Formation of di- d -fructofuranose-1,2′:2,1′-dianhydride by three novel inulin fructotransferases from the Nocardiaceae family

Abstract

Abstract 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 luteus , Nocardioides sp. JS614, and Nocardioidaceae bacterium Broad-1, were cloned and expressed in Escherichia coli . The recombinant IFTases from N. luteus ( Nolu IFTase), Nocardioides sp. JS614 ( No spIFTase), and N. bacterium Broad-1 ( Noba IFTase) 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- d -fructofuranose-1,2′:2,3′-dianhydride (DFA III)-forming IFTases. Nolu IFTase, No spIFTase, and Noba IFTase exhibited maximal activities at 55 °C, 50 °C, and 45 °C and were stable at 70 °C (for 15 min), 70 °C (187 min), and 55 °C (239 min), respectively. Furthermore, by comparing with our previously reported DFA I-forming IFTase, namely Cc IFTase, a probable mechanism for the formation of DFA I by the three new enzymes was speculated, and Cc IFTase will be selected for future structural resolution to illustrate the catalytic mechanism of DFA I-forming IFTases toward inulin.