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 alpha-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 alpha-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 alpha-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound beta-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.

Invertase immobilized on spacer-arm attached poly(hydroxyethyl methacrylate) membrane: Preparation and properties

“…3,24,25 Ulbrich et al 25 report that the activity of the immobilized preparation, especially in a covalently bound system, is more resistant than that of the soluble form against heat and denaturing agents.…”

Immobilization of Thiol Proteases onto Porous Poly(vinyl alcohol) Beads

“…As is well known, the activity of immobilized enzymes, especially in covalently bound systems, is more resistant against heat and denaturing agents than that of the soluble form. 20 The effects of temperature on the stability of the immobilized papain in PBS are shown in Table III. The immobilized papain is more stable than free ones in the range of higher temperatures.…”

How do additives affect enzyme activity and stability in nonaqueous media?

“…Immobilization of the catalyst has been shown to be advantageous to the improvement of catalytic rates and, in some cases, to the enhancement of the thermostability of the preparation (Blanco et al, 1992, Clark, 1994Ulbrich et al, 1986). Deleterious protein-protein interactions are suppressed and mass-transfer is facilitated.…”