Isolation and purification ofβ-galactosidase ofLactobacillus murinus CNRZ 313

Abstractβ-Galactosidase has been isolated inLactobacillus murinus CNRZ 313, and its properties have been studied. The enzyme was purified 292-fold by chromatography on Ultrogel ACA 34, DEAE-Sephadex A-50 columns, and by affinity chromatography in agarose-p-aminophenyl-β-d-thiogalactoside. The purified extract exhibited a single band following polyacrylamide gel electrophoresis (PAGE). Molecular weight was estimated to be 170,000 on an Ultrogel ACA 34 column. Maximum enzymatic activity was observed at 45°C and pH 7 in 50 mM phosphate buffer. The Km for ONPG and ONPG + 20 mM of lactose were 480 μM and 870 μM, respectively. The effect of different salts on the enzymatic activity was studied. An inhibitory effect was observed when using 10 mM CaCl2, and a stimulating effect when using 10 mM MgCl2. The latter protected the enzyme from thermal denaturation. Among the protective agents belonging to the sulfhydryl group that was tested, mercaptoethanol and dithiotreitol acted as activators, while glutathione and cysteine acted as inhibitors, of the enzymatic activity.p-β-Galactosidase activity was not observed.