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.
The glucose isomerase(GI) was a metal activating enzyme It was most activated by Co 2+ and Mg 2+ ,and Mg 2+ was the best activator,whether the glucose or the xylose was the substrate When the glucose was substrate,the dissociation constant of Mg 2+ GI,Co 2+ GI and Mn 2+ -GI was 115 μmol/L,40 μmol/L, and 15 μmol/L respectively. The maximum activity of Mg 2+ GI,Co 2- GI and Mn 2+ GI was 100%,85%,and 20% respectively. When the xylose was substrate,the order of dissociation constant and maximum activity of the metal enzymes was the same Ca 2+ was a competitive inhibitor versus Mg 2+ ( K i 7 4 μmol/L)or Co 2+ ( K i 99 μmol/L). Compared with Mg 2+ GI,the K m of Co 2+ GI was more,and the V M of Co 2+ GI less The process of activity recovery from apo GI to metal GI showed that it was slow and of two
