Purification and kinetic properties of sialidase from Clostridium perfringens.

Clostridium perfringens sialidase was isolated from a culture medium of bacterial cells by ammonium sulfate precipitation (42-85%), followed by purification through Sephadex G-75 gel chromatography, DEAE A-50 anion exchange chromatography, FPLC medium pressure anion exchange chromatography and finally FPLC medium pressure isochromatofocussing. From 9 l culture medium 1.17 mg sialidase was isolated with a specific activity of 295 U/mg. The enzyme appeared to be homogeneous by analytical polyacrylamide gel electrophoresis. The molecular mass was measured to be 66 kDa. Km values ranging from 0.6 to 1.6mM were determined for several oligosaccharides as substrates. The enzyme catalyzed transglycosylation reactions with methanol as a nucleophilic reagent competitive with water. In the enzymatic hydrolysis of the (3'-methoxyphenyl)glycoside of alpha-N-acetylneuraminic acid, increase of methanol concentration had no effect on the release of 3-methoxyphenol. This finding suggests that the formation of the enzyme-glycon intermediate is the rate-determining step for this substrate.