Enhancing some biochemical properties of Anoxybacillus gonensis glucose isomerase by genetic manipulation

Glucose isomerase (D-xylose ketol isomerase; EC 5.3.1.5) is an intracellular enzyme found in a number of bacteria that utilize xylose as carbon substrate for growth. Glucose isomerase converts D-xylose to D-xylulose in vivo and also catalyzes the conversion of D-glucose to D-fructose in vitro. The latter activity is used in industry for the production of high fructose corn syrup (HFCS). Therefore, glucose isomerase is one of the largest volume commercial enzymes used today.Gene coding for thermophilic glucose isomerase of Anoxybacilus gonensis G2T was isolated and its complete nucleotide sequence was determined. The gene has been cloned to plasmid vector pET-28a(+) and overexpressed in E. coli BL21(DE3). Several discrete regions were highly conserved throughout the amino acid sequences of all glucose isomerases. By examining this regions; W137F, V184T, V184S, H99Q, H99N, H99D, H99E, and H99L mutations were performed by site-directed mutagenesis techniques at cloned glucose isomerase. The wild type enzyme and mutant enzymes were purified by colon chromatography techniques. The biochemical properties of all glucose isomerases were characterized. All mutant enzymes compared with wild type enzyme for biochemical properties and kinetics parameters.The wild-type optimal temperature was 85°C and maximal activity was observed in a pH 6,5. Its Km and Vmax value was calculated as 146,08 ± 9,50mM and 43,72 ± 1,01 µmol/dk/mg protein respectively. W135 F mutation reduced Km value to 64,02 ± 2,10. V184T and V184S has not changed kinetic parameters. H99 mutations were generally increased Km value and reduced Vmax value. W135 F/ V184T mutation reduced Km value to 39,29 ± 2,87 mM.