Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Cloning, Expression, and Characterization of Bacillus sp. snu-7 Inulin Fructotransferase

  • Kim, Chung-Sei (School of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Hong, Chang-Ki (School of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Kim, Kyoung-Yun (School of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University) ;
  • Wang, Xiu-Ling (College of Life Science, Agricultural University of Hebei) ;
  • Kang, Su-Il (International Environmental Research Center, Gwangju Institute of Science and Technology) ;
  • Kim, Su-Il (School of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University)
  • Published : 2007.01.31
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Abstract

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.

Keywords

References

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