绿色木霉葡聚糖内切酶EGⅢ基因在大肠杆菌中的表达、定点突变及其动力学特性的研究

Eexpression in E. coli, site-directed mutagenesis and kinetics analysis of endoglucanase Ⅲ from Trichoderma viride

将绿色木霉葡聚糖内切酶EGⅢ基因亚克隆到表达载体pET-22b(+),构建重组质粒pET-egl3,转化到大肠杆菌BL21(DE3)。利用金属亲和层析对重组EGⅢ进行纯化,纯化后酶比活力达到6 U/mg蛋白,最适反应温度为60℃,最适pH为4.0。同时对EGⅢ催化区的氨基酸残基R130和E218进行定点饱和突变,各筛选到一株酶活有提高的突变子R130P和E218F,其比活力为野生型EGⅢ的2.8倍和3.45倍。突变酶E218F的Km提高了一倍,催化效率Kcat提高了5.4倍;而R130P的Km和Kcat没有明显变化。两个突变酶的最适酶解温度和pH分别都提高至65℃和4.4。

Endo-glucanase Ⅲ gene from Trichoderma viride was subcloned into expression vector pET-22b （ ＋ ）. A recombinant plasmid pET- egl3 was con.strueted and transformed into E. coli BL21 （DE3）. Recombinant protein Endoglucanase Ⅲ was purified by metal chelating affinity chromatography, and the specific activity of EGIII was 6 U/mg protein. Its optimal temperature and pH were 60 ℃ and 4.0, respectively. Then, the mutants R130P and E218F in catalytic region of EGⅢ, whose enzyme activities were improved obviously, were obtained using site-saturation mutagenesis. And the characteristics of the two mutations were investigated. Their specific activities were 2.8 and 3.45 times than that of the wild type,respectively. Furthermore, the Km and Kcat of E218F mutation were increased one time and 5.4 times more than that of the wild type, respectively. While, Krn and Kcat in R130P mutation had not any change. Compared with wild type, the optimal temperature and pH of the two mutated enzymes were both enhanced to 65℃ and 4.5, respectively.