Thermostabilization of Kluyveromyces marxianus beta-galactosidase by histidine: Physical studies

The beta-galactosidase from Kluyveromyces marxianus was purified from a commercial source (LactAid) to electrophoretic homogeneity. It was rapidly inactivated at 45 degrees C, but was stabilized against activity loss by histidine at low concentrations, Stabilization by histidine was enhanced in the presence of lactose. Differential scanning calorimetry of enzyme solutions showed two enthalpic peaks: a primary melting transition (T-m) of approximate to 51 degrees C and a secondary transition, at a higher temperature, whose existence and melting temperature were concentration-dependent and may be related to aggregation, Lactose, galactose and glucose increased the value of T-m, but histidine had no effect. Unfolding of the enzyme under isothermal conditions at 47.5 degrees C was followed by aggregation, Histidine delayed unfolding at temperatures from 43 degrees C to 46 degrees C, but not at 47.5 degrees C, Binding of histidine to the enzyme could not be detected by equilibrium dialysis or gel filtration, It was concluded that histidine stabilized the enzyme by delaying unfolding at temperatures below the melting transition.