STABILITY AND ENZYMATIC PROPERTIES OF ?-GALACTOSIDASE FROM KLUYVEROMYCES FRAGILIS

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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.