A novel di-D-furanose 1,2′:2,3′-dianhydride hydrolase (DFA-IIIase) from Duffyella gerundensis A4

The prebiotic fructan inulin can provide energy to organisms via several pathways. One pathway is that inulin fructotransferase (IFTase) firstly converted inulin to III-type difructose anhydride (DFA-III). Then DFA-III is hydrolyzed to inulobiose via difructose anhydride hydrolase (DFA-IIIase). However, only five DFA-IIIases have been reported to date and all of them are from Arthrobacter genus. Whether other microbes except Arthrobacter genus can utilize DFA-III through DFA-IIIase are unknown. In this work, a DFA-IIIase from Duffyella gerundensis A4 (D. gerundensis A4), abbreviated as DgDFA-IIIase, was cloned, expressed, purified, identified, and characterized. It was approximately 50 kDa assayed by SDS-PAGE and showed the highest catalytic activity for DFA-III at pH 6.0 and 35 °C with specific activity of 56 U mg-1. The enzyme was metal-independent and had a high pH stability but low thermostability, which kept only 28% of residual activity after incubation under 50 °C for 3 h. Moreover, Km and kcat/Km for DFA-III was 122 mM and 0.39 mM-1 s-1, respectively. The constructed model of DgDFA-IIIase showed that it has identical residues around substrate at active site with AcDFA-IIIase whose crystal structure has been revealed, indicating that DgDFA-IIIase probably adopts the same catalytic mechanism with the reported AcDFA-IIIase. The work finds that DFA-III can be catalyzed by DFA-IIIase from microorganism of non-Arthrobacter genus, which also extends the enzymatic sources of DFA-IIIase.