title: Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.
abstract: The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.
authors: Wenli
authors: W
authors: Zhang
authors: Dan
authors: D
authors: Fang
authors: Qingchao
authors: Q
authors: Xing
authors: Leon
authors: L
authors: Zhou
authors: Bo
authors: B
authors: Jiang
authors: Wanmeng
authors: W
authors: Mu
mesh_terms: D000595:Amino Acid Sequence; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D005632:Fructose / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008670:Metals; D011994:Recombinant Proteins; C003243:psicose; D005632:Fructose; D008345:Manganese; D002238:Carbohydrate Epimerases
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