Characterization of a thermostable inulin fructotransferase from Clostridium clostridioforme AGR2157 that produces difructose dianhydride I from inulin

Abstract

A gene encoding a difructose dianhydride I (DFA I)-forming inulin fructotransferase (IFTase) from Clostridium clostridioforme AGR2157 was cloned and extracellularly expressed in Escherichia coli. SDS-PAGE and gel filtration analysis of the purified enzyme showed molecular mass of 43 and 128kDa, respectively, indicating that the recombinant enzyme was a trimer with three identical subunits. The enzyme displayed the highest activity at pH 5.5 and 50°C with the specific activity of 2.076Umg−1, and it was stable under heat treatment of up to 80°C for 4h. In comparison with other reported IFTases (DFA I-forming), the recombinant IFTase from C. clostridioforme demonstrated the best thermostability. Km and Vmax were calculated to be 0.42mM and 2.69mMmin−1, respectively. The main product of the hydrolysis of inulin by purified enzyme was identified as DFA I according to nuclear magnetic resonance analysis, and the minor products were suggested to be sucrose (GF), 1-kestose (GF2), nystose (GF3), and fructofuranosyl nystose (GF4). The smallest fructo-oligosaccharide substrate was determined to be GF3. When 10, 50, and 100gL−1 of inulin were catalyzed by the purified enzyme, the maximal yields of DFA I were approximately 85%, 73%, and 71%, respectively.