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International Biodeterioration & Biodegradation 63 (2009) 600–606

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International Biodeterioration & Biodegradation

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Malachite green decolourization and detoxification by the laccase from anewly isolated strain of Trametes sp.

Manel Maalej-Kammoun a,1, Hela Zouari-Mechichi a,1, Lassaad Belbahri b, Steve Woodward c,Tahar Mechichi a,*

a Ecole Nationale d’Ingeieurs de Sfax, Route de Soukra Km 4.5, BP 1173, 3038 Sfax, Tunisiab Laboratory of Applied Genetics, School of Engineering of Lullier, Jussy, Switzerlandc University of Aberdeen, Institute of Biological and Environmental Sciences, Department of Plant and Soil Science, Cruickshank Building, St. Machar Drive,Aberdeen AB24 3UU, Scotland, UK

a r t i c l e i n f o

Article history:Received 7 March 2009Received in revised form31 March 2009Accepted 6 April 2009Available online 14 May 2009

Keywords:LaccaseDyesMalachite greenDecolourizationDetoxificationStability

* Corresponding author. Tel.: þ216 74 274 088; faxE-mail address: tahar.mechichi@enis.rnu.tn (T. Me

1 The authors have contributed equally to this wor

0964-8305/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.ibiod.2009.04.003

a b s t r a c t

The decolourization and detoxification of the triarylmethane dye Malachite green (MG) by laccase fromTrametes sp. were investigated. The laccase decolorized efficiently the dye down to 97% of 50 mg L�1

initial concentration of MG when only 0.1 U mL�1 of laccase was used in the reaction mixture. The effectsof different physicochemical parameters were tested and optimal decolourization rates occurred at pH 6and at temperatures between 50 and 60 �C. Decolourization of MG occurred in the presence of metal ionswhich could be found in textile industry effluent. 1-hydroxybenzotriazole (HBT) affected positively thedecolourization of MG. The presence of some phenolic compounds namely ferulic, coumaric, gallic, andtannic acids was found to be inhibiting for the decolourization at a concentration of 10 mM.The effect of laccase inhibitors in the decolourization of MG was tested with L-cysteine, and ethylenediamine tetra-acetic acid (EDTA) at concentrations of 0.1, 1 and 10 mM. It was demonstrated that L-cysteine and EDTA inhibited the decolourization starting from 1 mM concentration. However, for NaCla concentration of 100 mM was needed for the inhibition of laccase. The decolourization of MG resultedin the removal of its toxicity against Phanerochaete chrysosporium.The stability of the laccase toward temperature and HBT free radicals was also assessed during MGdecolourization. It was shown that laccase was stable at 50 �C but in the presence of the laccase mediatorHBT, the stability of the enzyme was severely affected resulting in a loss of 50% of the activity after 3 hincubation.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Approximately 10,000 different dyes and pigments areproduced annually worldwide and used extensively in the dye andprinting industries. Several of these dyes are very stable to light,temperature and microbial attack; many are also toxic. Syntheticdyes are chemically diverse, with those commonly used in industrydivided into azo, heterocyclic/polymeric structures or triphenyl-methanes (Gregory, 1993).

The triphenylmethane dye malachite green (MG) is extensivelyused as a biocide in aquaculture worldwide. It is highly effectiveagainst important protozoan and fungal infections of farmed fish(Hoffman and Meyer, 1974; Alderman, 1985). Aquaculture

: þ216 74 275 595.chichi).k.

All rights reserved.

industries have used malachite green extensively as a topicaltreatment in bath or flush methods, despite the potential for top-ically applied therapeutic agents to be absorbed and producesignificant internal effects. Malachite green is also used as a foodcolouring agent, food additive, and a medical disinfectant as well asa dye in the silk, wool, jute, leather, cotton, paper and acrylicindustries (Eichlerova et al., 2005). The compound has now becomehighly controversial, however, due to the risks it poses toconsumers of treated fish (Alderman and Clifton-Hadley, 1993)including its effects on the immune system and its genotoxiccarcinogenic properties (Rao, 1995). Approximately 10–14% of thetotal dye used in the dying process may be present in wastewater,causing serious pollution problems (Vaidya and Datye, 1982).Despite the existence of a variety of chemical and physical treat-ment processes, removal of the dye residues from the environmentis very difficult. A number of studies have focused on microor-ganisms capable of decolorizing and biodegrading these dyes

M. Maalej-Kammoun et al. / International Biodeterioration & Biodegradation 63 (2009) 600–606 601

(Wesenberg et al., 2003). In recent years, the possible utilization ofthe biodegradative abilities of some white rot fungi has shownsome promise. These fungi do not require preconditioning toparticular pollutants and, producing non-specific extracellular freeradical-based enzymatic systems, they can degrade to non detect-able levels or even completely eliminate a variety of xenobiotics,including synthetic dyes.

Many white rot fungi (e.g. Phanerochaete chrysosporium,Pleurotus ostreatus, Trametes versicolor) have been intensivelystudied in relation to lignolytic enzyme production and ability todecolorize complex dyes (Bumpus and Brock, 1988; Borchert andLibra, 2001; Moldes et al., 2003). This biodegradation capacity isassumed to result from the activities of numerous lignolytic andnon-specific enzymes secreted by these fungi, including ligninperoxidases (EC.1.11.1.14), manganese peroxidases (EC.1.11.1.13) andlaccases, of which laccases (EC 1.10.3.2) are the preferred targetenzymes (Kirk and Farrell, 1987). Laccases are used in variousbiotechnological and environmental applications (Riva, 2006),including the removal of toxic compounds from polluted effluentsthrough oxidative enzymatic coupling and precipitation ofcontaminants (Zille et al., 2005), or as biosensors for phenoliccompounds (Torrecilla et al., 2008). Laccases have been extensivelyused in delignification, demethylation, and bleaching of wood pulp(Bajpai, 2004; Bourbonnais et al., 1997; Camarero et al., 2007). Thecapacity of laccases to act on chromophore compounds has lead toapplications in industrial decolourisation processes (Champagneand Ramsay, 2007; Svobodova et al., 2008). The oxidation ofa reducing substrate by laccase typically involves formation ofa free (cation) radical after the transfer of a single electron to lac-case. The efficiency of this oxidative process depends on differencesin the redox potential between the reducing substrate and type 1Cu in laccase. Due to its rather low redox potential (0.5–0.8 V),laccase is able to attack only the phenolic moieties in the ligninpolymer, thus being less efficient than lignin peroxidases andmanganese-dependent lignin peroxidases in delignification andbleaching of pulp. As wood lignin macromolecules are composed ofphenolic (10–20%) and non-phenolic (80–90%) moieties, thecleavage of non-phenolic linkages is a necessary condition for lignindegradation. The substrate range of laccases can be expanded toinclude these non-phenolic compounds in the presence of smallmolecular weight mediators that are easily oxidized by the enzymeand in turn oxidize other substrates with redox potentials higherthan laccase or are of inappropriate size to fit the active centre ofthe enzyme. The advantage of the mediators, apart from acting aselectron shuttles between the enzyme and the substrates, is thatthey may follow an oxidation pathway different from that of theenzyme. Recent studies showed that laccase-mediator systems areable to oxidize non-phenolic lignin and even xenobioticcompounds (Morozova et al., 2007).

The aim of the present work was to examine the ability of crudelaccase preparations from Trametes sp. to decolorize and detoxifymalachite green in the presence of a mediator and to investigate thekinetics of this process. The stability of the enzyme towardtemperature and HBT radical was also investigated.

2. Material and methods

2.1. Chemicals

2,20-Azino-bis(3)-ethylbenzothiazoline-6-sulphonic acid (ABTS),2,6-dimethoxyphenol (DMP), 1-hydroxybenzotriazole (HBT) andphenolic compounds were obtained from Sigma–Aldrich. Thecationic basic dye malachite green oxalate (Basic Green 4), wasobtained from Panreac Co., Spain and used without further

purification. This dye was chosen as a model compound of triaryl-methane dyes.

2.2. Fungal strains, media and culture conditions

Trametes sp. CLBE55 and P. chrysosporium CLBE56, two newlyisolated fungal strains, were identified using ITS-sequence analysis.The fungal isolates are deposited in the culture collection of ourlaboratory. For short term conservation, isolates were maintainedon 2% malt extract and 1% agar plates, cultured at 30 �C and storedat 4 �C.

Laccase production by Trametes sp. was induced in basal liquidmedium (Munoz et al., 1997), containing (per litre): glucose, 10 g;peptone, 5 g; yeast extract, 1 g; ammonium tartrate, 2 g; KH2PO4,1 g; MgSO4$7H2O, 0.5 g; KCl, 0.5 g; trace element solution, 1 mL.The trace element solution comprised (per litre): B4O7Na2$10H2O,0.1 g; CuSO4$5H2O, 0.01 g; FeSO4$7H2O, 0.05 g; MnSO4$7H2O;0.01 g; ZnSO4$7H2O, 0.07 g; (NH4)6Mo7O24$4H2O, 0.01 g. The pH ofthe basal medium was adjusted to 5.5 before dispensing in 300 mlvolumes into 1 L Erlenmeyer flasks. After autoclaving at 105 kPa for20 min, 3 ml of homogenised mycelium were used for inoculationof the flasks. 150 mM of CuSO4 was added to the basal medium tostimulate the production of laccase. Cultures were incubated at30 �C on a rotary shaker (160 rpm).

2.3. Enzyme and protein assays

After centrifuging the medium, laccase activity was assayedusing 10 mM 2,6-dimethoxyphenol (DMP) in 100 mM ammoniumtartrate buffer, pH 5 (3469 nm ¼ 27,500 M cm�1, referenced to DMPconcentration) (Munoz et al., 1997). The reactions were carried outat room temperature (22–25 �C). One unit of laccase activity wasdefined as the amount of enzyme oxidizing 1 mmol of substratemin�1.

2.4. MG decolourisation by laccase

Unless otherwise indicated all experiments were performedusing 50 ml-disposable flasks in 5 ml final reaction volume. Thereaction mixture contained 100 mM tartrate buffer pH 5, 50 mg L�1

MG and 0.1 U mL�1 laccase from culture filtrate. The reaction wasinitiated by the addition of laccase and incubated in the dark at37 �C. The decolourisation of MG was followed by recording thespectra of the reaction mixture (between 400 and 700 nm) at30 min intervals, or by measuring absorbance at 600 nm. Allexperiments were performed in duplicate; controls did not containlaccase.

2.5. Effect of initial MG and enzyme concentration on MGdecolourisation by Trametes sp. laccase

The effect of MG concentration on decolourisation by laccasewas studied in a first experiment at initial concentrations of 5, 25,50, 100 or 200 mg L�1 in the reaction mixture, with 0.1 U mL�1

laccase in 100 mM tartrate buffer pH 5. The effect of enzymeconcentration on decolourisation was tested at different levels ofactivity (0.01, 0.05, 0.1, and 1 U mL�1) in a reaction mixture con-taining 50 mg L�1 MG. Absorbance of the reaction mixture wasrecorded at 60 min intervals.

2.6. Effect of pH and temperature on MG decolourization

To study the effect of pH on the decolourization of MG,50 mg L�1 of the dye was incubated at 37 �C in the presence of0.1 U mL�1 laccase at different pH values using the following

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Fig. 1. Effect of enzyme (a) and dye (b) concentrations on the decolourization of MG bythe laccase from Trametes sp. Enzyme concentrations: 1 U mL�1 (-); 0.1 U mL�1 (C);0, 05 U mL�1 (:); 0.05 U mL�1 (*). Dye concentrations: 25 mg L�1 (B); 50 mg L�1 (,);100 mg L�1 (6); 200 mg L�1 (>).

M. Maalej-Kammoun et al. / International Biodeterioration & Biodegradation 63 (2009) 600–606602

buffers: 100 mM citrate for pH 2 and 3, 100 mM tartrate for pH 4, 5and 6, phosphate for pH 7 and 8. The effect of temperature wasstudied by the incubation of 50 mg L�l MG in the presence of0.1 U mL�1 laccase at 20, 30, 37, 45 and 55 �C and pH 5.

2.7. Effect of phenolic compounds and HBT concentration on dyedecolourisation

The effect of aromatic compounds on dye decolourization wastested with o-vanillin, syringate, caffeate, gallate, tannic acid, p-coumarate, m-coumarate, ferulate and benzoate. All compoundswere used at a final concentration of 1 mM. The reaction mixturewas as described above (100 mM tartrate buffer pH 5, 50 mg L�1

MG, 37 �C and 0.1 U mL�1 laccase). To determine the effect of HBTon the decolourisation of MG, HBT concentration in the reactionmixture was varied between 0 and 4 mM (0, 1, 2, 3, 4 mM). Thereaction mixture was as described above (100 mM tartrate buffer,50 mg L�1 MG and 0.1 U mL�1 laccase).

2.8. Effect of metal ions and laccase inhibitors onthe decolourisation of MG

In order to determine the effect of metal ions on the decolour-isation of MG by laccase, 10 mM of: MnCl2, FeCl2, MgSO4, CaCl2,CuCl2 or H3BO3 were added to the reaction mixture which consistedof tartrate buffer 100 mM pH 5, 50 mg L�1 MG and 0.1 U mL�1

laccase followed by incubation at 37 �C. L-cysteine, Na2SO4, NaN3,NaCl and EDTA were tested as laccase inhibitors at concentrationsof 0.1, 1 and 10 mM. The reaction mixture consisted of tartratebuffer pH 5, 50 mg L�1 MG and 0.1 U mL�1 of laccase.

2.9. Capacity of laccase from Trametes sp. to inhibitthe toxicological effects of MG on fungi

2.9.1. Evidence for MG toxicity toward fungiToxicity of MG toward fungi was tested at concentrations of 5, 10

and 15 mg L�1, using both the Trametes sp. and P. chrysosporum.

2.9.2. Effect of pre-treatment of MG by laccase on thegrowth of fungi

The effect of the time of MG pre-treatment with laccase on theinhibitory effects against the growth of fungi was examined usingTrametes sp. and P. chrysosporium. MG was pre-treated with laccasefor between 0 and 6 h before incorporating into the culturemedium. Growth was determined by measuring radial growth ofthe fungi in Petri dishes over 7 days at 30 �C.

3. Results and discussion

3.1. Kinetics of MG decolourization by crude laccase from Trametes sp.

The ability of the laccase obtained from the recently isolatedTrametes sp. to decolourize MG was studied. Incubation of MG inthe presence of laccase from Trametes sp. resulted in a detectablereduction in absorbance at 595 nm of the reaction mixture within30 min of initiation. Absorbance at 595 nm continued to decreasewith time of incubation, associated with oxidation of the dye.Decolourization was 48% and 72% after 30 and 60 min of incubation,respectively, and complete decolourization occurred within 3 h.

3.2. Effect of enzyme and dye concentrations on thedecolourization of MG

At higher enzyme concentrations, decolourisation of the dyeoccurred rapidly (Fig. 1a), with approximately 80% decolourisation

within 2 h of initiation of the reaction. At enzyme concentrationslower than 0.05 U mL�1 no decolourisation of MG was observedeven after 5 h incubation. As the decolourisation obtained withenzyme concentrations 0.1 and 1 U mL�1 were very similar,0.1 U mL�1 was used in subsequent experiments.

At MG concentrations between 5 and 25 mg L�1, 91% dyedecolourisation occurred after 6 h of incubation (Fig. 1b). Atconcentrations of 100 mg L�1 and above, however, decolourisationdid not exceed 76%. This weak decolourisation may result frominhibition of the enzyme with excess of MG.

3.3. Effect of pH and temperature on MG decolourization

Decolourisation of MG occurred at pH 5 and 6, and was optimalat pH 6 (Fig. 2a). No decolourisation was observed at pH values of 2,3, 7 or 8. Slow decolourisation occurred at pH 4. Similar resultswere reported for purified laccase from Trametes trogii in ABTS andDMP (Zouari-Mechichi et al., 2005) and for RBBR (Mechichi et al.,2005). These results contrasted with those of Nyanhongo et al.(2001), however, who showed that the optimal pH for the decol-ourisation of the triarylmethane dyes acid violet 17 and basic red 9was between 3 and 4.5 for laccase from Trametes modesta, althoughdecolourisation did not exceed 65%. These differences in resultssuggest that the optimum pH for laccase catalysed oxidation maydepend on the type of dye used as substrate.

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Fig. 2. Effect of (a) pH and (b) temperature on the decolourization of MG by the laccaseof Trametes sp. pH values: 2 (C); 4 (:); 5 (A); 6 (-); 8 (*). Temperature values (�C):20 (,); 30 (þ); 37 (B); 45 (6); 55 (>).

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Fig. 3. Decolourization of MG in the presence of (a) metal ions and (b) laccaseinhibitors.

M. Maalej-Kammoun et al. / International Biodeterioration & Biodegradation 63 (2009) 600–606 603

The pH dependence for the decolourization of dyes by laccase ofthe Trametes sp. observed in the present work was similar to thatobserved for many other fungal laccases, acting on dyes and otherlaccase substrates (Eggert et al., 1996; Xu, 1996).

The rate of MG decolourisation increased with temperature,with an optimum (86%) at 55 �C (Fig. 3b). These results indicate thatthe rate of laccase catalysed decolourization of the dye increasedwith elevated temperatures up to 60 �C (data not shown).

3.4. Effect of metal ions and laccase inhibitors onMG decolourization

A previous study suggested that metal ions had little effect onlaccase stability at low concentrations (1–10 mM) (Zouari-Mechichiet al., 2005). As these properties may vary with different laccases,however, it is important to determine the effects of the presence ofsuch ions in the dye decolourization process.

In the presence of 10 mM Mg2þ, Mn2þ, Ca2þ, Fe2þ, B and Cu2þ

the decolourisation of MG by laccase from Trametes sp. was little orslightly (less than 25% inhibition) affected (Fig. 3a). These resultsare similar to those of Mechichi et al. (2005) for the decolourizationof Remazol brilliant blue R by laccase from T. trogii.

At 0.1 mM, EDTA had little effect on decolourization, althoughthe process was inhibited completely at 10 mM (Fig. 3b). Incontrast, cysteine affected the decolourisation process a concen-tration of 1 mM, completely inhibiting laccase activity at 10 mM.With NaCl, laccase activity was totally inhibited at 100 mM. When

NaN3 was added to MG solutions, the dye precipitated immediately(data not shown).

The most common laccase inhibitors used are dithiothreitol,thioglycolic acid, cysteine, diethyldithiocarbamic acid, EDTA, sodiumfluoride and sodium azide. These inhibitors are not laccase-specific,however, and their use in tests on phenoloxidases originates fromresults obtained with other metalloenzymes (Slomczynski et al.,1995).

3.5. Effect of HBT as redox mediator and phenolic compoundson MG decolourisation

Decolourisation rates increased with increasing HBT concen-trations from 1 to 5 mM, with an optimal concentration for dyedecolourisation of 4 mM. At this concentration, decolourisation was90% after incubation for 1 h (Fig. 4a).

These results agree with those of Nyanhongo et al. (2001) whodemonstrated that decolourisation of acid violet 17, a triaryl-methane dye, by laccase from T. modesta was enhanced 6 times inthe presence of HBT. The enhancement of dye decolourization inthe presence of HBT has also been reported for other laccases(Li et al., 1999).

Phenolic compounds have been studies in the past, to determinetheir possible role as natural laccase mediators (Camarero et al.,2005). In the current work, the effect of phenolic compounds onMG decolourisation was studied at concentrations of 1 mM. Amongthe phenolic compounds tested, benzoic acid, coumaric acid andcaffeic acid enhanced the kinetics of MG decolourisation, whereastannic, gallic, ferulic and hydroxycinnamic acids inhibited

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Fig. 4. Effect of (a) HBT and (b) phenolic compounds on the decolourization of MG.HBT concentrations: control (-); 1 mM (A); 2 mM (C); 3 mM (:); 4 mM (�).Phenolic compounds: BA: benzoic acid, SA: syringic acid, FA: ferulic acid, m-CA:m-coumaric acid, GA: gallic acid, VA: vanillic acid, CFA: caffeic acid, TA: tannic acid,p-CA: p-coumaric acid.

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Fig. 5. Stability of Trametes sp. laccase after incubation in the presence of MG and/orHBT at different temperatures (a) 50 �C, (b) 60 �C, (c) 70 �C. Laccase (C), laccase þ dye(:), laccase þ dye þ HBT (-), laccase þ HBT (*).

M. Maalej-Kammoun et al. / International Biodeterioration & Biodegradation 63 (2009) 600–606604

decolourisation. No effect was observed with o-vanillin or syringicacid (Fig. 4b).

Laccases are copper-containing enzymes that catalyse theoxidation of electron-rich substrates such as phenolic compounds.Laccase alone has a limited effect on bioremediation due to itsspecificity for the phenolic subunits in lignin. Recently however,Camarero et al. (2005) demonstrated that phenolic aldehydes,ketones, acids, and esters related to the three lignin units actedas laccase mediators, along with p-coumaric acid, vanillin, aceto-vanillone, methylvanillate, and above all, syringaldehyde andacetosyringone.

3.6. Stability of laccase at high temperatures and in the presenceof HBT

In the presence of HBT, Trametes sp. laccase lost approximately15% of the intial activity within 4 h at 60 �C; higher temperaturescaused a more rapid inactivation of the enzyme. In the presence ofHBT radicals, the loss in activity at 50 �C was almost linear andamounted 20% per hour (Fig. 5a). At 60 �C with HBT, the enzymelost its activity completely within 1 h (Fig. 5b). At 70 �C, the enzymelost 80% activity per hour (Fig. 5c). These results confirm that at

high temperatures, HBT negatively affects the activity of Trametessp. laccase.

The extent of inactivation with Trametes sp. observed in thiswork, however, was less than that reported by Li et al. (1999),who observed approximately 90% inactivation of laccase fromP. cinnabarinus within 2-h when incubated in the presence of either10-mM vanillic acid (VA) or HBT in 50-mM sodium acetate buffer atpH 4.5 at 30 �C.

In the presence of HBT, reactive compounds produce free radi-cals (Fabbrini et al., 2002a,b; d’Acunzo et al., 2002). The reactionmixture, therefore, will contain significant quantities of reactivespecies, including free radicals. Given that the mediators hadsignificant impacts on the stability of the enzyme, this evidence

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Fig. 6. Effect of MG (100 mg l�1 initial concentration) decolourization rate on thegrowth of (a) P. chrysosporium and (b) Trametes sp.: control (non treated MG) (*), MGdecolorized to: 50% (A), MG decolorized to 60% (-), MG decolorized to 70% (:), MGdecolorized to 84% (�), without MG (C).

M. Maalej-Kammoun et al. / International Biodeterioration & Biodegradation 63 (2009) 600–606 605

supports the hypothesis that laccase is subject to attack by freeradicals. Therefore, in a reacting system, where free-radical prod-ucts are generated continuously either through the transformationof a substrate or the continuous cycling of mediators, free radicalsmay play a critical role in laccase inactivation.

Collectively, these results suggest that mediators enhance theinactivation of laccase. As shown in sub-section 3.5, the use ofhigher concentrations of mediators may promote more rapidreactions, but can also cause higher degrees of enzyme inactivation.It is important, therefore, to limit mediator concentrations inreaction systems in order to maintain catalytic stability. This factorwill be particularly important in applications requiring long-termstability of a laccase-mediator system.

3.7. Detoxification of MG by the laccase of Trametes sp.

Malachite green is commonly used as a fungicide in aqua-culture (Alderman, 1985). When included in the culture medium(solid medium) at different concentrations, MG completelyinhibited the growth of P. chrysosporium, at concentrations of10 mg L�1 and above (data not shown). These results could beexplained by the fact that this fungus is highly sensitive to MG,even at very low concentrations of MG (Papinutti and Forchiassin,2004). In contrast, the Trametes sp. grew and decolourized the

dye at MG concentrations of 50 and 200 mg L�1. These resultsagree with those of Levin et al. (2003), who demonstrated thatP. chrysosporium did not grow in MG at concentrations in excessof 6 mg L�1 of dye. High laccase-titer producing fungi, however,including T. troggi and Fomes sclerodermeus could grow in muchhigher concentrations of MG.

Both P. chrysosporium and Trametes sp. grew on agar containinglaccase-treated MG (Fig. 6a, b). Trametes sp. was less sensitive thanP. chrysosporium to MG. Moreover, these results provide furtherevidence for the detoxification of MG by laccase.

The inhibition of growth of P. chrysosporium could be due itshigh sensitivity to MG and not to non laccase production (Podg-ornik et al., 2001) since laccase production has demonstrated forthis fungi in several studies (Srinivasan et al., 1995; Levin et al.,2004) and even enhanced by the addition of inducers in otherstudies (Gnanamani et al., 2006).

4. Conclusion

In terms of the overall decolourization performance, it is clearthat laccase from the incompletely characterised Trametes sp.showed high potential to transform malachite green to colourlesscompounds. The system appeared to provide a biocatalyst for thedecolourization of this dye.

MG was decolorized by the Trametes sp. laccase most efficientlyunder acid conditions (pH 5–6). Decolourization by this laccaseincreased with temperature to 50–60 �C and in the presence of HBTasa mediator. Some phenolic compounds, however, were not efficientmediators for Trametes sp. laccase. Moreover, treatment with Tra-metes sp. laccase reduced the toxicity of malachite green against fungi.

Trametes sp. laccase was stable at temperatures up to 60 �C,although the presence of HBT radicals in the reaction mixture hada serious negative impact on enzyme activity, particularly at highertemperatures.

It is evident that the laccase of Trametes sp. may be used fordecolourization of textile dyestuffs, particularly those containingtriarylmethane dyes, in effluent treatment, and bioremediation oras a bleaching agent.

Acknowledgements

This work was supported in part by a grant provided by IFS‘‘International foundation for science’’.

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