ORIGINAL ARTICLE High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications E. Uzan 1 , P. Nousiainen 2 , V. Balland 3 , J. Sipila 2 , F. Piumi 1 , D. Navarro 1 , M. Asther 1 , E. Record 1 and A. Lomascolo 1,4 1 UMR 1163 Biotechnologie des Champignons Filamenteux INRA-Universite ´ s de Provence et de la Me ´ diterrane ´ e, ESIL, Case 925, Marseille Cedex, France 2 University of Helsinki, Department of Chemistry, Laboratory of Organic Chemistry, Helsinki, Finland 3 Laboratoire d’e ´ lectrochimie mole ´ culaire, Universite ´ Paris Diderot, UMR CNRS 7591, Paris Cedex, France 4 Universite ´ de Provence, UMR 1163 Biotechnologie des Champignons Filamenteux INRA, Case 925, Marseille Cedex, France Introduction Plant polymers (cellulose and lignin) are the main source of renewable materials on earth. The worldwide produc- tion of cellulose and lignin in terrestrial ecosystems is of the order of 50 and 20 billion tons per year, respectively, but lignin has mostly remained a noncommercialized residue (source: European Biorenew Project, http://www. Keywords biodiversity, laccase, nonphenolic lignin model compounds, P. sanguineus, polyphenolic dyes, Pycnoporus coccineus. Correspondence Anne Lomascolo, UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, ESIL, 163 avenue de Luminy, Case 925, 13288 Marseille Cedex 09, France. E-mail: lomascolo@esil.univmed.fr 2009 ⁄ 0940: received 28 May 2009, revised 3 November 2009 and accepted 7 November 2009 doi:10.1111/j.1365-2672.2009.04623.x Abstract Aims: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with proper- ties suitable for the lignin-processing sector. Methods and Results: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percent- age of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68Æ7–97Æ5% similarity with other Polyporale laccases. The three laccases (59Æ5–62Æ9 kDa with 7–10% carbohydrate content) had high redox potentials (0Æ72–0Æ75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75–78°C and at pH 5–7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v ⁄ v). The best laccase-1- hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. Conclusions: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo- and pH stability, catalytic efficiency towards 2,2¢-azino-bis-[3-ethylthiazoline-6-sulfonate] and resistance to alco- holic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. Significance and Impact of the Study: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale-up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor-made enzymes. Journal of Applied Microbiology ISSN 1364-5072 ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 2199–2213 2199