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

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

influence on substrate specificity and stability to high temperature and pH. The gene encoding cellulolytic and xylanolytic enzymes are usually regulated by a repressor/inducer system in fungi. Xylanolytic transcriptional regulators have been reported in thermophilic fungi. The strong promoter MtPpdc (pyruvate decarboxylase) recently used for the overexpression of xylanases from *M. thermophila* ATCC42464 (MtXyr1). The extracellular xylanase activity of the recombinant was reported higher as compared to the wild type indicating the MtXyr1 is a positive regulatory factor for xylanase gene expression and its feasibility of improving xylanase production by overexpressing Mtxyr1 in *M. thermophila* represented an effective approach to increase total xylanase productivity [66]. Similarly, a new Nglycosylated site was created in the coding sequence by amino acid replacements for the expression of endoxylanase in *T. reesei* M2C38. For this purpose, amino acid Asn at position 131 associated with Thr/Ser at position 133 was inserted that resemble a conserved feature for family GH11 xylanases. The new created N-glycosylation site Asn-Xaa-Thr/Ser displayed 40% enhanced protein expression in comparison with wild type [67]. The downregulation of Cre1 plays an important role in enhancing enzyme production and also the silencing of CRE1 improves cellulase and xylanase expression. In *T. reesei* and other fungi, the key regulator of CCR (Carbon catabolite repression) is the Cys2His2-type transcription factor CRE1. The role of CRE1 in *M. thermophilia* was verified through RNAi and suggested the feasibility of improving cellulase production by modifying the expression of regulators in thermophilic fungi [68].

*Achaetomium* sp. Xz-8, with substantial xylanase activity. Substrate specificity and the hydrolysis analysis of purified recombinant revealed that XynC81 and XynC83 were moderate on beechwood xylan (67 and 69%, respectively) and birchwood xylan (45 and 52%, respectively), and weak on barley β-glucan (18 and 14%, respectively). Only XynC81 had detectable activity against insoluble wheat arabinoxylan (22%). Although enzymatic properties of XynC81 and XynC83 were similar but XynC81 with CBM 1 had activity against insoluble wheat arabinoxylan (22%), whereas XynC83 had not. This result further implied the importance of CBM in enzyme activity toward insoluble substrate. As all of the GH11 xylanases characterized so far, about 25% of them carry at least one CBM [73]. Unlike the serine/threonine/asparagine-rich linker sequence found in other fungal xylanases, the XynC81 linker sequence is extremely glycine-rich. Therefore, it could be predicted that CBMs take part in the action of cellulolytic enzymes toward insoluble

Moreover, some xylanases bears CBMs specific for cellulose, which probably assist indirectly localization of xylanase to the xylan substrate, since it is in close association with cellulose. The number of characterized fungal xylanases harboring

*funiculosum* XynB [75, 76], *Neocallimastix patriciarum* XynS20 [77], *Lentinula edodes* Xyn11A [78] and *Phanerochaete chrysosporium* XynB/XynB-1 [79]. Recently, a Xyl-11 from *Podospora anserina* harboring a C-terminal CBM1 efficiently supplemented the industrial cocktail produced by *T. reesei* by improving significantly the release of

The metal ion and chemical reagents had been proved to be one of the critical factors which affected the enzyme activity of xylanases. The effect of metal ions and chemical reagents on the xylanase activities has been determined on various

) and chemical reagents (SDS, β-mercaptoethanol, ethanol, Triton X-100, and

Triton X-100 were reported to enhance enzyme activity by 6.4–29.9% [81], while Fe3+, Cd2+, Hg2+, and Ba2+ completely suppressed the xylanase activity. Besides this, the enzyme had certain ability to resist the Fe2+, Mg2+, Ag2+, SDS, ethanol and SDS. Xylanase activity was not inhibited by chelating reagents such as EDTA and EGTA. Moreover, it is predicted that Ca2+ and Mg2+ ions enhance the enzyme activity by stabilizing the enzyme–substrate complex. In contrast, EDTA is a chelating agent and it removes ions from the enzymes, thus inhibits the enzyme activity [7]. More detailed studies are needed in order to understand the mechanistic effect of metal ions on enzyme activity. Similarly, a xylanase activity isolated from *Planococcus* sp.

As thermophilic enzymes are preferred over the mesophilic enzymes complements because of high temperatures, which had a great influence on many factors such as decreases contamination risk and viscosity of substrate [82]. In a study carried out with the effect of xylanase activity from *A. niger* DFR-5 on different temperature (between 20 and 60°C). The xylanase activity was increased with increase in temperature with maximum activity of at 40°C. On further increase in

, Co2+, Cr3+, Ni2+, Cu2+, Mg2+, Fe3+, Zn2+, Pb2+, and

, Mn2+, EDTA, β-ME, Cu2+ and

, Cr3+, Li+

, Cu2+, Hg2+, Pb2+, Zn2+, Fe3+, and Cr3+

, and Na<sup>+</sup> showed

CBM1 module is relatively reduced. It includes xylanases from *Penicillium*

reducing sugars upon hydrolysis of wheat straw [80].

, Ca2+, Li+

EDTA) at the standard condition. Ca2+, Pb2+, K+

*SL4* was enhanced by Ca2+ and β-mercaptoethanol. K<sup>+</sup>

resulted in an almost complete loss of activity [28].

little or no effect on xylanase activity. Ag<sup>+</sup>

**7.3 Temperature and pH**

substrates [74].

**7.2 Metals and chemicals**

, K+

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

metal ions (Na<sup>+</sup>

Ag+

**303**

In order to fulfill the demands of industrial requirements, gene mining and protein engineering are applied to develop thermostable xylanases. Although some of thermophilic xylanases were produced from thermophiles but their lower expression levels and specific activities making them unable to be applied efficiently. The higher specific activity with enhanced thermostability of xylanases is therefore needed through genetic engineering. Recently the thermostability of mesophilic xylanase (AuXyn10A from *Aspergillus usamii* E001), was improved through elucidation of some local structures affecting the thermostability of mesophilic xylanases in corresponding to thermophilic *Thermoascus aurantiacus*. The temperature optimum of the mutant was 10°C higher than that of AuXyn10A [69]. The thermostability and alkalophilicity of another endo-xylanase from *T. reesei* was improved by replacement of amino acids at different positions and the replacement of NH2 terminal amino acid sequence of *Thermomonospora fusca* along with the addition of some extra amino acids selected from N-terminus of *Clostridium acetobutylicum* xynB. All these strategies increased the thermophilicity and alkalophicility of the enzyme from 55 to 75°C and pH 7.5 to 9.0 respectively [70]. Similarly, the thermostability of mutant xylanase (Xyn10A\_ASPNG) was improved by 17.4°C [71].
