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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 plasmid pTKD GI1 by PCR. At the 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 strain showed that pBZGI1 in DH5α was much more stable than pTKD GI1 in K38 / pGP1 2. Induced at 42 ° C, 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 Sepharcrcyl S 300 column. It also showed that the thermostability of the purified GI did not decline though the undesired 15 amino acids present in N terminal was deleted.