Isolation and properties of beta-galactosidase of a strain of Lactobacillus helveticus isolated from natural whey starter.

beta-Galactosidase has been isolated from Lactobacillus helveticus of a strain isolated from natural starters for the manufacture of Argentine hard cheeses and its properties have been studied. The enzyme was purified 14-fold (by chromatography on DEAE-cellulose and Sepharose 6B-DEAE-cellulose columns and by affinity chromatography in agarose-p-aminophenyl-beta-D-thiogalactoside). The purified extract exhibited a single band following polyacrylamide gel electrophoresis. Maximum enzymatic activity was observed at 42 degrees C and pH 6.5 in 50 mM phosphate buffer. At pH values substantially different from the optimum, a positive cooperativity between substrate molecules was observed. The Km's for o-nitrophenylgalactoside (ONPG) and ONPG + 10 mM of lactose were 4.46 X 10(-5) and 8.9 X 10(-5) M, respectively. Glucose, galactose, galactose 6-phosphate, and lactate acted as noncompetitive inhibitors; MgCl2 protected the enzyme from thermal denaturation. The activation energy of enzymatic hydrolysis of ONPG was 11,400 cal/mol. The Mr was estimated to be 250,000. It is an oligomeric enzyme made of 4 subunits of Mr 65,000.