• Journal of Microbiology and Biotechnology
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• v.14 no.2
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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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• v.40 no.3
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• pp.184-188
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• 1997

A bacterial strain, producing extracellular inulin fructotransferase which converts inulin into di-D-fructofuranose 1,2':2,3' dianhydride(DFA III), was isolated from soil and presumed as Bacillus sp.. The highest production of the enzyme was obtained by using medium containing Jerusalem artichoke extract as carbon source, peptone as organic nitrogen source, and $NH_4H_2PO_4$, as inorganic source. Under optimum condition, the enzyme activity of the culture broth supernatant reached maximal 2.61 units/ml after cultivation for 45 hrs.

• Journal of Microbiology and Biotechnology
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• v.17 no.1
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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.