Isolation and characterization of a blood group A substance-degrading .ALPHA.-N-acetylgalactosaminidase from an Acremonium sp.

Abstract

A fungus isolated from soil was found to produce α-N-acetylgalactosaminidase in culture, this enzyme being the dominant glycosidase. The fungus was identified as an Acremonium sp., based on various taxonomical characteristics. The enzyme produced by the fungus was purified to electrophoretic homogeneity from the culture broth by procedures including ammonium sulfate fractionation, and chromatography on DEAE-Sephadex A-50, hydroxylapatite, Sephadex G-150 and Concanavalin A-Sepharose 4B. The molecular weight of the enzyme was estimated to be 55, 000 and 57, 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, and the enzyme appeared to have a monomer structure. The enzyme was most active at pH 4.0-4.5, and was stable at pH 6.0-7.5 and below 40°C. The Michaelis constant for p-nitrophenyl α-N-acetylgalactosaminide was 1.3 mM. The enzyme liberated the N-acetylgalactosamine from the bovine submaxillary glycoprotein, which had been desialyzed with neuraminidase. The enzyme also liberated the N-acetylgalactosamine from various blood type A active substances, including porcine gastric mucin, horse gastric mucin and human salivary mucin, which was ascertained by a hemagglutination inhibition test. The enzyme could change blood type A erythrocytes into blood type O erythrocytes, which was confirmed by a hemagglutination test.