嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

Expression and Enzymatic Characterization of Thermus thermophilus α-glucosidase Gene

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a(+)上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。

A α-glucosidase gene （hbg） was amplified with PCR from the total DNA of Thermus thermophilus and linked with pGEM-T vector. Itbg gene was inserted into the expression vector pET-28a （ ＋ ） and transformed into Escherichia coli BI21 （ DE3 ） with electroporation, finally the recombinant strain which could efficently secret recombinant α-glucosidase was obtained. Induced by IPTG, the expression products of hbg gene analysed by SDS-PAGE had a molecular mass of 59kD. The optimum temperature and optimum pH of the recombinant expression a-glucosidase were 95℃ and 5.0 respectively.