Improvement Expression And Characterization Of A Multi-domain Xylanase XynA

Degradation of lignocellulosic biomass, which is one of the most abundant renewable resources in the nature, has been extensively concerned in recent years because of the fossil energy crisis and the sharp increase of human population. However, biodegradation of lignocellulosic biomass is limited by its complicated structure. Lignocellulose is mainly composed of lignin, cellulose and hemicelluloses, and cellulose is combined with lignin closely by the cross-link of hemicelluloses. Thus, many kinds of glycoside hydrolases are required during the biodegradation of lignocellulosic biomassXylan, the predominant constituent of hemicelluloses, is a kind of heterogeneous polysaccharide which is mainly composed of xylose. Xylanase is one of the two key enzymes during biodegradation of xylan, as it can randomly cleave the β-1,4-D-xylosidic linkage within xylan backbone and catalyze the initial break down of xylan. Thus, the potential applications of xylanase cover a wide range of industrial sectors, such as paper and pulp industry, food industry, energy industry, pharmaceutical industry, etc.In this study, a xylanase gene(xyn A) was cloned from Thermoanerobacterium aotearoense SCUT27, a species isolated from hot spring in China. Based on amino acid sequence alignment, it was attested that the xylanase consisted of a N-terminal signal peptide, one glycoside hydrolase family 10 domain, four carbohydrate binding modules, and three surface layer homology domains. In this study, expression in E. coli showed that xylanase Xyn A from T. aotearoense SCUT27 is sensitive to proteases and a main hydrolytic fragment has a similar size with the truncated xylanase Xyn A?SLH without SLH domains and linker peptide at the C-terminus. In B. subtilis, five different versions of secretary expression vectors and a intracellular expression vector were constructed and transformed into B. subtilis 1012 wt. The change of expression host from Escherichia coli to Bacillus subtilis resulted in 2.1 and 4.1-fold increases of the total xylanase activity and specific activity for the truncated Xyn AΔSLH, respectively. By affinity chromatography using a Ni-NTA affinity column, 24 mg purified protein, which exhibiting xylanase activity of 379.8 U/mg, was obtained. The recombinant xylanase was optimally active at 80 oC and p H 6.5, and its kinetic parameters Km, Vmax and Kcat were determined to be 4.10 mg/m L, 813.0 U/mg and 1539.1 s-1, respectively. In addition, Xyn AΔSLH showed great thermostability in slightly acidic solution, and remained 90% of initial activity after pre-heated at 75 oC for 2 h. Thin layer chromatography results showed that xylotriose, xylotetraose and xylopentaose could be degraded by Xyn AΔSLH, xylobiose and branched xylotriose was the major products during the hydrolysis of beechwood xylan using the recombinant xylanase and a little xylose was produced as well. The addition of Xyn AΔSLH to the hydrolysis of sugarcane bagasse containing Cellic® Ctec2 could increase the yield of reducing sugars while Xyn AΔSLH could also catalyze the hydrolysis of sugarcane bagasse independently to generate xylooligosaccharides. Almost all of the xylan in the sugarcane bagasse could be converted into xylooligosaccharides by Xyn AΔSLH.