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Purification of a new isoform of laccase from a Marasmius quercophilus strain isolated from a cork oak litter (Quercus suber L)

     Laboratoire de Microbiologie, Service 452, U.M.R. C.N.R.S. 6116, Institut Méditerranéen d'Ecologie et de Paléoécologie, Faculté des Sciences et Techniques de St Jérôme, 13397, Marseille, Cedex 20, France

G. Gil
E. Ferre

     Laboratoire de Chimie, Biologie et Radicaux Libres, Service 532, U.M.R. C.N.R.S. 6517, Universités d'Aix-Marseille I et III, Marseille, France

	ABSTRACT

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A new isoform of laccase from Marasmius quercophilus is described in this study. The strain of this white-rot fungus was isolated for the first time on a cork oak litter. This isoform exhibited certain common properties of laccases (a molecular weight of 65 Kda, an optimum pH of 6.2 with syringaldazine). But this laccase has also particularly novel features: the best activity measured was observed at high temperatures (80 C) and this isoform was not inhibited with EDTA. Furthermore, this induced laccase was able to transform most of the aromatic compounds tested without the addition of mediators to the reaction mixture, and the transformation of certain chlorophenols (2-chlorophenol and 2,4-dichlorophenol) by a laccase isoform from M. quercophilus is reported here for the first time. We also demonstrate the importance of 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as a mediator since it allowed veratryl alcohol and p-hydroxybenzoic acid transformation. Moreover, new products of transformation were observed using the combination of ABTS with this isoform of laccase.

Key words: aromatic compounds, mediator, polyphenoloxidase, white-rot fungus

	INTRODUCTION

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Marasmius quercophilus is a white-rot fungus which usually colonizes evergreen oak litter, a typical Mediterranean ecosystem. In previous studies, we have already described the predominant role of this basidiomycete in lignin biodegradation (Tagger et al 1998, Farnet et al 2000); this fungus is involved in ligninolysis because of its production of phenoloxidases such as laccases (EC 1.10.3.2.). These enzymes are copper-containing oxidases which reduce oxygen to water, while oxidizing various phenolic compounds in a non-specific radical-based reaction. Thus, they have been studied extensively because of the reactions they catalyze and because of their potential involvement in biotechnology, such as kraft pulp bleaching or bioremediation.

The purpose of the present work was first to describe a new induced isoform of laccase produced by a M. quercophilus strain isolated from a different ecosystem, a cork oak (Quercus suber L.) litter. This strain was collected in a siliceous area of Provence in the Collobrières site (Var, France). The induction was performed using ferulic acid as described in a previous study (Farnet et al 1999). Second, we analyzed its potential for transforming condensed tannins, various other aromatic compounds involved in lignin metabolism, and also different chlorophenols. This allowed us to compare this potential for transforming aromatic compounds with that of another laccase produced by M. quercophilus but induced with p-hydroxybenzoic acid (Farnet et al 2000). Furthermore, studying the transformation of chlorophenols by this laccase is a first assessment of the potential involvement of a phenoloxidase produced by M. quercophilus in bioremediation. The degradation of recalcitrant xenobiotic compounds is especially useful since most of these aromatic pollutants are ubiquitous in terrestrial and aquatic ecosystems. These compounds have been used on a large scale as biocides (pesticides, wood preservatives, or precursors of herbicides) and have led to severe contamination problems. The non-specific ligninolytic enzymes excreted by white-rot fungi have provoked considerable interest in the past few years since this enzymatic system seems to be suitable for the degradation of various phenolic pollutants. Mediators such as 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), promazine, Remazol brilliant blue, or 1-hydroxybenzotriazole (HBT) were also used to extend the substrate range of laccases, as already reported by Bourbonnais et al 1997. Therefore, we also studied the effect of two mediators (ABTS and HBT) on laccase potential for degradation of these particularly recalcitrant compounds.

	MATERIALS AND METHODS

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Isolation of the strain – Strain C7 of Marasmius quercophilus was isolated from part of the Collobrières site (Var, France) using the rhizomorphic form of the fungus. A fungal cap culture was first made on a malt-agar medium, 20 g L^-1 (Bio Mérieux, Marcy l'Etoile, France) and chloramphenicol, 50 mg L^-1 (Sigma). The pure mycelial culture obtained was used to inoculate an agar medium (whole wheat flour, 20 g L^-1) which favors rhizomorph production. Then one rhizomorph was used to inoculate a malt-agar plate in order to obtain a pure dicaryotic culture.

Cultures for purification – Precultures were performed in 200 mL Erlenmeyer flasks containing 50 mL of malt extract medium (20 g L^-1) in static conditions at 30 C. They were inoculated with a plug of agar cultures (malt extract 20 g L^-1, agar 15 g L^-1). These liquid precultures were used to inoculate, with about 2 g of fresh fungus, two 2000 mL Erlenmeyer flasks containing 400 mL of the following medium: malt extract medium, 20 g L^-1, CuSO₄, 5 mg L^-1 and ferulic acid, 250 mg L^-1. The ferulic acid solution was sterilized by filtration on minisart filters (Sartorius AG, Germany) and then added to the culture medium. These cultures were incubated under an axial shaking (50 rpm) at 30 C for 15 d. Enzyme activity was measured by following the oxidation of syringaldazine [N,N'-bis-(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine] to quinone (^M = 65 000 M^-1·cm^-1) at 525 nm on a Kontron Uvikon 860 spectrophotometer. The reaction mixture contained 300 µL of the filtered culture medium, 2.7 mL of phosphate buffer 0.1 M, pH 5.7 and the reaction was initiated by adding 10 µL of syringaldazine 0.6% (w/v), diluted in methanol. The blank consisted of 300 µL of the filtered culture medium and 2.7 mL of the same phosphate buffer. One unit (U) of laccase activity is defined as the amount of enzyme that oxidizes 1 µmole of the substrate per minute.

Purification of the laccase isozyme – Cultures were filtered on a glass microfibre filter GF/D, 2.7 µm (Whatman, England). These filtered media were concentrated on an ultrafiltration cell, model 8200 (Amicon, Beverly, Massachusetts, U.S.A.) until the volume reached 15 mL. The membrane used (Amicon), was rated at 10 MW (10 kD) cut-off. This sample was then loaded on a Mono Q column. A step gradient system of NaCl in Tris-HCl buffer 10 mM pH 6 (0, 0.2, 0.4, 1 M) was used to elute active fractions. Laccase activity was detected using syringaldazine. The active fractions eluted as one peak were pooled and ultrafiltered as described above.

Effects of pH and temperature on laccase activity – Glycine buffer 0.1 M was used for pH range from 2.2 to 3.8, acetate buffer 0.1 M was used for pH range from 4 to 5 and phosphate buffer 0.1 M for pH range from 5.7 to 8. Laccase activity was detected with syringaldazine and ABTS 100 µM each and using 0.005 U of the purified fraction. The optimum temperature for laccase activity was determined using 0.005 U of the purified fraction in phosphate buffer 0.1 M pH 6.2 with syringaldazine 100 µM as the substrate and in glycine buffer 0.1 M, pH 2.6 with ABTS 100 µM as the substrate. In the last case, enzyme activity was measured by following the oxidation of ABTS at 420 nm (^M = 36 000 M^-1·cm^-1). Six temperatures were tested: 30, 40, 50, 60, 70, and 80 C. The thermal stability of laccase activity was studied using the temperatures mentioned above and after several incubation periods (1, 2, 3, 5, 7, 9, 24, and 36 h). Laccase activity was measured as described above.

Effects of inhibitors on laccase activity – Laccase activity was measured using 0.005 U of the purified enzyme in glycine buffer 0.1 M, pH 2.6 with ABTS 100 µM as the substrate. The inhibitors used at a final concentration of 1 mM were: sodium azide, EDTA, cystein, dithiothreitol and sodium diethyldithiocarbamate. All the chemical products were purchased from Sigma.

Determination of kinetic parameters – Syringaldazine and ABTS were used as substrates for the determination of the V_m and K_m which were calculated using the Lineweaver-Burk transformation of Michaelis-Menten equation. The concentrations ranged from 2 to 200 µM and from 0.5 to 50 µM for ABTS and syringaldazine respectively.

Electrophoresis analysis – Sodium dodecyl sulfate (SDS)—polyacrylamide gel electrophoresis (PAGE) were carried out according to Laemmli (1970) using 4% stacking gel and 12% separating gel at 220 V with the Mini-Protean II electrophoresis cell (Bio-Rad). Molecular masses of the two laccase isozymes were determined using high range molecular mass prestained standard (Bio-Rad). Protein bands were revealed using the Coomassie blue standard method.

HPLC analysis of aromatic compound degradation – HPLC system was equipped with a C18 column (Merck, 4.6 x 250 mm) in the following gradient system: solvent A, water/trifluoro acetic acid 0.1%/methanol 90/10 v/v, solvent B water/trifluoro acetic acid 0.1%/methanol 10/90 v/v, gradient = 0 to 25 min A 100% to B 100%, 25 to 40 min B 100%, (flow rate 1 mL min^-1; detection at 270 nm). The aromatic compound degradation was assayed using a reaction mixture (10 mL) containing ABTS or HBT 1 mM, aromatic compound 1000 mg L^-1 and 5 U of the laccase isozyme in phosphate buffer 0.1 M pH 6.2. The aromatic compounds used were: catechin, ellagic acid, gallic acid, tannic acid, p-coumaric acid, p-hydroxybenzoic acid, vanillic acid, veratryl alcohol, 2 chlorophenol, 2,4-dichlorophenol, and pentachlorophenol. All the aromatic compounds were purchased from Sigma. The same reaction was performed without mediators. Controls were also done using only ABTS or HBT with laccase, and using ABTS or HBT with the aromatic compound but without laccase. Samples were collected at 0, 2, 4, 8, 24, and 36 hour(s), and 20 µL of each sample was injected into the HPLC system. The decrease in aromatic compounds in the reaction mixture was estimated by measuring the peak area in UV absorbance, and 50 µg of benzoic acid was co-injected each time to be used as reference. All the experiments were repeated three times and the results are reported as means with standard deviations.

	RESULTS

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Production of laccases under inducing conditions and purification of the induced isoform – We obtained the maximum laccase activity after a 15-d incubation. A major induced band was stained using syringaldazine on SDS-PAGE since SDS does not inhibit laccase activity as already reported by Niku-Paavola et al (1990). This SDS-PAGE profile is different from that obtained in our previous study (Farnet et al 1999) with the strains collected in the La Gardiole de Rians site. By Mono Q column chromatography, the laccase isoform induced by ferulic acid was separated from the two other isoforms (Fig. 1). The purified enzyme gave a single band on SDS-PAGE when the gel was stained using the activity of the enzyme with syringaldazine as the substrate. Furthermore, we also observed a major protein band in denaturing conditions with a molecular weight of about 65 kda (Fig. 2).

View larger version (20K): [in this window] [in a new window]    FIG. 1. The elution profile of the laccase isoform using Mono Q exchange chromatography. Laccase activity (--) as well as protein concentration (– – –) are shown. The NaCl gradient is also indicated (———). The peak of the induced isoform is noted with the arrow

View larger version (96K): [in this window] [in a new window]    FIG. 2. SDS-PAGE profile of the purified extracellular laccase from Marasmius quercophilus. Molecular mass markers (Biorad) are shown in the left lane

View larger version (13K): [in this window] [in a new window]    FIG. 3. The pH optimum for the activity of the induced laccase from Marasmius quercophilus was evaluated using ABTS (--) or syringaldazine (--) as the substrate. A value of 100 was ascribed to the highest laccase activity (14.36 U/L and 11.103 U/L for syringaldazine and ABTS respectively) and the other activities were expressed as a percentage of this value

View larger version (31K): [in this window] [in a new window]    FIG. 4. Percentages of transformation of the aromatic compounds tested after a 4-h incubation with the purified enzyme alone (), and with the purified enzyme and ABTS (). The abbreviations used were: C, catechin; pCA, p-coumaric acid; EA, ellagic acid; GA, gallic acid; pBA, p-hydroxybenzoic acid; TA, tannic acid VA, vanillic acid ; VE, veratryl alcohol; 2CP, 2-chlorophenol; 2,4DCP, 2,4-dichlorophenol

View larger version (14K): [in this window] [in a new window]    FIGS. 5A and B. The transformation of aromatic compounds with the induced isoform of Marasmius quercophilus after a 2-hour incubation with HBT and ABTS as mediators using RP C18 HPLC column. The transformation of p-coumaric acid (a) and tannic acid (b) with HBT (———) or ABTS (--) are shown. The arrow points out the elution time of the peak of the aromatic compound alone

	DISCUSSION

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In this paper we described the biochemical properties of an induced laccase and its action on aromatic compounds. This study was particularly aimed at comparing its potential of transforming aromatic compounds with that of another laccase also produced by a Marasmius quercophilus strain but induced with p-hydroxybenzoic acid (Farnet et al 2000). Although the induced isoform studied exhibits new enzymatic properties, certain of its biochemical characteristics matched those common in laccases from white-rot fungi. One protein band on SDS-PAGE in denaturing conditions suggested that the isoform was monomeric. This isoform was strongly inhibited by sodium azide (Périé et al 1998). With cysteine, dithiothreitol, and sodium diethyldithiocarbamate, the decrease of the absorbance may be explained by the reducing potential of these compounds. The absorbance increased again after the addition of the potential inhibitor, which showed that the enzyme was still active. Thus, these reducing compounds may not directly affect the enzyme as already decribed by Majcherczyk et al (1999). Nevertheless, this isoform exhibited a higher stability towards EDTA and a higher optimal temperature (80 C) than those observed with laccases purified from white-rot fungi (Bollag and Leonowicz 1984, Yaropolov et al 1994). Furthermore, optimum pH differed markedly according to the substrate used:the optimum pH is 6.2 with syringaldazine and 2.6 with ABTS. Similar behavior has already been reported for certain fungal laccases. This has been explained by two phenomena: (i) the difference in redox potential between a reducing substrate and the type 1 copper in the active site of the enzyme and (ii) inhibition of the type 2 and type 3 coppers by hydroxide at higher pH (Wahleithner et al 1996, Xu 1997). We also observed two different Km for ABTS and syringaldazine (50 and 7.7 mM respectively). This suggests a different behavior of the enzyme for these substrates, which is correlated to their structure.

This study showed the wide range of subtrates that can be transformed by this new induced isoform of laccase. This isoform did in fact react towards various aromatic compounds without the addition of any mediators. Nevertheless, we have demonstrated the importance of ABTS as a mediator in combination with the isoform studied. HBT did not seem to be prevalent in the action of this isoform since it did not extend its enzymatic potential to other substrates in the way ABTS did with veratryl alcohol or p-hydroxybenzoic acid. Laccase reactivities towards veratryl alcohol vary depending on the white rot fungi from which they were isolated. The redox potential of laccases (between 0.5–0.8 V) is not likely to lead to the oxidation of non phenolic compounds such as veratryl alcohol. Structural differences in the type I copper center located in the activation site, which is involved in the redox potential of the enzyme, may explain these reactivity variations (Solomon et al 1992). Several studies have demonstrated that when ABTS is used as co-oxidant mediator, laccases are able to oxidise compounds which have redox potentials above that of the enzyme (Bourbonnais et al 1998, Majcherczyk et al 1999). The common mechanism which has already been reported involved a first stage where the laccase oxidizes the mediator, which next transforms the aromatic compounds tested. This phenomenon is based on the laccase oxidation of ABTS to a dication. Moreover, the use of HBT did not lead to new products. On the other hand, ABTS seemed to have a non-specific action towards aromatic compounds, which encouraged the formation of various new products. The induced laccase produced by the strain isolated from evergreen oak litter (Farnet et al 1999) did not have the same effect on the different substrates in the absence of mediators. When the enzyme reacted alone, the peak of the aromatic compound tested usually disappeared, suggesting polymerization. New peaks were observed only when mediators were added to the reaction mixture. Previous studies have shown the importance of adding mediators to prevent polymerization, since they promoted oxidative degradation of the oligomeric substances (Chen et al 2000). In the present study, the isoform seemed to transform the aromatic compounds instead of polymerizing them, since we found new peaks on the chromatography profiles, regardless of whether mediators were added. Thus, the products formed by the induced isoform of laccase seemed to be stable and did not lead to polymerization.

The non-degradable nature of pentachlorophenol (PCP) has already been reported in several studies. This particularly recalcitrant component was not transformed by a Coriolus versicolor laccase using a similar kind of experiment (Itoh et al 2000). PCP was usually transformed after longer periods of time (several days) as described by Aïken et al (1996) using static cultures of white-rot fungi such as Phanerochaete chrysosporium. McGrath and Singleton (2000) have also demonstrated the decrease of PCP levels in contaminated soils inoculated with this fungus after a 15-d incubation time. In future studies it would be of great interest to use several other phenolic mediators, since all radicals formed by laccase can potentially react as mediators leading to the oxidation of many kind of substrates. It would also be interesting to extend our analysis to PAH, where mediators would help to avoid steric hindrance effects.

This study showed that laccase is involved in the transformation of particularly recalcitrant aromatic compounds such as chlorophenols. Thus, the enzyme can potentially be applied in various detoxification processes such as pesticide removal or industrial phenolic wastewater treatments (Lante et al 2000, Robles et al 2000). Furthermore, we pointed out the prime role of ABTS as a mediator of laccase activity. Identifying the products of transformation will be the aim of our future research, in order to provide a better understanding of the enzymatic mechanism of laccase.

	FOOTNOTES

 
¹ Corresponding author, Email: amfarnet{at}netscape.net

Accepted for publication February 8, 2002.

	LITERATURE CITED

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Farnet, A.M. Articles by Ferre, E. Search for Related Content PubMed Articles by Farnet, A.M. Articles by Ferre, E. Agricola Articles by Farnet, A.M. Articles by Ferre, E.