Development of some properties of a recombinant glucose isomerase from Geobacillus caldoxylosilyticus Thermophilic by mutation

In this study, it was aimed to obtain a glucose isomerase which 1) work at higher temperature and 2) lower pH value, 3) has more affinity to glucose, more 4) pH and 5) thermal stable by performing four site-directed mutations. H99Q, V184T, D102N ve H99Q/D102N mutations were performed for Geobacillus caldoxylosilyticus TK4GI gene that was previously cloned to pET-28a(+) vector. The obtained mutant genes were overexpressed in a BL21(DE3)pLysE host cell. Mutant proteins were purified by nickel affinity chromatography. Biochemical characterization of all mutant enzymes was examined. The findings are that the mutations mentioned above induced 1) an increase in optimum temperatures and 3) Km values of mutant proteins but 2) a decrease at optimum pHs and Vmax values, 4) an increase in the pH stability at pH 6.0 compared to recombinant enzyme, 5) to be more stable in temperatures especially at 4 °C compared to literature. Also in general, the highest activities of mutant enzymes were observed in the presence of Co2+, Cu2+ and Mn2+ and mutant enzymes were more resistant to inhibition of some metal ions.In conclusion, optimum pH, optimum temperature, pH stability and stability at storage temperature value of recombinant enyzme was improved as aimed, but affinity to substrat and maximum enzyme activitiy slightly decreased. This study was supported by the grant from TUBITAK (109T985).