【摘要】 应用定点突变方法 ,在M2 2 2A突变的枯草杆菌蛋白酶E基因上进行E1 56S和V1 65I定点突变 .将突变基因插入大肠杆菌 枯草杆菌穿梭质粒 pBE 2中 ,在碱性和中性蛋白酶缺陷型的枯草杆菌DB1 0 4中进行表达 ,得到突变种 (M 2 2 2A ,E1 56S)和 (M 2 2 2A ,E1 56S ,V1 65I)蛋白酶E .性质测定表明 ,E1 56S突变使蛋白酶比活力增加 90 % ,并不影响酶的热稳定性和抗氧化性 .而V1 65I突变使蛋白酶比活力降低更多还原

【Abstract】 E156S and V165I mutation were introduced into subtilisin E gene by site-directed mutagenesis. The mutated gene fragments were recombined with pBE-2 which is a shuttle vector between E.coli and Bacillus subtilis. The recombinant plasmids were used to transform B.subtilisDB104,a mutant strain deficient in alkaline and neutral protease,then they were expressed. They were (M222A,E156S) and (M222A,E156S,V165I). The property analysis of these enzymes revealed that the Subtilisin E E156S substitution enhenced the hydrolysis K cat/K m by 90% while keeping thermal stability and oxidation-resistance unchanged,however the V165I mutation reduced the K cat/K m value.更多还原