用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度  

Changing the Optimum pH and Temperature of Glucose Isomerase by Means of Protein Engineering

作　　者：肖亚中[1] 伍传金[1] 龙凡[1] 牛立文[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》1995年第5期469-476,共8页

基　　金：国家高技术发展计划资助

摘　　要：用寡核苷酸诱导的定点突变方法构建了葡萄糖异构酶基因的突变体（Ｎ１８４Ｄ和Ａ１９８Ｃ）。含突变体的重组质粒ｐＴＫＤ－ＧＩ１（Ｎ１８４Ｄ）和ｐＴＫＤ－ＧＩ２（Ａ１９８Ｃ）在Ｅ．ｃｏｌｉＫ３８菌株中表达，用ＤＥＡＥ－ＳｅｐｈａｒｏｓｅＦＦ和ＳｅｐｈａｃｒｙｌＳ－３００ＨＲ柱层析分离纯化突变酶。与野生型葡萄糖异构酶比较实验表明：（１）突变酶Ｎ１８４Ｄ的最适ｐＨ值下降了１个单位；等电点下降了０．６个单位，酶活性为野生型的９８．２７％。（２）突变酶Ａ１９８Ｃ最适温度提高了８℃，酶活性为野生型的８６．９５％。对突变酶和野生型酶进行了动力学研究，并通过分子模拟，对实验结果进行了初步分析。The mntants(N184D & A1980)of glucose isomerase(GI)were obtained by in vitro site-directed mutagensis using synthetic primers.The mutant recombinant plasmid pTKD-GI1(N184D) & pTKDA-GI2(A198C) were expressed in E.coli K38and purified by DEAE-Sepharose FF and Sephaoryl S-300 HR column chromatography.The comparison experiments of these mutant proteins with wild-type GI indicated that:(1)for mutant enzyme N184D,the optimum pH and the isoeleotric point decreased about 1.0 and 0.6 unit respectively,and its enzymatic activity was be.27% of that of the wild-type enzyme;(2)for A198C,its optimum temperature increased 8℃ and the enzymatic activity was 86.95% of that of the wild-type enzyme.The experimental results were analyzed by kinetics studies and molecular modeling method.

关 键 词：葡萄糖异构酶 蛋白质工程 最适PH值 最适温度 

分 类 号：Q558.07[生物学—生物化学]