title: Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1.
abstract: Inulin fructotransferase (IFTase) is an important enzyme that produces di-d-fructofuranose 1,2':2,3' dianhydride (DAF III), which is beneficial for human health. Present investigations mainly focus on screening and characterizing IFTase, including catalytic efficiency and thermostability, which are two important factors for enzymatic industrial applications. However, few reports aimed to improve these two characteristics based on the structure of IFTase. In this work, a structural model of IFTase (DFA III-producing) from Arthrobacter sp. 161MFSha2.1 was constructed through homology modeling. Analysis of this model reveals that two residues, Ser-309 and Ser-333, may play key roles in the structural stability. Therefore, the functions of the two residues were probed by site-directed mutagenesis combined with the Nano-DSC method and assays for residual activity. In contrast to other mutations, single mutation of serine 309 (or serine 333) to threonine did not decrease the enzymatic stability, whereas double mutation (serine 309 and serine 333 to threonine) can enhance thermostability (by approximately 5 °C). The probable mechanisms for this enhancement were investigated.
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Shuhuai
authors.initials: S
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Food Science and Technology and ‡Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University , Wuxi, Jiangsu 214122, China.
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
mesh_terms: D020816:Amino Acid Motifs; D001173:Arthrobacter / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007444:Inulin / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation
chemical_list: D001426:Bacterial Proteins; D007444:Inulin; D006602:Hexosyltransferases
keywords: di-d-fructofuranose 1,2′:2,3′ dianhydride (DFA III); homology modeling; inulin; inulin fructotransferase (IFTase); thermostability