Identification, separation, and preliminary characterization of invertase and β galactosidase in Actinomyces viscosus

Abstract

The initial step of disaccharide dissimilation by A. viscosus serotype 2 strain M-100 was studied. Sucrase activity was found in the 3,000 x g particulate fraction and the 37,000 x g soluble fraction of the cells, whereas lactase activity was found almost exclusively in the 37,000 x g soluble fraction. Neither sucrase nor lactase activity was appreciable in the culture liquor. Sucrose phosphorylase, α-glucosidase, and polysaccharide synthesis activities were not observed in the soluble cell fraction. The sucrase was identified as invertase (EC 3.2.1.26; β-D-fructofuranoside fructohydrolase). The lactase was identified as β-galactosidase (EC 3.2.1.23; β-D-galactoside galactohydrolase). The enzymes in the 37,000 x g soluble fraction were separable by diethylamino-ethyl-cellulose chromatography, giving one β-galactosidase peak and one major and one minor invertase peak. Acrylamide gel electrophoresis showed different electrophoretic mobilities of the enzymes. The molecular weight of the β-galactosidase is about 4.2 x 105 and that of invertase is about 8.6 x 104. The β-galactosidase has a Km for lactose of about 6 mM and a pH optimum between pH 6.0 and 6.5. The major invertase component has a Km for sucrose of about 71 mM and a pH optimum between pH 5.8 and 6.3.