枯草芽孢杆菌β-甘露聚糖酶的克隆表达及重组酶性质研究

Cloning,Expression and Characterization of Recombinant β-mannanase from Bacillus subtilis

为实现对魔芋葡甘聚糖具有高度专一性的β-甘露聚糖酶基因的异源表达,从枯草芽孢杆菌G1中克隆出β-甘露聚糖酶基因Bsman A,将该基因与表达载体pACYCDuet-1连接并转化到E.coil BL21(DE3)中。结果表明:该β-甘露聚糖酶基因Bsman A序列全长为1098 bp,编码366个氨基酸;经Ni柱亲和层析纯化后,测得重组酶分子量大小为38 kD;酶学性质研究结果显示:该酶促反应的最适温度为60℃,最适p H为6.5,在温度50~70℃间,pH4.5~7.0的范围内能保持较好的稳定性。本研究实现了β-甘露聚糖酶的异源表达,为生物催化制备低聚甘露糖的工业化提供了新的选择。

To achieve the heterologous expression of beta-mannanase which is highly specific to Konjac glucomannan,the beta-mannanase gene Bsman A was cloned from Bacillus subtilis G1,linked to the expression vector pACYCDuet-1 and transformed into E.coil BL21( DE3).The results showed that the full length of the beta-mannanase gene Bsman A was 1098 bp and encoded 366 amino acids.After purification by Ni affinity chromatography,the molecular weight of recombinant enzyme was determined to be 38 kD. The results of enzymatic properties of recombinant beta-mannanase showed that the optimum temperature and p H for the enzymatic reaction were 60 ℃ and pH6.5.The stability of recombinant beta-mannanase could be maintained in the range of pH4.5~7.0 and temperature 50~70 ℃.In this study,the heterologous expression of beta-mannanase was accomplished,which would provide a new choice for the industrialization of biocatalytic preparation of mannose oligosaccharides.