Abstract：As the hydrolysis of xylan is an important step towards the utilization of hemicelluloses, xylanases are widely used in the pulp and paper, animal feed, and brewing industries. In this work, amino acid alignment of xylanase from Bacillus subtilis with acidophilic and acid-stable xylanase from glycosyl hydrolase family 11 was carried by the ClustalW program. Then the mutants of N63D, A143E and N63D/A143E of xylanase were constructed by oligonucleotide-based site-directed mutagenesis methods, using the recombinant plasmid pUC18-xyl11 as the template. The mutated genes were inserted into expression vector pGEX-6p-1. And then, the recombinant plasmids pGEX-6p-xyl11, pGEX-6p-xyl11N63D, pGEX-6p-xyl11A143E and pGEX-6p-xyl11N63D/A143E were transformed into Escherichia coli BL21 (DE3) for protein expression and purification. After induction with IPTG(isopropyl β-D-thiogalactoside), the recombinant enzymes were successfully expressed in soluble form. The wild type Xyl11 and three mutants were purified to high homogeneity by glutathione-affinity chromatography followed by 3C protease digestion. SDS-PAGE analysis showed that the molecular mass of Xyl11 was about 20 kD. Then kinetic parameters of Xyl11 and mutants were determined. The optimum pH of the purified enzyme was determined within a pH range of 3.0~8.5. Xyl11 displayed the maximum activity at pH 6.0, while single site mutant, i.e., Xyl11N63D and Xyl11A143E, with showed the maximum activity at pH 4.5 and pH 5.0, respectively. Like Xyl11A143E, the pH optimum was 5.0. In comparison with the wild type enzyme, all the mutants shifted the pH optimum towards acid pH. The kinetic parameters of the purified xylanase and its mutants were determined by measuring the enzymatic activity with various concentrations of beechwood xylan as the substrate. The Michaelis constant(Km), Maximum reaction rate(Vmax) and catalytic number(Kcat)of Xyl11 were 5.996 mg/mL, 360.615 μmol/(min.mg) and 1 054.429 /s, respectively. The Km, Vmax and Kcat of Xyl11N63D was 6.876 mg/mL, 326.289 μmol/(min.mg) and 15.013 /s, respectively. The Km, Vmax and Kcat of Xyl11A143E was 9.178 mg/mL, 212.959 μmol/(min.mg) and 6.544 /s, respectively. For the mutant Xyl11N63D/A143E, Km was 14.885 mg/mL, Vmax was 201.248 (min.mg) and Kcat was 4.742 /s. According to these data, the Km of the mutans were increased, however, the Vmax and Kcat were decreased, compared to the wild type enzyme. Totally, it could be concluded that the sites Asn63 and Ala143 are involved in the pH optimum and activity of xylanase from Bacillus subtilis. This work provides insight into the structure-function study of xylanases, and valuable hints for the protein engineering of xylanases in future study.
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