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Purification and characterization of a novel laccase from the ascomycete

Process Biochemistry 45 (2010) 507–513

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Puri?cation and characterization of a novel laccase from the ascomycete Trichoderma atroviride: Application on bioremediation of phenolic compounds
Hanen Chakroun a, Tahar Mechichi a,*, Maria Jesus Martinez b, Abdelha?dh Dhouib a, Sami Sayadi a
a b

?de Laboratoire des Bioproce ?s, Centre de Biotechnologie de Sfax BP ‘‘1177’’, 3018 Sfax, Tunisia ?gicas (CSIC), Ramiro de Maeztu 9, E-28040 Madrid, Spain Centro de Investigaciones Biolo



Article history: Received 4 August 2009 Received in revised form 31 October 2009 Accepted 12 November 2009 Keywords: Laccase Ascomycete Phenolic compounds Bioremediation

The extracellular laccase produced by the ascomycete Trichoderma atroviride was puri?ed and characterized and its ability to transform phenolic compounds was determined. The puri?ed laccase had activity towards typical substrates of laccases including 2,20 -azinobis-(3-ethylbenzthiazoline-6sulphonate) (ABTS), dimethoxyphenol (2,6-DMP), syringaldazine and hydroquinone. The enzyme was a monomeric protein with an apparent molecular mass of 80 kDa and an isoelectric point of 3.5. The pH optima for the oxidation of ABTS and 2,6-DMP were 3 and 5, respectively, and the optimum temperature was 50 8C with 2,6-DMP. The laccase was stable at slightly acidic pH (4 and 5). It retained 80% of its activity after 4 h incubation at 40 8C. Under standard assay conditions, Km values of the enzyme were 2.5 and 1.6 mM towards ABTS and 2,6-DMP, respectively. This enzyme was able to oxidize aromatic compounds present in industrial and agricultural wastewater, as catechol and o-cresol, although the transformation of chlorinated phenols required the presence of ABTS as mediator. ? 2009 Elsevier Ltd. All rights reserved.

1. Introduction Fungi of the genus Trichoderma have been studied mainly as biological control agents against a number of soilborn pathogens [1,2]. They are remarkable for their rapid growth, capability of utilizing diverse substrates, and resistance to noxious chemicals. They are often predominant components of the myco?ora in various soils, where they are signi?cant decomposers of woody and herbaceous materials, and are also necrotrophic against the primary wood decomposers [3]. The mycoparasitic activity of this organism is attributed to a production of cell wall-degrading enzymes such as chitinase and b-glucanase [4]. Several strains of the genus Trichoderma are being tested as alternatives to chemical fungicides [5]. A species of Trichoderma isolated from natural sources was described as a ligninolytic strain [6] and used in degradation of humic acid and solubilization of low-rank coal [7]. In addition, recently has been discovered cell wall-associated laccases in conidia of T. atroviride and T. harzianum [8]. However, none of these Trichoderma laccases have been characterized with respect to their substrate speci?city or amino acid sequence. The production of laccases by Trichoderma species is very interesting

? * Corresponding author. Present address: Ecole Nationale d’Ingenieurs de Sfax, Route de Soukra Km 4.5, BP 1173, 3038 Sfax, Tunisia. Tel.: +216 74 274 088; fax: +216 74 275 595. E-mail addresses: tahar.mechichi@enis.rnu.tn, tahar98@yahoo.com (T. Mechichi). 1359-5113/$ – see front matter ? 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.11.009

because it secretes very high amounts of cellulases and is currently used for the production of industrial enzymes. The combination of laccases with cellulases enlarges the application of such enzymes on many industries. In fact, Xu et al. [9] suggested that there is a synergistic deligni?cation effect between these two enzymes and consequently demonstrated their ef?ciency in enhancing newspaper deinking processes. In addition, laccases cooperate with other enzymes in the degradation of lignocellulosic materials as well as in several other functions including coal solubilization [8]. Thus, it was of interest to know whether Trichoderma strains with phenol oxidase activity (laccase activity) would be capable of more extensive natural substrate hydrolysis. Laccase (E.C., p-benzenediol:oxygen oxidoreductase) is a copper-protein belonging to a small group of enzymes denominated blue oxidas. Laccase is an oxidoreductase able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water [10]. Moreover, in the presence of primary substrates which act as electron transfer mediators, such as 2,20 -azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), or 1-hydroxybenzotriazole (1-HBT), the substrate range can be extended to non-phenolic compounds [11]. Laccase or laccase-like activity has been demonstrated in higher plants, some insects and a few bacteria [12]. However, most known laccases are from fungi, especially from the white rot fungi. Finally mention that although most laccases have been characterized from white rot basidiomycetes, other groups of fungi producing laccases but they have been studied to a much lesser extent [13].


H. Chakroun et al. / Process Biochemistry 45 (2010) 507–513 and 2 mL samples were applied to a Mono-Q-anion exchange column (Pharmacia HR 5/5) equilibrated with the same buffer. Retained proteins were eluted with 10 mM sodium tartrate buffer at pH 4.5 (buffer A) and 1 M NaCl in the same buffer (buffer B) at a ?ow rate of 1 mL min?1 with the following elution gradient (expressed as percentage of buffer B): 0%, 5 min; 0–20%, 30 min; 20–100%, 5 min and 100%, 2 min. 2.4. Properties of puri?ed laccase The molecular mass of the laccase was determined by SDS/PAGE and gel ?ltration. SDS/PAGE was performed with 10% polyacrylamide gels, using high-molecular-mass standards (Bio-Rad): myosin (200 kDa), b-galactosidase (116.25 kDa), Phosphorylase b (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). Gel ?ltration chromatography was carried out on analytic Bio-Sil1 SEC column (Bio-Rad), calibrated with thyroglobulin (670 kDa), IgG (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (13.5 kDa). 10 mmol L?1 phosphate buffer (pH 6.5) containing 150 mmol L?1 NaCl was used as the mobile phase at a ?ow rate of 1 mL min?1. Protein was N-deglycosylated with 125 mU mL?1 of endoglycosidase H (endo-H, Boehringer). SDS-PAGE of native and deglycosylated proteins was performed in 7.5% polyacrylamide gels using high-molecular-mass standards from Bio-Rad. Isoelectrofocusing was performed on 5% polyacrylamide gels with a thickness of 1 mm and a pH gradient from 2.5 to 5.5 (prepared with Pharmacia Ampholine by mixing 85% from pH 2.5 to 5 and 15% from pH 3.5 to 10). The anode and cathode solutions were 1 M phosphoric acid and 1 M sodium hydroxide, respectively. The pH gradient formed was measured on the gel by means of a contact electrode. Protein band after SDS-PAGE and isoelectrofocusing were stained with Coomassie blue R-250. Zymograms were obtained using 10 mM 2,6-DMP in 100 mM sodium tartrate buffer, pH 5, after washing the gels for 10 min with the same buffer [20]. The N-terminal sequence was determined by automated Edman’s degradation, using an Applied Biosystems Protein Sequencer Procise 492 cLC [21]. 2.5. Effect of temperature, pH and heavy metals on laccase activity Optimum temperature for the activity of the laccase was determined by carrying out the laccase assay with 2,6-DMP at selected constant temperatures ranging from 20 to 70 8C. In each case the substrate was preincubated at the required temperature. The pH optimum was determined at a ?xed assay temperature of 30 8C at various pH between 2 and 10 using citrate–phosphate–borate buffer (100 mM). The activity of laccase was tested in the presence of several metal ions including Cu2+, Mg2+, Mo6+, Ni2+, Co2+, Li+, Ca2+, Mn2+, Cd2+ and Al3+. Two concentrations were tested: 10 and 100 mM. The residual activities were determined using the 2,6-DMP assay. 2.6. Effect of temperature, pH and metal ions on laccase stability The effect of temperature on laccase stability was determined by incubating the laccase solution at various temperatures ranging from 4 to 60 8C and then determining the residual laccase activity with the 2,6-DMP assay method. The laccase solutions were incubated on sodium acetate buffer (100 mM, pH 4.5). The pH stability of the puri?ed laccase was conducted in citrate-phosphate-borate buffer (pH range between 2 and 9). The stability of laccase against several metal compounds, some of which are normally present in wastewater ef?uents, was assessed. For this, laccase was incubated with the salts of the above-mentioned ions at concentrations of 10 mM at 4 8C. Samples were collected after 24 h incubation, and residual activities were determined using the 2,6-DMP assay. 2.7. Substrate speci?city and effect of inhibitors Substrate oxidation by the laccase of T. atroviride was examined spectrophotometrically at the speci?c wavelength of each substrate in a sodium acetate buffer (100 mM, pH 4.5). The laccase concentration used for the oxidation of each substrate was the same (0.2 U mL?1). These substrates are ABTS, 2,6-DMP, syringaldazine, and hydroquinone. These substrates were used at a concentration of 5 mM. Effects of potential inhibitors of laccase activity were determined using 2,6-DMP (5 mM) as substrate in a sodium acetate buffer (100 mM, pH 4.5) and the presence of an inhibitor. The following substances are used as inhibitors: NaN3, NaBr, Lcysteine, p-coumarate, EDTA and SDS. Kinetic constants were determined for ABTS and 2,6-DMP in 100 mM sodium acetate buffer, pH 4.5. 2.8. Transformation of aromatic pollutants by laccase from T. atroviride Pollutant transformation by T. atroviride laccase was investigated in 100 mM sodium acetate buffer, pH 4.5, containing 500 mM 2,4-dichlorophenoxyacetic acid (2,4-D), 4-chlorophenol, o-cresol or catechol, and 300 mU mL?1 of enzyme. Tests

Fungal laccases are involved in various processes in nature including the biodegradation of lignin [14] and their application in the detoxi?cation of various aquatic and terrestrial pollutants and in the treatment of industrial wastewater has been suggested [15]. Phenol-polluted waters are widely produced as wastes of several industrial and agricultural activities. Phenolic compounds and their derivatives are considered priority pollutants because they are harmful toward living organisms, even at low concentrations [16]. Oxidoreductases are able to catalyze the transformation of several phenolic compounds through an oxidative coupling reaction. This results in the formation of less soluble, high molecular weight compounds that may be easily removed from water by sedimentation or ?ltration [17]. In this paper, a fungal strain belonging to ascomycete phylum and identi?ed as a T. atroviride strain was studied with respect to its laccase production. T. atroviride has been discovered by means of a screening developed by our research group to isolate Tunisian autochthonous fungal ?ora able to produce oxidative enzymes and belonging to a novel taxonomic groups [18]. We isolated and characterized the laccase produced by this strain, the single ligninolytic enzyme secreted by this fungus in the studied conditions, and we have examined its role in bioremediation of phenolic compounds: 2,4-dichlorophenoxyacetic acid, 4-chlorophenol, o-cresol and catechol. The aim of this work was to contribute to a better knowledge of the enzyme secreted by this fungus respect to its potential environmental applications.
2. Materials and methods 2.1. Fungal strain and culture condition The fungus used in this study was isolated in our laboratory and preserved as pure culture in the culture collection of ‘‘Centre de Biotechnologie de Sfax’’. The fungal strain denominated CTM 10476 was identi?ed as T. atroviride by the analysis of its internal transcribed spacer nucleotide sequence [18]. T. atroviride CTM 10476 was selected for this study because of ability to oxidize ABTS in solid medium. The culture was maintained on 2% malt extract agar plates grown at 30 8C and stored at 4 8C. T. atroviride precultures were initiated by placing small mycelium pieces from 3-day-old (30 8C) malt agar slants, in 500 mL conical ?asks containing 50 mL of malt extract broth. Flasks were incubated for 4 days at 30 8C and 160 rpm on an orbital shaker (Braun Certomat S, Germany). For laccase production, the mycelium obtained was fragmented in a Waring blender (Torrington, USA) for 30 s and served to inoculate (5%, v/v) the liquid media. The culture medium contained per liter, 10 g of glucose, 9 g of bacto-peptone, 2 g of ammonium tartrate, 1 g of KH2PO4, 0.5 g of MgSO4?7H2O, 0.5 g of KCl, 1 mL of trace elements solution. The trace elements solution composition per litre was as follows: 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 solution was adjusted to 5.5. The culture medium was supplemented with 300 mM CuSO4 to induce the laccase production. 2.2. Protein and laccase assays Extracellular protein was determined by Bradford method using Bio-Rad protein assay and bovine serum albumin as standard. Laccase activity was determined either by using 5 mM ABTS as the substrate in 100 mM acetate buffer, pH 4.5 (e436 = 29,300 M?1 cm?1) [19], or 5 mM 2,6-DMP in 100 mM acetate buffer, pH 4.0 (e469 = 27,500 M?1 cm?1) [20]. All enzyme assays were carried out at room temperature. One unit of enzyme activity was de?ned as the amount of enzyme oxidizing 1 mmol of substrate min?1. 2.3. Laccase puri?cation The 5-day culture medium of T. atroviride was passed through ?lter paper to remove fungal mycelia, after which the ?ltrate was concentrated by ultra?ltration (Filtron 5 kDa cutoff membrane) and dialysed against 10 mM sodium phosphate (pH 5.5). This crude enzyme preparation was applied to a Bio-Rad Q-Cartridge equilibrated with the same buffer at a ?ow rate of 1.5 mL min?1. Retained proteins were eluted for 170 min using the following NaCl gradient: 0–250 mM, 100 min; 250–1000 mM, 30 min and 1000 mM, 20 min. Fractions with the laccase activity were pooled and concentrated (Filtron Microsep, 3 kDa cutoff), and samples of 0.2 mL were applied to a Superdex 75 (Phamacia HR 10/30) column equilibrated with 10 mM sodium phosphate buffer (pH 5) containing 150 mM NaCl, at a ?ow rate of 0.4 mL min?1. The laccase peak fractions were pooled, concentrated (Filtron Microsep, 3 kDa cutoff), dialysed against 10 mM sodium tartrate buffer (pH 4.5),

H. Chakroun et al. / Process Biochemistry 45 (2010) 507–513


Fig. 1. Time course of laccase production (&) and proteins (~) in culture of T. atroviride. were carried out with and without addition of 0.5 mM ABTS as laccase mediator. The reaction was initiated with enzyme addition and incubated at 30 8C. Samples were withdrawn at 2 h intervals and subsequently analyzed by high-performance liquid chromatography (HPLC). The effects of the mediator concentration and reaction time were studied by the use of 500 mM 4-chlorophenol or 2,4-D in the presence of 0.25, 0.75, 1.25, 2.5 and 5 mM concentrations of ABTS for different times (from 4 to 24 h) with 1 U of T. atroviride laccase mL?1 in the buffer described above at 30 8C. 2.9. HPLC analysis Phenolic compounds were analyzed by HPLC (Shimadzu) equipped with a (LC10ATvp) pump and variable wavelength absorbance (SPD-10Avp) detector set at 280 nm. A 25 cm ? 4.6 mm C18 column with a 4.6 mm particle size was used. The ?ow rate was 0.8 mL min?1. The mobile phase used was 0.1% phosphoric acid in water (A) versus 80% acetonitrile in water (B) for a total running time of 50 min, and the gradient changed as follows (expressed as percentage of B): 20%, 20 min; 20– 80%, 5 min; 80–100%, 1 min; 100, 4 min; 100–20%, 15 min; 20%, 5 min. 2.10. Statistical analysis The data presented are the average of the results of two replicates with a standard error of less than 5%. Fig. 2. Estimation of the molecular mass (by SDS-PAGE 7.5%) (a) and isoelectric point on 5% polyacrilamide gel (b) of T. atroviride laccase: lane S, molecular mass standards; lane 1, deglycosylated laccase; lane 2, puri?ed laccase.

puri?ed. During the ?rst chromatographic step (Q-cartridge) the laccase activity was separated from the impurities, which include a brown pigment absorbing strongly at 280 nm. During Superdex 75 chromatography, laccase activity was detected as a symmetrical peak. Chromatography through the Mono-Q column resolved one laccase activity peak. At the end of the process, laccase was been puri?ed 42-fold with yield of 47% (Table 1). 3.3. Properties of the puri?ed T. atroviride laccase The molecular mass of T. atroviride laccase was estimated to be around 100 kDa by gel ?ltration chromatography and 80 kDa on SDS/PAGE, indicating that this enzyme is a monomeric protein. The molecular mass of deglycosylated laccase showed that it is a glycoprotein with 9% N-linked carbohydrate (Fig. 2a). Analytical IEF showed a pI of 3.5 (Fig. 2b). The N-terminal amino acid sequence of T. atroviride laccase was DVAMLPASGY. The homology search with FASTA in Swiss prot showed that the amino acid sequence is 88% homologous to the laccase 2 from Podospora anserina (accession number P78722) [23] and laccase 3 from Botrinia fuckeliana (accession number Q96UM2) [24]. Also, 77% similarity was found to the laccase from Neurospora crassa (accession number P06811) [25]. All of these fungi belong to the group of ascomycetes and no similarity was found to the laccases of basidiomycetes. 3.4. Effect of pH, temperature and heavy metals on laccase activity The effect of pH on the initial rate of ABTS and 2,6-DMP oxidation was studied in 100 mM citrate-borate-phosphate buffer

3. Results 3.1. Laccase production by T. atroviride The production of laccase by T. atroviride was performed in shake ?ask cultures at 30 8C and 160 rpm using 10 g L?1 glucose and 300 mmol L?1 copper. The fungus grew well and the production of laccase started after 4–5 day cultivation. Within 7 days, a laccase activity of 661 U L?1 was reached (Fig. 1). Then, laccase activity dropped sharply at 8-day-old culture. Compared to other fungal species for which a longer production time (12–30 days) is required [22], this property is of some interest from the industrial point of view. It is noteworthy that the presence of copper in culture medium is strictly required for the production of laccase. 3.2. Puri?cation of laccase The laccase was puri?ed to homogeneity from culture medium supplemented with CuSO4 (300 mM). Table 1 summarizes the protocol used to purify the laccase from T. atroviride (activity assayed against ABTS). Three chromatographic steps were necessary to purify the protein. Only one protein with laccase activity was
Table 1 Puri?cation of laccase from Trichoderma atroviride. Activity (mU) Culture ?ltrate Ultra?ltration Q cartridge Superdex 75 Mono-Q 148,000 133,056 104,706 90,000 70,574 Protein (mg) 90,000 49,788 20,196 5,000 1,019

Speci?c activity (mU mg?1) 1.6 2.7 5.2 18 69.3

Yield (%) 100 89.9 70.8 60.8 47.8

Puri?cation degree 1 1.6 3.2 11 42.1


H. Chakroun et al. / Process Biochemistry 45 (2010) 507–513

Fig. 3. Effect of pH and temperature on activity and stability of laccase from T. atroviride: (a) optimum pH using 2,4-DMP (~) and ABTS (&) as substrates; (b) optimum temperature using 2,4-DMP as substrate; (c) pH stability on pH 2 (~), pH 3 (&), pH 4 (~), pH 5 (&), pH 7 (^) and pH 9 (+); (d) thermal stability at 4 8C (&), 30 8C (~), 40 8C (&), 50 8C (~) and 60 8C (+).

(in the range from 2 to 10) (Fig. 3a). T. atroviride laccase was active in pH range between 2 and 6 with both ABTS or 2,6-DMP. The highest pH optimum, 5, was determined in oxidizing ABTS. The pH optimum for 2,6-DMP was within the range of 2–3. The laccase showed practically no activity at pH values higher than six with the two substrates tested. The effect of temperature on laccase activity was tested between 4 and 60 8C. Fig. 3b shows that the enzyme was almost active in the temperature range 35–55 8C, with maximum activity at 50 8C. The effect of several heavy metal cations on laccase activity, frequently found in the environment such as Cu2+, Mg2+, Mo6+, Ni2+, Co2+, Li+, Ca2+, Mn2+, and Al3+, was also assessed. For this purpose, laccase was incubated with the salts of the abovementioned ions at two concentrations: 10 and 100 mM and using 2,6-DMP as a substrate (Fig. 4a). It was found that for a concentration of 10 mM, laccase activity remained stable in the presence of all the metal ions tested. It is noteworthy that the addition of Cu2+ into the reaction mixture stimulated laccase activity, increasing it around 27 and 35% at 10 and 100 mM Cu2+ concentrations, respectively, compared to control. This may be due to the ?lling of type-2 copper binding sites with copper ions [26]. When the concentration was increased to 100 mM, the laccase activity decreased in the presence of all metals tested, in particular against Al3+, Mg2+ and Co2+ which have 95, 94 and 90% of inhibition. The other ions have a variable inhibiting effect: Ni2+ (46% inhibition) Mn2+ (65% inhibition), Mo6+ (73% inhibition), Ca2+ (75% inhibition) and Li+ (85% inhibition). 3.5. Effect of pH, temperature and heavy metals on laccase stability The study on pH stability of laccase carried out at 4 8C showed that laccase was stable at slightly acidic pH (4 and 5) and when the laccase was incubated for 6 h in pH 3, 50% of laccase activity was retained (Fig. 3c). This property could potentially be exploited in the textile industry where acidic

conditions predominate in wool dyeing. Thermostability was determined during 24 h incubation from 4 to 60 8C (Fig. 3d). This data indicated that the laccase was stable at 4 and 30 8C for

Fig. 4. Effect of heavy metals on: (a) laccase activity ((&) control (&) 10 mM and ( ) 100 mM) and (b) stability of laccase from T. atroviride at a concentration of 10 mM ((&) 0 h (&) 24 h).

H. Chakroun et al. / Process Biochemistry 45 (2010) 507–513 Table 2 Relative activity of puri?ed Trichoderma atroviride laccase towards different substrates. Substrate ABTS 2,6-DMP Syringaldazine Hydroquinone Concentration (mM) 5 5 0.5 5 Realtive activity (%) 100 75 60 47


Table 3 Kinetic constants of laccase from Trichoderma atroviride.

lmax (nm)
ABTS DMP 436 469

e (M?1 cm?1)
29,300 27,500

Km (mM) 2.5 1.6

Vmax (mU mg?1) 870 778

Vmax/Km 348 486.25 Fig. 5. Transformation (%) of 0.5 mM of 2,4-D, 4-chlorophenol, o-cresol and catechol with 0.3 U mL?1 of T. atroviride Laccase in the absence (&) and presence (&) of ABTS. Incubation was performed at 30 8C, pH 5 for 24 h.

24 h, whereas 80% of its activity was retained after 4 h incubation at 40 8C. In the present investigation the laccase retained 50% of its activity after 1 h incubation at 60 8C and 96 min incubation at 50 8C. The stability of laccase activity against metal compounds was assessed by incubating laccase with these metals (Cu2+, Co2+, Ca2+, Cd2+, Mg2+, Mn2+, Mo6+, Ni2+, Li+ and Al3+) at a concentration of 10 mM (Fig. 4b). It was found that at this concentration, laccase activity remained stable for 24 h against all the metal ions tested except for Al3+ and Mo6+, which reduce the stability of the enzyme 77 and 56%, respectively, while the addition of Cu2+ increase the stability. The stability of laccase makes this enzyme an ef?cient agent in the treatment of wastewater containing heavy metals. 3.6. Substrate speci?city Table 2 presents the relative activities of puri?ed laccase from T. atroviride with various substrates. The highest relative activity was obtained with ABTS. The other typical substrates for laccases, 2,6-DMP, syringaldazine and hydroquinone were also oxidized by this enzyme. The kinetic constants of puri?ed enzyme were determined on 2,6-DMP and ABTS, two substrates frequently used in laccase speci?city studies. As shown in Table 3, all the kinetic parameters suggest that the enzyme has a higher af?nity towards 2,6-DMP than ABTS.
Table 4 Effect of various inhibitors on oxidation of 2,6-DMP by puri?ed Trichoderma atroviride laccase. Inhibitor EDTA Concentration (mM) 0.1 1 5 0.1 1 5 0.1 1 5 0.1 1 5 0.1 1 5 0.1 Inhibition (%) 5 13 81 58 80 100 0 0 0 3 33 33 0 24 42 100

3.7. Effect of inhibitors The effect of different inhibitors on T. atroviride laccase activity is shown in Table 4. As expected the most potent inhibitors assessed was sodium azide (NaN3). Total laccase inhibition was observed with 0.1 mM sodium azide and 5 mM L-cycteine. EDTA, pcoumaric acid and SDS caused some inactivation, but NaBr did not have any inhibition effect on this laccase under the test conditions. 3.8. Transformation of aromatic pollutants by laccase from T. atroviride Transformation of 2,4-D, 4-chlorophenol, o-cresol and catechol by the laccase from T. atroviride in the absence and presence of ABTS was monitored by HPLC. Catechol and o-cresol showed similar behaviour (Fig. 5). Indeed, after 24 h incubation, catechol and o-cresol were completely transformed either in the presence or the absence of ABTS. However, the transformation of 2,4-D and 4-chlorophenol was less ef?cient. In fact, 79 and 72% of



p-Coumaric acid



Fig. 6. Transformation of 4-chlorophenol (a) and 2,4-D (b) by 1 U mL?1 laccase of T. atroviride in the presence of various concentrations of ABTS. Incubations were performed at 30 8C, pH 5 for 4 and 24 h, respectively.


H. Chakroun et al. / Process Biochemistry 45 (2010) 507–513

untransformed 2,4-D and 4-chlorophenol, respectively, were still present in the reaction mixture after 24 h incubation in the absence of ABTS. The disappearance of these chlorinated phenols was somewhat enhanced by the presence of ABTS (50 and 45% transformation, respectively, after 24 h incubation). In order to improve the transformation of chlorophenols, the concentration of the laccase mediator ABTS was varied. The in?uence of the ABTS concentrations on the transformation of 4-chlorophenol with 1 U laccase mL?1 (4 h treatment) is shown in Fig. 6a. The transformation of chlorophenol was increased with increasing the ABTS concentrations. It reached 100% by the addition of 1.25 mM ABTS for 4-chlorophenol. Whereas, 2,4-D was weakly transformed compared to 4-chlorophenol. In fact, the transformation rate does not exceed 60% with all the range of ABTS concentration studied after 24 h incubation (Fig. 6b). 4. Discussion T. atroviride has previously been reported to produce chitinase,

b-glucanase and esterase [8]. The presence of laccase in T.
¨ atroviride has been demonstrated by Assavanig et al. [27], Holker et al. [8] and Gochev and Krastanov [28]. The onset of laccase activity in T. atroviride occurred on day 4 and reached its maximum on day 7 and then the rate of enzyme production declined gradually. A similar pro?le was observed in the cultures of T. harzianum with a maximum laccase activity reached on day 4 [29]. T. atroviride laccase was puri?ed and biochemically characterized. It showed a molecular mass of 80 kDa on SDS-PAGE. It falls within the molecular mass range reported for many fungal laccases which can range from 50 to 90 kDa [30]. This molecular mass is similar to those reported from the Trichoderma sp. laccase [27], T. harzianum laccase [29] and the ascomycete Melanocarpus albomyces laccase [31] which have a molecular mass of approximately 71, 79 and 80 kDa, respectively. The comparison of the N-terminal amino acid sequence with other fungal laccases showed that the T. atroviride laccase was similar to other ascomycetes’ laccases. In contrast, the homology with basidiomycete laccases was low. This result supports the theory that the gene of ascomycete and basidiomycete laccases have evolved signi?cantly after the phylogenetic divergence of the two groups of fungi [23,31]. Unlike many fungi that often produce several laccase isoforms [32–34], T. atroviride produced only one laccase isoform with a pI of 3.5 in the selected culture conditions. This pI is similar to that of the laccase from the new laccase-producing fungus designated strain I-4 [35] which has an acidic isoelectric point. T. atroviride laccase was able to oxidize the typical laccase substrates ABTS, 2,6-DMP, syringaldazine and hydroquinone. The highest activity was observed on ABTS, as reported for many other laccases like those from Maugnielle sp. [36] and Trichophyton rubrum LKY-7 [37]. T. atroviride laccase was strongly inhibited by the typical laccase inhibitor sodium azide, but it was not sensitive to NaBr, EDTA, p-coumaric acid and SDS. In fact the binding of NaN3 to the type 2 and 3 copper sites affects internal electron transfer, thus inhibiting the activity of the laccase. These ?ndings are in keeping with the general properties of laccase from a diverse range of fungal sources. The pH optimum of the T. atroviride laccase depended on the substrate. The laccase exhibited a low pH optimum (in the range of 2–3) with ABTS as a substrate, like many other fungal laccases [38,39]. Using 2,6-DMP, the pH optima was 5. The difference in pH optima for ABTS and 2,6-DMP is typical for laccases and it re?ects the difference in oxidation mechanism with different substrates [40]. The stability of T. atroviride laccase was also good in slightly acid pH values, over 85 and 75% of the activity remained after 24 h incubation at pH 5 and 4, respectively. This

stability is rare among fungal laccases, for example laccases from M. albomyces [31], Pleurotus ostreatus [41], P. erengii [42] and Perenniporia tephropora [43] have good stability at neutral and slightly alkaline pH values. Temperature pro?les of T. atroviride laccase activity usually do not differ from other laccases with optima temperature between 50 and 70 8C [13] and stability at temperature below 50 8C [44]. The interest in laccases has increased in recent years because of its ability to oxidize a variety of xenobiotic compounds in the presence of synthetic or natural redox mediators [45]. The effect of laccases from different sources on the transformation of phenolic compounds has been extensively studied [16,46]. We showed in the present study that T. atroviride laccase is also ef?cient for the transformation of phenolic compounds. In fact, the transformation of catechol and o-cresol was practically complete after 24 h incubation without addition of mediator (Fig. 6). Nevertheless, in the case of chlorophenols the presence of ABTS is required for the oxidation by T. atroviride laccase. Indeed, 1.25 mM ABTS concentration provides complete transformation of 4-chlorophenol and the removal of 60% of 2,4-D after 4 and 24 h incubation, respectively. The use of ABTS to enhance the transformation of chlorinated phenol has been previously reported [47]. As widely reported in the literature [48], laccase transformation of phenols is less ef?cient with increasing the molecular weight of the substituent and its position on the aromatic ring with respect to the OH groups. The results found in this study are in agreement with this trend. Acknowledgments This research has been funded by a Cooperation project between Spain and Tunisia (31p/02—Spanish ‘‘Ministerio de Asuntos Exteriores’’) and in part by a grant from ‘‘Contrats Programmes MRSTCD’’ Tunisia. We are grateful to Professor Hafedh Mejdoub Sfax Faculty of Science, Tunisia for N-terminal sequence determination and to Mr Chedhli Bouzid for technical assistance. References
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