G138P定点突变对葡萄糖异构酶热稳定性的改善  

Mutation of G138P Enhanced the Thermostability of D glucose Isomerase

作　　者：朱国萍[1] 滕脉坤[1] 伍传金[1] 杭俊[1] 王淳[1] 徐冲[1] 王玉珍[1] 

机构地区：[1]中国科学技术大学生命科学学院

出　　处：《生物化学与生物物理学报》1998年第6期607-610,共4页

基　　金：国家"863"计划;中国科学技术大学结构生物学青年实验室资助

摘　　要：通过分子设计，确定Ｇｌｙ１３８为改善葡萄糖异构酶（ＧＩ）热稳定性的目标氨基酸。用双引物法对ＧＩ基因进行体外定点突变，构建了ＧＩ突变体Ｇ１３８Ｐ。含突变体的重组质粒ｐＴＫＤ－ＧＩＧ１３８Ｐ在Ｅ．ｃｏｌｉＫ３８菌株中表达。ＧＩＧ１３８Ｐ与野生型ＧＩ比较实验表明：（１）ＧＩＧ１３８Ｐ的热失活半衰期约是野生型ＧＩ的２倍；（２）ＧＩＧ１３８Ｐ的最适反应温度提高了１０～１２°Ｃ；（３）ＧＩＧ１３８Ｐ的比活与野生型ＧＩ相当。初步分析认为，Ｐｒｏ取代１３８位的Ｇｌｙ后，可能由于引入了一个吡咯烷环，该侧链刚好能够充填由于Ｇｌｙ１３８无侧链基团而留下的空洞，使蛋白质空间结构更具刚性，从而提高了酶的热稳定性。In order to enhance the thermostability of D glucose isomerase(GI), Gly 138 was decided to be the target to be replaced by molecular design. The mutant G138P was obtained by in vitro site directed mutagenesis of GI gene. The recombinant plasmid pTKD GI containing mutant site was expressed in E.coli K38 strain. The comparison experiments of GIG138P with wild type GI showed that: (1)The half time of GIG138P was as about two times as that of the wild type. (2)The optimum temperature of GIG138P was increased by 10～12 ℃. (3)The specific activity of GIG138P was similar to the wild type GI. We suppose, based on the above facts, that the substitution of Pro for Gly at position 138 introduced a pyrrolidine ring, which could just fill perfectly the empty hole leaved by Gly 138 which has no side chain and could make the protein structure more rigid, therefore the mutant G138P enhanced the thermostability of SM33GI.

关 键 词：葡萄糖异构酶 蛋白质工程 热稳定性 最适反应温度 

分 类 号：Q550.2[生物学—生物化学]