K253R和N184V点突变对葡萄糖异构酶热稳定性的影响  

Effects of Site-specific Mutagenesis of K253 and N184V on the Thermostability of D-Glucose Isomerase

作　　者：伍传金[1] 肖亚中[1] 龙凡[1] 滕脉坤[1] 王淳[1] 崔涛[1] 

机构地区：[1]中国科学技术大学生物系,安徽大学生物系

出　　处：《生物化学与生物物理学报》1996年第3期272-278,共7页

基　　金：国家"863"计划资助

摘　　要：用人工合成的寡核苷酸突变引物，以双引物法于重组Ｍ１３ｍｐ１９载体上进行定点突变，分别得到了葡萄糖异构酶的突变体ＧＩＫ２５３Ｒ和ＧＩＮ１８４Ｖ。测定它们的热稳定性，并将之与野生型酶比较，结果表明：（１）ＧＩＫ２５３Ｒ于７０℃、８０℃下的热稳定性小于野生型，但在７０℃、１ｍｏｌ／ＬＬ－鼠李糖中，两者的失活速度相近。另外；ＧＩＫ２５３Ｒ的比活是野生型的１．５倍。（２）ＧＩＮ１８４Ｖ的比活和热稳定性都远低于野生型，Ａｓｎ１８４为酶活性中心构象的维持所必需．本文还根据动力学数据和分子结构模型对以上结果作了初步分析．In Order to enchance the thermostability of D-Glucose isomerase (GI), its mutants, GIK253R and GIN184V, were obtained with the double primer method of site-directed mutagenesis . Then their biochemical properties were assayed and compared with wild-type GI. The results showed that: (1)The mutant K253R was less stable than the wild-type GI at 70℃ and 80℃. But in 1M L-Rhaminose at 70℃, they had similar rate of heat inactivition. Futhermore, the mutant K253R has higher specific activily than the wild-type GI. (2) The mutant N184V had much less thermostability and specific activity as compared with the wild-type GI. These results were explained by their kinetic parameters and crystal structure model.

关 键 词：葡萄糖异构酶 定点诱变 蛋白质工程 热稳定性 

分 类 号：Q558.02[生物学—生物化学]