Purification and characterization of w137f/v184s and w137f/v184t double mutant anoxybacillus gonensis g2 xylose isomerase

This study includes purification and characterization of Xylose Isomerase (XI) of Anoxybacillus gonensis G2 with double mutations (W137F/V184S and W137F/V184T). For XI W137F/V184S, Km, Vmax and Kcat/Km values were calculated as Km= 91,13 ± 10,54 mM; Vmax= 31,71 ± 1,1 µmol/dk/mg protein; Kcat/Km = 1,161. Also for XI W137F/V184T, Km, Vmax and Kcat/Km values were calculated as Km= 25,94 ± 3,73 mM; Vmax=7,66 ± 0,37 µmol/dk/mg protein; Kcat/Km = 0,985. The optimum temprature and optimum pH for both mutant XI were determined as 85°C and pH 7,5 respectively. At 70°C, after 30 minutes XI W137F/V184S lost its activity rapidly. At 80°C, after 30 minutes XI W137F/V184T lost half of its activity. XI W137F/V184S was stable at pH 8, while XI W137F/V184T was stable at alkaline pH values. The maximum activity of XI W137F/V184S and XI W137F/V184T were observed in presence of 10 mM Mg+2 and 10 mM Mn+2 respectively. Ca+2, Hg+2, Ni+2, Zn+2, Fe+2 and Cu+2 were determined as inhibitors for activities of XI W137F/V184S and XI W137F/V184T.Key Words: Anoxybacillus gonensis, D-xylose isomerase, mutation, enzyme purification, characterization