Improvoment of kinetic and some biochemical parameters of glucose isomerase from anoxybacillus gonensis g2t by site directed mutagenesis g33d, n138g, t144s, v293i, v32i, e373k and v380i

Glucose isomerase is one of the three highest tonnaged enzymes with along amylase and protease in the World. Glucose isomerase catalyses conversion isomerization of D-glucose to D-fructose, and D-xylose to D-xylulose. Isomerization of D-glucose to D-fructose has a commercial importance in the production of HFCS. Because of this importance, many new GIs have been isolated and characterized to date. The coding gene of glucose isomerase from Anoxybacillus gonensis G2T was cloned to pET-28a(+) vector and GI was produced and characterized. To improve biochemical properties and kinetic parameters of the enzyme, G33D, N138G, T144S, V293I, V32I, E373K and V380I mutations were performed on the wild-type enzyme by site directed mutagenesis. All mutant enzymes and wild-type enzyme were purified by column chromatography methods. All characterized enzymes were compared with each other and with wild-type enzyme in terms of their biochemical and kinetic parameters. As a result of the characterization studies, mutations of G33D, V293I, V32I, E373K and V380I did not give any significant change in the biochemical properties and kinetic parameters of the wild-type enzyme. An increase was observed in the Vmax, kcat, and kcat/ Km values of the wild-type enzyme by N138G and T144S mutantations. The Vmax value of N138G mutant enzyme was calculated as 44,24 ± 0,27 μmol / min / mg protein and the Vmax value of T144S as 34,59 ± 0,26 μmol / min / mg protein. Increases were observed in the thermal stability of the G33D and T144S mutant enzymes compared to wild-type enzyme.