Cloning, isolation and characterization of D-glucose (D-Xylose) isomerase gene from Anoxybacillus gonensis

SUMMARY Cloning, Isolation and Characterization of D-Glucose (D-Xylose) Isomerase Grene from Anoxybacillus gonensis 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). The major uses of HFCS are in the beverage, baking, canning, and confectionery industries. The use of glucose isomerase is expensive, because it is an intracellular enzyme and large quantities are needed to compensate for the high Km for glucose. Therefore, it is important to immobilize glucose isomerase for its industrial applications. Anoxybacillus gonensis G2T has glucose isomerase gene and activity. This work describes the cloning, isolation, and characterization of the glucose isomerase (E.C 5.3.1.5) from Anoxybacillus gonensis G2T' A 530 bp part of the xylA gene coding for glucose isomerase from Anoxybacillus gonensis G2 was cloned and sequenced. The glucose isomerase optimal temperature was 85°C and maximal activity was observed in pH 6,5.. It was found that the enzyme was stable in the range of pH 5-9,5 at 4°C for 300 hours. After incubation at 4°C and 30°C for 300 hours, the enzyme saved 80 % of its activity. It was determined that the enzyme had a lower Km (15,24 mM) for glucose than most of the glucose isomerases. It was observed that the enzyme thermostability at 85°C increased in the presence of metallic cation Co+2 and decreased by the effect of Mn+2, while it was not affected by Mg+2. It was found that at least one of the divalent metallic cations Co+2, Mg+2 or Mn+2 is essential for glucose isomerase activity Also, it was recorded that the maximum activiy of the glucose isomerase was in the presence of metallic cation Co+2. The experiment conducted with the metallic cations Cd+2, Ca+2, Sn+2, Hg+2, Ni+2, Zn+2, Fe+2, and Cu+2 shown that these divalent metallic cations inhibated the actvity of the enzyme. The weights of subunits of molecule were calculated as aproximatelly 43.000 Daltons. In the light of all data it has been suggested that the enzyme's biocatalytic properties proved to be one of the important industrial enzymes. Key Words: Anoxybacillus gonensis, Glucose Isomerase, Xylose Isomerase, HFCS vn