葡萄糖异构酶基因工程菌的改造初探  

Preliminary Study on Reformation of Engineered Bacteria E.coli of Glucose Isomerase

作　　者：徐冲[1] 左军[1] 廖军 杨永辉[1] 陶丽梅[1] 朱国萍[1] 伍传金[1] 滕脉坤[1] 王玉珍[1] 

机构地区：[1]中国科学技术大学生命科学学院,合肥230027

出　　处：《中国生物化学与分子生物学报》1999年第4期674-676,共3页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家"863"高科技计划资助

摘　　要：In order to realize stable overexpression of mutant glucose isomerase(GI)gene from Streptomyces diastaticus No.7 M1033 in E.coli, a 1.2 kb fragment containing the intact coding sequence of the protein was amplified specifically from plasmid pTKD GI1 by PCR.At the same time,45 bp unnecessary sequence at GI gene upstream was deleted.The amplified fragment was inserted into the expression vector pBV220 to obtain the recombinant plasmid pBZGI1,which was introduced into E.coli DH5α.Data gathered from passage of the generations of the strains showed that pBZGI1 in DH5α was much more stable than pTKD GI1 in K38/pGP1 2.Induced at 42℃,pBZGI1 overexpressed the mutant GI,which accounted for about 55% of total soluble proteins and was purified through heat treatment,DEAE Sepharose FF column and Sephacrcyl S 300 column.It also showed that the thermostability of the purified GI didn’t decline though the undesired 15 amino acids present in N terminal was deleted.In order to realize stable overexpression of mutant glucose isomerase(GI)gene from Streptomyces diastaticus No.7 M1033 in E.coli, a 1.2 kb fragment containing the intact coding sequence of the protein was amplified specifically from plasmid pTKD GI1 by PCR.At the same time,45 bp unnecessary sequence at GI gene upstream was deleted.The amplified fragment was inserted into the expression vector pBV220 to obtain the recombinant plasmid pBZGI1,which was introduced into E.coli DH5α.Data gathered from passage of the generations of the strains showed that pBZGI1 in DH5α was much more stable than pTKD GI1 in K38/pGP1 2.Induced at 42℃,pBZGI1 overexpressed the mutant GI,which accounted for about 55% of total soluble proteins and was purified through heat treatment,DEAE Sepharose FF column and Sephacrcyl S 300 column.It also showed that the thermostability of the purified GI didn't decline though the undesired 15 amino acids present in N terminal was deleted.

关 键 词：葡萄糖异构酶 基因工程菌 改造 

分 类 号：Q78[生物学—分子生物学] TQ925.5[轻工技术与工程—发酵工程]