Specificity of phenolic disazo dyes in relation to transformation by laccase Grac ¸a M.B. Soares a , M.T. Pessoa Amorim a , Ana Maria Oliveira-Campos b , Radim Hrdina c , Maria Costa-Ferreira d, * a Department of Textile Engineering, University of Minho, 4800 Guimara ˜es, Portugal b Center of Pure and Applied Chemistry, University of Minho, 4700 Braga, Portugal c Department of Technological Chemistry, University of Pardubice, Studentska ´ 95, Pardubice, Czech Republic d Bioengineering and Bioprocessing Unit, Department of Biotechnology, National Institute for Industrial Engineering and Technology (INETI), Est. Pac ¸o do Lumiar, Lisbon, Portugal Received 30 October 2001; accepted 19 December 2001 Abstract Four novel disazo dyes were synthesized and characterized using FTIR and NMR spectroscopy. These water-soluble dyes had a hydroxyl group in the para position of the phenolic ring in relation to the azo bond, whereas the other substituents on the phenolic ring and/or between the two azo bonds varied. The transformation of the dyes was studied using purified fungal laccase. The rank order in which the dyes were transformed was IV  III  I. Dye II was not a laccase substrate. As both dye I and II had a carboxylic substituent on the phenolic ring but the ring structures between the two azo linkages were different, we suggest that the latter also influenced laccase catalysis. Both of the redox mediators tested, violuric acid and N-hydroxybenzotriazole, enhanced the laccase reaction with violuric acid, being generally more effective. The results show that the chemical structure of both the phenolic ring as well as the ring distal to the phenolic moiety affect the reaction kinetics. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Biotransformation; Disazo; Laccase; Syntheses 1. Introduction The ability of basidiomycetous fungi to degrade or de- colorize a broad range of recalcitrant compounds such as polyaromatic hydrocarbons, polychlorinated biphenyls and certain dyes has been recognized for some time [1– 4]. Azo dyes are not readily degraded by aerobic bacterial fermen- tation. Under anaerobic conditions, azo reductases usually cleave azo dyes into the corresponding amines, many of which are potentially carcinogenic dyes [5]. In contrast, fungal phenol oxidases have much broader substrate spec- ificity [6,7]. Studies involving lignin peroxidase [8,9] and Mn peroxidase type systems [10 –12] have validated the use of these enzymes for dye decolourisation. The phenoloxidase laccase (benzenediol:oxygen oxi- doreductase, EC 1.10.3.2) has also been studied, in part because of its many potential applications (reviewed in 13, 14). Laccases are multi-Cu oxidases that catalyse the oxi- dation of a range of reducing dyes with the concomitant reduction of oxygen. The recent explosive interest in laccase is in part a consequence of the finding by Bourbonnais and Paice [15] that the substrate range of laccase can be ex- panded to include non-phenolic dyes in the presence of small molecular weight redox mediator. Within the context of azo dye biotransformation, Chivukula and Renganathan [16] reported the oxidation of phenolic monoazo dyes by a commercial partially purified laccase from P. oryzae. Pac- zczynski and co-workers [17,18] have also examined the pathways for the biodegradation of monoazo and one disazo dye in P. chrysosporium and S. chromofuscus. However, these studies did not deal with laccase and instead, a key role for Mn peroxidase and lignin peroxidase was described. In a recent study, Schliephake et al. [19] reported the deg- radation of the disazo dye Chicago Sky Blue by a purified laccase from P. cinnabarinus, although this study did not include redox meditors. The aim of the present work was to examine the reac- tivity of four novel phenolic disazo dyes toward purified laccase. This laccase was obtained from a commercial lac- case formulation as previously described [20]. Novel dyes * Corresponding author. Tel: + 351 21 716 5141; fax:+ 315 21 71636 36. E-mail address: maria.ferreira@mail.ineti.pt (M. Costa-Ferreira). www.elsevier.com/locate/enzmictec Enzyme and Microbial Technology 30 (2002) 607– 612 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S0141-0229(02)00005-4