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

Optimal conditions for lactase extraction with 2% chloroform from the yeast Kluyveromyces marxianus NCYC 111 grown on whey included pH 6.8, 25C, and 10 h of treatment. The enzyme was purified l 1-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 52C. The enzyme was inactivated in 4 rain at 56C and retained 50% of its activity after 90 min at 50C. Michaelis-Menten constants were 3.1 mM on o-nitrophenyl-/3-D-galactopyranoside and 25 mM on lactose. Hydrolysis o-nitrophenyl-/3-D-galactopyranoside was inhibited competitively by methyl-/3-D-galactoside (inhibition constant 58 mM), galactose (110 mM), ribose (111 mM), and lactose (52 raM). Pchloromercuribenzoate inhibited noncompetitively, 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 37C. Kluyveromyces marxianus represents a potential source of lactase for reduction of lactose in milk and sweet whey.