beta-GLYCOSIDASE OF Thermus thermophilus KNOUC202 : GENE AND BIOCHEMICAL PROPERTIES OF THE ENZYME EXPRESSEDIN Escherichia coli

摘要

The p-glycosidase gene of nermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202/]-gly) was composed of 1296 bp encoding a P-glycosidase (KNOUC202p-glycosidase) of 431 a.a., belonging to the family i ol giyc-osyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino termmal methiomne excisea in E. CO// JM109(DE3). The enzyme hydrolyzed p-glycosides whose glycone are galactose, glucose and tucose well, however showed no or very low activity on p-D-glycosides whose glycone are disacchandesana xyiose k,,, of the enzyme for the hydrolysis of p-Nph-p-D-Glc/j was lower than those p-Nph-p-D-4ja ONPG, however for p-Nph-B-D-Glcp was highly lower than those forp-Nph-P-D-Ga and OKFO resulting in the catalytic efTiciencyfA: JKJ for the hydrolysis of/)-Nph-p-D-Glc/J much higher than those tor /)-Nph-p-D-Gal/j and ONPG. Optimum pH and optimum temperature of the enzyme were PH 5.4 and 90deg C. The enzyme has high thermostability, not losing its activity at 80deg C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with of 100.0 +- 0.031 deg C in 0.02 M Tris-HCl buffer of pH 8.2. The p-glycosidase produced a disaccharide composed of galactose as transglycosylation byproduct during hydrolysis of lactose.