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KMID : 0545120040140030584
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 3 p.584 ~ p.592
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Cloning and Expression of Thermostable β-Glycosidase Gene from Thermus filiformis Wai33 A1 in Escherichia coli and Enzyme Characterization
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Kang SK
Cho KK/Ahn JK/Kang SH/Han KH/Lee HG/Choi YJ
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Abstract
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A thermostable b-glycosidase gene tfi b-gly was cloned from the genomic library of Thermus filiformis Wai33 A1. tfi b-gly consists of 1296 bp nucleotide sequence and encodes a polypeptide of 431 amino acids. It shares a strong amino acid sequence similarity with the b-glycosidases from other Thermus spp. belonging to the glycosyl hydrolase family 1. In the present study the enzyme was overexpressed in Escherichia coli BL21 (DE3) using the pET21b(+) vector system. The recombinant enzyme was purified to homogeneity by heat treatment and a Ni2+-affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) showed that the recombinant Tfi b-glycosidase was a monomeric form with molecular mass of 49 kDa. The temperature and pH range for optimal activity of the purified enzyme were 80- 90oC and 5.0- 6.0 respectively. Ninety-three percent of the enzyme activity was remained at 70oC after 12 h and its half-life at 80oC was 6 h indicating that Tfi b-glycosidase is highly thermostable. Based on its Km or Kcat/Km ratio Tfi b-glycosidase appeared to have higher affinity for b-D-glucoside than for b-D-galactoside however Kcat for b-D-galactoside was much higher than that for b-Dglucoside. The activity for lactose hydrolysis was proportionally increased at 70oC and pH 7.0 without substrate inhibition until reaching 250 mM lactose concentration. The specific activity of Tfi b-glycosidase on 138 mM lactose at 70oC and pH 7.0 was 134.9 U/mg. Consequently this newly cloned enzyme appears to have a valuable advantage of conducting biotechnological processes at elevated temperature during milk pasteurization in the production of low-lactose milk.
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