Studies on the thermal inactivation of immobilized enzymes

Abstract

The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for α-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, K_M and E_a values in silica-bound α-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound α-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound β-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated.