Enzyme and Microbial Technology 40 (2007) 1732–1738 Enzymatic reduction and oxidation of fibre-bound azo-dyes S. Pricelius a , C. Held a , S. Sollner b , S. Deller b , M. Murkovic c , R. Ullrich d , M. Hofrichter d , A. Cavaco-Paulo e , P. Macheroux b , G.M. Guebitz a,∗ a Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria b Department of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria c Department of Food Chemistry and Technology, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria d Chair of Environmental Biotechnology, International Graduate School Zittau, Germany e Department of Textile Engineering, University of Minho, 4800 Guimaraes, Portugal Received 12 August 2006; received in revised form 3 November 2006; accepted 3 November 2006 Abstract A new customer and environmental friendly method of hair bound dye decolouration was developed. Biotransformation of the azo-dyes Flame Orange and Ruby Red was studied using different oxidoreductases. The pathways of azo dye conversion by these enzymes were investigated and the intermediates and metabolites were identified and characterised using UV–vis spectroscopy, high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Laccase from Pycnoporus cinnabarinus, manganese peroxidase (MnP) from Nematoloma frowardii and the novel Agrocybe aegerita peroxidase (AaP) were found to use a similar mechanism to convert azo dyes. They N-demethylated the dyes and concomitantly polymerized them to some extent. On the other hand the mechanism for cleavage of the azo bond by azo-reductases of Bacillus cereus and B. subtilis was based on reduction of the azo bond at the expense of NAD(P)H. © 2006 Elsevier Inc. All rights reserved. Keywords: Azo dye; Hair; Azoreductase; Laccase 1. Introduction Azo dyes are widely used in industry and daily life, for example, as components in permanent hair dyes. Additionally, azo-compounds represent the largest class of dyes applied in the textile, food, and cosmetic industries. Azo dyes used as components in permanent hair dyes can only be decolorized by drastical methods such as chemical bleaching using hydro- gen peroxide. However, this treatment may cause hair damage and health problems. Hence, the use of these methods requires extreme precautions in order to prevent these adverse effects. Therefore, it would be useful to develop a customer friendly and safe treatment method avoiding the use of hazardous chem- icals. Enzymes that specifically oxidize or reduce azo-bonds and, thereby, decolorize dyed hair could be an elegant alterna- tive since they can be used under mild conditions and they do not have negative effects both on hair and skin. ∗ Corresponding author. E-mail address: guebitz@tugraz.at (G.M. Guebitz). Azo dyes are quite resistant to biological degradation [1] but, nevertheless, decolorization in aerobic, anaerobic, anoxic or combined treatment processes have been reported [2–5]. Several mircroorganisms produce enzymes which reductively cleave the azo bond at the expense of a reducing agent, typically NAD(P)H. The occurrence of these so-called azo-reductases has been reported for a number of bacteria. Recently, we have shown that an azo-reductase from a thermoalkalophilic Bacillus sp. was able to reduce a large structural variety of systematically substituted azo dyes [6]. The non-specificity of the azo-reductase reaction was also demonstrated in several reports on the decoloriza- tion of azo dyes by sewage sludge under anaerobic conditions [1,7,8]. It seems that almost all azo compounds tested are bio- logically reduced under anaerobic conditions, although there are some indications that metal-ion-containing dyes sometimes have lower decolorization rates [9]. Cell extracts show usually much higher rates for anaerobic reduction of azo dyes than preparations of resting cells do [4]. This has generally been explained by the low permeability of the cell membranes for the azo-compounds [10]. Azo-dyes are also degraded efficiently under aerobic condi- tions by wood-rotting fungi (e. g. Phanerochaete chrysosporium, 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.11.004