Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Purification of Exoinulinase from Bacillus sp. snu-7

  • Kim, Kyoung-Yun (School of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Koo, Bong-Seong (BioNgene Co. Ltd.) ;
  • Jo, Do-Hyun (Department of Molecular Science and Technology, Ajou University) ;
  • Kim, Su-Il (School of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University)
  • Published : 2004.04.01
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Abstract

A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at $OD_{600}$ 1.2 and $30^{\circ}C$ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product. $K_m$ and $k_{cat}$, for the hydrolyses of inulin and sucrose were $2.28\pm0.08$ mM and 358.05$\pm$20.38 $min^{-l}$, and 22.02$\pm$0.41 mM and 4619.11$\pm$215.12 $$min^{-1}, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and $50^{\circ}C$.

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