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International audience; Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80°C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds; Deux isozymes de laccases ont été obtenues à partir d'une culture liquide induite de Marasmius quercophi-lus en utilisant l'acide p-hydroxybenzoïque comme inducteur. Les isozymes constitutives et induites ont toutes deux un poids moléculaire de 60 kDa mesuré en électrophorèse sur gel de polyacrylamide. En électrofocalisation, nous avons observé trois isozymes pour l'enzyme constitutive (pI 4, 4.2, 4.4) et quatre pour l'isozyme induite (pI 4.75, 4.85, 4.95, 5.1). Nous avons observé certaines différences entre ces deux isozymes; l'activité spécifique de l'isozyme induite était deux fois plus importante que celle de l'isozyme constitutive et deux pH optimum (5 et 6) ont été notés pour l'isozyme induite (seulement un, pH 5, pour l'isozyme constitutive). Les deux enzymes avaient la même stabilité ther-mique et la même température d'activité maximale (80°C). De plus, la réactivité de ces deux laccases vis-à-vis des composés aromatiques était semblable. L'utilisation de médiateurs a augmenté la gamme des substrats oxydés par les laccases étudiées. Différents produits de dégradation ont été observés selon le médiateur utilisé. Quand seule la laccase est utilisée, la diminution du signal correspondant au composé aromatique et l'absence d'autres pics à différentes lon-gueurs d'ondes, suggèrent une polymérisation des composés aromatiques.

A white-rot basidiomycete, Marasmius quercophilus , was isolated from an evergreen oak litter. This fungus, which very thoroughly bleaches the whole leaf area, produces laccases, a property which has not been studied before. Except on a ground leaf medium, we did not reveal phenoloxidases other than laccases. M. quercophilus is able to transform many aromatic compounds which are by-products of lignin and tannin metabolism. We observed, for example, that lignin monomers, namely p -coumaryl, coniferyl, and sinapyl alcohols are oxidized at different rates by the extracellular crude enzyme extract. The highest production of laccases is obtained with shaking cultures on malt extract supplemented with Tween 80 (0.1%) and CuSO 4 (0.5 mg l −1 ). These enzymes have an optimum activity at 80°C and a stable activity at 40°C for 18 h. The extracellular crude enzyme extract medium contains three isoenzymes with pI values of 3.8, 3.5, and 3.2. It is able to significantly reduce the kappa number (21.2%) of a kraft pulp without inclusion of any mediator.

The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak ( Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzyme's pH optimum was 4.5, the optimal temperature was 75°C, and the K m was 7.1 μM. The structural gene, lac1 , was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.

In this work we tried to caractérisé biological activity of the formed litter into two QueAcut ilex ecosystems. The methods used showed that respiratory and alkaline phosphatase activities are different in the two ecosystems. The other enzymatic activities (acid phosphatases , ureases and catechol-oxygenases ) are very high in all ecosystems and do not allow to distinguish between them. No nitrogenasic activity could be revealed., Le but de ce travail a été de caractériser l 'activité biologique dans la litière de Chêne vert en deux stations. Les méthodes utilisées ont mis en évidence des différences dans les activités respiratoires et phosphatase alcalins. Les autres activités enzymatiques (phosphatases acides , uréases et catéchol-oxygénases) sont très élevées et ne permettent pas une distinction entre les stations. Aucune activité nitrogénasique n'a été décelée., Chabert Alain, Le Petit J., Loisel Roger, Matheron Robert, Tagger S. Dégradation biologique de la litière de chêne vert dans deux forêts de la région provençale (Var, France). In: Ecologia mediterranea, tome 10 n°1-2, 1984. pp. 171-181.