화학공학소재연구정보센터
Enzyme and Microbial Technology, Vol.37, No.6, 655-662, 2005
Cloning, expression, and enzyme characterization of thermostable beta-glycosidase from Thermus flavus AT-62
A gene encoding thermostable P-glycosidase from genomic library of Thermus flavus AT-62 (ATCC 33923) was cloned, sequenced, and expressed in Escherichia coli. The gene, designated as tat beta-gly, consists 1296 bp of nucleotides and encoded a polypeptide of 431 amino acids. Tat beta-gly showed a strong amino acid sequence similarity to those of beta-glycosidases from other Thermus species belonging to the glycosyl hydrolase family 1. The enzyme was overexpressed in E. coli BL21(DE3) with pET21b(+) vector system and purified to homogeneity by heat precipitation and Ni2+-affinity chromatography. The recombinant enzyme was monomeric with a molecular mass of 49 kDa. The temperature and pH range for optimal activity of the purified enzyme were 80-90 degrees C and 5.0-6.0, respectively. At 70 degrees C, the enzyme was highly thermostable that could maintain 94% of its activity over 12 h. Tat P-glycosidase showed higher affinity for P-D-glucoside than for P-D-galactoside regarding as its K-m or K-cat/K-m ratio, however V-max and K-cat for beta-D-galactoside were much higher than those for beta-D-glucoside. The enzyme activity displayed a constant increasing behavior for lactose hydrolysis without substrate inhibition until 250 mM lactose was added at 70 degrees C and pH 7.0. This study suggests that the newly cloned enzyme has a strong potential to utilize in lactose milk production during milk pasteurization. (c) 2005 Elsevier Inc. All rights reserved.