The paper proposes the method of continuous monitoring of inactivation of immobilized enzyme. The principal aim was to design a rigorous inactivation experiment eliminating any differences between the procedure of inactivation and activity measurement. The inactivation of invertase covalently immobilized on bead cellulose, that was induced by the extreme pH, was investigated by means of flow calorimetry. So called enzyme thermistor, primarily designed for determination of metabolite concentration,1 was used for a continuous thermometric measurement2 of activity of the enzyme that was packed in a small column. During the study of the inactivation kinetics, it was observed a probable enzyme relaxation or partial regeneration by substrate (sucrose). The enzyme relaxation/regeneration during the activity measurement was registered by on-line monitoring of the reaction rate. This effect can seriously impair the result of a periodic measurement of the enzyme activity when the phase of inactivation in substrate-free conditions is separated from the enzyme activity measurement. This drawback was eliminated by the method developed in this study.

[1] Danielsson, B., in R.M. Lafferty and E. Maier (Editors), Enzyme Technology, Springer-Verlag, Berlin, 1983, p. 195.

[2] Stefuca, V. and Gemeiner, P., in T. Scheper (Editor) Advances in Biochemical Engineering/Biotechnology, Vol. 64, Springer - Verlag, Berlin Heidelberg, 1999, p. 71.