The critical problem in the identification of enzyme inactivation is to properly relate the phenomenon of the loss of enzyme activity with the undergoing changes in the structure of enzyme molecule. As a substantial improvement of the analysis of inactivation mechanisms, a method of simultaneous evaluation of inactivation experiments carried out at several temperatures have been presented [1]. A further improvement of the identification of inactivation mechanisms can be then sought in integrating the evaluation of data obtained by different techniques. The benefits of the suggested approach are demonstrated on the case study of thermal inactivation of yeast invertase utilizing the measurements of enzyme activity, heat flow in differential scanning calorimetry and concentrations of macromolecular species detected by size-exclusion chromatography.

Twelve inactivation curves were measured for varying temperature in the range of 50-70°C and initial enzyme concentration from 0,1 to 1 g/L. Four differential scanning calorimetry (DSC) measurements were made at the heating rates of 1 or 0.5 K/min, respectively. Furthermore, the samples taken at different periods of activity measurements were analysed for their molecular weight distribution using size exclusion chromatography. The multi-response evaluation was applied for all activity and DSC experiments. The simultaneous evaluation of all data allowed the identification of the mechanisms including several reactions. The parameter estimation provided the values of the coefficients of the Arrhenius equations of individual rate constants as well as the reaction enthalpies of these reactions. The statistical discrimination of these mechanisms was supported by the semiquantitative comparison of the modelling results with the results obtained by size-exclusion chromatography.

[1] Vrábel, P., Polakovic, M., Štefuca, V., Báleš, V., Enyme Microb. Technol., 20, 348-354, 1997.