Identification of a Novel Di-D-Fructofuranose 1,2’:2,3’ Dianhydride (DFA III) Hydrolysis Enzyme from Arthrobacter aurescens SK8.001

Abstract

Previously, a di-D-fructofuranose 1,2’:2,3’ dianhydride (DFA III)-producing strain, Arthrobacter aurescens SK8.001, was isolated from soil, and the gene cloning and characterization of the DFA III-forming enzyme was studied. In this study, a DFA III hydrolysis enzyme (DFA IIIase)-encoding gene was obtained from the same strain, and the DFA IIIase gene was cloned and expressed in Escherichia coli. The SDS-PAGE and gel filtration results indicated that the purified enzyme was a homotrimer holoenzyme of 145 kDa composed of subunits of 49 kDa. The enzyme displayed the highest catalytic activity for DFA III at pH 5.5 and 55°C, with specific activity of 232 U mg-1. K m and V max for DFA III were 30.7 ± 4.3 mM and 1.2 ± 0.1 mM min-1, respectively. Interestingly, DFA III-forming enzymes and DFA IIIases are highly homologous in amino acid sequence. The molecular modeling and docking of DFA IIIase were first studied, using DFA III-forming enzyme from Bacillus sp. snu-7 as a template. It was suggested that A. aurescens DFA IIIase shared a similar three-dimensional structure with the reported DFA III-forming enzyme from Bacillus sp. snu-7. Furthermore, their catalytic sites may occupy the same position on the proteins. Based on molecular docking analysis and site-directed mutagenesis, it was shown that D207 and E218 were two potential critical residues for the catalysis of A. aurescens DFA IIIase.

Structural and Functional Basis of Difructose Anhydride III Hydrolase, Which Sequentially Converts Inulin Using the Same Catalytic Residue

“…H65-7 has no activity toward inulin . Other DFA-IIIases’ ,, activity toward inulin was not studied. However, we found that AcDFA-IIIase was also able to decompose inulin under the same conditions as those used for hydrolyzing DFA-III.…”

“…After centrifugation (18 000 g , 4 °C, 20 min) and filtration using a 0.22 μm filter, the filtrate was analyzed by the Sugar-Pak I column (Waters, Milford, MA) with an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) and a refractive index detector. The standard marker of inulobiose was synthesized by our previously obtained DFA-IIIase . The amount of AcDFA-IIIase or its mutant that produces 1 μmol inulobiose per minute at pH 6.5 and 55 °C was defined as one unit of activity.…”

“…To gain energy from fructan for cell growth, DFAs are assimilated into cell and hydrolyzed by difructose anhydride hydrolase (DFAase) to inulobiose. To date, only four DFA-III hydrolases (DFA-IIIases) have been reported. − Although the enzymatic mechanisms of degradation of the fructans inulin and levan into DFAs have been well investigated, , how DFAs are further degraded into inulobiose by DFAase is elusive. DFA-III has so many physiological functions that its products have been on the market since 2014.…”

Metabolic diversity of the emerging pathogenic lineages of Klebsiella pneumoniae

“…The putative inulin utilization region consists of three co-oriented genes. It comprises a gene encoding an enzyme similar to an inulinfructotransferase (65.71% similarity) previously described in Arthrobacter (Yu et al, 2015).…”

Carbon Dioxide as a Traceless Caramelization Promotor: Preparation of Prebiotic Difructose Dianhydrides (DFAs)-Enriched Caramels from d-Fructose

“…Their unique cyclic structure confers on DFAs much higher enzymatic and chemical stability as compared with classical fructosyl derivatives such as sucrose. , The growing interest in the prebiotic properties of DFAs has led to the search for new methodologies allowing the preparation of single isomers or DFA-enriched products. A method based on the enzymatic catalytic degradation of fructans was one of the first studied. − More recently, the enzymatic synthesis of a DFA diastereomer from sucrose, namely, α- d -fructofuranose β- d -fructofuranose 1,2′:2,3′-dianhydride, by coupling a sucrose fructosyltransferase and an inulin fructotransferase, has also been reported . Major drawbacks of these syntheses are the low thermal stability, the substrate dependence of enzymes, and the high cost of these processes.…”