Influence of Structure on Dye Degradation with Laccase Mediator Systems EVA ALMANSA a , ANDREAS KANDELBAUER a,b , LUCIANA PEREIRA a,c , ARTUR CAVACO-PAULO c and GEORG M. GUEBITZ a * a Department of Environmental Biotechnology, Graz University of Technology, Petersgasse 12, A-8010, Graz, Austria; b Department of Applied Research, Funder Industrie Gesellschaft m.b.H., Klagenfurterstrasse 87-89, A-9300 St. Veit Glan, Austria; c Department of Textile Engineering, University of Minho, 4800 Guimaraes, Portugal (Received for publication 16 June 2004; Revised manuscript accepted 29 October 2004) A new laccase was purified from Trametes hirsuta IMA2002. The laccase had a molecular mass of 62 kDa and an isoelectric point of pH 7. It had an optimum pH of 3.0 and an optimum temperature of 558C. The laccase was quite stable at 308C and pH 4.0 with a half-life of more than 100 hours. On ABTS, syringaldazide, and DMP the laccase showed K M and K cat values of 75, 12 and 37 mM and 64, 83 and 54 s 1 , respectively. The structu- rally diverse commercial dyes Indigo Carmine, Lanaset Blue 2R, Diamond Black PV 200 and Diamond Fast Brown were oxidized by the laccase. While the rate and extent of decolorization of the latter dye was signifi- cantly enhanced by the presence of different types of mediators, the structurally similar azo-dye Tartrazine was not oxidized. Lanaset Blue 2R, a commercial textile dye containing an anthrachinoid structural fragment acted similarly to anthrachinone sulfonic acid by strongly enhancing the rate of the decolorization reac- tion. Twenty two model azo-dyes based on the molecular framework of 2,7-dihydroxy-1-phenylazonaphtalene-3,6- disulfonic acid were synthesized and the kinetics of their laccase-catalyzed decolorization was studied. Hy- droxy-substituted dyes were the most susceptible to enzyme/mediator action. All reactions were well de- scribed by Michaelis  /Menten-like kinetics and the Hammett free energy linear relationship could be suc- cessfully applied to describe the influence of dye structure (substituents on the aromatic ring) on decolor- ization. Strongly electron withdrawing substituents such as a nitro-group in the meta -position ( /0.7) resulted in positive s-constants whereas electron donating groups such as para -methyl (  /0.3) resulted in negative values for s-constants. Keywords: Laccase; Mediators; Bioremediation; Textile Dye deco- lorization; Enzyme kinetics; Structure  /reactivity relationship INTRODUCTION Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) catalyze the removal of a hydrogen atom from the hydroxyl group of ortho and para -substi- tuted mono- and poly-phenolic substrates and from aromatic amines by one-electron abstraction, to form free radicals, capable of undergoing further depoly- merization, repolymerization, demethylation or qui- none formation (Thurston, 1994; Yaropolov et al ., 1994). The rather broad substrate specificity of laccases may be additionally expanded by addition of redox mediators such as ABTS, HBT, TEMPO (Fabbrini et al ., 2002), polyoxometalates (Tavares et al ., in press) and osmium-based redox polymers (Barriere et al ., 2004) or compounds secreted by lignolytic fungi (Eggert et al ., 1996; Johannes and Majcherczyk, 2000a). Laccases are involved in the biodegradation of lignins and a great number of white-rot fungi have been reported to produce the lignin degrading enzymes laccase, lignin peroxidases and manganese peroxidases, or at least, one of these enzymes. Based on their natural role in lignin depolymerization laccases can be used for bleaching of wood (Bour- bonnais and Paice, 1992; Paice et al ., 1995) and non- wood pulps (Camarero et al ., 2004). Laccases have also found several applications in bioremediation. The enzymes render phenolic com- pounds less toxic via degradation or polymerization reactions and/or cross-coupling of pollutant phenols ISSN 1024-2422 print/ISSN 1029-2446 online # 2004 Taylor & Francis Ltd DOI: 10.1080/10242420400024508 * Corresponding author. E-mail: guebitz@tugraz.at Biocatalysis and Biotransformation, 2004 VOL. 22 (5/6). pp. 315  /324