The first di-D-fructofuranose 1,2′:2,3′-dianhydride hydrolase (DFA-IIIase) from the genus of non-Arthrobacter (pathogen Salmonella enterica subsp. enterica serovar Mbandaka): Molecular cloning, expression, identification, and characterization

Abstract

The prebiotic polysaccharide inulin belongs to fructan and is extensively used in food industry. It can provide energy to organisms via several pathways. One pathway is catalyzed by inulin fructotransferase (IFTase) to III-type difructose anhydride (DFA-III) that has many health-promoting functions. DFA-III is further converted to inulobiose, a kind of fructo-oligosaccharide, via DFA-III hydrolase (DFA-IIIase). However, only five DFA-IIIases from Arthrobacter genus have been reported to date. Whether other microbes except Arthrobacter genus can utilize DFA-III through DFA-IIIase is unknown. Against this background, in this work, a DFA-IIIase from pathogen Salmonella enterica subsp. enterica serovar Mbandaka (Salmonella Mbandaka), abbreviated as SmDFA-IIIase, was purified, identified, and characterized after gene cloning, heterologous expression in Escherichia coli (E. coli) system. This metal-independent enzyme showed the highest activity of 791 U mg−1 under the optimal reaction conditions (pH 6.5 and 50 °C). The Km of SmDFA-IIIase was assayed as 367 mM and the yield of inulobiose reached 29%. SmDFA-IIIase probably adopts the same catalytic mechanism with the previously reported DFA-IIIase. The work extends the enzymatic sources of DFA-IIIase from microorganism of non-Arthrobacter genus and the enzyme showed a higher specific activity and thermostability compared with the reported DFA-IIIases, which has a potential to produce prebiotic inulobiose in industry in the future. However, the exploration in this work also reminds that we need to pay more attention when use or store DFA-III because the proliferation and contamination of Salmonella probably occur based on SmDFA-IIIase found here.