Enzyme and Microbial Technology 39 (2006) 141–148 Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme H´ ela Zouari-Mechichi a , Tahar Mechichi a , Abdelhafidh Dhouib a , Sami Sayadi a , Angel T. Mart´ ınez b , Maria Jesus Mart´ ınez b,∗ a Centre de biotechnologie de Sfax BP “K” 3038, Tunisia b Centro de Investigaciones Biol´ ogicas (CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain Received 10 April 2005; received in revised form 9 November 2005; accepted 15 November 2005 Abstract A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes trogii, was selected in a broad plate screening because of its ability to degrade commercial dyes. In liquid cultures using a glucose–peptone medium, the sole ligninolytic activity detected was laccase. The highest laccase levels were obtained in presence of CuSO 4 as inducer (around 20000 U/l). Two isoenzymes, were purified using anion-exchange and size-exclusion chromatographies. Both isoenzymes are monomeric proteins, with M w around 62 kDa and isoelectric points of 4.3 and 4.5, showing similar stability at pH and temperature, optimum pH and substrate specificity. The highest oxidation rate was obtained at pH 2 and 2.5 for ABTS and DMP, respectively. They were stable up to 50 ◦ C for 24 h and the stability was higher at alkaline pH. Activity increased by the addition of 10 mM Ni, Mo or Mn but it was not affected by Cd, Al, Li and Ca. Identical N-terminal sequences were determined in both laccases. The crude enzyme, as well as the purified laccase, was able to decolorize dyes from the textile industry. © 2006 Elsevier Inc. All rights reserved. Keywords: Fungi; Basidiomycete; Enzymes; Industrial dyes; Trametes trogii 1. Introduction White rot fungi are believed to be the most effective lignin- degrading microbes in nature. They produce different kinds of extracellular oxidoreductases, including laccases [26], peroxi- dases [10] and oxidases producing H 2 O 2 [18]. These enzymes are involved in the degradation of lignin [20], but also of other aromatic recalcitrant compounds causing environmen- tal problems. Both laccases and peroxidases can catalyze the one-electron oxidation of aromatic lignin units, resulting in var- ious non-enzymatic reactions. The peroxidases have the highest redox potential, being able to catalyze directly the oxidation of non-phenolic compounds. However, the use of oxygen (a non- limiting electron acceptor) by the laccases makes these enzymes more adequate for industrial and environmental applications. ∗ Corresponding author at: Centro de Investigaciones Biol´ ogicas, Consejo Superior de Investigaciones Cient´ ıficas, Ramiro de Maeztu 9, E-28040 Madrid, Spain. Tel.: +34 918 373 112/34 915 611 800; fax: +34 915 360 432/34 915 627 518. E-mail address: mjmartinez@cib.csic.es (M.J. Mart´ ınez). Laccase is an enzyme secreted by the most of the lignin degrading basidiomycetes [23] and it has been reported as an essential enzyme for lignin degradation in fungi without per- oxidases [15]. This enzyme catalyzes the oxidation of a wide number of phenolic compounds and aromatic amines but its substrate range have been extended to non-phenolic compounds in the presence of low molecular mass compounds acting as mediators [5,14]. Most of the studies have been carried out with laccases from eukaryotes, principally with enzymes secreted by basidiomycetes being their distribution in prokaryotes more recently reported [7]. The textile industry, by far the most avid user of synthetic dyes, is in need of ecologically efficient solutions for its colored effluents. Wastewaters from textile industries are a complex mix- ture of many polluting substances such as organochlorine-based pesticides, heavy metals, pigments and dyes. Dye effluents are poorly decolorized by conventional biological wastewater treat- ments and may be toxic for the microorganisms present in the treatment plants due to their complex aromatic structures. Fur- thermore, following anaerobic digestion, nitrogen-containing dyes are transformed into aromatic amines that are more toxic and mutagenic than the parent molecules. To overcome these 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.11.027