Substrate specificity and other properties of the β-d- galactosidase from Aspergillus niger

β-d-Galactosidase from Aspergillus niger was purified by conventional techniques, including the repeated use of chromatography on hydroxylapatite. The final preparation represented a 112-fold purification, with a 22% yield. The specific activity of the purified enzyme was 72 μmol of d-galactose released/min/mg of protein, using p-nitrophenyl β-d-galactopyranoside as the substrate. The substrate specificity of the enzyme was studied by using saccharides having structural linkages similar to those found in naturally occurring glycoconjugates. At substrate concentrations of 5mm, the β-d-galactosidase efficiently hydrolyzed β-Gal- 1→OC₆H₄NO₂-p, β-Gal-(1→3)-Gal, β-Gal0(1→3)-β-Gal-1→OC₆H₄NO₂-p, and β-Gal-(1→3)-α-Gal-1→OC₆H₄NO₂-p, at rates of 63, 53, 65, and 29 μmol/min/mg of protein, respectively. Slower hydrolysis was observed for β-Gal-(1→4)-β- Glc, β-Gal-(1→4)-β-GlcNAc-1→OC₆H₄NO₂-p, and β-Gal-(1→6)-β-GlcNAc- 1→OC₆H₄NO₂-p, with rates of 10, 13 and 9 μmol/min/mg of protein, respectively. Poorly hydrolyzed, at rates 1/300th of that of β-Gal-1→OC₆H₄NO₂-p, were synthetic substates having d-galactose attached β-(1→3)- to either GalNAc or GlcNAc. The K_m value for β-d-galactosidase with β-Gal-(1→4)-β-GlNAc- 1→OC₆H₄NO₂-p was ∼20 times that with β-Gal-1→OC₆H₄NO₂-p. The β-d-galactosidase of A. niger has a molecular weight of 300,000, as demonstrated by gel-filtration chromatography. Sodium dodecyl sulfate-poly(acrylamide)-gel electrophoresis indicated a single subunit having a molecular weight of 130,000.