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

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Abstract
Optimal conditions for lactase extraction with 2% chloroform from the yeast K. marxianus NCYC 111 grown on whey included pH 6.8, 25.degree. C and 10 h of treatment. The enzyme was purified 11-fold after acetone and ammonium sulfate precipitations. The MW was 280,000. Optimum pH and temperature for enzyme activity were 6.2 and 45.degree.-52.degree. C. The enzyme was inactivated in 4 min at 56.degree. C and retained 50% of its activity after 90 min at 50.degree. C. Michaelis-Menten constants were 3.1 mM on o-nitrophenyl-.beta.-D-galactopyranoside and 25 mM on lactose. Hydrolysis of o-nitrophenyl-.beta.-D-galactopyranoside was inhibited competitively by methyl-.beta.-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 SH groups in the enzyme and their possible involvement in lactase activity. The enzyme (at 0.59 mg protein/ml) hydrolyzed 50% of the lactose in milk and whey solutions at 6% of concentration in 5 h at 37.degree. C. K. marxianus represents a potential source of lactase for reduction of lactose in milk and sweet whey.