Natural and recombinant fungal laccases for paper ... - Springer Link

Appl Microbiol Biotechnol (2004) 64: 346–352 DOI 10.1007/s00253-003-1468-3

BIOTECHNOLOG ICA L PROD UCTS A ND PRO CESS ENGINE ERIN G

C. Sigoillot . E. Record . V. Belle . J. L. Robert . A. Levasseur . P. J. Punt . C. A. M. J. J. van den Hondel . A. Fournel . J. C. Sigoillot . M. Asther

Natural and recombinant fungal laccases for paper pulp bleaching Received: 10 July 2003 / Revised: 17 September 2003 / Accepted: 19 September 2003 / Published online: 5 November 2003 # Springer-Verlag 2003

Abstract Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l−1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (Km) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using C. Sigoillot (*) . E. Record . J. L. Robert . A. Levasseur . J. C. Sigoillot . M. Asther UMR 1163 INRA/Université de Provence de Biotechnologie des Champignons Filamenteux, IFR 86 BAIM, Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, C.P. 925, 13288 Marseille Cedex 09, France e-mail: [email protected] Tel.: +33-4-91828605 Fax: +33-4-91828601 V. Belle . A. Fournel UPR 9036 de Bioénergétique et Ingénierie des Protéines– CNRS 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France P. J. Punt . C. A. M. J. J. van den Hondel Department of Applied Microbiology and Gene Technology, TNO Nutrition and Food Research Institute, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, The Netherlands

laccases expressed in P. cinnabarinus or A. niger with 1hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

Introduction Laccases (EC 1.10.3.2) are blue multicopper oxidoreductases first described in 1883 by Yoshida (1983) and are widespread and found in numerous plants, bacteria and fungi (Gianfreda et al. 1999). Although the biological function of laccases is still unclear, they have a wide range of industrial applications, including dye decolorization, wine clarification, bioremediation, drug analysis, ethanol production and paper pulp bleaching (Mayer and Staples 2002). Laccases can be classified as low or high redox potential oxidoreductases (Xu et al. 1996). The highest redox potential laccases are mainly produced by white rot fungi, such as Trametes versicolor (Reinhammar 1984) or Pycnoporus cinnabarinus (Eggert et al. 1996). Variation in laccase redox potentials are due to differences in the protein structure which are more or less favorable to electron transfer (Solomon et al. 2001; Kanbi et al. 2002). The four copper atoms of the laccases are distributed in one mononuclear (T1) center and one trinuclear (T2–T3) center. T1 copper, responsible of the characteristic blue color of the enzyme, is the primary electron acceptor during oxidation of a substrate (Ducros et al. 1987, 1998). Electron capture by this copper is enhanced by a favorable electronic environment, due to the amino acid residues involved in the copper binding (Solomon et al. 2001). Next, electrons are transferred to the two-electron-acceptor, the T2–T3 center. This trinuclear center accepts these electrons with the simultaneous reduction of a molecule of

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dioxygen (Thurston 1994). This reaction allows the oxidation of phenolic compounds, like the polyphenols or methoxy-substituted monophenols found in paper pulp (Call and Mücke 1997). Nonphenolic compounds can also be oxidized in the presence of a redox mediator, such as 2,2′-azino-bis-(3-ethyltiazoline-6-sulfonate) (ABTS), which allows the oxidation of higher redox potential substrates (Bourbonnais et al. 1998; Johannes and Majcherczyk 2000). The main bottlenecks in the use of laccases in industrial processes are the large-scale availability and production costs of the enzymes. Genetically modified strains could be developed to increase production. In our laboratory, we isolated a monokaryotic strain from P. cinnabarinus described as a laccase hypersecretory fungus in the presence of chemical inducers, such as ferulic acid and 2,5-xylidine (Herpoël et al. 2000). In this case, two isoenzymes, LacI and LacII, were produced (Otterbein et al. 2000). The laccase I gene, lacI, was isolated, characterized (GenBank accession number AF170093) and expressed in an Aspergillus niger strain under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter (Record et al. 2002). In this study, laccase production yield was comparable to wild-type expression and did not require the presence of a chemical inducer. In the present work, we expressed the lacI gene in A. oryzae, a filamentous fungal host known to overproduce homologous and heterologous proteins of industrial interest. Laccases were purified and characterized by biochemical and electron paramagnetic resonance (EPR) spectroscopy to understand some aspects of structure/function relationships; and their industrial interest for paper pulp bleaching was tested and discussed.

Materials and methods Strains, media and culture conditions The A. oryzae ATCC 16868 pyrG strain (pyrg−) was used for heterologous expression. After co-transformation with vectors containing respectively the pyrG gene and laccase cDNA, A. oryzae was grown on selective solid minimum medium without uridine (Punt and van den Hondel 1992). Also used in this study were P. cinnabarinus ss3 (already described as a laccase hypersecretory strain; Herpoël et al. 2000) and the best recombinant Aspergilli laccase producers: A. niger 450 (deposited in the “Banque de Ressource Fongique de Marseille”; Record et al. 2002) and A. oryzae transformants.

pAB4-1 containing the pyrG selection marker, in a 5:1 ratio. Transformants were selected for uridine prototrophy. Cotransformants containing expression vectors were selected as described in the following section.

Screening of transformants Agar plate assays on selective minimal medium (Punt and van den Hondel 1992) with 200 µM ABTS were used for the selection of transformants secreting laccase. Plates were incubated for 10 days at 30°C and checked for the development of a green color. In order to screen the laccase production, cultures were performed in selective liquid minimal medium (Punt and van den Hondel 1992). The cultures were monitored for 12 days at 30°C in a shaker incubator (200 rpm) and the pH was adjusted to 5.0 daily with 1 M citric acid.

Laccase assays Laccase activities were determined by monitoring the change in adsorption at 420 nm (A420), using a Uvikon spectrophotometer (Kontron Instrument, Milan, Italy), during the oxidation of 500 µM ABTS to the corresponding radical (ε420=36 mM−1 cm−1; Herpoël et al. 2000). Enzyme activity was expressed in international units (IU). One activity unit was defined as the quantity of enzyme which leads to the transformation of 1 µmol of ABTS min−1. For the stability and optimal pH determination, syringaldazine (17 µM) was used as a substrate to monitor the production of colored quinone at 530 nm (ε530=65 mM−1 cm−1; Yaver et al. 1996). Assays were performed in triplicate and at least at two enzyme dilutions. Standard deviations did not exceed 10% of the average values.

Laccase production P. cinnabarinus ss3 inoculum was obtained from 7-day-old nonagitated precultures, grown in Roux flasks containing 200 ml of medium, according to Sigoillot et al. (1999), and incubated at 30°C. The strain of P. cinnabarinus was cultivated in bioreactor according Herpoël et al. (2002). Inocula of the Aspergilli strains were obtained from 5-day-old agitated preculture grown in baffled Erlenmeyer flasks containing 200 ml of screening medium (Record et al. 2002). Media were inoculated with 2×105 spores ml−1; and 600 ml of these precultures were ground for 1 min with a Turax T25 blender (Janke and Kunkel, Staufen, Germany) at 10,000 rpm. For Aspergilli bioreactor cultures, the medium contained (per liter): 10 g glucose, 6 g NaNO3, 0.5 g KCl, 28 g Na2HPO4 , 0.5 g MgSO4 and trace elements. The pH was adjusted and maintained at 5.0 with a 1 M citric acid solution. Laccase productions were performed in a 12-l (working volume) bioreactor at 30°C and stirred at 150 rpm. Activities reached 2,800 IU l−1 for the natural strain, as compared wit 11,000 IU and 8,000 IU l−1 for A. niger and A. oryzae, respectively.

Laccase purification Vector construction and Aspergilli transformation The laccase gene, lacI, from P. cinnabarinus (GenBank accession number AF 170093) was cloned in the vector pLac1-B. The 21 amino acids (aa) of the laccase signal peptide were replaced by the 24-aa glucoamylase preprosequence from A. niger. In this construction, the A. nidulans glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) promoter, the 5′-untranslated region of the gpdA mRNA and the A. nidulans trpC terminator were used to drive the expression of the laccase-encoding sequence. Fungal cotransformation was carried out basically as described by Punt and van den Hondel (1992), using each of the laccase expression vectors and

Culture media were harvested on the optimal laccase production day. The mycelium was removed using a press filter. The filtrate was ultrafiltrated using a 10-kDa Pelicon, further clarified on a GF/F glass fiber filter (Whatman, Maidstone, UK) and stored at −20°C. An aliquot of the culture filtrates was diafiltrated with 25 mM sodium acetate buffer (pH 5.0), using a 10-kDa polyethersulfone membrane (Amicon system; Millipore, Bradford, USA), in order to remove salts from the culture medium. Concentrated proteins were loaded onto a DEAE-CL6B ion-exchange column (25×300 mm column; Amersham Pharmacia Biotech, Orsay, France). The loaded gel was washed with three column volumes of 25 mM sodium

348 acetate buffer, pH 5.0, then the bound proteins were eluted with 800 ml of a linear gradient of NaCl (0–500 mM) in 25 mM sodium acetate buffer (pH 5.0) at 1 ml min−1 and 10-ml fractions were collected. Blue-colored laccase fractions were checked for electrophoretic homogeneity and used for biochemical characterization.

Protein analysis The protein concentration was determined according to Lowry et al. (1951), using bovine serum albumin as standard. Sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDSPAGE) on 13% gel (Laemmli 1970) was used to determine the purity and molecular mass of the laccases (100 µg of each purified protein were loaded on the SDS-PAGE gel). Isoelectric focusing (IEF) in the pH range 3.0–9.0 was performed on a Phast system, using a precast gel (PhastGel IEF; Amersham Pharmacia Biotech). Protein bands were stained with Coomassie blue, following the procedure of the manufacturer. For N-terminal analysis, purified laccases were submitted to the Edman′s degradation, using an Applied Biosystems 476A apparatus. Western blot analysis were performed as described by Bonnarme et al. (1994). The primary antibodies raised against the laccases were detected using alkaline phosphatase-conjugated goat anti-rabbit antibodies (Roche Molecular Biochemical, Meylan, France) at dilutions of 1/25,000 and 1/4,000. Alkaline phosphatase activity was detected using the 5-bromo-4-chloro-3-indoyl-phosphate nitro blue tetrazolium assay.

Spectroscopic measurements and redox titrations Redox titrations of the laccases were performed in triplicate at 24°C in 50 mM sodium phosphate buffer (pH 5.5) containing 100 µM protein solutions. The reduction extent of the T1 copper center was determined by monitoring the A608 variation with a Kontron 932 spectrophotometer (Kontron, Meylan, France). Redox potentials were measured witha combined Pt-Ag/AgCl/KCl (3 M) microelectrode adapted to the 1-cm-thick quartz cuvette. Since proteins were not totally oxidized in their as-prepared state, it was necessary to add a small amount of a saturated solution of iridium chloride to reach a high potential stable level. Redox potentials were adjusted byion of 20 mM sodium dithionite solution in thepresence of the following mediators, each at 10 µM: Fe(II/III)-Tris-(1,10-phenantroline) (+1,070 mV), 1,1′-dicarboxylic acid ferrocene (+644 mV) and monocarboxylic acid ferrocene (+530 mV). The midpoint potential, E°′, was determined by fitting the experimental data to the Nernst equation, using a least-squares regression. X-band EPR spectra were recorded at 100 K using an ESP 300E Bruker spectrometerfitted with an ESR 900 helium-flow Oxford Instrument cryostat. For spin quantitation, the second integral value of the

spectrum recorded in non saturatingconditions was compared with thatgiven by a CuSO4 standard recorded at the same temperature.

Paper pulp treatment The unbleached wheat straw Kraft pulp used in this study was provided by the Gaoyou mill (Gaoyou, China) and its initial Kappa number was 22.6. Experiments were carried out in triplicate, using 10 g of oven-dried pulp. All treatments were performed in a stainless steel vessel preheated in a water bath to the required temperature, with mechanical agitation (marine propeller, 150 rpm). Enzymatic treatments in the presence of 1-hydroxybenzotriazole (HBT) as redox mediator were performed in conditions close to those described by Herpoël et al. (2002), except for the xylanase treatment step, which was performed at pH 5.0 in 100 mM acetate buffer. Trials without the addition of laccase were performed as a control and all treatments were performed at least in triplicate.

Paper pulp analysis The Kappa number (from which the percentage lignin content can be roughly estimated by multiplying Kappa number by a factor of 0.15) was measured according to ISO 302 (Record et al. 2002). The content in β-O-4-linked lignin sub-units were determined by the thioacidolysis method using treated pulp, as described by Lapierre et al. (1986). The degradation yield of guaiacyl and syringyl monomers was determined in relation to their content in the untreated sample. All trials were performed in triplicate.

Results Transformation of Aspergillus strains, screening and laccase production The lacI gene of the main laccase expressed by P. cinnabarinus was cloned and sequenced. The gene was integrated in an expression vector to transform A. niger (Record et al. 2002) and A. oryzae. The Aspergilli strains were transformed with a mixture containing the plasmid pAB4.1 (containing the PyrG gene) and the vector containing lacI cDNA from P. cinnabarinus. Transformants were selected for their ability to grow in minimum medium without uridine and containing ABTS, resulting in a green zone around lac+ clones. Colored zones on

Table 1 Purification of laccase I from Pycnoporus cinnabarinus and purification of the same laccase expressed in Aspergillus niger and A. oryzae. IU International units of activity Species

Purification stage

P. cinnabarinus

Crude extract Ultrafiltration DEAE Crude extract Ultrafiltration DEAE Crude extract Ultrafiltration DEAE

A. niger

A. oryzae

Volume (ml) 300 35 20 1,000 30 20 700 46 9

Protein (mg) 619 68 8.5 2,365 385 16 1,881 344 12

Total activity (IU) 2,783 2,919 1,989 7,118 5,796 2,253 5,643 4,468 1,339

Specific activity (IU mg−1) 4.5 43 234 3 15 140 3 13 109

Recovery (%) 100.0 104.0 71.5 100.0 81.4 31.6 100.0 79.2 23.7

Purification (x-fold) 1.0 9.5 52.0 1.0 5.0 47.0 1.0 4.3 36.0

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Fig. 1 SDS/PAGE (with Coomassie blue stain; A) and Western blot (B) analyses of purified products of the lacI gene, using Pycnoporus cinnabarinus (lane 1), Aspergillus niger (lane 2) or A. oryzae (lane 3) as the expression host. For immunodetection, antibodies raised against P. cinnabarinus laccase were used. Lane Sd Molecular mass standards

plates were not observed in the case of the control transformants lacking the laccase gene. Thirty positive clones were grown in liquid medium and then assayed at the optimal day of laccase production. Results for laccase activity ranged over 1,900–8,400 IU l−1 (day 12), and the best A. oryzae clone was selected in order to study the time-course of the laccase activity. During fungal culture, the mycelial dry weight increased until day 6 and reached a maximum of 15–18 g l−1 by day 12. The laccase activity reached a maximum of 8,400 IU l−1 on day 11, at the end of the culture.

enzymes (AIGPVADLTL), confirming that the signal peptide was correctly cleaved in the recombinant laccases. The main biochemical characteristics of the three purified laccases were determined using ABTS as the substrate (Table 2). Most of these characteristics were similar, except for the isoelectric point of the A. niger laccase (3.7), which was slightly less acidic than the other ones (3.5). These laccases were also different concerning their Km, kcat and the activation energy calculated according to Arrhenius′ law. The wild-type laccase had a better affinity for ABTS than the recombinant laccases. The normalized amplitude of the absorption band measured at λmax=608 nm as a function of the potential for the three laccases is displayed in Fig. 2. Experiments led to the following E°′ values: 810±10 mV for P. cinnabarinus laccase, 760±10 mV for A. niger laccase and 735±10 mV for A. oryzae laccase. The integrity of the three laccases studied by UV/visible absorption spectrophotometry was controlled by recording the EPR spectrum of a fully oxidized sample. Spin quantitations were performed in order to ensure that the observed EPR signal reflected the state of the full population of molecules present in the sample. The integrated intensity of the signal gave 1.7 spins per protein, a value consistent with the presence of two paramagnetic copper centers per molecule. No difference between the EPR signatures of the three laccases could be observed. The EPR spectrum of the P. cinnabarinus enzyme is shown in Fig. 3. The EPR signature of the T1 center was typical, with a g// value equal to 2.18 and a parallel hyperfine splitting A// value equal to 86×10−4 cm−1. Paper pulp-bleaching ability of laccases

Purification and study of the biochemical characteristics of the three laccases Laccases were purified for biochemical, physico-chemical characterization and functional studies. The main step was ion exchange chromatography giving specific activities of 109, 140 and 234 IU mg−1 for A. oryzae, A. niger and P. cinnabarinus, respectively (Table 1). SDS-PAGE analysis confirmed the purity of the enzymes (Fig. 1A). Each laccase exhibited a single band around 70 kDa. Western blot analysis (Fig. 1B) using antibodies raised against the P. cinnabarinus laccase confirmed the similarities between the natural and recombinant laccases. Moreover, the Nterminal amino acid sequences were identical for the three

The bleaching efficiency of each laccase type was studied in order to determine the feasibility of their use in paper pulp delignification. The results of enzymatic treatments are shown in Fig. 4. The laccase expressed in A. niger has the same efficiency in delignification as the wild-type laccase (close to 50% compared with the control trial without laccase), whereas the laccase expressed in A. oryzae seems not to have a delignifying effect. These results were confirmed by thioacidolysis analysis (Fig. 4B), which showed that the guaiacyl and syringyl sub-units linked with β-O-4 bonds were not degraded by the laccase expressed in A. oryzae.

Table 2 Main biochemical characteristics of laccase I from P. cinnabarinus and characteristics of the same laccase expressed in A. niger and A. oryzae, using 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonate) as substrate Species P. cinnabarinus A. niger A. oryzae

Molecular mass (kDa) 70 70 70

pI 3.5 3.7 3.5

Optimal temperature (°C) 65 65 65

Optimal pH 4.5 4.0 4.5

Km (µM) 41 55 55

kcat (min−1) 16,300 9,800 7,600

Ea (kJ mol−1) 36 30 42

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Fig. 3 Electron paramagnetic resonance spectrum given by the P. cinnabarinus laccase recorded at 100 K. The T1 hyperfine splittings A// and one of the T2 hyperfine lines are indicated. The experimental conditions were: microwave frequency 9.345 GHz, microwave power 10 mW, modulation amplitude 2 mT, modulation frequency 100 kHz

Fig. 2 Redox potential measurement at 608 nm of the P. cinnabarinus (A), A. niger (B) and A. oryzae (C) laccases. The solid lines show the best fit of the experimental data given by oneelectron Nernst curves Fig. 4A, B Effect of P. cinnabarinus (Pc), A. niger (An) and A. oryzae (Ao) laccases on the delignification of wheat straw chemical pulp in the presence of 1-hydroxybenzotriazole as a redox mediator. A Kappa number measurement. B Thioacidolysis determination of guaiacyl (white columns) and syringyl (gray columns) residual subunits. Treatments and measurements were performed at least in triplicate. WL Trial without laccase

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Discussion In this work, laccase I from P. cinnabarinus and the corresponding laccases expressed in A. niger and A. oryzae were compared in order to select the best production system for pulp and paper applications. Concerning production levels, the culture of A. oryzae yields 80 mg of recombinant laccase l−1, which is comparable with A. niger production (70 mg l−1; Record et al. 2002). When compared with previous work on laccase production in A. oryzae, these results are better than those obtained with the laccase from Myceliophthora thermophila (11–19 mg l−1; Berka et al. 1997) and are in the same range as the laccase from Coprinus cinereus (8– 135 mg l−1; Yaver et al. 1996). However, the natural production of laccase by P. cinnabarinus reached the best production level (145 mg l−1; Herpoël et al. 2000). The recombinant laccases presented specific activities lower than the natural one, i.e. close to 100 IU, against 200 IU mg−1. The diminution of this parameter could be explained by differences due to heterologous maturation, resulting in the decrease in laccase activity. This observation is supported by measurement of the Km and kcat of each enzyme. Recombinant laccases presented a higher Km and a lower kcat than the P. cinnabarinus laccase, which implies a decrease in substrate/enzyme affinity and molar specific activity. Activation energies, calculated according to Arrhenius′ law, were similar to those previously reported (Gianfreda et al. 1999) but some differences between the three enzymes were observed, corroborating the previous observations. Redox potential measurement of the three laccases highlights the differences due to the host-specific processing. P. cinnabarinus laccase exhibited a very high redox potential (810 mV). Interestingly, this redox potential is higher than those reported for laccases from P. cinnabarinus strains, which generally measured around 750 mV (Li et al. 1999). However, recombinant laccases presented a lower redox potential (760 mV for laccase expressed in A. niger, 735 mV for laccase expressed in A. oryzae). These differences, even if the EPR spectra were unchanged, might be explained by changes in the enzyme structure affecting its redox potential (Pionteck et al. 2002). To a lesser extent, A. oryzae and A. niger expression probably results in differences in protein processing (maturation, folding and/or glycosylation) and consequently induces slight shifts in molecular structure. In this way, the redox potential that depends on T1 copper coordination could be modulated, even if the primary structure is similar. A laccase mediator system (LMS) treatment (Call and Mücke 1997) using HBT, a redox mediator well known for its high delignification potential (Fabbrini et al. 2002) was evaluated in order to compare the efficiency of each laccase for pulp and paper bleaching. Sequential treatment of wheat straw Kraft pulp in the order xylanase/LMS/ alkaline extraction allowed a delignification close to 75% for P. cinnabarinus and A. niger laccases. These results were similar to those obtained by Herpoël et al. (2002) using the P. cinnabarinus laccase for delignification.

Thioacidolysis analysis corroborated these observations. The content of syringyl and guaiacyl units in paper pulp was drastically reduced in both cases. In contrast, laccase produced in A. oryzae seems to have no delignifying effect. The results of thioacidolysis analysis confirmed that syringyl and guaiacyl units are not attacked by this LMS. This lack of efficiency could be explained by the lower redox potential of A. oryzae laccase. Bourbonnais et al. (1998) used cyclic voltamperometry to demonstrate that, for potentials lower than 750 mV, the HBT could not be oxidized and delignification could not occur. Results obtained with the laccase produced by A. oryzae are in good agreement with these observations. In conclusion, P. cinnabarinus laccase was successfully produced by heterologous expression using Aspergilli hosts. The laccases secreted by the three expression systems showed some physico-chemical and biochemical differences. The A. niger laccase was more similar to the natural one than the laccase produced by A. oryzae. A. oryzae recombinant enzyme seemed to be incorrectly matured and showed a redox potential lower than expected. The efficiency of the recombinant laccases on paper pulp bleaching was compared with that of the natural form. A. niger laccase was demonstrated to be as efficient as the P. cinnabarinus laccase, showing that a recombinant enzyme-based technology could be considered in order to reduce process costs. However, A. oryzae laccase was not able to be used for delignification when HBT was used as a redox mediator. This result was partially explained by the difference in redox potentials. Acknowledgements We thank P. Mansuelle (UMR 6560, IFR Jean Roche, Marseille, France) for the N-terminal sequence analysis. This research was supported by the EU Quality of Living Resources programme (Project QLK3-99-590, Fungal metalloenzymes oxidizing aromatic compound of industrial interest) and GIS-EBL (Région Provence Alpes Côte d’Azur and Conseil Général des Bouches-duRhône, France). C.S. is grateful to the Conseil Régional ProvenceAlpes-Côte d’Azur, France, the Institut National de la Recherche Agronomique and Tembec-Tarascon S.A. for a PhD scholarship.

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