Journal of Inorganic Biochemistry 92 (2002) 75–81 www.elsevier.com / locate / jinorgbio Enzymatic degradation of 2,6-dichlorophenol by horseradish peroxidase: UV–visible and mass spectrophotometric characterization of the reaction products * Enzo Laurenti, Elena Ghibaudi, Gianpiero Todaro, Rosa Pia Ferrari ` Dipartimento di Chimica I.F.M., Universita di Torino, Via P . Giuria 7, 10125 Turin, Italy Received 27 March 2002; received in revised form 12 June 2002; accepted 17 June 2002 Abstract The reaction mechanism of the oxidation of 2,6-dichlorophenol (2,6-DCP) by horseradish peroxidase (HRP) and H O has been 2 2 investigated and the reaction products have been characterized by UV–visible and mass spectrometry. Evidence for the dimerization of 2,6-DCP to 3,39,5,59-tetrachloro-4,49-dihydroxybiphenyl and the subsequent fast oxidation of this product to the corresponding 3,39,5,59-tetrachlorodiphenoquinone have been collected. The reaction rate was found to decrease markedly as soon as the pH was raised, with a clear inflection point at pH(6.6–6.9; it also resulted independent from H O concentration. Since the pK for 2,6-DCP is 6.80, the 2 2 a reaction rate might be influenced by the protonation state of the substrate.  2002 Elsevier Science Inc. All rights reserved. Keywords: Chlorophenol; Horseradish peroxidase; Enzymatic degradation 1. Introduction easier than microorganism manipulation; besides, enzyme concentration is not affected by bacterial growth [11] and Polychlorinated phenols belong to a family of highly the specificity of isolated enzymes is higher if compared toxic [1–4] and potentially carcinogenic compounds [5,6] with that of intact organisms or even with chemical that can be found in waste waters as persistent environ- methods. Moreover, their employment in the selective mental pollutants; they result from several man-made removal of hazardous compounds makes subsequent treat- activities such as coal conversion, water disinfection, ments of waste waters easier, if required. uncontrolled use of herbicides, pesticides and fungicides, Due to their proven ability to oxidize a large number of wood pulp and paper manufacturing [7,8]. Chlorophenols substituted phenols [12–14], peroxidases are the most are widely distributed in the environment and they can be widely employed enzymes in the field. Many data con- found also in water and soils of non-industrial regions. cerning the oxidation of chlorophenols by lignin per- Several different methods have been suggested in order oxidase [7], soybean peroxidase [11], lactoperoxidase [8] to remove phenols and chlorophenols from industrial waste and some peroxidase-like models [15,16] can be found in waters: adsorption on activated carbon, solvent extraction, the literature. Although, the most employed enzyme in the microbial degradation, chemical oxidation, etc.; unfortuna- field is by far horseradish peroxidase (HRP, EC 1.11.1.7) tely, all of them suffer from shortcomings such as high due to its relatively high availability and low cost as well cost, incomplete detoxification or conversion into even as its ability to oxidize a wide range of substrates [9,17– more hazardous products [9,10]. 20]. In most recent years, a new approach to this problem has The degradation of chlorophenols by peroxidases been tried, based on enzymatic degradation. Compared to proceeds through the generation of phenoxyl radicals; conventional biological methods, this strategy looks advan- these can diffuse out from the active site of the enzyme tageous since handling and storage of isolated enzymes is and react with other substrate molecules, giving rise to oligomers or polymers [9] that are much less water-soluble than the original monomers; as a consequence, they *Corresponding author. Tel.: 139-11-670-7516; fax: 39-11-670-7855. E-mail address: rosapia.ferrari@unito.it (R. Pia Ferrari). precipitate out of the solution and can be separated by 0162-0134 / 02 / $ – see front matter  2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134(02)00488-9