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 degrees 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 degrees C for 1 h. The K-m and V-max were estimated to 12.93 mM and 2.89 mu mol 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 gl(-1) of inulin were hydrolyzed by the purified IFTase, the DFA III yield reached 81%, 72%, and 67%, respectively.