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.
[1]付正，张华山，曾小英，等．嗜热真菌木聚糖酶1YNA 的表达和定点突变[J]．湖北农业科学，2011，50（7）：1488-1493． [2]李勇如，李秀婷，朱运平，等．枝链霉菌 S. rameus L2001 产木聚糖酶对馒头品质的影响[J]．食品科学，2012，33（17）：25-29． [3]卢毅弘，彭彦峰，周玉玲，等．造纸用木聚糖酶的基因工程研究进展[J]．氨基酸和生物资源，2014,36（3）：1-6． [4]王雅珍，李秀婷，孙宝国．微生物酸性木聚糖酶及其应用的研究进展[J]．食品与发酵工业，2012，38（8）：107-113． [5]王启修，张兆敏，张磊，等．日粮添加木聚糖酶对肉鸡小肠葡萄糖吸收及其转运载体基因表达影响[J]．农业生物技术学报，2005,13（4）：497-502． [6]杨浩萌，柏映国，李江，等．木聚糖酶 XYNB 的 N46D 突变、表达及酶学性质变化[J]．中国生物化学与分子生物学报，2006，22（3）：204-211． [7]杨小军，杨凤霞，罗定媛，等．木聚糖酶对肉仔鸡养分利用和消化器官发育的影响[J]．动物营养学报，2010，22（1）：157-162． [8]Collins T，Gerday C，Feller G，et al．Xylanases，xylanase families and extremophilic xylanases[J]．FEMS Microbiology，2005，29(1)：3-23． [9]Fushinobu S, Ito K, Konno M, Wakagi T, Matsuzawa H. Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH[J]. Protein engineering, 1998, 11(12): 1121-1128. [10]Joshi M D, Sidhu G, Pot I，et al. Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase[J]．Journal of Molecular Biology，2000，299(1)：255-279． [11]Kongsted J, Ryde U, Wydra J, Jensen JH. Prediction and rationalization of the pH dependence of the activity and stability of family 11 xylanases[J]. Biochemistry, 2007, 46(47): 13581-13592. [12]Ratanakhanokchai K，Kyu K L，Tanticharoen M．Purification and properties of a xylan-binding endoxylanase from alkaliphilic Bacillus sp. strain K-1[J]． Applied and environmental microbiology，1999， 65(2)：694-697． [13]Vieira D S, Degrève L, Ward R J．Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation[J]．Biochimica et Biophysica Acta (BBA)-General Subjects，2009， 1790(10)：1301-1306．
