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

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

International Biodeterioration & Biodegradation 63 (2009) 600–606 Contents lists available at ScienceDirect International Biodeterioration & Biodegr...

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International Biodeterioration & Biodegradation 63 (2009) 600–606

Contents lists available at ScienceDirect

International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod

Malachite green decolourization and detoxification by the laccase from a newly 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’Inge´ieurs de Sfax, Route de Soukra Km 4.5, BP 1173, 3038 Sfax, Tunisia Laboratory of Applied Genetics, School of Engineering of Lullier, Jussy, Switzerland 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 b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 March 2009 Received in revised form 31 March 2009 Accepted 6 April 2009 Available online 14 May 2009

The decolourization and detoxification of the triarylmethane dye Malachite green (MG) by laccase from Trametes sp. were investigated. The laccase decolorized efficiently the dye down to 97% of 50 mg L1 initial concentration of MG when only 0.1 U mL1 of laccase was used in the reaction mixture. The effects of different physicochemical parameters were tested and optimal decolourization rates occurred at pH 6 and at temperatures between 50 and 60  C. Decolourization of MG occurred in the presence of metal ions which could be found in textile industry effluent. 1-hydroxybenzotriazole (HBT) affected positively the decolourization of MG. The presence of some phenolic compounds namely ferulic, coumaric, gallic, and tannic 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 ethylene diamine tetra-acetic acid (EDTA) at concentrations of 0.1, 1 and 10 mM. It was demonstrated that Lcysteine and EDTA inhibited the decolourization starting from 1 mM concentration. However, for NaCl a concentration of 100 mM was needed for the inhibition of laccase. The decolourization of MG resulted in the removal of its toxicity against Phanerochaete chrysosporium. The stability of the laccase toward temperature and HBT free radicals was also assessed during MG decolourization. It was shown that laccase was stable at 50  C but in the presence of the laccase mediator HBT, the stability of the enzyme was severely affected resulting in a loss of 50% of the activity after 3 h incubation. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Laccase Dyes Malachite green Decolourization Detoxification Stability

1. Introduction Approximately 10,000 different dyes and pigments are produced annually worldwide and used extensively in the dye and printing industries. Several of these dyes are very stable to light, temperature and microbial attack; many are also toxic. Synthetic dyes are chemically diverse, with those commonly used in industry divided into azo, heterocyclic/polymeric structures or triphenylmethanes (Gregory, 1993). The triphenylmethane dye malachite green (MG) is extensively used as a biocide in aquaculture worldwide. It is highly effective against important protozoan and fungal infections of farmed fish (Hoffman and Meyer, 1974; Alderman, 1985). Aquaculture * Corresponding author. Tel.: þ216 74 274 088; fax: þ216 74 275 595. E-mail address: [email protected] (T. Mechichi). 1 The authors have contributed equally to this work. 0964-8305/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2009.04.003

industries have used malachite green extensively as a topical treatment in bath or flush methods, despite the potential for topically applied therapeutic agents to be absorbed and produce significant internal effects. Malachite green is also used as a food colouring agent, food additive, and a medical disinfectant as well as a dye in the silk, wool, jute, leather, cotton, paper and acrylic industries (Eichlerova et al., 2005). The compound has now become highly controversial, however, due to the risks it poses to consumers of treated fish (Alderman and Clifton-Hadley, 1993) including its effects on the immune system and its genotoxic carcinogenic properties (Rao, 1995). Approximately 10–14% of the total 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 treatment processes, removal of the dye residues from the environment is very difficult. A number of studies have focused on microorganisms capable of decolorizing and biodegrading these dyes

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(Wesenberg et al., 2003). In recent years, the possible utilization of the biodegradative abilities of some white rot fungi has shown some promise. These fungi do not require preconditioning to particular pollutants and, producing non-specific extracellular free radical-based enzymatic systems, they can degrade to non detectable 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 intensively studied in relation to lignolytic enzyme production and ability to decolorize complex dyes (Bumpus and Brock, 1988; Borchert and Libra, 2001; Moldes et al., 2003). This biodegradation capacity is assumed to result from the activities of numerous lignolytic and non-specific enzymes secreted by these fungi, including lignin peroxidases (EC.1.11.1.14), manganese peroxidases (EC.1.11.1.13) and laccases, of which laccases (EC 1.10.3.2) are the preferred target enzymes (Kirk and Farrell, 1987). Laccases are used in various biotechnological and environmental applications (Riva, 2006), including the removal of toxic compounds from polluted effluents through oxidative enzymatic coupling and precipitation of contaminants (Zille et al., 2005), or as biosensors for phenolic compounds (Torrecilla et al., 2008). Laccases have been extensively used in delignification, demethylation, and bleaching of wood pulp (Bajpai, 2004; Bourbonnais et al., 1997; Camarero et al., 2007). The capacity of laccases to act on chromophore compounds has lead to applications in industrial decolourisation processes (Champagne and Ramsay, 2007; Svobodova et al., 2008). The oxidation of a reducing substrate by laccase typically involves formation of a free (cation) radical after the transfer of a single electron to laccase. The efficiency of this oxidative process depends on differences in the redox potential between the reducing substrate and type 1 Cu 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 lignin polymer, thus being less efficient than lignin peroxidases and manganese-dependent lignin peroxidases in delignification and bleaching of pulp. As wood lignin macromolecules are composed of phenolic (10–20%) and non-phenolic (80–90%) moieties, the cleavage of non-phenolic linkages is a necessary condition for lignin degradation. The substrate range of laccases can be expanded to include these non-phenolic compounds in the presence of small molecular weight mediators that are easily oxidized by the enzyme and in turn oxidize other substrates with redox potentials higher than laccase or are of inappropriate size to fit the active centre of the enzyme. The advantage of the mediators, apart from acting as electron shuttles between the enzyme and the substrates, is that they may follow an oxidation pathway different from that of the enzyme. Recent studies showed that laccase-mediator systems are able to oxidize non-phenolic lignin and even xenobiotic compounds (Morozova et al., 2007). The aim of the present work was to examine the ability of crude laccase preparations from Trametes sp. to decolorize and detoxify malachite green in the presence of a mediator and to investigate the kinetics of this process. The stability of the enzyme toward temperature 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) and phenolic compounds were obtained from Sigma–Aldrich. The cationic basic dye malachite green oxalate (Basic Green 4), was obtained from Panreac Co., Spain and used without further

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purification. This dye was chosen as a model compound of triarylmethane dyes. 2.2. Fungal strains, media and culture conditions Trametes sp. CLBE55 and P. chrysosporium CLBE56, two newly isolated fungal strains, were identified using ITS-sequence analysis. The fungal isolates are deposited in the culture collection of our laboratory. For short term conservation, isolates were maintained on 2% malt extract and 1% agar plates, cultured at 30  C and stored at 4  C. Laccase production by Trametes sp. was induced in basal liquid ˜ oz et al., 1997), containing (per litre): glucose, 10 g; medium (Mun 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 of the basal medium was adjusted to 5.5 before dispensing in 300 ml volumes into 1 L Erlenmeyer flasks. After autoclaving at 105 kPa for 20 min, 3 ml of homogenised mycelium were used for inoculation of the flasks. 150 mM of CuSO4 was added to the basal medium to stimulate the production of laccase. Cultures were incubated at 30  C on a rotary shaker (160 rpm). 2.3. Enzyme and protein assays After centrifuging the medium, laccase activity was assayed using 10 mM 2,6-dimethoxyphenol (DMP) in 100 mM ammonium tartrate buffer, pH 5 (3469 nm ¼ 27,500 M cm1, referenced to DMP ˜ oz et al., 1997). The reactions were carried out concentration) (Mun at room temperature (22–25  C). One unit of laccase activity was defined as the amount of enzyme oxidizing 1 mmol of substrate min1. 2.4. MG decolourisation by laccase Unless otherwise indicated all experiments were performed using 50 ml-disposable flasks in 5 ml final reaction volume. The reaction mixture contained 100 mM tartrate buffer pH 5, 50 mg L1 MG and 0.1 U mL1 laccase from culture filtrate. The reaction was initiated by the addition of laccase and incubated in the dark at 37  C. The decolourisation of MG was followed by recording the spectra of the reaction mixture (between 400 and 700 nm) at 30 min intervals, or by measuring absorbance at 600 nm. All experiments were performed in duplicate; controls did not contain laccase. 2.5. Effect of initial MG and enzyme concentration on MG decolourisation by Trametes sp. laccase The effect of MG concentration on decolourisation by laccase was studied in a first experiment at initial concentrations of 5, 25, 50, 100 or 200 mg L1 in the reaction mixture, with 0.1 U mL1 laccase in 100 mM tartrate buffer pH 5. The effect of enzyme concentration on decolourisation was tested at different levels of activity (0.01, 0.05, 0.1, and 1 U mL1) in a reaction mixture containing 50 mg L1 MG. Absorbance of the reaction mixture was recorded 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 L1 of the dye was incubated at 37  C in the presence of 0.1 U mL1 laccase at different pH values using the following

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buffers: 100 mM citrate for pH 2 and 3, 100 mM tartrate for pH 4, 5 and 6, phosphate for pH 7 and 8. The effect of temperature was studied by the incubation of 50 mg Ll MG in the presence of 0.1 U mL1 laccase at 20, 30, 37, 45 and 55  C and pH 5.

a

2.7. Effect of phenolic compounds and HBT concentration on dye decolourisation

Decolourization (%)

602

2.8. Effect of metal ions and laccase inhibitors on the decolourisation of MG In order to determine the effect of metal ions on the decolourisation of MG by laccase, 10 mM of: MnCl2, FeCl2, MgSO4, CaCl2, CuCl2 or H3BO3 were added to the reaction mixture which consisted of tartrate buffer 100 mM pH 5, 50 mg L1 MG and 0.1 U mL1 laccase followed by incubation at 37  C. L-cysteine, Na2SO4, NaN3, NaCl and EDTA were tested as laccase inhibitors at concentrations of 0.1, 1 and 10 mM. The reaction mixture consisted of tartrate buffer pH 5, 50 mg L1 MG and 0.1 U mL1 of laccase. 2.9. Capacity of laccase from Trametes sp. to inhibit the toxicological effects of MG on fungi

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The effect of aromatic compounds on dye decolourization was tested with o-vanillin, syringate, caffeate, gallate, tannic acid, pcoumarate, m-coumarate, ferulate and benzoate. All compounds were used at a final concentration of 1 mM. The reaction mixture was as described above (100 mM tartrate buffer pH 5, 50 mg L1 MG, 37  C and 0.1 U mL1 laccase). To determine the effect of HBT on the decolourisation of MG, HBT concentration in the reaction mixture was varied between 0 and 4 mM (0, 1, 2, 3, 4 mM). The reaction mixture was as described above (100 mM tartrate buffer, 50 mg L1 MG and 0.1 U mL1 laccase).

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Time (h) 2.9.1. Evidence for MG toxicity toward fungi Toxicity of MG toward fungi was tested at concentrations of 5, 10 and 15 mg L1, using both the Trametes sp. and P. chrysosporum. 2.9.2. Effect of pre-treatment of MG by laccase on the growth of fungi The effect of the time of MG pre-treatment with laccase on the inhibitory effects against the growth of fungi was examined using Trametes sp. and P. chrysosporium. MG was pre-treated with laccase for between 0 and 6 h before incorporating into the culture medium. Growth was determined by measuring radial growth of the 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 isolated Trametes sp. to decolourize MG was studied. Incubation of MG in the presence of laccase from Trametes sp. resulted in a detectable reduction in absorbance at 595 nm of the reaction mixture within 30 min of initiation. Absorbance at 595 nm continued to decrease with 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 the decolourization of MG At higher enzyme concentrations, decolourisation of the dye occurred rapidly (Fig. 1a), with approximately 80% decolourisation

Fig. 1. Effect of enzyme (a) and dye (b) concentrations on the decolourization of MG by the laccase from Trametes sp. Enzyme concentrations: 1 U mL1 (-); 0.1 U mL1 (C); 0, 05 U mL1 (:); 0.05 U mL1 (*). Dye concentrations: 25 mg L1 (B); 50 mg L1 (,); 100 mg L1 (6); 200 mg L1 (>).

within 2 h of initiation of the reaction. At enzyme concentrations lower than 0.05 U mL1 no decolourisation of MG was observed even after 5 h incubation. As the decolourisation obtained with enzyme concentrations 0.1 and 1 U mL1 were very similar, 0.1 U mL1 was used in subsequent experiments. At MG concentrations between 5 and 25 mg L1, 91% dye decolourisation occurred after 6 h of incubation (Fig. 1b). At concentrations of 100 mg L1 and above, however, decolourisation did not exceed 76%. This weak decolourisation may result from inhibition 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 optimal at pH 6 (Fig. 2a). No decolourisation was observed at pH values of 2, 3, 7 or 8. Slow decolourisation occurred at pH 4. Similar results were reported for purified laccase from Trametes trogii in ABTS and DMP (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 decolourisation of the triarylmethane dyes acid violet 17 and basic red 9 was between 3 and 4.5 for laccase from Trametes modesta, although decolourisation did not exceed 65%. These differences in results suggest that the optimum pH for laccase catalysed oxidation may depend on the type of dye used as substrate.

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

The pH dependence for the decolourization of dyes by laccase of the Trametes sp. observed in the present work was similar to that observed for many other fungal laccases, acting on dyes and other laccase 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 that the rate of laccase catalysed decolourization of the dye increased with elevated temperatures up to 60  C (data not shown). 3.4. Effect of metal ions and laccase inhibitors on MG decolourization A previous study suggested that metal ions had little effect on laccase stability at low concentrations (1–10 mM) (Zouari-Mechichi et al., 2005). As these properties may vary with different laccases, however, it is important to determine the effects of the presence of such 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 or slightly (less than 25% inhibition) affected (Fig. 3a). These results are similar to those of Mechichi et al. (2005) for the decolourization of Remazol brilliant blue R by laccase from T. trogii. At 0.1 mM, EDTA had little effect on decolourization, although the process was inhibited completely at 10 mM (Fig. 3b). In contrast, cysteine affected the decolourisation process a concentration of 1 mM, completely inhibiting laccase activity at 10 mM. With NaCl, laccase activity was totally inhibited at 100 mM. When

control

Fe

Mg

Mn

Ca

Cu

B

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

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, sodium fluoride and sodium azide. These inhibitors are not laccase-specific, however, and their use in tests on phenoloxidases originates from results obtained with other metalloenzymes (Slomczynski et al., 1995). 3.5. Effect of HBT as redox mediator and phenolic compounds on MG decolourisation Decolourisation rates increased with increasing HBT concentrations from 1 to 5 mM, with an optimal concentration for dye decolourisation of 4 mM. At this concentration, decolourisation was 90% after incubation for 1 h (Fig. 4a). These results agree with those of Nyanhongo et al. (2001) who demonstrated that decolourisation of acid violet 17, a triarylmethane dye, by laccase from T. modesta was enhanced 6 times in the presence of HBT. The enhancement of dye decolourization in the presence of HBT has also been reported for other laccases (Li et al., 1999). Phenolic compounds have been studies in the past, to determine their possible role as natural laccase mediators (Camarero et al., 2005). In the current work, the effect of phenolic compounds on MG decolourisation was studied at concentrations of 1 mM. Among the phenolic compounds tested, benzoic acid, coumaric acid and caffeic acid enhanced the kinetics of MG decolourisation, whereas tannic, gallic, ferulic and hydroxycinnamic acids inhibited

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FA m-C A GA VA CF A

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Decolourization (%) 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.

decolourisation. No effect was observed with o-vanillin or syringic acid (Fig. 4b). Laccases are copper-containing enzymes that catalyse the oxidation of electron-rich substrates such as phenolic compounds. Laccase alone has a limited effect on bioremediation due to its specificity 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 acted as laccase mediators, along with p-coumaric acid, vanillin, acetovanillone, methylvanillate, and above all, syringaldehyde and acetosyringone.

3.6. Stability of laccase at high temperatures and in the presence of HBT In the presence of HBT, Trametes sp. laccase lost approximately 15% of the intial activity within 4 h at 60  C; higher temperatures caused a more rapid inactivation of the enzyme. In the presence of HBT radicals, the loss in activity at 50  C was almost linear and amounted 20% per hour (Fig. 5a). At 60  C with HBT, the enzyme lost its activity completely within 1 h (Fig. 5b). At 70  C, the enzyme lost 80% activity per hour (Fig. 5c). These results confirm that at

c Residual activity (%)

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Time (h) Fig. 5. Stability of Trametes sp. laccase after incubation in the presence of MG and/or HBT 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 (*).

high temperatures, HBT negatively affects the activity of Trametes sp. laccase. The extent of inactivation with Trametes sp. observed in this work, however, was less than that reported by Li et al. (1999), who observed approximately 90% inactivation of laccase from P. cinnabarinus within 2-h when incubated in the presence of either 10-mM vanillic acid (VA) or HBT in 50-mM sodium acetate buffer at pH 4.5 at 30  C. In the presence of HBT, reactive compounds produce free radicals (Fabbrini et al., 2002a,b; d’Acunzo et al., 2002). The reaction mixture, therefore, will contain significant quantities of reactive species, including free radicals. Given that the mediators had significant impacts on the stability of the enzyme, this evidence

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a

dye at MG concentrations of 50 and 200 mg L1. These results agree with those of Levin et al. (2003), who demonstrated that P. chrysosporium did not grow in MG at concentrations in excess of 6 mg L1 of dye. High laccase-titer producing fungi, however, including T. troggi and Fomes sclerodermeus could grow in much higher concentrations of MG. Both P. chrysosporium and Trametes sp. grew on agar containing laccase-treated MG (Fig. 6a, b). Trametes sp. was less sensitive than P. chrysosporium to MG. Moreover, these results provide further evidence for the detoxification of MG by laccase. The inhibition of growth of P. chrysosporium could be due its high sensitivity to MG and not to non laccase production (Podgornik et al., 2001) since laccase production has demonstrated for this fungi in several studies (Srinivasan et al., 1995; Levin et al., 2004) and even enhanced by the addition of inducers in other studies (Gnanamani et al., 2006).

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Time (d) Fig. 6. Effect of MG (100 mg l1 initial concentration) decolourization rate on the growth of (a) P. chrysosporium and (b) Trametes sp.: control (non treated MG) (*), MG decolorized to: 50% (A), MG decolorized to 60% (-), MG decolorized to 70% (:), MG decolorized to 84% (), without MG (C).

In terms of the overall decolourization performance, it is clear that laccase from the incompletely characterised Trametes sp. showed high potential to transform malachite green to colourless compounds. The system appeared to provide a biocatalyst for the decolourization of this dye. MG was decolorized by the Trametes sp. laccase most efficiently under acid conditions (pH 5–6). Decolourization by this laccase increased with temperature to 50–60  C and in the presence of HBT as a mediator. Some phenolic compounds, however, were not efficient mediators for Trametes sp. laccase. Moreover, treatment with Trametes 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 had a serious negative impact on enzyme activity, particularly at higher temperatures. It is evident that the laccase of Trametes sp. may be used for decolourization of textile dyestuffs, particularly those containing triarylmethane dyes, in effluent treatment, and bioremediation or as a bleaching agent. Acknowledgements

supports the hypothesis that laccase is subject to attack by free radicals. Therefore, in a reacting system, where free-radical products are generated continuously either through the transformation of a substrate or the continuous cycling of mediators, free radicals may play a critical role in laccase inactivation. Collectively, these results suggest that mediators enhance the inactivation of laccase. As shown in sub-section 3.5, the use of higher concentrations of mediators may promote more rapid reactions, but can also cause higher degrees of enzyme inactivation. It is important, therefore, to limit mediator concentrations in reaction systems in order to maintain catalytic stability. This factor will be particularly important in applications requiring long-term stability 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 aquaculture (Alderman, 1985). When included in the culture medium (solid medium) at different concentrations, MG completely inhibited the growth of P. chrysosporium, at concentrations of 10 mg L1 and above (data not shown). These results could be explained 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

This work was supported in part by a grant provided by IFS ‘‘International foundation for science’’. References Alderman, D.J., 1985. Malachite green: a review. Journal of Fish Diseases 8, 289–298. Alderman, D.J., Clifton-Hadley, R.S., 1993. Malachite green: a pharmacokinetic study in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 16, 297–311. Bajpai, P., 2004. Biological bleaching of chemical pulps. Critical Review in Biotechnology 24, 1–58. Borchert, M., Libra, J.A., 2001. Decolorization of reactive dyes by the white rot fungus Trametes versicolor in sequencing batch reactors. Biotechnology and Bioengineering 75, 313–321. Bourbonnais, R., Paice, M.G., Freiermuth, B., Bodie, E., Borneman, S., 1997. Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Applied and Environmental Microbiology 63, 4627–4632. Bumpus, J.A., Brock, B.J., 1988. Biodegradation of crystal violet by the white rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology 54, 1143–1150. Camarero, S., Ibarra, D., Martınez, M.J., Martınez, A.T., 2005. Lignin derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Applied and Environmental Microbiology 71, 1775–1784. Camarero, S., Ibarra, D., Martı´nez, A.T., Romero, J., Gutie´rrez, A., del Rı´o, J.C., 2007. Paper pulp delignification using laccase and natural mediators. Enzyme and Microbial Technology 40, 1264–1271. Champagne, P.P., Ramsay, J.A., 2007. Reactive blue 19 decolouration by laccase immobilized on silica beads. Applied Microbiology and Biotechnology 77, 819–823.

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