Purification and characterization of neuraminidase from Clostridium perfringens

Clostridium perfringens cells were cultivated on a large scale using an automatic system. Neuraminidase secreted by the cells into the culture medium was purified 380 000-fold by: precipitation with ammonium sulfate between 50 and 85% saturation, filtration on Sephadex G-75, electrophoresis on polyacrylamide gel, and by isoelectric focusing. Three enzyme fractions with different migration rates were obtained by preparative disc electrophoresis in polyacrylamide gel, and five fractions with isoelectric points between pH 4.7 and 5.4 were observed after isoelectric focusing. This microheterogeneity disappeared after denaturation of the enzyme in 0.1% sodium dodecylsulfate or 8M urea. The isoelectric point of the denatured enzyme corresponded to pH 4.3. All enzyme fractions were identical with regard to their immunological and kinetic properties; they had the same molecular weights. The origin of the different "conformers" of neuraminidase is discussed. The existence of genuine isoenzymes could largely be excluded. The yield of neuraminidase was 65%, which corresponded to about 10 mg of pure enzyme from 100 l of culture medium. The enzyme was free of protease and various other glycosidase activities. The neuraminidase preparation appeared not to be contaminated by other proteins as judged by electrophoretic analysis using either the native enzyme or the enzyme denatured by sodium dodecylsulfate or urea; ultracentrifugation; chromatography on Sephadex G-200; and immunological methods. The molecular weights of the native or denatured enzyme were found to be in the range between 60 000 and 69 000 (on an average 63 750) using four independent methods. The existence of subunits of neuraminidase was excluded. The neuraminidase exhibited a spec. act. of 580 or 615 U/mg protein with glycopeptides from edible birds' nests or sialyllactose, respectively, as substrates. Additional kinetic properties and the UV-absorption spectrum of the enzyme are described.