Kinetic modelling of the thermal inactivation of an industrial β-galactosidase from Kluyveromyces fragilis

The thermal and operational inactivation of a commercial β-galactosidase from Kluyveromyces fragilis (Lactozym, from Novozymes) was studied in several buffered solutions usually employed to study the activity of this enzyme. Some previous experiments have been done to understand the effect of the composition of the buffers on the enzyme stability. Afterwards, data obtained at temperatures from 25 to 50 °C were fitted by several kinetic models. Discrimination among the kinetic models was performed considering the temperature as a constant or as a variable. When using a 50 mM phosphate buffer pH 7.5, first-order reaction model is able to fit inactivation data, while a more complex model, involving two consecutive first-order reactions, was chosen for the lacteous buffer BM, with and without lactose. In both cases, the final form of the enzyme was totally inactive. Both lactose and mercaptoethanol have proved to be stabilisers of the enzyme, increasing half-life four to five times or twice, respectively. The intermediate forms of the enzyme during the inactivation process proved to have an activity, which, surprisingly, was higher at higher temperatures when lactose was present.