Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704 |
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| Authors: | Wenli Zhang Dan Fang Qingchao Xing Leon Zhou Bo Jiang Wanmeng Mu |
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| Institution: | 1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of China.; 2. Roquette America, Keokuk, Iowa, United States of America.; University of South Florida College of Medicine, United States of America, |
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| 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%. |
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