Immobilization of horseradish peroxidase on cinnamic carbohydrate esters

Horseradish peroxidase (HRP) was immobilized by adsorption on totally cinnamoylated derivates of D-glucosone or D-manitol. The polymerization and cross-linking of the derivates initially obtained was achieved by irradiation in the ultraviolet region, where these prepolymers show maximum sensitivity. Immobilization of HRP on these supports involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The apparent Michaelis constants of soluble HRP acting on HO2 (51.48 muM) and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS, 92.75 muM) were calculated first to serve as reference for comparing the values obtained for the immobilized enzyme. The affinity of HRP for both enzyme-substrates was slightly lower for the immobilized enzyme than for its soluble counterpart with apparent Michaelis constants of 108.6 and 126.6 muM for H2O2 and ABTS, respectively. The immobilized enzyme was also more resistant to inactivation by hydrogen peroxide and to heat at neutral pH. The stability and durability were also described for the immobilized HRP. The results show that cinnamic carbohydrate esters represent an appropriate support for peroxidase immobilization and could be used for several peroxidase applications. (C) 2003 Elsevier Ltd. All rights reserved.