Beta-glycosidase of Thermus thermophilus KNOUC202: gene and biochemical properties of the enzyme expressed in Escherichia coli.
The beta-glycosidase gene of Thermus thermophilus KNOUC202 was cloned, expressed in Escherichia coli JM109(DE3), and the enzyme was purified and characterized. The gene (KNOUC202/beta-gly) was composed of 1296 bp encoding a beta-glycosidase (KNOUC202beta-glycosidase) of 431 a.a., belonging to the family 1 of glycosyl hydrolase. The gene was expressed as monomer of 430 a.a. with amino terminal methionine excised in E. coli JM109(DE3). The enzyme hydrolyzed beta-glycosides whose glycone are galactose, glucose and fucose well, however showed no or very low activity on beta-D-glycosides whose glycone are disaccharides and xylose. kcat of the enzyme for the hydrolysis of p-Nph-beta-D-Glcp was lower than those for p-Nph-beta-D-Galp and ONPG, however K(m) for p-Nph-beta-D-Glcp was highly lower than those for p-Nph-beta-D-Galp and ONPG resulting in the catalytic efficiency(k(cat)/K(m)) for the hydrolysis of p-Nph-beta-D-Glcp much higher than those for p-Nph-beta-D-Galp and ONPG. Optimum pH and optimum temperature of the enzyme were pH 5.4 and 90 degrees C. The enzyme has high thermostability, not losing its activity at 80 degrees C for 2 h in 0.05 M Na-phosphate buffer of pH 6.8 with T(m) of 100.0 +/- 0.031 degrees C in 0.02 M Tris-HCl buffer of pH 8.2. The beta-glycosidase produced a disaccharide composed of galactose as transglycosylation byproduct during hydrolysis of lactose.
Department ofAgricultural Sciences, Korea National Open University, Seoul 110- 797, Republic of Korea.
E S
ES
Nam
M S
MS
Kim
H B
HB
Lee
J K
JK
Ahn
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli; D005798:Genes, Bacterial; D006868:Hydrolysis; D007785:Lactose / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics*; D001617:beta-Glucosidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D007785:Lactose