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KMID : 0545120040140020344
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Journal of Microbiology and Biotechnology
2004 Volume.14 No. 2 p.344 ~ p.349
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Cloning, Expression, and Purification of Exoinulinase from Bacillus sp. Snu-7
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KIM, KYOUNG-YUN
KOO, BONG-SEONG/JO, DOHYUN/KIM, SU-IL
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Abstract
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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. oli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at OD, 1.2 and 30℃ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60kDa 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±0.08 mM and 358.05±20.38 min^(-1), and 22.02±0.41 mM and 4619.11±215.12 min^(-1), respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and 50℃.
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KEYWORD
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