Acrylamide-2-hydroxyethyl methaacrylate (AAm-HEMA) copolymer was used for covalent coupling of Horseradish peroxidase (HRP). Hydroxyl and amino groups were activated using glutaraldehyde and p-benzoquinone, respectively, before coupling of the enzyme. Activation of both amino and hydroxyl groups gave a synergistic effect and the increase in activity of bound enzyme was more than two-fold. The storage stability of covalently bound enzyme was 60% after 3 weeks whereas free enzyme lost all activity in 2 weeks time indicating better stability of the immobilized system. The covalently bound enzyme lost 50% activity after eight cycles. When the substrate concentration was varied and enzyme concentration was kept constant the kinetic parameters were observed to be K-m = 8 X 10(-5) and V-max = 1.53 for free enzyme and K-m = 8.14 x 10(-5) and V-max = 2.0 for covalently bound enzyme. When the substrate concentration was kept constant and enzyme concentration was varied, K-m and V-max values were observed to be 4 x 10(-11) and 0.45 for free enzyme and 4.25 x 10(-11) and 0.55 for covalently bound enzyme indicating no conformational change during immobilization. Phenol oxidation was carried out using a fixed bed reactor of dimension 17 cm x 1 cm at: flow rate 0.5 cm(3)/min, temperature 45 degreesC and l/d ratio of 6. (C) 2004 Elsevier Ltd. All rights reserved.