绿色木霉葡聚糖内切酶Ⅰ基因的定点突变及酶学性质的研究

Research of the characters of site-directed mutagenesis of Trichoderma viride endoglucanase Ⅰ

将绿色木霉葡聚糖内切酶EGⅠ基因eg1亚克隆到表达载体pSE380,构建出重组质粒pSE380-eg1,转化到大肠杆菌JM109中表达,利用金属亲和层析对EGI进行纯化,纯化后的酶比活力达到3.8U/mg,其最适反应温度为48℃,最适pH为5.2。同时对EGⅠ的氨基酸残基G291位进行随机定点突变,筛选到一株突变酶G291S,其比活力为野生酶的2.2倍,Km值为野生酶的0.66倍,最适反应温度和最适反应pH值未发生改变。

The gene of endoglucanse I from Trichoderrna viride was subcloned on prokaryotic expressive vector pSE380 in order to construct the recombinant expression plasmid called pSE380-egl, which was transformed into Escherichia coli JM109 and expressed enzyme with the activity of endoglucanase. EGI was purified by metal affinity chromatography, and the specific activity of purified EGI was 3. 8 U/mg, the optimum temperature and optimum pH were 480C and 5.2, respectively. A mutation enzyme retained activity was selected, the mutation was determined by sequencing and made sure the G291 （Gly） transformed to Ser. The CMCase of specific activity of mutation enzyme was 2. 02 times of wild type. Furthermore, the Km of G291S mutation was 0. 66 times of wild type, and G291S mutation wasn＇t change in the optimum temperature and optimum pH.