• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.344-349
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• 2004

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 $50^{\circ}C$.

• Applied Biological Chemistry
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• 제40권3호
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• pp.184-188
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• 1997

Inulin을 가수분해하여 di-D-fructofuranose dianhydride(DFA)를 생성하는 inulin frucroransferase를 분비하는 균주를 토양으로부터 분리하였으며, Bacillus sp로 동정하였다. 본 효소에 의하여 생성되는 DFA는 TLC 와 HPLC로 분석한 결과, fructose 두 분자가 ${\beta}\;1,2':{\alpha}2,3'$ 결합을 한 DFA III로 동정되었다. 본 균주는 탄소원으로 1.5% 돼지감자즙, 유기 질소원으로 1.0% peptone, 무기 질소원으로 $0.27%\;NH_4H_2PO_4$를 사용했을 떼 효소 생산이 2.709 units/ml로 최대였으며, inulin을 탄소원으로 사용할 경우는 1.0% yeast extract, $0.2%\;NaNO_3$를 첨가했을 때 효소 생산이 2.245units/ml로 최대를 나타내었다. 본 균주를 최적 액체 배지에서 72시간 배양한 결과 배양 45시간에 2.61 units/ml 최대 활성을 보였다.

• Journal of Microbiology and Biotechnology
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• 제17권1호
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• pp.37-43
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• 2007

A gene encoding inulin fructotransferase (di-D-fructofuranose 1,2': 2,3' dianhydride [DFA III]-producing IFTase, EC 4.2.2.18) from Bacillus sp. snu-7 was cloned. This gene was composed of a single, 1,353-bp open reading frame encoding a protein composed of a 40-amino acid signal peptide and a 410-amino acid mature protein. The deduced amino acid sequence was 98% identical to Arthrobacter globiformis C11-1 IFTase (DFA III-producing). The enzyme was successfully expressed in E. coli as a functionally active, His-tagged protein, and it was purified in a single step using immobilized metal affinity chromatography. The purified enzyme showed much higher specific activity (1,276 units/mg protein) than other DFA III-producing IFTases. The recombinant and native enzymes were optimally active in very similar pH and temperature conditions. With a 103-min half-life at $60^{\circ}C$, the recombinant enzyme was as stable as the native enzyme. Acidic residues and cysteines potentially involved in the catalytic mechanism are proposed based on an alignment with other IFTases and a DFA IIIase.