Cloning, Expression, and Characterization of a D-Psicose 3-Epimerase from Clostridium cellulolyticum H10

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli. The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as D-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co2+. The optimum pH and temperature for enzyme activity were 55 degrees C and pH 8.0. The half-lives for the enzyme at 60 degrees C were 6.8 h and 10 min when incubated with and without Co2+, respectively, suggesting that this enzyme was extremely thermostable in the presence of Co2+ but readily inactivated without metal ion. The Michaelis-Menten constant (K-m), turnover number (k(cat)), and catalytic efficiency (k(cat)/K-m) values of the enzyme for substrate n-psicose were estimated to be 17.4 mM, 3243.4 min(-1,) and 186.4 nM min(-1), respectively. The enzyme carried out the epimerization of D-fructose to D-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential D-psicose producer.