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Cloning, Expression, and Purification of Exoinulinase from Bacillus sp. snu-7
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  • Cloning, Expression, and Purification of Exoinulinase from Bacillus sp. snu-7
저자명
Kim. Kyoung-Yun,Koo. Bong-Seong,Jo. Do-Hyun,Kim. Su-Il
간행물명
Journal of microbiology and biotechnology
권/호정보
2004년|14권 2호|pp.344-349 (6 pages)
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한국미생물생명공학회
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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기타언어초록

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 $2.28pm0.08$ 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 $50^{circ}C$.