Differences in the Hydrolysis of Lactose and Other Substrates by β-D-Galactosidase from Kluyveromyces lactis

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The hydrolysis of o-nitrophenyl galactopyranoside and lactose by β-D-galactosidase from Kluyveromyces lactis was enhanced by the addition of Mg2+ and Mn2++, but the rates of activation by each metal on both substrates were not the same. The Co2+, Zn2+, and Ni2+ activated the o-nitrophenyl galactopyranoside hydrolyzing activity of the enzyme, but these same metals inhibited the lactose-hydrolyzing activity. The addition of Mg2+ and EDTA to the assay buffer increased the hydrolysis of o-nitrophenyl galactopyranoside and lactose at different rates. The responses of o-nitrophenyl galactopyranoside and lactose to the enzyme activity were different as a function of pH. The hydrolyzing activity toward both substrates also was influenced by the concentration of the phosphate in the assay buffer. However, the profile of the enzyme activity toward each substrate was different as a function of concentration. Because the assay of β-galactosidase using o-nitrophenyl galactopyranoside is fast and convenient, the estimation of lactose-hydrolyzing activity of the enzyme has frequently been made based on the assay of o-nitrophenyl galactopyranoside hydrolysis. As shown in this study, a slight change in the conditions of the assay system and the enzyme application may cause changes in the ability of the enzyme to hydrolyze both lactose and o-nitrophenyl galactopyranoside. The change in o-nitrophenyl galactopyranoside-hydrolyzing activity is not always consistent with that of the lactose-hydrolyzing activity under the given condition, which may cause an inaccurate estimation of the enzyme activity in the enzyme preparation as well as in actual applications of the enzyme.