Stability of horseradish peroxidase immobilized on different cinnamic carbohydrate esters

Abstract

AbstractFour types of organic polymer were used to cover 3 mm diameter glass beads for subsequent use in immobilizing horseradish peroxidase (HRP). These supports were the totally cinnamoylated derivatives of D‐glucosone (GSOCN), D‐sorbitol (SOTCN), ethyl‐D‐glucopyranoside (EGSCN) or inuline (INCN). In some assays, a partially cinnamoylated derivative, 2,3,4,6‐tetracinnamoyl‐D‐glucopyranoside (GPSTCN) (obtained from the EGSCN derivative by hydrolysis in acidic medium) was used. Polymerization and cross‐linking of the derivatives obtained initially was carried out by irradiation in the ultraviolet region, where these polymers show the greatest degree of sensitivity. The enzyme was immobilized by adsorption to the support. Immobilized enzymatic activity varied with the incubation time used (2–21 h), depending on the chemical nature of the immobilization support. The affinity of HRP for H2O2 and 2,2′‐azinobis(3‐ethylbenzothiazolinesulfonic acid) (ABTS) was slightly lower in the case of the enzyme immobilized on the GSOCN, SOTCN and EGSCN derivatives than in the case of the soluble enzyme, as can be seen from the corresponding apparent kinetic constants ($K_{\rm m}^{\rm H_{2}O_{2}}$ and $K_{\rm m}^{\rm ABTS}$). However, the affinity of HRP immobilized on the INCN derivative for these substrates was higher than that shown by the enzyme in solution. In turn, the enzyme immobilized on all the supports was more resistant than the soluble form to inactivation by H2O2 and by heat at neutral pH. When the stability and durability of the immobilized HRP were analysed, the cinnamoylated derivatives prepared were seen to function as suitable supports for immobilizing peroxidase and to be suitable for industrial application of the enzyme. Copyright © 2004 Society of Chemical Industry