Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning and Expression of Thermostable $\beta$-Glycosidase Gene from Thermus filiformis Wai33 A1 in Escherichia coli and Enzyme Characterization

  • Kang, Sang-Kee (School of Agricultural Biotechnology, Seoul National University) ;
  • Cho, Kwang-Keun (Department of Animal Resource Technology, Chinju National University) ;
  • Ahn, Jong-Kun (Department of Agricultural Science, Korea National Open University) ;
  • Kang, Seung-Ha (School of Agricultural Biotechnology, Seoul National University) ;
  • Han, Kyung-Ho (Biotechnology Research Team 1, Institute of Bioscience ＆ Biotechnology, Dae-Woong Pharmaceutical Co., LTD) ;
  • Lee, Hong-Gu (School of Agricultural Biotechnology, Seoul National University) ;
  • Choi, Yun-Jaie (School of Agricultural Biotechnology, Seoul National University)
  • Published : 2004.06.01
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Abstract

A thermostable $\beta$-glycosidase gene, tfi $\beta$-gly, was cloned from the genomic library of Thermus filiformis Wai33 A1. ifi $\beta$-gly consists of 1,296 bp nucleotide sequence and encodes a polypeptide of 431 amino acids. It shares a strong amino acid sequence similarity with the $\beta$-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 $Ni^{2+}$-affinity chromatography. Polyacrylamide gel electrophoresis (PAGE) showed that the recombinant Tfi $\beta$-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- $90^{\circ}C$ and 5.0-6.0, respectively. Ninety-three percent of the enzyme activity was remained at $70^{\circ}C$ after 12 h, and its half-life at $80^{\circ}C$ was 6 h, indicating that Tfi $\beta$-glycosidase is highly thermostable. Based on its K_m$, or $K_{cat}K_m$, ratio, Tfi $\beta$-glycosidase appeared to have higher affinity for $\beta$-D-glucoside than for $\beta$-D-galactoside, however, $K_{cat} for \beta$-D-galactoside was much higher than that for $\beta$-D-glucoside. The activity for lactose hydrolysis was proportionally increased at $70^{\circ}C$ and pH 7.0 without substrate inhibition until reaching 250 mM lactose concentration. The specific activity of Tfi TEX>$\beta$-glycosidase on 138 mM lactose at $70{^\circ}C$ 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.

Keywords

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