Gene cloning and enzymatic properties of hyperthermostable β-glycosidase from Thermus thermophilus HJ6(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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A microorganism (strain HJ6) producing extracellular β-glycosidase was isolated from a hot springs located in Arima-cho, Hyogo, Japan. The cells were long-rods (2-4μm) about 0.4μm in diameter, and formed yellow-colored colonies, like most other strains of the genus Thermus. The pH and temperature for optimal growth were 6.5 and 80℃. Thus, the HJ6 strain displayed a higher optimal temperature than other described Thermus sp. The gene encoding β-glycosidase (TtβGly) was cloned, sequenced, and comprised of 1296 nucleotides encoding a protein (431 amino acids) with a predicted molecular mass of 48.7kDa. TtβGly was expressed in Escherichia coll cells, and the recombinant protein was purified to homogeneity. The optimal temperature and pH for β-glycosidase activity were found to be 90℃ and 8.5, respectively. The half-life of heat inactivation was about 30min at 95℃ indicating that TtβGly had higher thermostability than β-glycosidases from other Thermus sp. The results of the kinetics experiment indicated that β-D-fucoside and β-D-glucoside were better substrates of TtβGly than β-D-galactoside. The catalytic efficiency (k_<cat>/K_m) of TtβGly at 80℃ increased 70-fold to that at 40℃, indicating that this enzyme was activated at high temperatures. Thin layer chromatography showed that the enzyme had transglycosylation activity at high temperature and that various transfer products were formed in the reaction with lactose or cellobiose.