Effects of the Ionic Environment on the Stability of Kluyveromyces lactis β-Galactosidase

Abstract

The effect of several cations on the stability of soluble and whole cell Kluyveromyces lactis lactase was studied in buffer solutions as a function of cation concentration and temperature. From several divalent cations tested at 45°C in phosphate buffer (pH 6.6), only Mn2+ (0.1 to 0.2 mmol/L) and to a lesser extent Mg2+ (2.5 to 5.O mmol/L), protected the enzyme against thermal deactivation, while Zn2+ (10-3 mmol/L) and Ca2+ (10-4 mmol/L) deactivated the lactase. Stability was also dependent on the potassium concentration, but was not affected by sodium ions (20 to 40 mmol/L). Studies in barbital buffer (pH 6.6) showed a strong deactivation effect of Ca2+ (5 to 13 mmol/L). Based on data of the thermal deactivation kinetics, it could be estimated that at low temperatures (<20°C) the lactases will exhibit high stability even without the addition of Mg or Mn ions (half life >1 year). Deactivation by Ca2+, Zn2+ or Ca2+ was markedly dependent on the temperature and highly susceptible to small changes of the cation concentration. At 5°C the deactivation rate was significantly reduced. Kinetic data of the initial rate of lactose hydrolysis showed that the ionic effect on enzyme stability was not correlated with changes in the enzymatic activity. The whole cell lactase showed higher stability than the soluble product anti was less sensitive to deactivation by cations. Enzyme activity after deactivation by Ca2+ could be almost restored by washing the cells with phosphates. Activity after deactivation by Zn+was partially restored with chelating agents such as EDTA, and no significant restoration occurred after deactivation by Ca2+.