β- d -Phosphogalactoside Galactohydrolase from Streptococcus cremoris HP: Purification and Enzyme Properties

Abstract

β- d -phosphogalactoside galactohydrolase (β-PGal) was isolated and purified from cell-free extracts of Streptococcus cremoris HP to apparent homogeneity to gel electrophoresis. Using the chromogenic o -nitrophenol-β- d -galactopyranoside-6-phosphate as substrate, the purified enzyme exhibited a specific activity of 18.71 U/mg of protein and K m and V max values of 5.88 × 10 −4 M and 23.8 μmol of o -nitrophenol liberated per min per mg of protein, respectively. d -Galactose-6-phosphate was a weak competitive inhibitor of β-PGal. Activity was relatively heat resistant and was maximal from pH 5.0 to 8.0 and over a temperature range of 45 to 52 C. Dithiothreitol, ethylenediaminetetraacetic acid, and citrate stimulated β-PGal activity, whereas Mg 2+ , Li 1+ , and p -hydroxymercuribenzoate were inhibitory. Molecular weight of the enzyme was estimated at 6.76 × 10 4 . Amino acid composition was similar to other β-phosphogalactosidases previously investigated, with the exception that the S. cremoris enzyme contains a small amount of half cystine.