一株纤维降解菌β-葡萄糖苷酶基因的克隆与表达及酶学性质分析

Cloning, expression and enzymatic properties analysis of β-Glucosidase gene from a fiber-degrading bacteria

试验对1株娄彻氏链霉菌(B5)的β-葡萄糖苷酶(高活性纤维降解酶)基因进行克隆,通过构建高效表达的载体实现基因表达,对所得表达产物的酶学性质进行测定分析,为饲用纤维素酶的开发利用提供参考。通过PCR扩增B5的Egl基因的完整CDS序列,构建pET-32a-egl表达载体,转化到BL21(DE3)大肠杆菌感受态细胞。试验成功构建了高产β-葡萄糖苷酶的基因工程大肠杆菌,实现了β-葡萄糖苷酶的分泌表达,其分子量约4.1×10^-4 U,酶学特性分析表明,在40℃、pH值8、底物浓度为3.0×10^-2 mol/L时,酶活力最高,为1085 U/mL。

Theβ-glucosidase(highly active fiber degrading enzyme)gene of a strain of Streptomyces loucherii(B5)was cloned,and gene expression was achieved by constructing an efficient expression vector,and the enzymatic properties of the resulting expression product were determined and analyze to provide a theoretical reference for the development and utilization of feed cellulase.The complete CDS sequence of Egl gene of B5 was amplified by PCR,and the expression vector pet-32 a-egl was constructed and transformed into e.coli competent cells of BL21(DE3).The results showed that the gene engineering escherichia coli with high yield of beta-glucosidase was successfully constructed,and the secretion expression of beta-glucosidase was realized.The molecular weight was about 4.1×10^-4 U.Enzymatic characteristic analysis showed that the enzyme activity reached its maximum value,as high as 1085 U/ml,at 40℃,pH 8 and substrate concentration of 3.0×10^-2 mol/L.