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

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。
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 5mmol/L Mg2+ could increased 50% β-glucosidase activity