Purification and Characterization of Endo-α-N-acetylgalactosaminidase from Alcaligenes sp.

Abstract

A gram negative bacterium isolated from soil was found to produce an endo-α-N-acetylgalactosaminidase in culture. The microorganism was identified as Alcaligenes sp. from various bacteriological characteristics. The enzyme was purified from the culture fluid by fractionation with ammonium sulfate, and column chromatographies on DEAE-Sephadex A-50 and hydroxylapatite. The molecular weight of the enzyme was estimated to be 160, 000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH was found to be 4.5 and the stable pH range was 4.5-6.5. The Km values were 3.7mM and 3.2mM with asialofetuin and asialo κ-casein glycopeptide as substrates, respectively. The enzyme released the disaccharide, Galβ1→3GalNAc, from glycoproteins possessing serine or threonine O-glycosidic linkages. The enzyme production was highly induced by porcine gastric mucin added to the medium.