7号淀粉酶链霉菌M1033菌株葡萄糖异构酶在大肠杆菌中的高效表达  

Overexpression of Streptomyces diastaticus No.7 Strain M1033 Glucose Isomerase in Escherichia coli

作　　者：崔虹[1] 刘咸安 李澄清[1] 伍传金[1] 王玉珍[1] 王淳[1] 牛立文[1] 徐洵[1] 崔涛[1] 

机构地区：[1]中国科学技术大学生物系

出　　处：《生物工程学报》1996年第S1期102-105,共4页Chinese Journal of Biotechnology

基　　金：国家高技术项目资助

摘　　要：从已克隆的含７号淀粉酶链霉菌Ｍ１０３３菌株产葡萄糖异构酶基因的质粒ｐＵＢ中，用ＤｄｅＩ和Ｋｐｎｌ酶解，分离出酶结构基因，以平端方式与大肠杆菌质粒ｐＴ７７连接，构建了重组表达质粒ｐＴＫＤＧＩ。将ｐＴＫＤＧＩ转化到特异大肠杆菌寄主Ｋ３８，经４２℃热诱导，所产葡萄糖异构酶占菌体可溶性蛋白３５％。通过ＤＥＡＥ－Ａ５０柱和Ｇ－１５０柱层析，发酵液可获电泳纯蛋白３４ｍｇ／Ｌ。The gene encoding thermostable glucose isomerase in Streptomyces diastaticus No.7 strain M1033 was cloned into vector pUC resulting shuttle plasmid pUB.The pUB was digested by DdeI and KpnI to obtain structural glucose isomerase gene without its regulation sequence.Then the fragment was subcloned into expressed vector pT7 7 to construct expression plasmid pTKD GI,which was introduced into E.coli K38 which containing T7 polymerase gene.The glucose isomerase gene was overexpressed in E.coli .The expressed protein accounted for about 35% of total soluble fraction.The solution was applied to a DEAE A 50 column,elution with a linear (0.25～0.5mol/L) NaCl gradient and G 150 column wash with 0.15mol/L NaCl in same buffer.We obtain 34mg pure glucose isomerase per liter culture,which was a band in SDS PAGE (purity>95%).Its specific activity was identify of pure enzyme of strain M1033.

关 键 词：7号淀粉酶链霉菌M1033菌株 葡萄糖异构酶(木糖异构酶) 高效表达 

分 类 号：Q558[生物学—生物化学]