Bacillus altitudinis SYBC hb4碱性β-葡萄糖苷酶基因的克隆表达及酶学性质的研究

Cloning,Experssion and Characterization of Alkali-Stable β-Glucosidase from Bacillus altitudinis SYBC hb4

为获得耐碱性β-葡萄糖苷酶,进一步研究碱性β-葡萄糖苷酶的酶学性质。从实验室已有蜂蜜中筛选出一株耐碱性的高地芽孢杆菌Bacillus altitudinis SYBC hb4,根据Bacillus altitudinis SYBC hb4中β-葡萄糖苷酶(bglA)基因序列设计一对引物,通过PCR扩增技术,获得β-葡萄糖苷酶基因(bglA),将扩增后的bglA与质粒pColdII构建重组表达载体pColdII-bglA,并转化至大肠杆菌E.coli BL21(DE3)中表达。重组表达的β-葡萄糖苷酶酶活可达12.40 U/mL;该重组β-葡萄糖苷酶最适反应温度为60℃;最适反应pH值为pH 8.0;5 mmol/L的Mg^(2+)可使酶活提高50%左右。本实验所获得的碱性β-葡萄糖苷酶比已报道的重组酶在碱性条件下稳定性更好。

In order to obtain alkali-stableβ-glucosidase and study its enzyme characterization,an alkali-stable strain Bacillus altitudinis SYBC hb4was screened and isolated from native honey.Based on theβ-glucosidasecoding sequences from Bacillus altitudinis SYBC hb4,design up and downstream oligonucleotide primers were designed,and the target gene(bglA)was amplified by using PCR.The expression vector pColdII-bglA was constructed by subcloning the target gene into plasmid pColdII,and then transformed into E.coli BL21(DE3)for heterogeneous expression.The expressionβ-glucosidase acticity reached to12.40U/mL.The optimum temperature and pH of the recombinantβ-glucosidase were60℃and8.0,respectively.And5mmol/L Mg2+could increased50%β-glucosidase activity.