Purification and Some Properties of UDP-N-Acetylglucosamine 4-Epimerase from Bacillus subtilis

Abstract

UDP-N-Acetylglucosamine 4-epimerase (EC 5.1.3.7) was purified from a cell extract of Bacillus subtilis by protamine sulfate treatment, ammonium sulfate fractionation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, and Sephadex G-150. The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 62,000 by gel filtration. The enzyme had an optimal pH in the range of 7.0 to 9.0, and was active at 30~35°C in the absence of added NAD and more active at 40~45°C in the presence of NAD. The enzyme activity was highly stimulated by small amounts of NAD. The estimated Km values were 4.4 mM for UDP-N-acetylglucosamine and 0.13mM for NAD. No UDP-glucose 4-epimerase activity was found in the purified enzyme. The enzyme was inhibited by high concentrations of glucosamine and inhibition by glucosamine was competitive with UDP-N-acetylglucosamine. The inhibition constant was 37.0 mM. N-Acetylglucosamine did not inhibit the enzyme.