title: 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. Km and Vmax 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.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.
authors.forename: Shuhuai
authors.initials: S
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Stressler
authors.affiliation: University of Hohenheim, Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, Garbenstr. 25, 70599, Stuttgart, Germany.
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China.
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
mesh_terms: D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004187:Disaccharides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D013696:Temperature
chemical_list: D004187:Disaccharides; C087758:difructose anhydride III