1559 Research Article Received: 20 February 2009 Revised: 23 April 2009 Accepted: 23 April 2009 Published online in Wiley Interscience: 28 May 2009 (www.interscience.wiley.com) DOI 10.1002/jctb.2224 Fate of horseradish peroxidase during oxidation of monobrominated phenols Vered Cohen-Yaniv and Carlos G. Dosoretz * Abstract BACKGROUND: Peroxidase-catalyzed polymerization of phenols is accompanied by substantial enzyme precipitation with reaction products. The enzyme fate during the polymerization of monobromophenols by horseradish peroxidase (HRP) was studied. Enzyme fate was simultaneously monitored by protein, total nitrogen mass balance and gel electrophoresis (SDS-PAGE) analysis of both soluble and precipitate fractions. RESULTS: SDS-PAGE analysis revealed that molecular weight bands of protein in the precipitate shifted upwards toward higher molecular weights, compared with protein control. When co-polymerization was practiced higher HRP precipitation occurred compared with polymerization of a single substrate, regardless of substrate combination applied. Addition of polyethylene glycol (PEG) to the reaction mixture decreased the extent of HRP precipitation. At 2 mmol L -1 H 2 O 2 , corresponding to the stoichiometric equivalent concentration, 50% precipitation occurred after 1 h (∼70% after 24 h) compared with 97–98% (∼100% after 24 h) without PEG. Nevertheless, further increase of H 2 O 2 increased HRP precipitation regardless of PEG (85% at 4 mmol L -1 and 95% at 5 mmol L -1 ). The lowest degree of enzyme inactivation was observed for metabromophenol, which displayed the lowest transformation yield, compared to the other congeners. CONCLUSIONS: Results from SDS-PAGE indicate that an interaction stronger than hydrophobic, resisting the denaturative conditions, may take place between HRP and the reaction products, suggesting the occurrence of a covalent link between them. Oxidation was enhanced by inclusion of PEG, which partially suppressed product-dependent inactivation. The extent of enzyme inactivation depends on the substrate used, while highest inactivation occurred when co-polymerization was practiced. c  2009 Society of Chemical Industry Keywords: halogenated phenols polymerization; horseradish peroxidase; enzymatic polymerization; product inactivation INTRODUCTION Peroxidases are heme enzymes that catalyze the oxidation of a wide variety of aromatic compounds (including phenols) by utilizing H 2 O 2 as an electron acceptor. The phenols are oxidized to phenoxy radicals that couple to form polymeric and oligomeric products. 1,2 It is thought that the increase of molecular weight increases the hydrophobicity and decreases the solubility of forming products that eventually precipitate, and therefore can easily be separated from the aqueous media. Hence, when applied to water treatment, the peroxidase-catalyzed polymerization process is expected to reduce the toxicity of the water contaminated by toxic phenolic compounds. An intrinsic characteristic of peroxidase-catalyzed oxidation of phenolic compounds is enzyme inactivation during the reaction, resulting in significant activity loss and incompletion of the reaction. 3,4 This is a further substantial obstacle to the implementation of the technology. The catalytic capability of the enzyme depends on its chemical and physical environment, whereas enzyme inactivation limits the productivity of the reaction. Three pathways for horseradish peroxidase (HRP) inactivation during phenol polymerization have been proposed. The first is associated with excess peroxide at limited reducing substrate concentration, which results in enzyme susceptibly to suicide inactivation. 5–8 Two other inactivation pathways involve inactivation by-products. 9 Studies have shown that immediately after the initiation of phenol polymerization, the enzyme was inactivated by the products formed in the course of the reaction. 4,6,9,10 Although the fate of the enzyme due to product inactivation is still uncertain, both enzyme precipitation due to hydrophobic interaction with precipitating polymeric products, i.e. adsorption 9–11 and direct inactivation due to phenoxyl radical intermediates attack 3,4,7,9,11,12 are thought to be involved. Whether or not radical attack ends in covalent binding is still unknown. Qing et al. 9 reported that at reaction conditions below 7.5 mmol L −1 of both phenol and peroxide, a severe heme loss occurs, i.e. near 50% heme loss. At these conditions peroxide inactivation was negligible because HRP inactivation was less than 2%. Therefore it was suggested that heme loss was predominantly caused by a radical attack. A pseudo-steady-state kinetic model of the HRP–H 2 O 2 –aromatic compound system was reported by Buchanan et al. 11 for phenol removal in a batch reactor within a concentration range of 0.5–6 mmol L −1 . It was concluded that the predominant inactivation mechanism was due to free radical ∗ Correspondence to: Carlos G. Dosoretz, Division of Environmental, Water and Agricultural Engineering, Technion-Israel Institute of Technology, Haifa 32000, Israel. E-mail: carlosd@tx.technion.ac.il Division of Environmental, Water and Agricultural Engineering, Technion-Israel Institute of Technology, Haifa, Israel J Chem Technol Biotechnol 2009; 84: 1559–1566 www.soci.org c  2009 Society of Chemical Industry