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

摘要

In order to obtain alkali-stable β-glucosidase and study its enzyme characterization,an alkali-stable strain Bacillus altitudinis SYBC hb4 was screened and isolated from native honey.Based on the β-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 expression β-glucosidase acticity reached to 12.40 U/mL.The optimum temperature and pH of the recombinant β-glucosidase were 60 ℃ and 8.0,respectively.And 5mmoYL Mg2+ could increased 50％β-glucosidase activity.%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Mg2+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.