Anne Marie Farnet Da Silva

A C Chevremont

G Gil

S Gastaldi

Elisée Ferré

A M Farnet
email: m.farnet@univ-cezanne.fr

E Ferre

Alkylphenol oxidation with a laccase

Keywords:

Introduction

Many natural and synthetic chemical compounds exhibit estrogenic activities. These are known as endocrine disrupting compounds, EDCs [START_REF] Mc Lachland | Estrogens in the Environment II[END_REF] and include bisphenol A (2,2-bis(4-hydroxy-phenol)propane), various alkylphenols or triclosan (5-chloro-2(2,4-dichloro-phenoxy)phenol). These compounds have significant ecotoxicological effects in aquatic environments contaminated with wastewaters in intensively urbanised areas [START_REF] Svenson | Removal of estrogenicity in Swedish municipal sewage treatment plant[END_REF][START_REF] Peck | Sediments ara major sinks of steroidal estrogens in two United Kingdom rivers[END_REF][START_REF] Schiliro | The endocrine disrupting activity of surface waters and of wastewater treatment plant effluents in relation to chlorination[END_REF][START_REF] Nagarnaik | Concentrations and mass loadings of hormones, alkylphenols, and alkylphenol ethoxylates in healthcare facility wastewaters[END_REF]. For instance, these molecules may affect sex ratio changes or reduce fecundity of fish [START_REF] Nagler | High incidence of a male-specific genetic marker in phenotypic female Chinook salmon from the Columbia River[END_REF][START_REF] Chowen | Temporal and spatial occurence of female Chinook salmon carrying a male-specific genetic marker in the Columbia River watershed[END_REF]. Alkylphenols, such as p-nonylphenol and p-octylphenol, are environmentally persistent molecules with estrogenic activities: they are produced via the microbial transformation of alkylphenol polyethoxylates in sewage treatment plants [START_REF] Tollefsen | Estrogenicity of alkylphenols and alkylated non-phenolics in a rainbow trout (Oncorhynchus mykiss) primary hepatocyte culture[END_REF]. Lignin-degrading enzymes i.e. phenoloxidases (laccases, Mn-peroxidases or lignin-peroxidases) have been extensively used to transform many aromatic pollutants such as PAHs (polycyclic aromatic hydrocarbons), chlorophenols or EDCs [START_REF] Tanaka | Treatment of nonylphenol with laccase in a rotating reactor[END_REF][START_REF] Saito | Detoxification of bisphenol A and nonylphenol by purified extracellular laccase from a fungus isolated from soil[END_REF][START_REF] Torres-Duarte | Halogenated pesticide transformation by a laccase-mediator system[END_REF][START_REF] Uhnakova | Biodegradation of brominated aromatics by cultures and laccase of Trametes versicolor[END_REF]. These enzymes, mainly produced by white-rot fungi, are involved in the transformation of natural aromatic compounds. Moreover, their non-specific oxidation makes the transformation of pollutants with similar aromatic structure possible. Thus these enzymes, which have a broad substrate specificity, have been extensively tested for biotechnological applications [START_REF] Ghosh | Laccase-catalyzed removal of 2, 4-dimethylphenol from synthetic wastewater: effect of polyethylene glycol and dissolved oxygen[END_REF].

Marasmius quercophilus, a white-rot fungus which was isolated from Mediterranean ecosystems such as Quercus ilex or Quercus suber litters, mainly produces laccases. Recent studies have demonstrated their potential in transforming aromatic pollutants such as PAHs or chlorophenols [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF][START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. For instance, these laccases were able to oxidise anthracene to anthaquinone and to remove chloride from chlorophenols. Thus the laccases produced by this fungus seem to exhibit an oxidative potential which can be used towards various aromatic pollutants. This potential can be of major importance in litters colonised by this fungus since it can be involved in detoxification of natural environments. Furthermore these enzymes can easily be used in biotechnological applications since they can be produced in high amounts, no cofactors are needed for the reaction they catalyse and they are very stable at quite high temperatures (80% of remaining activity after 48 h at 40 °C, [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF].

Here, we examine the potential modification of various alkylphenols using the laccase of M. quercophilus in liquid cultures or using the purified laccase with or without a common laccase mediator, ABTS. We partially describe the mechanisms of oxidation of laccase with or without ABTS via the products of p-toctylphenol oxidation which were determined using mass spectrometry.

Materials and methods

Isolation of the strain

M. quercophilus strain 19 was isolated from an area of the site of La Gardiole de Rians, Var, France (10 000 m 2 ) using the rhizomorphic form of the fungus. First, a fungal cap culture was made on a malt-agar medium, 20 g L À1 (Bio Mérieux, Marcy l'Etoile, France) and chloramphenicol, 50 mg L À1 (Sigma). Second, the pure mycelial culture obtained was used to inoculate an agar medium (whole wheat flour, 20 g L À1 ) which favours rhizomorph production. Then one rhizomorph was used to inoculate a malt-agar plate to obtain a pure dicaryotic culture.

Partial purification of the laccase of M. quercophilus

The partial purification was performed as described by [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF]. Pre-cultures were realised in 200 mL Erlenmeyer flasks containing 50 mL of malt extract liquid medium, 20 g L À1 (MEL) in static conditions at 28 °C. They were inoculated with a plug of agar cultures (15 g L À1 ) with malt extract (20 g L À1 ). These liquid pre-cultures were used to inoculate two 3000 mL Erlenmeyer flasks with 800 mL of malt extract medium (20 g L À1 ), Tween 80, 0.05%, CuSO 4 , 5 mg L À1 . They were incubated at 25 °C for 12 d under axial shaking. Enzyme activity was measured by monitoring the oxidation of syringaldazine [N,N 0 -bis- (3,5-dimethoxy-4-hydroxybenzylidene)hydrazine] to its quinone (e M : 6.5 Â 10 4 M À1 cm À1 ) at 525 nm [START_REF] Harkin | Use of syringaldazine for detection of laccase in sporophores of wood rotting fungi[END_REF] on a spectrophotometer Kontron Uvikon 860 (Serlabo Technologies, Entraigues, France). The assay contained 100 lL of the culture medium, 2.5 mL of acetate buffer 0.1 M, pH 4.5 and 10 lL of syringaldazine 0.6% (w/v), diluted in methanol. The blank consisted of 100 lL of the culture medium and 2.5 mL of the same acetate buffer. Peroxydase activities were also measured using H 2 O 2 and MnSO 4 . When laccase activity was detected, cultures were filtered on a glass microfibre filter GF/D, 2.7 lm (Whatman, England). Then, the filtered culture medium was concentrated in dialysis tubes rated at 12 kDa (32 mm diameter, Poly Labo) using polyethyleneglycol. The samples from the different tubes were pooled using acetate buffer 10 mM, pH 4.5 in a final volume of 15 mL. The concentrated filtered medium was loaded on an ion-exchange Mono Q column in fast performance liquid chromatography (FPLC) equilibrated with phosphate buffer 0.1 M pH 6.0. Laccase was eluted with a step gradient (0.2 M, 0.4 M, 2 M NaCl) for 30 min at a flow rate of 1 mL min -1 [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF]. Laccase activity was measured as described above to detect fractions containing laccase. Laccase purity was checked on a polyacrylamide gel electrophoresis (PAGE) carried out according to [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 4% stacking gel and 7.5% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Bio-rad) and protein was stained using the Coomassie Blue standard method.

Cultures of M. quercophilus with alkylphenols

Cultures, previously inoculated with 10 mL of preculture of strain 19, were performed for 8 or 15 d on a rotary shaker (200 rpm) at 25 °C in 1 L-Erlenmeyer flasks with 200 mL of MEL including 50 mg of p-octylphenol, 4-(1,1,3,3-tetramethylbutyl)phenol, (p-t-octylphenol) or p-nonylphenol. From day four, laccase activities were measured every day with syringaldazine as the substrate as described above. Cultures with 800 mL of MEL and 200 mg of alkylphenols were also performed. Different control cultures without mycelium were performed: control cultures after autoclaving and control cultures incubated in the same conditions and for both incubation periods assess alkylphenol evaporation during sterilisation or incubation time, respectively. Each experiment was performed in three replicates. Induction experiments were also performed using pre-cultures with 200 mL of MEL with p-octylphenol (50 mg L À1 ). These pre-cultures were incubated for 8 d at 25 °C and then were used to inoculate cultures with 200 mL of MEL with p-octylphenol (250 mg L À1 ).

For all these experiments, cultures were filtered and both the mycelium and the supernatant were extracted three times with 200 mL of dichloromethane. The solvent was then evaporated and the solid extract solubilised in 1 mL of ethanol. Thirty microliter of dilution 10 À3 were injected in the HPLC system. Alkylphenol transformation was calculated as percentages using calibration curve described previously.

Sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE)

For all the cultures with alkylphenols, SDS-PAGE were performed in order to check whether any laccase isoforms were induced. Electrophoresis was carried out according to [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 4% stacking gel and 12% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Bio-Rad). For laccase activity staining, p-phenylenediamine (0.1%) was used as the substrate in acetate buffer 0.1 M, pH 4.

Statistical analysis

The non-parametric U-test of Mann-Whitney was used to separate significantly different means (P < 0.05) of remaining alkylphenols under the different experimental conditions. These analyses were performed using SPSS 11.0 statistical software (SPSS Inc., Chicago, IL).

Analysis of alkylphenol transformation with high performance liquid chromatography (HPLC)

HPLC system was equipped with a C18 Reverse Phase column (Merck, 4.6 Â 250 mm) in the following gradient system: solvent A, (water/trifluoro acetic acid 0.1%)/acetonitrile 90/10 v/v, solvent B (water/trifluoro acetic acid 0.1%)/acetonitrile 95/5 v/v, gradient = 5-15 min, A 100% to B 100%; 15-25 min B 100% (flow rate 1 mL min À1 ). The alkylphenols tested were transformed realised using the laccase extract. The reaction mixture (5 mL) contained ABTS 1 mM, alkylphenol 250 mg L À1 and 5 U of laccase in acetate buffer 0.1 M pH 4.5 and 10% ethanol. The reaction mixtures were incubated at 30 °C for 6 h in the dark. An extraction with 5 mL of dichloromethane was performed for each reaction mixture prior to injection. The organic phase was evaporated and the products of extraction were dissolved in 1 mL of acetonitrile (30 lL injected). The alkylphenols used were: p-octylphenol, p-t-octylphenol and p-nonylphenol. All alkylphenols were purchased from Sigma. The same reactions were performed without mediators. Controls were also realised using only ABTS with laccase and using ABTS with the alkylphenol but without laccase. All these controls were incubated at 30 °C for 6 h. p-Hydroxybenzoic acid was used as reference in each injection. A calibration curve was also performed for alkylphenol concentrations ranging from 50 to 400 lM. Results were expressed as percentages of alkylphenol disappearance.

MS analysis

Mass spectrometry was performed with a Spectrophotometer QStar Elite (Applied Biosystems SCIEX) equipped with an electrospray atmospheric pressure ionisation (ESI) source in the positive ion monitoring mode. The ESI conditions were set as follows: electrospray voltage (ESV), 5500 V, orifice voltage (OR), 70 V, gas nebulisation pressure, 20 psi (138 kPa), collision energy (CE), 15 eV.

Results and discussion

M. quercophilus is a white-rot basidiomycete whose laccases have been involved in the transformation of various organic pollutants, mainly chlorophenols and PAH such as anthracene [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid reactivity towards natural and xenobiotic aromatic compounds[END_REF][START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. Here, we investigate the potential of these enzymes to transform different alkylphenols using both liquid cultures and a partially purified isoform of laccase.

Different culture conditions were used to test alkylphenol transformation in liquid cultures with M. quercophilus. Both liquid medium and mycelium were extracted to check whether any adsorption of alkylphenols occurred on the hyphae. Liquid cultures of 200 mL containing alkylphenols at 200 mg L À1 were performed. Under these conditions, the percentages of transformation were particularly high after 15-d incubation with p-t-octylphenol and p-nonylphenol (81 ± 5% and 91 ± 6% respectively), while the weaker percentage was obtained with p-octylphenol (37.0 ± 3.5%). The transformation of these molecules was also tested in cultures of 800 mL with 200 mg L À1 of alkylphenols. After a 21-d incubation, the percentages of disappearance of p-octylphenol, p-nonylphenol and p-t-octylphenol were 100%, 92% and 90%, respectively (data not shown). Thus, the partial solubility of alkylphenols in water did not affect their transformation by the M. quercophilus laccase. No solvents were added in the culture medium since their toxicity towards the fungus may have altered its growth. It is also noteworthy that the only phenoloxidase activity detectable in the culture medium was that of laccase, which shows that neither Mn-peroxidase nor lignin-peroxidase were responsible for this transformation.

In order to enhance p-octylphenol transformation, laccase induction via pre-cultures with p-octylphenol (50 mg L À1 ) were performed. These pre-cultures were then used to inoculate the cultures to test p-octylphenol transformation (250 mg L À1 ). This time, the percentages of disappearance were 91.0 ± 0.5% and 97 ± 1% after a 8-d and a 15-d incubation, respectively (Table 1). Thus inducing pre-cultures significantly enhances the percentage of p-octylphenol transformation. Laccase induction has been reported under different conditions such as antagonisms between microorganisms [START_REF] Velazquez-Cedeno | Interspecific interactions with Trichoderma longibrachiatum induce Pleurotus ostreatus defence reactions based on the production of laccase isozymes[END_REF] or when cultures are complemented with aromatic compounds [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white-rot fungus Marasmius quercophilus[END_REF]. Induction can either lead to the production of new isoforms of enzymes or enhance the production of the constitutive isoform with an increase in laccase activity as a result. Here, laccase was produced for a longer time in cultures inoculated with pre-cultures induced with p-octylphenol (Fig. 1a). Laccase activity indeed increased after 11-and 14-d incubations in induced cultures while very low activity was observed after an 11-d incubation. SDS-PAGE did not reveal new laccase isoforms when p-octylphenol was added to the culture medium: only one isoform was produced (R f = 0.47) whatever the experimental conditions (Fig. 1b). However, on lane b, with induced pre-cultures, the laccase band is strongly stained, showing higher activity under these culture conditions and for the same quantity of proteins in the samples loaded into the gel. Thus, this result suggests that, in our study, the production of the constitutive laccase isoform was enhanced, leading to higher p-octylphenol transformation yields. [START_REF] Tanaka | Treatment of nonylphenol with laccase in a rotating reactor[END_REF] have found that laccase from Trametes sp. and Pycnoporus coccineus decrease the amount of p-nonylphenol, p-octylphenol and bisphenol A adsorbed on sea sand and the products of oxidation of p-nonylphenol are polymers. [START_REF] Sakuyama | Oxidative degradation of alkylphenols by horseradish peroxidase[END_REF] have also observed polymers from the oxidation of alkylphenols with Horseraddish peroxidise.

Here, whatever the experimental conditions used, no products of transformation were observed in the extract from cultures. In order to identify the oxidation products, we also tested the transformation of these compounds using the purified isoform of laccase with and without mediators. In these experiments, acetone was used as a solvent for alkylphenols since a previous study has shown that a concentration of 10% of acetone alters weakly M. quercophilus laccase activity [START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. After laccase purification from M. quercophilus cultures on MEL one band on SDS-PAGE (data not shown) was obtained with a molecular weight of 60 KDa as described previously [START_REF] Farnet | Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. Without ABTS, alkylphenol oxidation by laccase was weak: the percentages of transformation were 37 ± 2% and 20.0 ± 1.5% for both p-t-octylphenol and p-nonylphenol and for p-octylphenol respectively. When ABTS was added to the reaction mixture, oxidation yields were 92 ± 1%, 67 ± 2% and 47.0 ± 1.5% for p-t-octylphenol, p-nonylphenol and p-octylphenol respectively. Thus, the use of mediators leading to chemical oxidation strongly increases the yields of the reaction. [START_REF] Cabana | Preparation and characterization of crosslinked laccase aggregates and their application to the elimination of endocrine disrupting chemicals[END_REF] tested the oxidation of p-nonylphenol and bisphenol A with laccases from Coriolopsis polyzona. They identified oligomers as products of oxidation with both the endocrine disrupting chemicals tested. Here, MS analysis of p-octylphenol products of oxidation (Fig. 3a) indeed show a compound of m/z 411 which can be assigned to dimers of p-octylphenol (C 28 H 43 O 2 ). This compound is indeed ionised in two fragments with m/z 299 and 187: the loss of two fragments of m/z 112 (411 À 112 = 299 and 299 À 112 = 187) may be explained by the loss of ethylene and 1-hexene from the octyl chain from p-octylphenol. This mechanism can be extrapolated for both dimers of p-octylphenol (C-C and C-O dimers). This result can be of major importance since steric hindrance of the dimers obtained may limit the biological effects of these molecules. [START_REF] Cabana | Preparation and characterization of crosslinked laccase aggregates and their application to the elimination of endocrine disrupting chemicals[END_REF] have demonstrated that the estrogenic effect of nonylphenol and bisphenol A dramatically decreases after oxidation with laccases. [START_REF] Tamagawa | Removal of estrogenic activity of 4-tert-octylphenol by ligninolytic enzymes from white rot fungi[END_REF] have also observed this result with the oxidised products of p-t-octylphenol after laccase and Mn-peroxydase oxidation. Thus, though laccase oxidation does not lead to the degradation of such compounds, estrogenic effects of alkylphenols on organisms may decrease because of their structure modification via polymerisation. We also tested alkylphenol oxidation using ABTS as a mediator of laccase activity. This mediator was chosen since previous studies [START_REF] Pickard | Polycyclic aromatic hydrocarbon metabolism by white-rot fungi and oxidation by Coriolopsis gallica UAMH 8260 laccase[END_REF][START_REF] Baiocco | Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems[END_REF][START_REF] Saito | Detoxification of bisphenol A and nonylphenol by purified extracellular laccase from a fungus isolated from soil[END_REF] have shown that ABTS is more efficient than HBT (hydroxybenzotriazole) most probably because of the high redox potential of this molecule. Whatever the alkylphenol tested, the reaction mixture became purple and the chromatogram from HPLC revealed a compound absorbing at 550 nm (Fig. 2). HPLC-MS-MS was used to identify this compound which has a mass of 476 m/z (C 23 H 30 O 4 N 3 S 2 ). The mass spectrum of this fragment contained only one fragment of 244 m/z. Äkerström et al. (2007) have studied the oxidation of tyrosin with ABTS Á+ and found a similar purple compound (451 m/z). They show that the mass spectrum of ABTS Á+ contains three fragments of 244, 259 and 272 m/z and suggest that the purple compound combines both the fragment from ABTS Á+ (244 m/z) and tyrosine. This result suggests that, in our study, ABTS Á+ reacts with alkylphenols and that this reaction leads to a purple product. The structure of the fragment of 244 m/z described by Äkerström et al. (2007) is presented in Fig. 3b and indicates the presence of a p-conjugated system which explains the purple colour of the compound. Fig. 3b also suggests the probable structure of the purple compound (476 m/z) produced from the reaction between ABTS Á+ and p-octylphenol. Thus, this study shows that ABTS Á+ interacts with the substrate which has to be oxidised and this reaction produce another molecule. This result should be taken into account when ABTS is used as a mediator since it leads to byproducts and affects oxidation yields.

Previous studies have demonstrated that ABTS cannot really be considered as a relevant substrate for laccase activities since the oxidised form, ABTS Á+ , is highly reactive and used as a mediator of laccase oxidation [START_REF] Terrón | Tannic acid interferes with the commonly used laccase-detection assay based on ABTS as the substrate[END_REF][START_REF] Farnet | Polycyclic aromatic hydrocarbon transformation with laccases of a white-rot fungus isolated from a Mediterranean schlerophyllous litter[END_REF]. Thus, abiotic reactions may occur during enzyme measurement, leading to a misinterpretation of the results. The present study confirms that, in complex environments such as soils or litters where many a b 1 2 molecules can interact with the enzyme substrate or the product of oxidation, laccase activities and those of other phenoloxidases should not be measured with ABTS.

Fig. 1 .Fig. 2 .

 12 Fig. 1. (a) Laccase activity in cultures of Marasmius quercophilus with p-octylphenol (200 mg L À1 ). Cultures were inoculated: (j) with standard pre-cultures or (h) with precultures induced with p-octylphenol (50 mg L À1 ). (b) SDS-PAGE patterns of laccase isoform from cultures of Marasmius quercophilus on malt extract without (1) and with (2) p-octylphenol. The arrow indicates the laccase isoform stained with p-phenylenediamine.

Table 1

 1 Percentages of transformation of alkylphenols in cultures of Marasmius quercophilus after different incubation times.

		% of Transformation
	Incubation time (d) p-Octylphenol	p-t-	p-
			Octylphenol	Nonylphenol
	8	15.0 ± 1.5 (57 ± 1 a ) 69.0 ± 1.5	62 ± 4
	15	37.0 ± 3.5 (97 ± 1 a ) 81 ± 5	91 ± 6
	21	65 ± 0.8	90 ± 2	92 ± 1

a % of transformation of p-octylphenol obtained with cultures inoculated with induced pre-cultures.

Acknowledgments

We would like to thank Mrs. Marjorie Sweetko for her very helpful assistance in English language. This work was financially supported by Naturatech (France).


Anne Marie Farnet Da Silva

G Gil

Elisée Ferré

A M Farnet
email: m.farnet@univ.cezanne.fr

E Ferre

M R C N R S 6116

M R C N R S 6180

Effects of pollutants on laccase

Keywords: Halide, Laccase, Metal ions, Organic solvent

come

Introduction

Laccases are blue copper phenoloxidases which are mainly produced by white-rot fungi but which are also expressed in bacteria or plants. In natural ecosystems these enzymes oxidize phenolic compounds from lignin while reducing oxygen to water. They raised great interest because of the reaction they catalyse: their action is nonspecific and thus they can oxidize various substrates. Thus these enzymes have been extensively used in biotechnologies for the transformation of aromatic compounds such as dyes, aromatic pollutants or in wastewater treatments [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid : reactivity towards natural and xenobiotic aromatic compounds[END_REF][START_REF] Rodrı ´guez | Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase[END_REF][START_REF] Jaouani | Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater[END_REF][START_REF] Rodrı ´guez Couto | Influence of redox mediators and metal ions on synthetic acid dye decolourization by crude laccase from Trametes hirsuta[END_REF].

Our studies have focused on laccases from the white-rot fungus Marasmius quercophilus, which is a basidiomycete colonizing Mediterranean ecosystems such as evergreenor cork-oak litters. We have shown that this fungus is involved in carbon recycling [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid : reactivity towards natural and xenobiotic aromatic compounds[END_REF] in these particular ecosystems submitted to great environmental pressures such as drought and edaphic conditions (little organic matter available, schlerophyllous litters). In these specific conditions, M. quercophilus plays an important role in organic matter recycling since it produces laccases which are involved in phenol degradation which is a major limiting factor in carbon recycling because of the structure of these molecules. Thus, studying the effect of environmental pollutants on the activity of such enzymes is of great 0045-6535/$ -see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016All rights reserved. doi:10. /j.chemosphere.2007.06.086 .06.086 importance in order to assess the resilience potential of Mediterranean ecosystems, already weakened by natural environmental conditions. Here we describe the effect of metal ions, which are common soil pollutants with strong toxic potentials. We wanted to investigate whether such ions may inhibit laccase activity and, thus, have an effect on carbon recycling in forest litter. We have also tested organic solvent and halide salt effects on laccase reaction. The effect of solvents on laccase activity may be of great interest since most of aromatic pollutants can only be dissolved in organic solvents. Thus laccase involvement in xenobiotic transformation has to be monitored using cosolvents and sometime using biphasic reaction mixtures [START_REF] Bogdanovskaya | Peculiarities of direct bioelectrocatalysis by laccase in aqueous-nonaqueaous mixtures[END_REF]. Many studies have depicted laccase activity in various experimental conditions, but few have already gathered data about the actions of these various compounds. Thus, in this study we want to explain more precisely the inhibition observed since a weaker activity may have various causes depending on experimental conditions.

Materials and methods

Isolation of the strain

Strain 19 was isolated from a part of the site of La Gardiole de Rians, Var, France (1 ha) using the rhizomorphic form of the fungus [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white-rot fungus Marasmius quercophilus[END_REF]. A fungal cap culture was firstly grown on a malt extract agar medium (MEA), 20 g l À1 (Bio Me ´rieux, France) and chloramphenicol, 50 mg l À1 (Sigma). Secondly, the pure mycelial culture obtained was used to inoculate an agar medium (whole wheat flour, 20 g l À1 ) which favours rhizomorph production. Then one rhizomorph was used to inoculate a MEA plate to obtain a pure dicaryotic culture.

Culture conditions and partial purification of laccase

Pre-cultures were performed in 200 ml Erlenmeyer flasks with 50 ml of MEA (Bio Me ´rieux, Marcy l'Etoile, France) 20 g l À1 and CuSO 4 , 5 g l À1 . They were inoculated with a plug (1 cm diameter) of a fungal culture grown on MEA (20 g l À1 ) and incubated at 25 °C for 4 days. These pre-cultures were used to inoculate two 3 l-Erlenmeyer-flasks with 400 ml of MEA 20 g l À1 and CuSO 4 , 5 g l À1 . Cultures were incubated at 25 °C for 5 days. Enzyme activity was measured by following the oxidation of syringaldazine [N, N 0 -bis-(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine] to quinone (e M = 65 000 M À1 cm À1 ) at 525 nm in acetate buffer 0.1 M, pH 4.0 on a spectrophotometer Kontron Uvikon 860. One unit (U) of laccase activity is defined as the amount of enzyme that oxidises 1 lmole of the substrate per minute. The reaction mixture contained 500 ll of the medium, 10 ll of syringaldazine (0.6% in methanol) and 2.5 ml of acetate buffer. Cultures were filtered on a glass microfibre filter GF/D, 2.7 lm (Whatman, England). The filtered medium was concentrated using dialysis tubes (Cellu Sep, VWR, France) rated at 10 MW (10 kD) cutoff. A total volume of 100 ml was obtained. The concentrated filtered medium was loaded on an ion-exchange Mono Q in fast performance liquid chromatography (FPLC) equilibrated with phosphate buffer 0.1 M pH 6.0 [START_REF] De ´deyan | Biochemical and molecular characterization of a laccase from Marasmius quercophilus[END_REF]. Laccase was eluted with a step gradient (0.2 M, 0.4 M, 2 M NaCl) for 30 min at a flow rate of 1 ml min À1 . Laccase purity was checked on a PAGE (polyacrylamide gel electrophoresis) carried out according to [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] using 4% stacking gel and 7.5% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Biorad) and protein was stained using the Coomassie Blue standard method.

Effects of organic solvents on laccase activity. Laccase activity was measured in acetate buffer, 0.1 M, pH 4, with syringaldazine or ABTS as substrates at a final concentration of 4.5 mM with 10 ll of the purified enzyme in a final volume of 1 ml. Organic solvents (methanol, ethanol, acetone, acetonitrile and dioxan) were used at final concentrations of 5%, 10%, 15%, 20%, 30%, 50%, 70% and 80% in the reaction mixture. The reaction was followed for two minutes, immediately after adding the solvent into the reaction mixture. A control test was performed without solvent and using syringaldazine or ABTS as substrates as described above. The amount of laccase activity measured was calculated as a percentage of the activity in the control test. Each experiment was repeated twice. Results are expressed as percentage averages with standard deviations for both experiments.

Effect of metal ions and halides on laccase activity. Laccase activity was measured as described above with metal ions solubilised in acetate buffer 0.1 M, pH 4, at different concentrations (10 lM, 50 lM, 0.25 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10 mM, 15 mM and 20 mM) depending on the metal ion tested [START_REF] Robles | Characterisation of laccase activity produced by the hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH32[END_REF][START_REF] Lorenzo | Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decoulorise dyes[END_REF]. Laccase activity was measured immediately after the addition of the metal ion tested and a control without metal ion was also performed. The amount of laccase activity measured was calculated as a percentage of the activity in the control test. The ions tested were: Cu 2+ as CuCl 2 , CuSO 4 , Mn 2+ as MnCl 2 , MnSO4, Hg 2+ as HgCl 2 , Ca 2+ as CaCl 2 and Na 2+ as NaCl. To test the abiotic effect of Fe 2+ on the quinone of syringaldazine, a control test was realized by adding 30 ll of a solution of Fe SO 4 (20 mM final concentration) after a 2 min reaction between laccase and syringaldazine and a spectral scan from 200 nm to 700 nm was performed. To test the abiotic oxidation of Fe 3+ on syringaldazine, Fe (NO 3 ) 3 and Fe 2 (SO 4 ) 3 at 20 mM were added to syringaldazine, 4.5 mM without laccase and a spectral scan from 200 nm to 700 nm was realized.

The effect of halide ions on laccase activity was measured as described above using the following concentrations: 5 mM, 10 mM, 15 mM, 20 mM and 25 mM. A control test without halide ions was also performed. Furthermore, I À oxidation was measured under the same experimental conditions but with NaI concentrations of 100 mM, 200 mM and 400 mM. Each experiment was repeated twice. Results are expressed as percentage average with standard deviations for both experiments.

Results and discussion

Ion exchange chromatography allowed us to purify the constitutive isoform of laccase from strain 19 (Fig. 1) produced under these culture conditions: malt extract, 20 g l À1 , CuSO 4 , 5 mg l À1 [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white-rot fungus Marasmius quercophilus[END_REF]. We obtained a purification factor of 324 (calculated using specific activities) and a total yield of 81% (using total activities). Thus, this one step purification allowed us to reach a satisfactory yield of purification in order to assess the effects of the pollutants selected, on this isoform activity. As described previously, the M. quercophilus strains isolated from the site of La Gardiole have similar electrophoretic patterns of laccase isoenzymes [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white-rot fungus Marasmius quercophilus[END_REF]. Thus, this study sustains our previous observations: the constitutive isoform found here has a similar molecular weight of 60 kDa (Fig. 1) than that observed with strain 17 [START_REF] Farnet | Purification, partial characterization and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17[END_REF] which was collected at the same time in La Gardiole de Rians (Var, France). Intraspecific isoenzyme homogeneity has been observed for many fungal species such as Agaricus bisporus [START_REF] Kerrigan | Extracellular laccases: biochemical markers for Agaricus systematics[END_REF].

In this study, we tested the effect of various solvents on this laccase isoform activity. The inhibiting concentration where 50% of laccase activity remains (IC 50) was 75% with ethanol, 65% with acetone, 60% with methanol, 55% with acetonitrile, 45% with dioxane when syringaldazine was used as a substrate. IC 50 was 60% with ethanol, 50% with acetonitrile, 40% with methanol and acetone and 30% with dioxane when ABTS was used as a substrate. When laccase activity was measured with syringaldazine as a substrate, an increase in activity with ethanol, methanol, acetonitrile and acetone was observed in a range from 0 to 40% of solvent (Fig. 2). When solvent were added at 50% and more, laccase activity decreased except for ethanol (the decrease started at 60% of ethanol). With dioxan, laccase activity increased when 5% of solvent were used, was stable in a range from 10% to 30% and decreased when higher concen-trations of this solvent were used. Thus, ethanol was found to be the weakest inhibiting solvent (50% of laccase activity remained with 70% of ethanol) while dioxan exhibited the strongest effect. When laccase activity was measured with ABTS, a greater inhibition was observed since the decrease in enzyme activity started at 25% of solvent concentration whatever the solvent used (Fig. 2). For this concentration, with syringaldazine as a substrate, the percentage of activity ranged from 87 to 220% and with ABTS, from 65 to 95%. [START_REF] Robles | Characterisation of laccase activity produced by the hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH32[END_REF] have shown a greater decrease in laccase activity with methanol, ethanol, acetonitrile and acetone at 25%, respectively 63.6%, 73%, 16% and 29.5% of remaining activity as well as in the study of [START_REF] Cambria | Production, purification, and properties of an extracellular laccase from Rigidoporus lignosus[END_REF] who have found an inhibition of 50% of laccase activity when methanol was used at 50% (in our study, with 50% of methanol, about 100% of laccase activity remains when syringaldazine was used as the substrate). This result may be explained by the fact that these laccases are produced by other fungal species and thus, M. quercophilus laccase seems to exhibit a greater stability when used with solvents. This result suggests the particular resistance of this laccase towards organic solvents. Furthermore, certain organic solvents improve the solubilisation of substrates such as syringaldazine which are commonly solubilized in methanol. In the case of ABTS, which is solubilized in water, laccase activity did not increase (laccase activity was not higher than that of the control test) and the effect of the solvent used on the enzyme itself may be observed. Thus, when the substrate used is more apolar, the effect of solvent on substrate solubilization is greater than that on the enzyme at least for solvent concentration below 30%. This result can be correlated with the fact that the quinone of syringaldazine produced by laccase was also more stable when solvents were used. Indeed, in the control test without solvent, the absorbance at 525 nm quickly decreased after a 2 min -reaction suggesting that the quinone produced may have polymerized (Fig. 3). When the reaction was performed with 20% of methanol, the absorbance at 525 nm did not decrease through time but remained constant (absorbance at 525 nm was monitored for 10 min). Polymerization seems to be stopped since the quinone is solubilized in methanol. The instability of laccase oxidation-products in aqueous medium have been shown in previous studies [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid : reactivity towards natural and xenobiotic aromatic compounds[END_REF][START_REF] Mustafa | Phenolic colorants obtained by enzymatic synthesis using a fungal laccase in a hydro-organic biphasic system[END_REF]. Thus, the use of hydro-organic mixtures in enzymatic reactions involving apolar molecules is of great interest since the solubilization of substrate is improved and the product of the reaction can be found in the organic phase. In further studies, apolar solvents should be used in a biphasic reaction mixture in order to check whether this methodology allows a final-product extraction in the organic phase while the reaction occurs.

We have also shown that dioxan and acetone, which are the most apolar solvents used in this study, were the strongest inhibiting solvents. With acetone, the inhibition was easily observable with ABTS (solubilized in water) while with syringaldazine, acetone improved quinone solubilization which affected enzyme inhibition observation. This result is in agreement with the work of [START_REF] Rodakiewicz-Nowak | Effect of various water-miscible solvents on enzymatic activity of fungal laccases[END_REF], who have described the effects of acetonitrile, acetone and dimethyl sulfoxide (DMSO) on laccase activity and found that hydrophobicity was the major factor involved in the enzyme inhibition because of enzyme denaturation. They have also found that laccase inhibition with organic solvents strongly depends on the substrate used to detect laccase activity since the inhibition can be competitive or mixed. In the case of a competitive inhibition, the effects observed may vary depending on the nature of the substrate and, thus, on the degree of competition with the organic solvent. Another mechanism which should be taken into account is that laccases can aggregate in solution and be present as a non-active resting form depending on various parameters such as protein concentrations, temperature and oxygenation [START_REF] Schleev | Autoreduction and aggregation of fungal laccase in solution phase: possible correlation with a resting form of laccase[END_REF]. Thus the concentration of native fully-active laccase is not similar to the total concentration of the enzyme. Solvents may also have an effect on enzyme aggregation and therefore may influence the native fully-active enzyme concentration in the solution. Thus, in order to assess laccase inhibition with organic solvents, the combination of different mechanisms must be taken into account: the inhibition observed may vary depending on the substrate used, the oxidation product solubility in solvent may lead to an apparent absence of inhibition, protein may be denaturated with different solvent concentrations depending on the enzyme studied. Pre-incubation of laccase in solvents should be performed to assess the effect of enzyme denaturation on its activity.

With metal ions, we have shown variable responses of laccase activity depending on the ions and the substrate used. IC 50 was of 13 mM with HgCl 2 , of 1 mM with MnCl 2 , CaCl 2 , NaCl and of 0.5 mM with CuCl 2 when syringaldazine was used as a substrate and of 25 mM with MnCl 2 , CuCl 2 , NaCl and of 17 mM CaCl 2 when ABTS was used as a substrate. With syringaldazine, Fe(NO 3 ) 3 and Fe 2 (SO 4 ) 3 induced an increase in quinone production while FeSO 4 led to a quick decrease in absorption. The control test with syringaldazine and Fe(NO 3 ) 3 or Fe 2 (SO 4 ) 3 without the enzyme, suggests that an abiotic oxidation of the substrate occurs since quinone production was observed (data not shown). Furthermore, when FeSO 4 was added one or two minutes after syringaldazine oxidation started (Fig. 4), the spectral scan showed than the quinone of syringaldazine was transformed but did not lead to the initial compound (a new peak at 300 nm was observed).

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When sulphate salts such as CuSO 4 and MnSO 4 were tested, an increase in laccase activity occurred (Fig. 5). Laccase production enhancement when CuSO 4 was added in fungal culture has been extensively described [START_REF] Farnet | Effects of copper and aromatic inducers on the laccases of the white-rot fungus Marasmius quercophilus[END_REF][START_REF] Baldrian | Copper and cadmium increase laccase activity in Pleurotus ostreatus[END_REF][START_REF] Lorenzo | Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decoulorise dyes[END_REF] which may be explained by gene-expression induction. However when added to a purified enzyme, CuSO 4 provoked various effects depending on the laccase tested: [START_REF] Lorenzo | Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds[END_REF] have found a great laccase inhibition (about 40% of inhibition with 20 mM of CuSO 4 ) with a laccase from Trametes versicolor while [START_REF] Baldrian | Copper and cadmium increase laccase activity in Pleurotus ostreatus[END_REF] have shown that laccase activity increases and remains stable with a CuSO 4 concentration of 50 mM. [START_REF] Munoz | Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties and participation in activation of molecular oxygen and Mn 2+ oxidation[END_REF] have demonstrated that semiquinone produced by laccases can be transformed to quinone via autooxidation involving oxygen. The superoxide anion produced in this reaction is then reduced to hydrogen peroxide. In our study, Cu 2+ or Mn 2+ may favour the role of oxygen and may be reduced to Cu + or Mn + leading to an apparent increase in laccase activity. This result points out that the laccase isoform studied remains active in the presence of certain toxic pollutants and that this activity can even be enhanced by certain ions. Thus, this enzymatic system seems to be strongly involved in ecosystem resilience since carbon recycling linked to phenol degradation would be weakly affected under these stress conditions.

We also tested the effects of halide salts on laccase activity and we observed the following inhibition order, F À , I À , Cl À , Br À with F À as the strongest inhibitor (Fig. 6). IC50 was of 20 mM with NaBr, 7.5 mM with NaCl, 3.3 mM with NaI and 2.7 mM with NaF. This result is consistent with previous studies on laccases from different fungi [START_REF] Jung | Purification and characterisation of laccase from wood-degrading fungus Trichophyton rubrun LKY-7[END_REF][START_REF] Kim | Impact of reaction conditions on the laccasecatalysed conversion of bisphenol A[END_REF]. Inhibition mechanism of laccases by halide ions has been described by [START_REF] Naki | Inhibition mechanism of Polyporus versicolor laccase by halide ions[END_REF]: chloride and bromide ions act as competitive inhibitors with the electron donor while F À acts as a non-competitive inhibitor. Furthermore, the degree of inhibition of laccases by halides seems to be linked to the availability of copper atoms (type 2 and type 3) in the active site [START_REF] Abadulla | Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta[END_REF]. In the case of NaI, we showed that laccase was able to oxidize I À to I 2 (Fig. 7) since a strong yellow coloration appeared (absorption at 350 nm). Concentrations ranging from 0.1 M to 0.4 M were used in order to favour I 2 production monitoring at 350 nm. Thus the inhibition observed when NaI was added to laccase and syringaldazine seems to be due to the use of I À as a substrate. Laccase inhibition by chloride salts was also stated in the experiment involving metal salts. CuCl 2 , CaCl 2 , MnCl 2 , NaCl and HgCl 2 indeed showed a strong inhibition towards laccase activity (Figs. 5 and8). From an ecological point of view, laccase inhibition with NaCl is noteworthy since this would mean that carbon recycling may be altered under particular environmental conditions. For instance, in Mediterranean littoral areas where saline concentrations in litters can reach higher levels, carbon turn-over may be less effective. Further studies should investigate whether laccase activities decrease in such ecosystems or whether microbial adaptation may have led to laccase isoforms which are less inhibited by chloride salts. Mediterranean ecosystems are submitted to anthropic and natural pressures. This work contributes to a better understanding of how a laccase isozyme produced in such fragile ecosystems may be affected by environmental pollutants. Overall, laccase activity was weakly affected by the solvents tested (most of the activity remains for a concentration of 30% whatever the solvent used). Interestingly we also found that the laccase isoform studied here was not drastically affected by the metal ions tested compared to other studies. Moreover, certain ions, such as Cu 2+ or Mn 2+ , enhanced laccase activity. Actually, chloride ions were the most inhibiting ions, which may have a significant importance in littoral ecosystems. Further studies should investigate whether this enzymatic system may also be involved in environmental detoxification by oxidizing aromatic pollutants. We have already tested laccase activity towards chlorophenols [START_REF] Farnet | Purification of a laccase from Marasmius quercophilus induced with ferulic acid : reactivity towards natural and xenobiotic aromatic compounds[END_REF] and we should also analyse laccase oxidizing potential with other widespread aromatic pollutants such as polycyclic aromatic hydrocarbons.

Fig. 1 .

 1 Fig. 1. Electrophoretic profile of the purified constitutive laccase from Marasmius quercophilus strain 19 on SDS-PAGE. Molecular weight markers on the left line (kDa).

Fig. 2 .

 2 Fig. 2. Percentage of remaining laccase activity using syringaldazine (a) or ABTS (b) as a substrate with solvent concentrations ranging from 0% to 80%. Solvents used were: methanol (j) ethanol (Ç), acetone (•), acetonitrile (•), dioxane (m).

Fig. 3 .

 3 Fig. 3. Stability of syringaldazine quinone, syringaldazine oxidation with laccase in acetate buffer 0.1 M, pH 4.0 with (-) or without (-) 20% of methanol is followed at 525 nm for 10 min.

Fig. 4 .

 4 Fig. 4. Transformation of syringaldazine quinone with FeSO 4 , ( ) syringaldazine, (--) syringaldazine and laccase, (---) syringaldazine and laccase, 1 min after adding FeSO 4 , ( ) syringaldazine and laccase, 2 min after adding FeSO 4 .

  Fig. 5. Percentage of remaining laccase activity using syringaldazine as a substrate with metal salt concentration ranging from 0 to 20 mM depending on the metal salt used : CuCl 2 (j), CuSO4 (r), MnCl 2 (--), MnSO4 (m) HgCl 2 (•), CaCl 2 (d), NaCl (h).

Fig. 6 .Fig. 7 .

 67 Fig.6. Percentage of laccase remaining activity using syringaldazine as a substrate with different halide concentrations: NaBr (j), NaCl (r), NaI (•), NaF (m).

Fig. 8 .

 8 Fig.8. Percentage of remaining laccase activity using ABTS as a substrate with metal salt concentration ranging from 0 to 20 mM depending on the metal salt used: CuCl 2 (j), MnCl 2 (h), HgCl 2 (r), CaCl 2 (•), NaCl (m).

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