Cloning of $\beta$-Glucosidase Gene from Streptomyces coelicolor A3(2) and Characterization of the Recombinant $\beta$-Glucosidase Expressed in Escherichia coli

Streptomyces coelicolor A3(2)로 부터 $\beta$-Glucosidase 유전자 클로닝 및 재조합 효소의 특성

  • Kim, Jae-Young (Department of herbal medicine, Basic Science Institute, Hoseo University) ;
  • Kim, Bong-Kyu (BMIC, Division of Bioscience and Biotechnology, Konkuk University) ;
  • Yi, Yong-Sub (Department of herbal medicine, Basic Science Institute, Hoseo University) ;
  • Kang, Chang-Soo (Department of Biological Science, Hoseo University) ;
  • Ahn, Joong-Hoon (BMIC, Division of Bioscience and Biotechnology, Konkuk University) ;
  • Lim, Yoong-Ho (BMIC, Division of Bioscience and Biotechnology, Konkuk University)
  • 김재영 (호서대학교 한방화장품과학과 기초과학연구소) ;
  • 김봉규 (건국대학교 생명공학과 생명/분자정보학센터) ;
  • 이용섭 (호서대학교 한방화장품과학과 기초과학연구소) ;
  • 강창수 (호서대학교 생명과학과) ;
  • 안중훈 (건국대학교 생명공학과 생명/분자정보학센터) ;
  • 임융호 (건국대학교 생명공학과 생명/분자정보학센터)
  • Received : 2009.01.12
  • Accepted : 2009.05.25
  • Published : 2009.06.28

Abstract

The $\beta$-glucosidase gene from Streptomyces coelicolor A3(2) was cloned and expressed in Escherichia coli. The ORF consisted of 1377 nucleotides encoding 51 kDa in a predicted molecular weight. Effects of pH indicated that the $\beta$-glucosidase showed similar activity using $\alpha$-pNPG($\rho$-nitrophenyl-$\alpha$-D-glucopyranoside), $\beta$-pNPG($\rho$-nitrophenyl-$\beta$-D-glucopyranoside), and $\beta$-pNPF($\rho$-nitrophenyl-$\beta$-D-fucopyranoside) at range of pH 3 to 10, and high activity using $\beta$-pNPGA ($\rho$-nitrophenyl-$\beta$-D-galactopyranoside) from pH 5 to 10, especially, 3.3 times higher activity at pH 9. Effects of temperature indicated that the $\beta$-glucosidase showed low activity using $\alpha$-pNPG, $\beta$-pNPG, and $\beta$-pNPF from $20^{\circ}C$ to $70^{\circ}C$, and increased activity using $\beta$-pNPGA from $30^{\circ}C$ to $50^{\circ}C$, 1.8 times higher activity at $50^{\circ}C$ than at $30^{\circ}C$. According to activity determination of other substrates, the enzyme was active on daidzin, genistin, and glycitin, inactive on esculin and apigenin-7-glucose. The EDTA and DTT as reducing agents inhibited $\beta$-glucosidase activity, but SDS and mercaptoethanol did not inhibit. Monovalent or divalent metal ions such as $MnSO_4$, $CaCl_2$, KCl, and $MgSO_4$ did not inhibited $\beta$-glucosidase activity. $CuSO_4$ and NaCl showed low inhibition, and $ZnSO_4$ inhibited 3.3 times higher than control.

Keywords

$\beta$-glucosidase;deglycosylation;Streptomyces coelicolor A3(2);substrate specificity

Acknowledgement

Supported by : 농촌진흥청

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