Stability and Enzymatic Properties of ?-Galactosidase From Kluyveromyces Fragilis

Abstract

Kluyveromyces fragilis β‐galactosidase purified to electrophoretic, chromatographic and immunochemical homogeneity was used. The enzyme specifically required potassium ions for stability; MnCl2 increased the stability. The enzyme was maximally stable at pH 6.5 to 7.5; stability was markedly less at pH's below 6.5 and above pH 8.5 at 37°C. Temperature denaturation followed first order kinetics with an activation energy for denaturation of 56 kcal/mol. Maximum activity was achieved in the presence of 5mM KCl. In potassium phosphate buffer, the enzyme was further activated by Mn2+, Mg2+, Co2+ and Zn2+; Mn2+, at 0.1 mM, gave the highest activation. None of these ions activated the enzyme in Tris buffer and> 0.1 mM Zn2+ caused complete loss of activity. Activity was completely inhibited by ethylenediaminetetraacetate and partially restored by addition of MnCl2. p‐Chloromercuribenzoate caused rapid loss of activity which could be restored by dithiothreitol. Iodoacetamide, N‐ethylmaleimide and sodium tetrathionate did not inactivate the enzyme. The enzyme was specific for β‐galactosides. Km's for o‐nitrophenyl β‐D‐galactopyranoside and lactose were 2.72 and 13.9 mM, respectively, at pH 6.6 D‐Galactono‐1, 4‐lactone was a good competitive inhibitor (Ki=0.17 mM). pH optimum for hydrolysis of o‐nitrophenyl β‐D‐galactopyranoside was 6.2–6.4. Vmax for this substrate was dependent on two ionizable groups of pKa of 6.13 and 6.51 while Vmax/Km was dependent on two ionizable groups of pKa of 6.39 and 7.23. Activation energy for hydrolysis of o‐nitrophenyl β‐D‐galactopyranoside at pH 7.0 was 9.1 kcal/mol in the range 20–40°C.

Purification and properties of a β-galactosidase with potential application as a digestive supplement

“…The pH vs activity profile of both the crude and purified enzyme was similar to that previously reported for Kluyveromyces-derived β-galactosidases (13,27,33). The profile (pH optimum of 6.8, with 50% or greater residual activity between pH 5.6 and 7.8) suggests that it would be suitable for lactose hydrolysis in the small intestine.…”

“…The enzyme was most efficient in catalyzing the hydrolysis of PNPG with ONPG and lactose in that order. However, the kinetic constants of the enzyme in hydrolyzing lactose compare favorably with those reported for other microbial β-galactosidases (13,40).…”

“…Enzyme Assay β-Galactosidase activity assay was based on the method of Mahoney and Whitaker (13). Ortho-nitrophenyl-β-d-galactopyranoside (ONPG) substrate was dissolved to a final concentration of 5 mM in 200 mM potassium phosphate buffer, pH 6.8.…”

Development and Evaluation of Whole‐Cell Yeast Lactase for Use in Dairy Processing

“…4, 1986 much broader and lower optimum (pH 5.6 -6.0), and consequently greater acid stability than generally reported for (partially) purified lactase (Wendorff and Amundson, 1971;Mahoney and Whitaker, 1977;Novo Enzymes, 1982).…”

Enzyme-Assisted Process for Production of Superior Quality Vanilla Extracts from Green Vanilla Pods Using Tea Leaf Enzymes

“…α-Glucosidase enzyme activity was measured in TLEE using the method of Ye-Yun et al (2005). Galactosidase enzyme activity was measured as described by Mahoney and Whitaker (1977). Unit activity was defined as micromoles of oNP released per minute.…”