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 (Tm) and the urea midtransition concentration (Cm) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn2+-bound enzyme showed higher Tm and Cm with 67.3°C and 5.09 M. The Michaelis-Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) 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%. © 2013 Zhang et al.