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Sami Sayadi

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Enzymatic oxidative transformation of phenols by Trametes trogii laccases

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TENT 655317

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Printed: 27/1/2012

Environmental Technology Vol. 00, No. 00, Month 2012, 1–9

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Enzymatic oxidative transformation of phenols by Trametes trogii laccases Hanen Chakroun, Mohamed Bouaziz, Abdelhafidh Dhouib and Sami Sayadi* Laboratoire des Bioprocédés Environnementaux, Pôle d’Excellence Régional AUF-LBPE, Centre de Biotechnologie de Sfax, Université de Sfax, Sfax, Tunisia (Received 25 August 2011; final version received 22 December 2011 )

Q1

The removal of toxic phenolic compounds from industrial wastewater is an important issue to be addressed. Their presence in water and soil has become a great environmental concern, and effective methods for their removal need to be addressed. The feasibility of applying laccases for the degradation of phenolic compounds has received increasing attention. In the present work, the transformation of five phenolic compounds (catechol, hydroxytyrosol, tyrosol, guaiacol and p-coumaric acid), the main constituents of a typical wastewater derived from an olive oil factory, by Trametes trogii laccases was studied at concentrations ranging between 0.2 and 1.6 mM. High-performance liquid chromatography analysis showed high degradation rates of phenolic compounds by T. trogii laccases. Independently of the used concentration, a complete transformation of guaiacol, p−coumaric acid, hydroxytyrosol and tyrosol occurred after 1 h of incubation. The transformation of catechol depends on its initial concentration. The liquid chromatography-mass spectrometry analysis showed that laccases catalysed transformation of p-coumaric acid and tyrosol, resulting in the formation of phenolic dimers. No reduction of enzyme activity has been observed during the oxidation of all phenolic compounds. These results suggest that the studied laccases were capable of efficiently removing phenolic compounds, as well as catalysing the production of novel phenolic dimers. Keywords: laccase; phenols, oxidative transformation; removal, polymerization

1. Introduction Phenolic compounds are common in agroalimentary, pharmaceutical, paper mills, oil refineries, coal conversion, petrochemistry, pesticide and dye industrial discharges and in wastewater treatment plants [1,2]. Some phenols are known to be toxic to humans and both terrestrial and aquatic ecosystems. Therefore, they represent a serious environmental problem and must be efficiently eliminated, to preserve the environment. Currently, many physical and chemical methods are available for phenol-rich effluent decontamination, including adsorption on activated carbon [3], chemical oxidation [4,5], solvent extraction [6,7] and electrochemical oxidation [8,9]. These conventional methods of dephenolization are often expensive, incomplete, applicable in a limited range of concentrations and generate hazardous by-products [10]. A microbial degradation alternative has been proposed as cheaper and environmentally less aggressive [11,12]. Moreover, enzymes used for phenolic compound degradation has attracted an increasing attention [13]. The underlying mechanism of pollutant removal involves their enzymatic oxidation to free radicals or quinones that subsequently undergo polymerization and partial precipitation [14,15]. Extracellular oxidoreductases, such as peroxidase, laccase or tyrosinase, are mostly of microbial origin and are involved in oxidative coupling processes of phenols [16–22]. The effect of laccases from different origins and under both free and immobilized forms ∗ Corresponding

author. Email: [email protected]

ISSN 0959-3330 print/ISSN 1479-487X online © 2012 Taylor & Francis http://dx.doi.org/10.1080/09593330.2012.655317 http://www.tandfonline.com

on the transformation of phenolic compounds has been extensively studied [14,18,23–25]. Laccases were used in a variety of applications, such as to remove toxic compounds from aquatic and terrestrial systems, to produce and to treat beverages, as analytical tools and as bio-sensors to estimate the quantity of phenols in natural juices or the presence of other enzymes [26]. These enzymes are multi-copper phenol oxidases that catalyse the oxidative coupling of phenolic compounds, resulting in their polymerization [13]. This results in the formation of high molecular weight compounds, which are less soluble and may be easily removed from the water by sedimentation or filtration. This phenomenon may alter the toxicity of the phenolic pollutant [23]. The objective of this study was to verify whether phenolic compounds (catechol, hydroxytyrosol, tyrosol, guaiacol and p-coumaric acid) may be treated with Trametes trogii laccases for detoxification purposes and to highlight the economic efficiency of this catalytic process by the emphasis of the stability of the enzyme and the high proportion of the residual activity after the catalytic reaction. 2. Materials and methods 2.1. Chemicals Catechol, p-coumaric acid, guaiacol, dimethoxyphenol (2,6-DMP) and tyrosol were obtained from Sigma Chemical

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Co. (St. Louis, MO). Hydroxytyrosol was purified in our laboratory as described previously [27]. 2.2. Enzyme preparation The fungus used in this study was deposited in the German Collection of Microorganisms and Cell Cultures under reference DSM 17786 and identified as Trametes Trogii [28]. T. trogii laccases were produced on glucosepeptone medium containing 300 μM CuSO4 , as described by Dhouib et al. [28] with a slight modification. Laccase isoenzymes were purified by the filtration of the culture broth supernatant at maximum extra-cellular laccase activity. The filtrates were saturated with ammonium sulphate at 80% and centrifuged at 10,000 g for 30 min. The precipitate was resuspended in 10 mM phosphate buffer (NaH2 PO4 , Na2 HPO4 ), pH 5.5, and extensively dialysed against the same buffer. Samples were applied to a Mono-Q anionexchange column (Bio-Rad, 1 × 15 cm) equilibrated with 10 mM sodium phosphate buffer, pH 5.5 at a flow rate of 0.3 ml min−1 . Fractions with laccase activity were pooled, concentrated and stored at −20◦ C for later applications. Laccase activity was assayed at 25◦ C using 5 mM 2,6-DMP in 100 mM acetate buffer, pH 4.0 (ε469 = 27, 500 M−1 cm−1 ). One unit of enzyme activity (U) was defined as the amount of enzyme oxidizing 1 μmol of substrate per minute. 2.3. Phenol transformation Initial concentrations of phenols of 0.2, 0.4, 0.8 and 1.6 mM in 100 mM citrate/phosphate buffer, pH 5.0, were incubated at 25◦ C for 3 h in the presence of laccases (1 U ml−1 in 10 mM sodium phosphate buffer, pH 5.5) in 25 ml flasks. Samples were taken periodically from two replicates during the incubation and analysed for residual phenol concentration and laccase activity. To determine the residual concentration of phenols, the enzymatic reaction was stopped by lowering the pH to 2 by concentrated acetic acid and centrifuging the samples at 12,000 g for 15 min. The supernatants were filtered through 0.45 μm and analysed by high-performance liquid chromatography (HPLC). The removal of phenols was determined by the difference between levels in the experimental assay and in a control (a substrate solution lacking the enzyme). For residual enzymatic activity, 1 ml of samples was immediately assayed using 2,6-DMP. 2.4. HPLC analysis Phenolic compounds were analysed by HPLC using Shimadzu apparatus equipped with a (LC-10ATvp) pump and variable wavelength absorbance (SPD-10Avp) detector set at 280 nm. A 25 cm × 4.6 mm C18 column with a 4.6 μm particle size was used. The flow rate was 0.8 ml per minute. 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% to 80%, 5 min; 80% to 100%, 1 min; 100%, 4 min; 100% to 20%, 15 min and 20%, 5 min.

2.5.

Liquid chromatography-mass spectrometry analysis

Mass spectrometry was used as the major tool for the assessment of polymerization, and analysis was often performed on the collected fractions of the transformation of tyrosol and p−coumaric acid. The liquid chromatography-mass spectrometry (LC-MS)/MS experiments were carried out with an Agilent 1100 LC system consisting of degasser, binary pump, auto sampler and column heater. The column outlet was coupled to an Agilent MSD Ion Trap XCT mass spectrometer equipped with an ESI ion source. Data acquisition and mass spectrometric evaluation was carried out on a personal computer with data analysis software (Chemstations). For the chromatographic separation a Zorbax 300Å Extend-C-18 Column (2.1 mm × 150 mm ) was used. The Q2 column was held at 95% solvent A (0.1% formic acid in water) and 5% solvent B (0.1% formic acid in ACN) for 1 min, followed by an 11 min step gradient from 5% B to 100% B, then it was kept for 4 min with 100% B; finally, the elution was achieved with a linear gradient from 100% B to 5% B in 2 min. The flow rate was 200 μl per minute and the injection volume was 5 μl. The following parameters were employed throughout all MS experiments. For electrospray ionization with positive ion polarity the capillary voltage was set to 3.5 kV, the drying temperature to 350◦ C, the nebulizer pressure to 40 psi and the drying gas flow to 10 l per minute. The maximum accumulation time was 50 ms, the scan speed was 26,000 m z−1 s−1 (ultra scan mode) and the fragmentation time was 30 ms.

2.6. Phytotoxicity Phytotoxicity, suggested by Zucconi et al. [29], was employed using seeds of Lycopersicon esculentum. Phenolic compounds at concentrations of 0.2, 0.4, 0.8 and 1.6 mM and their intermediate degradation products were placed in Petri dishes. Ten seeds of L. esculentum were distributed in the dishes, equally spaced. The plates were incubated at 30◦ C for 120 hours. After this time, the number of germinated seeds was counted and the elongation of the roots was measured. The germination index (%GI) was calculated through the following equation:

GI =

Rootlet’s length in sample Rootlet’s length in water ×

Germination in sample × 100 Germination in water

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3

Figure 1. Transformation of catechol (a), guaiacol (b), hydroxytyrosol (c), p-coumaric acid (d) and tyrosol (e) at different incubation times and different initial phenol concentrations [0.2 mM (•), 0.4 mM (), 0.8 mM () and 1.6 mM (∗)]. Solutions were incubated at 30◦ C and pH 5.0 with 1 U of laccase. Error bars represent standard deviation.

3. Results and discussion Trametes trogii laccases were demonstrated to have an oxidizing ability on phenolic compounds that are commonly found in industrial emissions. Figure 1 reports the transformation rate (%) of catechol, guaiacol, hydroxytyrosol, p-coumaric acid and tyrosol at different incubation times and different initial concentrations ranging from 0.2 to 1.6 mM. Tests were performed by using 1 U ml−1 laccase (2,6-DMP) in citrate/phosphate buffer pH 5.0 and at 30◦ C. Independently of the used initial concentration, a total conversion of the guaiacol, hydroxytyrosol, p-coumaric acid and tyrosol was obtained after one hour of incubation (Figure 1(b)–(e)). Catechol showed different behaviours with a degree of conversion that is function of its initial concentration (Figure 1(a)). Indeed, when the initial concentration of catechol increased, its conversion yield decreased, leading to a lengthening of the time required to reach 100% of conversion to 3 h.

According to the results obtained by Gianfreda et al. [25] with a laccase from Rhus vernificera, catechol was transformed more easily than hydroxytyrosol and tyrosol, in the order listed. However, the transformation of these compounds was less efficient compared to the results found with T. trogii laccases. In fact, 30% of untransformed hydroxytyrosol and catechol and more than 65% of tyrosol were still present in the reaction mixture after 24 h incubation with 3 U ml−1 laccase (2,2 -azinobis(3-ethylbenzo6-thiazolinesulfonic acid) (ABTS)) from Rhus vernificera. Our experimental results showed that 1 U laccase (2,6DMP) corresponds to approximately 2.8 U laccase (ABTS). In addition, in the studies monitored by Gianfreda et al. [25], the enhancement of phenol transformation requires the increase of the unit of laccase utilized. For example, a complete oxidation of catechol occurred with 6 U of laccase (ABTS) after 24 h. In our study, the use of 1 U ml−1 laccase (2,6-DMP) allows a complete transformation of all

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Figure 2. The HPLC images of phenolic compounds before and after treatment with T. trogii laccases: 1U Lac ml−1 ; 0.5 h; 1.6 mM: catechol (a), guaiacol (b), hydroxytyrosol (c), p-coumaric acid (d) and tyrosol (e).

phenolic compounds tested after 1 h incubation. This could indicate a higher efficiency of T. trogii laccases. In addition, the reaction mixtures achieved different colours, which intensified with time. Mixture colours ranged from a bright yellow to intense yellow for catechol and hydroxytyrosol at 3 h incubation. Light to dark brown colours were observed for guaiacol solutions. Guaiacol showed a similar behaviour at each concentration tested and each time of incubation: it was converted up to 99.9%. This finding suggests that in the range of concentration used we were not reaching a saturation concentration of guaiacol. The formation of a solid material was observed, especially with tyrosol and guaiacol. The oxidative coupling reaction monitored by laccases results in the formation of less soluble, high molecular weight compounds that may be easily removed from water by sedimentation or filtration. In fact, during the oxidative coupling of phenol, for example, aryloxy or phenolate radicals are formed by the removal of one electron and a proton from the hydroxyl group. Then, the resulting phenolate radicals interact to yield stable dimerized and polymerized products [30]. The production of low molecular weight products, such as dimers and trimmers, might be of interest in the exploitation of laccases

for the selective formation of new products and is a goal of synthetic relevance. HPLC images of the phenolic compounds (1.6 mM) before and after the treatment with T. trogii laccases (1 U ml−1 ) during 0.5 h of incubation in citrate/phosphate buffer pH 5.0 and at 30◦ C indicated that during the removal of catechol, hydroxytyrosol and guaiacol no additional peaks were detected (Figure 2(a)–(d)). This result suggests that these three compounds were transformed and their phenolic properties detectable at 280 were completely removed Q3 after their incubation with T. trogii laccases. However, the transformation of tyrosol and p-coumaric acid was concretized by the disappearance of their characteristic peaks at 15.9 and 35.3 min of elution time, respectively, and the appearance of a new peak at higher elution time at 36.8 and 39.8 min, respectively, corresponding to new compounds with a decreased polarity (Figure 2(d) and (e)). To obtain some additional information on the nature of the collected HPLC peaks corresponding to the transformation products of tyrosol and p-coumaric acid, liquid chromatographyelectrospray ionization mass spectrometry was carried out. In the case of tyrosol, the mass spectrum displayed peaks at 273 [M–H]− (Figure 3(a)). These m/z values suggest the

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Figure 3.

5

(a) ESI-MS spectrum of tyrosol dimer. (b) ESI-MS2 spectrum of molecular ion at m/z 273.

presence of tyrosol dimers. The ESI-MS2 spectrum of the molecular ion at m/z 273 showed fragment ions at m/z Q1 255, 243 and 228 (Figure 3(b)) . The product ion at m/z 255 can be explained by the loss of a water molecule. The ionic species at m/z 243 could be produced by the loss of units based on −CH2 OH and the fragment at 228 could be justified by a loss of the −CH2 −CH2 −OH fragment. We tentatively assign this molecule as the tyrosol dimer, which is to our knowledge reported for the first time using a laccase enzyme. The mass spectra of the p-coumaric acid transformation product in negative ion mode showed the molecular ion [M–H]− m/z 325 (Figure 4(a)), and ions at m/z 281, 255 and 237. The MS/MS base ion m/z 281 resulting from

loss of 44 Da (CO2 ), and other fragment ions (m/z 255 [MH–C2 H2 CO2 ]− and m/z 237 [M-H–2 CO2 ]− ), all of which suggest that peak P2 is a p-coumaric acid dimer derivative. The ESI-MS2 experiment of the base product ion at m/z 281 (Figure 4(b)) produced only one ion at m/z 237, by losing 44 Da, which is obviously corresponding to the loss of two neutral molecules of the CO2 group . It has Q1 been demonstrated that the loss of CO2 from the [M–H]− ion is due to the contraction of the ring [31]. The results described above, whose fragmentation mechanism of mass spectrometry confirmed and agreed well with the hypothesis of the structure of a p-coumaric acid dimer, were previously described [32]. Indeed, the ability of phenoloxidases to polymerize phenols and mediate covalent binding of these

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Figure 4.

H. Chakroun et al.

(a) ESI-MS spectrum of p-coumaric acid dimer. (b) ESI-MS2 spectrum of molecular ion at m/z 281.

chemicals to humic substances is well documented (for both free and immobilized laccase) [14,23,33]. Laccases from Rhizoctonia praticola and Trametes versicolor were shown to be able to polymerize different phenolic contaminants, such as chlorophenols, bromophenols, methylphenols and methoxyphenols [17]. The different laccase mediated transformation pathways of phenolics could be explained by their molecular structures. Indeed, the molecular structure of the substrate strongly influences its redox potential, thus determining the higher or lower ability to be oxidized by the enzyme [25]. In fact, catechol and hydroxytyrosol have two hydroxyl groups in the aromatic ring that facilitate the cleavage of the aromatic ring during the oxidation by T. trogii laccases. However, for tyrosol and p-coumaric acid, having only one hydroxyl group in the aromatic ring, laccases catalyse the

formation of polymeric products. This suggests that compounds having one hydroxyl group in the aromatic ring polymerize more readily than those having two hydroxyl groups. For evaluating the fate of laccase after its contact with the tested phenolic compounds, activity assays were performed in the supernatants represented by each data point of Figure 1, under standard conditions using 2,6-DMP as the test substrate. Enzymatic activity was not affected by the increase of the substrate concentrations (data not shown). Moreover, the amount of active enzyme was almost constant throughout the reaction time and no reduction of laccase activity was detected. In contrast, an increase between 20% and 40% of laccase activity was measured, which suggests the possible recycling of the enzyme: for instance, degradation can be performed by successive additions of phenolic

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7

Figure 5. Phytotoxicity of the phenolic compounds: 0.2 mM (a); 0.4 mM (b); 0.8 mM (c);and 1.6 mM (d) before and after 3 h laccase treatment. Error bars represent standard deviation.

compounds. Gianfreda et al. [24] observed also this activation of laccase and obtained 18% and 60% increases of laccase activity with tyrosol and hydroxytyrosol, respectively. This increase of laccase activity was considered as a contrasting and not easily understandable result because, usually, the inactivation of the enzyme by the products cannot be excluded. Indeed, during the oxidative transformation of phenolic compounds, permanent inactivation through interactions of the enzyme with free radical products and through entrapment in polymer precipitates give Q4 rise [34]. Phytotoxicity of untreated and laccase-treated phenolic compounds for concentrations ranging between 0.2 and 1.6 mM on Lycopersicon esculentum seeds was performed to evaluate the variation of the degree of toxicity. Phytotoxicity of phenolic compounds was shown to be concentration dependant (Figure 5). Moreover, for guaiacol and catechol a highly significant inhibition of seed germination was evident from a concentration of 0.4 mM. Hydroxytyrosol, tyrosol and p-coumaric acid exhibited limited phytotoxicity, since the GI is more than 50% for all concentrations tested. Pair-wise comparison of GI values obtained on untreated and laccase-treated phenolic compounds showed that the enzymatic treatment exerted a positive effect on the germination rate, being particularly evident for guaiacol and catechol. Furthermore, laccase treatment for each phenolic compound improved the seed germinability compared to

the control. Previous studies emphasized the contribution of certain monomeric phenols in the phytoxicity associated with olive mill wastewaters (OMWs) and dry olive residue [35,36]. In addition, Capasso et al. [37] demonstrated the phytotoxic effect of catechol and tyrosol extracted from OMWs. Several studies, devoted to investigate the potential of laccase to detoxify OMWs, have demonstrated its higher efficiency with almost all phenolic compounds, which simulate this waste. In fact, Aranda et al. [36] suggested the importance of phenol removal by laccase treatment in the elimination of the phytotoxicity of olive mill dry residues. Furthermore, Casa et al. [38] demonstrated that germinability inhibition due to OMWs can be significantly reduced using fungal laccase, which is able to remove OMW phenolic compounds. In addition, Font et al. [39] found a correlation between laccase production and toxicity reduction in the treatment of industrial pulp mill wastewater, which is rich in aromatic compounds with the white rot fungus Trametes versicolor. On the other hand, Tsioulpas et al. [40] demonstrated that the phenolic compounds were more toxic in the laccase-treated OMWs than in untreated ones. This is in line with previous findings by Martirani et al. [41], which reported that some of the oxidation products of the laccase reaction were more toxic than the original phenolics. Therefore, there are no general rules for the transformation and detoxification of phenolic compounds by laccases.

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4. Conclusion In this paper we have shown the usefulness of T. trogii laccases for the removal of phenolic compounds in high yield and under mild reaction conditions. The fate of laccase after its catalytic action was also investigated. Laccase activity did not show any decrease when a corresponding significant removal of phenol was measured. These results encourage the possible use of T. trogii laccases to remove aromatic pollutants in purposeful applications. Furthermore, the biooxidation of tyrosol and p-coumaric acid, affording dimeric products, is interesting for the biocatalytic production of novel polyphenolics and related compounds. Acknowledgements The present research study was supported by the Ministry of Higher Education and Scientific Research (MHESR) of Tunisia under the Contract Program of the Environmental Bioprocesses Laboratory, Centre of Biotechnology Sfax. We thank Mr Adel Gargoubi for technical assistance with the HPLC analysis. We also thank Mr Hedi Issawi for the mass spectrometry facilities. Q8 References [1] Y. Wu, K.E. Taylor, N. Biswas, and J.K. Bewtra, Kinetic model for removal of phenol by Horseradish peroxidase with PEG, J. Environ. Eng. 5 (1999), pp. 451–458. [2] P.C. Pradu and C. Udayasoorian, Decolorization and degradation of phenolic paper mill effluent by native white rot fungus Phanerochaete chrysosporium, Asian J. Plant Sci. 4 (2005), pp. 60–63. [3] T.S. Anirudhan, S.S. Sreekumari, and C.D. Bringle, Removal of phenols from water and petroleum industry re?nery ef?uents by activated carbon obtained from coconut coir pith, Adsorption 15 (2009), pp. 439–451. [4] S. Azabou, W. Najjar, A. Gargoubi, A. Ghorbel, and S. Sayadi, Catalytic wet peroxide photo-oxidation of phenolic olive oil mill wastewater contaminants part II. Degradation and detoxification of low-molecular mass phenolic compounds in model and real effluent, Appl. Catal. B 77 (2007), pp. 166–174. [5] W. Najjar, S. Azabou, S. Sayadi, and A. Ghorbel, Catalytic wet peroxide photo-oxidation of phenolic olive oil mill wastewater contaminants part I. Reactivity of tyrosol over (Al–Fe) PILC. Appl. Catal. B 74 (2007), pp. 11–18. [6] N. Allouche, I. Fki, and S. Sayadi, The use of olive mill wastewaters as a cheap source of natural antioxidants: biological activities of a continuous solvent extract and purified hydroxytyrosol, Polyphenols Actualites 23 (2003), pp. 16–19. [7] Z. Lazarova and S. Boyadzhieva, Treatment of phenolcontaining aqueous solutions by membrane-based solvent extraction in coupled ultrafiltration modules, J. Chem. Eng. 100 (2004), pp. 129–138. [8] S. Khoufi, F. Aloui, and S. Sayadi, Treatment of olive mill wastewater by combined process electro-Fenton reaction and anaerobic digestion, Water Res. 40 (2006), pp. 2007–2016. [9] F. Kaplan, A. Hesenov, B. Gözmen, and O. Erbatur, Degradations of model compounds representing some phenolics in olive mill wastewater via electro-Fenton and photoelectronFenton treatments, Environ. Technol. 32 (2011), pp. 685– 692.

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