Rapid procedures for determination of endo- N-acetyl-α- d-galactosaminidase in clostridium perfringens, and of the substrate specificity of exo-β- d-galactosidases

Abstract

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates, β-Gal-(1→3)-α-GalNAc-1→OPh and, β-Gal-(1→3)-α-GalNAc-1→OC 6H 4-NO 2- o or -p to β-Gal-(1→3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo- N-acetyl-α- d-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates β-Gal-(1→3)-β-GlcNAc-1→-OC 6H 4NO 2- p, β-Gal-(1→4)-β-GlcNAc-1→OC 6H 4NO 2- p, and β-Gal-(1→6)-β-GlcNAc-1-OC 6H 4NO 2- p, was developed for the rapid identification of the linkage specificity of exo-β- d-galactosidases from any source via a coupled reaction with N-acetyl-β- d-hexosaminidase.