Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

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 Mn 2+ as optimum cofactor for activity. In addition, Mn 2+ 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 Mn 2+ -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%.