In this work, three novel genes encoding di-D-fructofuranose-1,2':2,1'-dianhydride (DFA I)-forming inulin fructotransferases (IFTases) from Nocardiaceae family, including Nocardioides hums, Nocardioides sp. JS614, and Nocardioidaceae bacterium Broad-1, were cloned and expressed in Escherichia coli. The recombinant IFTases from N. luteus (NolulFTase), Nocardioides sp. JS614 (NosplFTase), and N. bacterium Broad-1 (NobalFTase) were purified, identified, and characterized. SDS-PAGE analysis showed that they had molecular weights of approximately 41-42 kDa, while gel filtration analysis indicated that their native molecular weights ranged from 50 to 62 kDa, suggesting that the three enzymes may be monomers. Their optimum pH values ranged from 5.5 to 6.0, similar to other DFA I-forming IFTases or di-b-fructofuranose-1,2':2,3'-dianhydride (DFA III)-forming IFTases. NoluIFTase, NospiFTase, and Nobair I ase exhibited maximal activities at 55 degrees C, 50 degrees C, and 45 degrees C and were stable at 70 degrees C (for 15 min), 70 degrees C (187 min), and 55 degrees C (239 min), respectively. Furthermore, by comparing with our previously reported DFA I-forming IFTase, namely CcIFTase, a probable mechanism for the formation of DFA I by the three new enzymes was speculated, and CcIFTase will be selected for future structural resolution to illustrate the catalytic mechanism of DFA I-forming IFTases toward inulin.