Appl Microbiol Biotechnol (2005) 68: 251–258 DOI 10.1007/s00253-004-1860-7 APPLIED MICROBIAL AND CELL PHYSIOLOGY Albert Kollmann . François-Didier Boyer . Paul-Henri Ducrot . Lucien Kerhoas . Claude Jolivalt . Isabelle Touton . Jacques Einhorn . Christian Mougin Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent enhancers of laccase production in Trametes versicolor ATCC 32745 Received: 18 October 2004 / Revised: 19 November 2004 / Accepted: 19 November 2004 / Published online: 13 January 2005 # Springer-Verlag 2005 Abstract Numerous chemicals, including the xenobiotic 2,5-xylidine, are known to induce laccase production in fungi. The present study was conducted to determine whether the metabolites formed from 2,5-xylidine by fungi could enhance laccase activity. We used purified laccases to transform the chemical and then we separated the metab- olites, identified their chemical structure and assayed their effect on enzyme activity in liquid cultures of Trametes. versicolor . We identified 13 oligomers formed from 2,5- xylidine. (4E)-4-(2,5-dimethylphenylimino)-2,5-dimethyl- cyclohexa-2,5-dienone at 1.25×10 -5 M was an efficient inducer, resulting in a nine-fold increase of laccase activity after 3 days of culture. Easily synthesized in one step (67% yield), this compound could be used in fungal bioreactors to obtain a great amount of laccases for biochemical or biotechnological purposes, with a low amount of inducer. Introduction Numerous xenobiotics, including anthropogenic or natural compounds, are known to induce laccase production in filamentous fungi belonging to different classes: Ascomy- cetes (Neurospora sp.), Deuteromycetes (Botrytis sp.) and Basidiomycetes (Pycnoporus sp., Trametes sp.; Gianfreda et al. 1999). Induction is commonly used at the laboratory or industrial scale in order to obtain large amounts of enzymes for biochemical (i.e. structural; Bertrand et al. 2002), biotechnological (i.e. bioconversion and biotrans- formation; Jolivalt et al. 2000) and environmental (i.e. biomarkers for ecotoxicity assessment; Mougin et al. 2002, 2003) purposes. Several isomers of xylidines (dimethylanilines) act as laccase inducers (Soden and Dobson 2001). They have been used for more than 40 years to produce high yields of enzyme in liquid fungal cultures (Fahraeus et al. 1958; Bollag and Leonowicz 1984). In these cultures (i.e. the white-rot T. versicolor), laccase induction was concomitant with a red coloration of the culture medium. The compounds responsible for that colour have never been characterised. Indeed, oxidation of several poly-methylated anilines by peroxidase has been studied but, as far as we know, no in- formation is available for 2,5-xylidine (Baker and Saunders 1974 and references cited therein). Previous work performed in our laboratory showed that 2,5-xylidine (2,5-dimethyla- niline) was a substrate for fungal laccases (Bertrand et al. 2002) and that its enzymatic conversion led to products with a colour similar to that observed in T. versicolor cul- tures undertaken in presence of 2,5-xylidine. Apart from their use in fungal cultures, xylidines are mainly used in industry as dyes. In addition, they are toxic components of rocket fuels (Kallas et al. 2003). It is thus likely that xylidines could also be transformed in soils from military bases through bacterial and fungal activity, providing a new and interesting in situ bioremediation method. The objectives of the present paper are: (1) to isolate and identify the products formed from 2,5-xylidine by fungal laccases and (2) to assess the potential of these transfor- mation products to induce fungal laccase production and so possibly provide a new efficient laccase inducer. A. Kollmann . F.-D. Boyer . P.-H. Ducrot . L. Kerhoas . I. Touton . J. Einhorn . C. Mougin (*) Unité de Phytopharmacie et Médiateurs Chimiques, INRA, Route de Saint-Cyr, 78026 Versailles Cedex, France e-mail: mougin@versailles.inra.fr Tel.: +33-1-30833102 Fax: +33-1-30833119 C. Jolivalt ENSCP, Laboratoire de Synthèse sélective organique et Produits naturels, UMR CNRS 7573, 11 rue P. et M. Curie, 75231 Paris Cedex 05, France