2* and *cbh2*. Promoter of the *T. reesei xyn2* gene encodes the endo-1,4-β-xylanase II (Xyn2). Gene expression cassettes with the *xyn2* and *cbh2* promoters were introduced simultaneously into a *T. reesei* strain (EC-21), which

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

produced XynB under the egl2 promoter [58]. Expression of the *xynB* gene under all three different promoters resulted in improvements of the enzyme [58]. Two novel genes of family GH11 xylanases *xyn5* and *xyn6*, isolated from the thermophilic filamentous fungus *Acrophialophora nainiana,* were successfully expressed in an industrially-exploited fungal host *T.* reesei [46]. Moreover, the beneficial aspect of this fungus use for recombinant gene expression is the secretion of proteins into the growth medium and consequent gene products achieved comparatively straightforward. However, degradation of recombinant gene products also occurs due to the secreted acidic proteases into the cultivation medium [58].

#### **5.3 Yeast expression system**

to reduce the inclusion bodies, several strategies could be used such as regulation of the protein synthesis rate, co-expression of chaperone genes and empowering the secretion of proteins into the periplasm. However, the control rate of protein synthesis can be achieved by altering the promoter to regulate the level of expression, fusing the target gene to another gene, and adjusting the growth conditions, such as

Although previously it has been reported that the expression of the xylanase genes usually cannot be functionally expressed in *E. coli* due to some factors including the repetitive appearance of rare codons and the requirement for specific post translational modifications such as disulfide bond formation and glycosylation [51] and also it require N-glycosylation whereas *E. coli* can only perform simple O-glycosylation [52]. However, recently recombinant xylanase of family GH10 (XYN) from *Thermoanaerobacterium thermosaccharolyticum* (DSM 571) was successfully overexpressed in *E. coli* (strain BL21) [53]. Similarly, another xylanase gene (xyn10B) encoding the endo-xylanase from *Thermotoga thermarum*, was successfully cloned and expressed in *E. coli* (strain BL21) and exhibits the thermostability at high temperature [54]. These finding indicated that *E. coli* might be an effective and suitable host for the expression of xylanases. Furthermore, xylanase (gene xynA) from thermophilic fungus *Thermomyces lanuginosus* exhibits the activity endo-xylanase of GF11 and the expression of optimized sequence of xynA in *E. coli* was found to be a high level. However, the recombinant XynA was mainly found in inclusion bodies, and only a small proportion was soluble and active [55]. For this purpose, a strategy was exposed to overcome inclusion-body formation, an expression plasmid named pHsh exhibit a synthetic heat-shock (Hsh) promoter, in which gene expression is regulated by an alternative sigma factor (σ32). pHsh derivative was constructed by fusing a signal peptide to xynA2 gene, eases to export the recombinant protein to periplasm

pH and temperature of the medium [9].

*Biotechnological Applications of Biomass*

and xylanase was successfully produced in a soluble form [56].

Filamentous fungus is the promising organism for protein expression and its production by fermentation has a long history in industrial area. Even developed other expression systems for recombinant protein expression, fungal expression system also considered an appropriate candidate for the expression [9]. Natural capability of fungal expression system to secrete large amounts of proteins into the medium gave an advantage to this expression system. Furthermore, it has feasibility for functional expression of other xylanases from remote sources by using of native

xylanase expressing machinery [7, 9]. Most of the xylanase genes have been expressed in fungi under homologous expression system and frequently used fungus as expression hosts are *T. reesei*, *A. niger* and *Aspergillus oryzae* [57]. *T. reesei* system relies on the integration of the transforming DNA into the fungal genome, which results in excellent stability of transformants. The vectors construct provided a variety of N and C-terminal modifications that facilitate gene product processing and purification. In *T. reesei*, the most frequent choice of a promoter used for recombinant gene expression is the *cbh1* (cellobiohydrolase 1) gene encoding the cellulose. For high level expression of recombinant proteins in *T. reesei* is to use a variety of strong promoters that simultaneously transcribe the target gene, instead of one promoter with multiple copies of genes, which might be leads to the depletion of specific transcription factors for the promoter. Recently *T. reesei* strain expressed the recombinant bacterial xylanase XynB under the promoters of *egl2* (endoglucanase 2), *xyn2* and *cbh2*. Promoter of the *T. reesei xyn2* gene encodes the endo-1,4-β-xylanase II (Xyn2). Gene expression cassettes with the *xyn2* and *cbh2* promoters were introduced simultaneously into a *T. reesei* strain (EC-21), which

**5.2 Fungal expression system**

**300**

Yeasts considered as excellent and attractive host for the expression of heterologous proteins and offer many advantages over the other established expression systems especially in protein maturation [59]. The methylotrophic yeast *Pichia pastoris* is an established protein expression host for the production of industrial enzymes. It can be grown to very high cell densities, produces high titer of recombinant proteins and ability to secrete proteins into fermentation media thus it is a very useful expression host, especially when scaling up to industrial process [60]. *P. pastoris* can be expressed intercellularly and provides extra benefits over the other expression systems such as ability to perform eukaryotic post-translational modifications, glycosylation, proper folding of the proteins [61]. Moreover, the most significant feature the of this expression host are due to the availability of strong and regulatory promoter of alcohol oxidases AOX1, involved in the methanol utilization pathway which provided exceptionally high levels of heterologous recombinant protein [62]. Because of all such features, the expression of xylanase genes in *P. pastoris* preferred mostly and provides high yield of recombinant xylanases expression under methanol induction. The enzyme activity of xylanase was reported 3676 U mL<sup>1</sup> for the gene product of xylB from *A. niger*, when expressed under AOX1 in *P. pastoris*. In fact, this is one of the highest expressions of recombinant xylanase expressed from *P. pastoris* reported [63]. Similarly, Cheng et al. [61] and Chantasingh et al. [64] also attained high xylanase activity (342.2 U mL<sup>1</sup> and 238.5 mg mL<sup>1</sup> , respectively) under this promoter (67-fold and 4-fold) higher recombinant xylanase activity, compared to the native fungal xylanases. The gene coding xylanase (*xynS14*) from a thermophilic xylan degrading actinomycetes *Actinomadura* S14, were expressed in both *E. coli* and *P. pastoris* [65]. The specific activity of purified recombinant xylanase from *P. pastoris* transformants was approximately 2.4-fold higher than that of purified recombinant xylanase from *E. coli* transformants*,* suggesting that *P. pastoris* is a better host for expression of recombinant XynS14*.* Although both recombinant XynS14 showed approximately the same basic properties, such as substrate specificity, optimal pH and temperature, stability for pH and temperature, and effects of EDTA and metal ions, whereas XynS14 (*P. pastoris*) showed higher specific activity and kinetic values (*V*max and *K*cat) than XynS14 (*E. coli*) [65]. These finding suggested the glycosyl chains present in XynS14 (*P. pastoris*) stabilized the enzyme and the enzymes were folded properly in *P. pastoris*. Cloning and expression of another xylanase gene belongs to family GH11 from *T. fusca* NTU22, also reported higher yield and thermostability than the original strain [61].
