Characterization of a new tyrosinase from Pycnoporus species with high potential for food technological applications S. Halaouli 1 , Mi. Asther 1 , K. Kruus 2 , L. Guo 3 , M. Hamdi 4 , J.-C. Sigoillot 1 , M. Asther 1 and A. Lomascolo 1 1 UMR 1163 INRA-Universite´ de Provence de Biotechnologie des Champignons Filamenteux, IFR 86 de Biotechnologie Agro- Industrielle de Marseille, Marseille Cedex 09, France, 2 VTT Biotechnology, Tietotie 2, Espoo, Finland, 3 Systematic Mycology and Lichenology Laboratory, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China, and 4 Laboratoire de Microbiologie Alimentaire et des Proce´de´s de l’Environnement, Institut National des Sciences Applique´es et de Technologie, Tunis, Tunisia 2004/0403: received 8 April 2004, revised 9 July 2004 and accepted 12 August 2004 ABSTRACT S. HALAOULI, MI. ASTHER, K. KRUUS, L. GUO, M. HAMDI, J.-C. SIGOILLOT, M. ASTHER AND A. LOMASCOLO. 2004. Aims: Tyrosinase production by Pycnoporus cinnabarinus and Pycnoporus sanguineus was screened among 20 strains originating from various geographical areas, particularly from tropical environments. The tyrosinase from the most efficient strain was purified and characterized and tested for food additive applications. Methods and Results: Monophenolase and diphenolase activities of tyrosinase were measured from cell lysate from the 20 Pycnoporus strains, for 8–10 days of cultivation. The strain P. sanguineus CBS 614.73 showed the highest productivity (45Æ4 and 163Æ6Ug )1 protein per day for monophenolase and diphenolase respectively). P. sanguineus CBS 614.73 tyrosinase was purified from concentrated cell lysate, anion-exchange, size-exclusion and hydroxyapatite chromatography, with a final yield of 2% and a purification factor of 35–38. The pure enzyme was a monomere with a molecular mass of 45 kDa and it showed four isoforms or isoenzymes with pI between 4Æ5–5. No N-glycosylation was found. The N-terminal amino acid sequence was IVTGPVGGQTEGAPAPNR. The enzyme was shown to be almost fully active in a pH range of 6–7, in a large temperature range (30–70°C), and was stable below 60°C. The main kinetic constants were determined. The tyrosinase was able to convert p-tyrosol and p-coumaric acid into hydroxytyrosol and caffeic acid, respectively, and it could also catalyse the cross-linking formation of a model protein. Conclusions: Among the genus Pycnoporus, known for the production of laccase, the strain P. sanguineus CBS 614.73 was shown to produce one other phenoloxidase, a new monomeric tyrosinase with a specific activity of 30 and 84 U mg )1 protein for monophenolase and diphenolase respectively. Significance and Impact of the Study: This study identified P. sanguineus CBS 614.73 as a potential producer of a tyrosinase which demonstrated effectiveness in the synthesis of antioxidant molecules and in protein cross-linking. Keywords: antioxidant, cross-linking, polyphenol oxidase, Pycnoporus sanguineus, tyrosinase. INTRODUCTION Tyrosinase or polyphenol oxidase (monophenol, o-diphe- nol:oxygen oxidoreductase, EC 1.14.18.1) is a copper- containing enzyme that is widely distributed throughout micro-organisms, plants and animals (Robb 1984). This enzyme catalyses the ortho-hydroxylation of monophenols (monophenolase activity) and the subsequent oxidation of o-diphenols to o-quinones (diphenolase activity), both using molecular oxygen. According to the mechanism proposed by Lerch (1981) and supported by the work of Solomon and Lowery (1993), the product released from the active site is Correspondence to: Lomascolo Anne, UMR 1163 INRA de Biotechnologie des Champignons Filamenteux, IFR 86 de Biotechnologie Agro-Industrielle de Marseille, ESIL, 163 Avenue de Luminy, Case 925, 13288 Marseille Cedex 09, France (e-mail: lomascolo@esil.univ-mrs.fr). ª 2004 The Society for Applied Microbiology Journal of Applied Microbiology 2005, 98, 332–343 doi:10.1111/j.1365-2672.2004.02481.x