Partial Purification and Characterization of β-D-Galactosidase from Kluyveromyces marxianus

Abstract

Optimal conditions for lactase extraction with 2% chloroform from the yeast Kluyveromyces marxianus NCYC 111 grown on whey included pH 6.8, 25°C, and 10 h of treatment. The enzyme was purified 11-fold after acetone and ammonium sulfate precipitations. The molecular weight was 280,000. Optimum pH and temperature for enzyme activity were 6.2 and 45 to 52°C. The enzyme was inactivated in 4 min at 56°C and retained 50% of its activity after 90 min at 50°C. Michaelis-Menten constants were 3.1 mM on o-nitrophenyl-β-D-galac-topyranoside and 25 mM on lactose. Hydrolysis o-nitrophenyl-β-D-galactopy-ranoside was inhibited competitively by methyl-β-D-galactoside (inhibition constant 58 mM), galactose (110 mM), ribose (111 mM), and lactose (52 mM). P-chloromercuribenzoate inhibited non-competitively, and its activity was blocked by dithiothreitol, indicating sulphydryl groups in the enzyme and their possible involvement in lactase activity. The enzyme (at .59 mg protein/ml) hydrolyzed 50% of the lactose in milk and whey solutions at 6% of concentration in 5 h at 37°C. Kluyveromyces marxianus represents a potential source of lactase for reduction of lactose in milk and sweet whey. © 1982, American Dairy Science Association. All rights reserved.