Bioconversion of inulin to difructose anhydride III by a novel inulin fructotransferase from Arthrobacter chlorophenolicus A6

Abstract The gene encoding difructose anhydride III (DFA III)-forming inulin fructotransferase (IFTase) from Arthrobacter chlorophenolicus A6 was cloned and overexpressed in Escherichia.coli . The recombinant IFTase (DFA III-forming) was purified using one-step nickel affinity chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration analyses, the enzyme showed a homotrimeric form composed of three identical subunits, each with an apparent molecular mass of 43 kDa. The maximum catalytic activity was shown at 65 °C and pH 5.5, and the specific activity was measured as 902 U mg -1 . The enzyme showed remarkable thermostability and over half of the initial activity was retained after incubation at 80 °C for 1 h. The K m and V max were estimated to 12.93 mM and 2.89 μmol min -1  ml -1 , respectively. After complete hydrolysis of inulin for DFA III production by the purified enzyme, the produced minor products contained sucrose (GF), 1-kestose (GF 2 ), and nystose (GF 3 ). The smallest substrate was identified to be GF 3 . When 50, 100, and 200 g l -1 of inulin were hydrolyzed by the purified IFTase, the DFA III yield reached 81%, 72%, and 67%, respectively.