Cloning and expression of thermostable beta-glycosidase gene from Thermus nonproteolyticus HG102 and characterization of recombinant enzyme

The gene coding for beta -glycosidase (EC 3.2.1.21) from Thermus nonproteol yticus HG102 was cloned and expressed in Escherichia coli. The gene open re ading frame was 1311 bp, and it codes for 437 amino acids. The deduced amin o acid sequence of the enzyme showed identity with members of the glycosyl hydrolase family I. The enzyme had high content of Arg and Pro. The recombi nant enzyme was purified to homogeneity with heat precipitation, ammonium s ulfate precipitation, DEAE-cellulose (DE52) chromatography, and prepared sl ab polyacrylamide gel electrophoresis. The enzyme was monomeric and had a m olecular mass of 48,900 Daltons and a pI of 5.2. The enzyme showed optimum activity at pH 5.6 and 90 degreesC. It catalyzed the hydrolysis of beta -D- glucoside, beta -D-galactoside, beta -D-fucoside, and beta -D-mannoside. Li neweaver-Burk plots showed that the k(cat)/K-m ratio for beta -D-glucoside and beta -D-fucoside was higher than for beta -D-mannoside and beta -D-gala ctoside. The enzyme was extremely thermostable, with a half-life of 2.5 h a t 90 degreesC, and was stable over a wide range of pH. It also had transgly cosidic activity at high temperature.