Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.

Improvement of the catalytic properties of fungal laccases is a current challenge for the efficient bioremediation of natural media polluted by xenobiotics. We developed the heterologous expression of a laccase from the white-rot fungus Trametes versicolor in the yeast Yarrowia lipolytica as a first step for enzyme evolution. The full-length cDNA consisted of a 1,561-bp open reading frame encoding lacIIIb, a 499-amino-acid protein and a 21-amino-acid signal peptide. Native and yeast secretion signals were used to direct the secretion of the enzyme, with the native signal yielding higher enzyme activity in the culture medium. The level of laccase activity secreted by the transformed yeast was similar to that observed for the non-induced wild-type strain of T. versicolor. The identity of the recombinant enzyme was checked by Western blot and matrix-assisted laser desorption/ionization time-of-flight analysis. Electrophoresis separation in native conditions indicated a molecular mass of the recombinant protein slightly higher (5 kDa) than that of the mature T. versicolor laccase IIIb, suggesting a limited excess of glycosylation. The laccase production level reached 2.5 mg/l (0.23 units/ml), which is suitable for engineering purpose.

Retention processes play a major role in the fate and impact of organic contaminants in soils. The main goal of this study was to determine the influence of soil hydrophobic properties on the retention of diuron by using plots of a long-term experiment in Versailles. We selected seven plots with pH 3.4 to 8.2 and low organic content. Sorption isotherms were obtained on soil slurries and kinetic measurements of diuron sorption were performed on undisturbed soil samples. The results showed that the Freundlich coefficient k f decreased as pH increased and that the K oc coefficient, k f /TOC, was linearly related to the contact angle measured on the clay fraction over a wide range of pH. A low initial adsorption rate and low adsorption equilibrium were observed for the plot treated with NaNO 3 . In this case, the structure in micro-aggregates was assumed to limit the accessibility of adsorption sites. The CaCO 3 and CaO treated plots had similar organic matter contents, pHs, CECs and bulk densities, although their < 2 mm:C ratio differed. The higher retention of diuron in the CaCO 3 plot is attributed to the higher hydrophobicity of the < 2 mm fraction, probably masking part of the permanent clay negative charges. Thus, in addition to the different treatments, organic matter composition and distribution should also be included as specific soil properties. We conclude that the sorption properties of pesticides such as diuron can be a good tool to obtain greater understanding of soil properties and the degree of soil hydrophobicity/hydrophilicity in particular.

The biotransformation of nonylphenol was investigated in an agricultural soil treated with a mixture of 14 C-labelled and unlabelled surfactant. It was then studied in soil samples amended with sludges spiked with the mixture of chemicals. Nonylphenol amount in all samples of soil and soil/sludge mixtures was 40 mg kg -1 . In the soil free of sludge, the half-life of nonylphenol was found to be 4 d. When the soil was amended with sludge from the city of Ambares, France, it was about 16 d. In the soil amended with sludge from Plaisir, a 8-day lag phase was observed before the transformation starts, and nonylphenol half-life exceeded 16 d. In each case, nonylphenol transformation resulted in mineralization as well as stabilization of the chemical as bound residues within the soil. Further, some strains of filamentous fungi were isolated from the soil/sludge mixtures and identified to belong to the Mucor and Fusarium species. Most of them were able to efficiently transform nonylphenol in liquid cultures. In addition, the ligninolytic basidiomycete Trametes versicolor was able to catalyze partly the conversion of nonylphenol into carbon dioxide. Laccases purified from T. versicolor cultures are enzymes involved in nonylphenol oxidative coupling leading to oligomerization.

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