Two protein bands with laccase activity were found after PAGE of culture liquid or mycelium extract of Pleurotus eryngii, grown on glucose-ammonium tartrate-yeast extract medium with and without inducers. A major and a minor laccase band were observed in the basal medium. The intensity of the major band (laccase I) did not change after the addition of inducers. However, the minor band (laccase II), characterized by higher electrophoretic mobility, was strongly induced by wheat-straw alkalilignin and vanillic and veratric acids. Laccase activity in the basal medium had an optimum pH of 4.5 and was stable from pH 3 to 10 during 24 h at room temperature. This enzyme had wide substrate specificity on hydroquinones, methoxy-substituted monophenols, and aromatic amines. In general, laccase activity was found only with compounds having a redox potential lower than 0.5 mV. The highest activity was obtained with methoxy- and methyl-substituted p-hydroquinones and aromatic diamines. Some activity also occurred with the aliphatic compound 3,5-cyclohexadiene-1,2-diol.

This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer 'ITS' regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml -1 within 15 days of incubation.

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Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [ 14 C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn 2؉ to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.