Xylanase Gene From Bacillus Agaradhaerens Expression In Escherichia Coli

Xylan is a major constituent of plant cell walls, is the second largest storage capacity of renewable resources. The backbone of xylan may carry various substitutents like arabinose and glucuronic acid, Depending on the complexity of xylan, complete degradation of xylan requires the concerted action of several enzymes. Xylanase is also known as endo-1,4-β-D-xylanase, it can undo the β-1,4-glycosidic linkages of the xylan backbone, then produce short chain xylo-oligosaccharides of various length, so the endo-p-xylanase is key of the microbial xylanolytic systems. Xylanases also find applications in the production of ethanol, textile, animal feed, fruit juices, aroma, paper and pulpindustries.In this study, we amplified the gene of xylanase from Bacillus agaradhaerens by PCR. The gene of xylanase was cloned and expressed in E.coli researched about enzymatic properties, the main research results were as follows:The xylanase gene was amplified by PCR from the genomic DNA of Bacillus agaradhaerens. The length of the xylanase gene was 632bp, encoding 210 amino acid, which has no signal peptide sequence. Analysis the gene of xylanase reveals it had a conserved domain which was Glyco-hydro-11, hence it belongs to family G11 xylanase.The xylanase gene was cloned, ligated to the expression vector pET-22b for building the recombinant expression vector, then transformed into E.coli BL21, using IPTG to induced the expression of the recombinant protein. By optimizing the medium inducing conditions and other factors, the recombinant protein activity can be achieved 3729U/mL from the supernatant, which was 2.3 times as big as the original one.The enzymatic properties of the recombinant xylanase showed:the optimum pH and optimum temperature of the recombinant enzyme were 8.0 and 55℃. The influence of metal ions on xylanase activity showed Zn2+、Fe3+、Ca2+、Cu2+ could inhibit the activity of the recombinant xylanase. We found no metal ions could improve the activity of recombinant xylanase, Suggesting that no metals are needed for enzumatic reactions.