Enzyme and Microbial Technology 36 (2005) 478–486 Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater Atef Jaouani a , Francisco Guill´ en b , Michel J. Penninckx a , Angel T. Mart´ ınez b , Mar´ ıa Jes ´ us Mart´ ınez b,∗ a Laboratoire de Physiologie et Ecologie Microbiennes, Universit´ e Libre de Bruxelles, C/O Institut Pasteur, 642 Rue Engeland, B-1180, Bruxelles, Belgium b Centro de Investigaciones Biol´ ogicas, Department of Microbiologia Molecular, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain Received 27 April 2004; accepted 11 November 2004 Abstract In a previous work was reported the ability of Pycnoporus coccineus to decolorize olive oil mill wastewaters (OOMW) without an additional carbon source. We studied the composition of the enzymatic system involved in the process. The fungus secreted only laccase under the different culture conditions studied even in presence of compounds promoting the production of peroxidases. The highest laccase levels were attained in Cu 2+ –ethanol medium (100,000 U/l after 45 incubation-days). A single isoenzyme was purified with a yield of 79%. This laccase is a glycoprotein (8% N-linked carbohydrate) with a molecular mass and pI of 61.5kDa and 3.7, respectively. The highest oxidation rate was obtained around pH 3.5 for ABTS and DMP and the highest DMP oxidation at 60 ◦ C. The enzyme was stable at pH 7 at room temperature and showed a half-life of 8 and 2 h at 50 and 60 ◦ C, respectively. The treatment of OOMW with the laccase showed similar results to those reported with the fungus indicating that laccase plays an important role in the degradative process. The high levels of laccase secreted by P. coccineus and its stability suggest that it could be a useful tool for this and other environmental applications. © 2004 Elsevier Inc. All rights reserved. Keywords: OOMW; Fungi; Enzymes; Phenoloxidases 1. Introduction Mediterranean countries are responsible for more than 95% olive oil world production. During the process of extrac- tion of oil from olives, large amounts of dark liquid effluents called olive oil mill wastewaters (OOMW) are generated. These effluents result from the mixture of “vegetation wa- ter” coming from the olives, and water added during the pro- cess. They are characterized by low pH and high organic load including lipids, pectin, polysaccharides and polyphe- nols [34,43]. Due to the low cost and high mineral content of these residues, they could be used as organic fertilizers, although several authors have observed phytotoxic effects in plants [7]. Conventional physico-chemical processes to ∗ Corresponding author. Tel.: +34 918373112; fax: +34 915360432. E-mail address: mjmartinez@cib.csic.es (M.J. Mart´ ınez). detoxify OOMW, including simple evaporation or reverse os- mosis and ultrafiltration, are expensive and scarcely effective [13]. As an alternative to the conventional methods, microor- ganisms able to degrade the aromatic compounds present in OOMW are being investigated. Since the major aromatic compounds present in OOMW are phenolic and similar to those derived from lignin biodegradation, most of these stud- ies have been focused on degradation and decolourization of OOMW by ligninolytic fungi [1,17,26,30,44]. These fungi have developed a nonspecific oxidative system to degrade lignin and other aromatic recalcitrant compounds, including extracellular oxidoreductases, low molecular weight metabo- lites and activated oxygen species [46]. The extracellular en- zymes involved in the degradative process include different kinds of laccases [27], peroxidases [5,6,31] and oxidases pro- ducing H 2 O 2 [20,25]. However, the simultaneous presence of these three groups of enzymes is a non common fact in all 0141-0229/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2004.11.011