Abstract

Abstract

Background and Objective: Although biochemically similar, two laccase enzymes isolated from basidiomycetes (Trametes sp.) showed differences in their affinity to two types of phenolic compounds, interacting stronger with diphenols (catechol and caffeic acid), compared to interactions with benzenetriols (pyrogallol and gallic acid). Catalytic efficiency of Trametes pubescens laccase was detected 4-5 times higher than determined for commercial laccase (Trametes versicolor). In this study, the interactions of the two immobilized enzymes with di and triphenols were examined by various electrochemical techniques.

Material and Methods: Following electrochemical techniques: cyclic voltammetry, chronoamperometry and differential pulse voltammetry were used in this study. Experiments were carried out in varying substrate concentrations. Activity and sensitivity of the two alternative laccase – based biosensors were compared using DPV and chronoamperometry.

Results and Conclusion: Constant potential amperometric measurements indicated that the biosensor produced with Trametes pubescens laccase was much more active than biosensor based on laccase from Trametes versicolor when interacting with caffeic and gallic acids. The phenolic content of three different herbal extracts was evaluated with the developed laccase biosensors and results were found to be similar to those from chromatographic analysis used as a reference method. Therefore, biosensors can be used for rapid testing of phenolic content in real samples.

1. Introduction

Daily intake of antioxidant-rich foods and drinks is considered an important factor of a healthy regimen [1]. Antioxidants, the physiological role of which is to scavenge reactive oxygen species thus preventing oxidative damage of living cells [2] are micro-ingredients of plant cells such as those of fruits, vegetables, cereals and herbs. From natural antioxidants, phenolic acids are especially important not only because their antioxidant activity is within the highest ones, but also because they can rapidly be digested and adsorbed by gastrointestinal tract [3]. Due to their exceptional pharmacological, nutritional and wellbeing effects on humans, a wide spectrum of analytical methods for the assay of phenolic acids has been developed. Chromatographic analytical techniques receive extensive application for the quantification of these antioxidants in a variety of food samples [4-6]; however the required time-consuming sample preparation procedures stimulated the advance of various optical methods such as visible spectroscopy and fluorometry [7], or even paper-based colorimetric sensors [8]. Because of the susceptibility of phenolic acids to participate in redox processes, a range of electroanalytical methods such as voltammetry [9,10], pulse voltammetry [11-15] and amperometric detection [9,10,16,17] have been developed.

Modern electrochemical approaches for the quantify-cation of phenolic acids include phenolic acid detection with electrodes modified with advanced materials such as polymers [4], nitrogen doped carbon [12], carbon nanotubes [18,19] and gold nanostructures [10]. The use of nanostruc-tured materials or composites for electrode modification offers enhanced selectivity of the determination due to either pronounced electrocatalytic effect or greatly enhanced electrode surface area [10].

Assessment of antioxidant capacity through biosensing is a novel trend in contemporary studies [9,11,20-22]. Numerous authors report an improved selectivity of the analysis when using biosensing systems for the assessment of antioxidant content [23]. Copper –containing enzymes laccase or tyrosinase were the primary choice for developing biosensing systems for the analysis of phenols [24-26]; however, whole-cells [27] and DNA-based [28] elements for molecular recognition have also been used for this purpose. Due to the formation of colored products of biocatalyzed transformations of phenolic compounds, most of the highlighted biosensing platforms rely on optical detection principle.

Biosensors with electrochemical detection for phenolic antioxidants analysis, reported in current literature [24], require nanostructured electrode surfaces and sophisticated bioreceptor immobilization protocol. Unlike these, a simple enzyme attachment to an unmodified glassy carbon electrode is discussed, which ensures electrochemical response sensitive enough to guarantee phenolic acid assay at micromolar concentrations. Therefore, the focus of the present study was to develop and optimize an electrochemical method based on two identically prepared laccase biosensors for the determination of di- and trihydroxy aromatic compounds – one bearing commercial laccase from Trametes versicolor, and another laccase – isolated from T. pubescens, which is used seldom in biosensor development. Laccases are widely used in a variety of industrial cycles such as pulp and paper production, wastewater treatment (e.g. from olive-oil mills and textile industry), brewing and food industries, pharmacy or in the construction of fuel cells [25] and biosensors for the quantification of di-substituted aromatic compounds [29].

Laccases are complex enzymes with more than one active site, which embeds three copper clusters (type T1, T2 and T3) differing in both function, and spectroscopic character-istics [25,26]. The reaction mechanism of laccases involves uptake of one electron from the substrate- a hydrogen donor, which is oxidizing to form radical with concomitant 4-electron reduction of molecular oxygen to form two water molecules [26]. The T1 copper site is responsible for binding the aromatic substrate to be oxidized via 1-electron pathway, while the T2-T3 copper cluster binds molecular oxygen and catalyzes its 4-electron reduction to water [26]. Electrons are transferred from T1 site to T2-T3 trinuclear cluster through internal molecular electron transfer. The difference between laccases from T. versicolor and T pubescens is linked to their amino acid sequences. Despite these belong to the family of fungal laccases, variations are seen in their primary structures that may affect their catalytic efficiency, substrate binding specificity, and thermal stability. Thus, amino acids surrounding the less conservative T1 copper-binding site and the overall folding of the enzymes might differ partially, which may result in differences in catalytic efficiency of the laccase manifested as substantially different catalytic constants.

Here reported biosensors function on the following principle: laccases electrocatalytically reduce the dissolved in the working medium oxygen, thus generating reductive current, which is enhanced in the presence of phenol derivatives that act as electron shuttles (mediators) between the enzyme active site and electrode surface. Gallic and caffeic acids are two representatives of phenolic acids, the positive effects of which are commented not only in terms of their antioxidant abilities, but also with respect to their potential anti-inflammatory [30] and anticancer [31] pharmacological activity that was the major reason for their use as laccase substrates in this study. As a demonstration of the applicability of biosensing method, a series of three different types rich in phenolic compounds herbal extracts were analyzed for their phenolic content and results were compared with those from high-performance liquid chromatography (HPLC) analysis.

1. Materials and Methods

2.1. Reagents

Laccase (Е.С. 1.10.3.2, polyphenol oxidoreductase) enzymes from T. versicolor (Fluka, USA) and T. pubescens (a generous gift from Prof. Roland Ludwig, Department of Food Science and Technology, BOKU University of Natural Resources and Life Science, Vienna, Austria) were with homogeneous specific enzyme activities of 21 and 46 U mg^-1, respectively. One unit is the amount of enzyme necessary for the oxidation of 1.0 μmol of ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) per min at pH 4 and 30 °C. Laccases were dissolved in 0.05 M sodium-citrate buffer, pH 4, in such quantity to form enzyme solutions with concentrations of 950 U.ml^-1. The two enzymes were used without further purification.

Catechol, resorcinol, pyrogallol, caffeic acid, gallic acid, ABTS and reagents for the preparation of buffer solutions (sodium citrate, citric acid monohydrate and NaClO₄) were of analytical grade (Acros, Belgium) and used without further purification. All stock solutions of the enzyme substrates were prepared with a concentration of 10 mM.

2.2. Enzyme immobilization

Enzyme electrodes were prepared based on commercial glassy carbon electrodes (2-mm diameter; Metrohm, Utrecht, The Netherlands). Prior to modification, electrodes were polished with 0.05 µm alumina slurry on a polishing cloth (Kulzer, Hanau, Germany), water-rinsed and cleaned using ultrasonication in ultrapure water for 1 min for at least two consecutive times.

Enzyme immobilization was carried out as follows [29]: 2 ml of enzyme solution were drop-cast on the electrode surface. Then, a 4-µl drop of the binder (Nafion 117 diluted with ultrapure water to 0.2%) was applied. Surface was dried at room temperature (RT). The two types of laccases were immobilized on the electrode surfaces identically and the amount of the immobilized enzyme in terms of enzyme units was equal.

After electrochemical measurements, enzyme electrodes were rinsed with ultrapure water and refrigerated at 4°C, when not in use. Regeneration of the working enzyme electrodes could be carried out after the mechanical removal of the enzyme-polymer layer via polishing procedure and following the above steps.

2.3. Electrochemical measurements

All electrochemical experiments were performed in a conventional single compartment three-electrode cell with working volume of 10 ml, connected to a computer-controlled electrochemical workstation Autolab PGSTAT 302 N (Metrohm-Autolab, Utrecht, The Netherlands) controlled by NOVA 2.1.6 software. Either a modified with enzyme glassy carbon electrode, or an enzyme– free electrode (for control experiments) was used as working electrode. A Ag|AgCl, sat. KCl (Metrohm, Utrecht, The Netherlands) was the reference and a platinum foil was the auxiliary electrode. If not otherwise specified, all reported potentials were stated against this reference electrode (Ag|AgCl, sat. KCl electrode) [29]. Cyclic voltammetry was run at scan rates of 5–20 mV.s^-1. Volt-ampere curves (voltammograms) were obtained in both background electrolyte - 10 ml of citrate buffer (pH 4, containing 0.1 M NaClO₄) and in the presence of enzyme substrates with a stock concentration of 10 mM until a 30 mM concentration was achieved in the cell.

Amperometric detection has been carried out at a constant potential of -0.2 V through successive additions of aliquots of 10 mM substrate stock solutions (typically from 20 to 500 ml) to 10 ml of the electrolyte in the cell. Chronoamperometric detection was carried out under constant stirring at 500 rpm.

Differential pulse voltammograms were recorded both in the absence and presence of studied compounds over the potential range from +0.6 to –0.6 V at a scan rate of 10 mV s^-1, pulse duration of 50 ms and an amplitude of 0.025 V, as optimized in the authors’ previous studies [29,34].

Data analysis was implemented with Origin Pro 8.5 software. Non-linear regressions and statistics were carried out using embedded software module for enzyme kinetics.

2.4. Herbal extracts preparation

Herbal extracts were prepared as follows: 20 g of dry herbal mixtures were added to 1 l of pure water and boiled for 20 min at atmospheric pressure (~101 kPa). Then, the herbal extract was set to cool down to RT, filtered through nylon cloth 6.6, packed in 50 ml sealed containers and refrigerated at 4˚C until analyses. Three types of herbal extracts were subjected to electrochemical and HPLC analyses, further referred as PM1, PM3 and PM7. The three types of herbal extracts consisted of following herbs:

PM1: Geranium sanguineous, Arctostaphylos uva-ursi, Betula alba, Polygonum hydropiper, Achillea millefolium;

PM3: Crataegus monogyna, Equisetum arvense, Geranium sanguineum, Urtica dioica; and

PM7: Fragaria vesca, Hypericum perforatum, Calendula arvensis, Frangula alnus, Polygonum hydropiper.

These herbal combinations were selected due to their use as healing teas (pharmaceutical products) with high antioxidant content.

2.5. Chromatographic analysis

The HPLC analysis has been carried out as a referent method for phenolic acid quantification, as follows: The phenolic acid composition of the extracts was assessed using chromatographic system (Shimadzu, Japan), which consist of auto sampler (Nexera X2, SIL- 30AC); CTO-20AC column and SPD-20A UV detector (Shimadzu, Japan). Analysis was carried out using column Metitaranea Sea RP-18e (150 mm × 4.6 mm × 2 μm) (Teknokrom, Spain), mobile phase of 4% acetic acid and 100% AcCN (80:20), flow rate of 0.65 ml.min^-1, λ = 280 nm and temperature of 35 ˚C. Results were analyzed using Lab-Solution Nexera-XR-RF software and the standard phenolic acids (gallic and caffeic acids) [32,33].

The content of the phenolic compounds was calculated against a standard line constructed with its solutions at concentrations ranging from 500 µg.ml^-1 to 2.5 µg.ml^-1 of the corresponding phenolic acid with a correlation coefficient of R² > 0.9991. Total phenolic content was calculated by summing the content of each determined phenolic compound and re-calculating the total phenolic content in equivalents of gallic acid.

1. Results and Discussion

3.1. Studies of two different laccases in di – and trihydroxy aromatic compounds present by cyclic voltammetry (CV)

Electrochemical behavior of the two types of laccase-based bioelectrodes was probed using cyclic voltammetry (CV) in the absence and presence of both laccase substrates – oxygen and phenolic compounds. Voltammetric studies have shown that for the two types of laccase biosensors, reductive wave starts at potentials more negative than -0.25 V in aerated buffer solutions (i.e. in the presence of oxygen), which was not seen in deaerated solutions. It is well known that laccase is a metalloprotein capable of exchanging electrons with underlying electrode surfaces directly [34] without the need for additional electron shuttles (mediators). The efficiency of the electrical communication between the electrode and laccase depends on the enzyme orientation and distance between its active site and electrode surface [34]. Most probably, the negatively charged Nafion membrane electrostatically repulsed the negatively charged laccase active site, this way orienting the enzyme to electrode surface. The latter conformation is favorable for the electron exchange with the underlying electrode, which was manifested by a reductive wave appearing on the CV in the presence of molecular oxygen. Therefore, voltammetric studies verified the ability of immobilized laccase to carry out bioelectrocatalytic O₂ reduction to water molecules, thus proving that enzymes were electrochemically active.

Comparison of the CVs of the enzyme electrode recorded in aerated solutions in the absence and presence of pyrogallol and catechol as substrates (Fig. 1) revealed laccase-catalyzed oxidation of the two phenols to semi-quinones, followed by electrochemical regeneration of the oxidized products. When resorcinol was tested as laccase substrate, the resulting voltammograms did not show interactions between either of the immobilized enzymes, as no reduction of the product of its enzyme-catalyzed oxidation was noticed (Fig. S1, Supplementary information). These phenolic compound-depending differences in the performance of the two types of laccase biosensors were due to the difference between catechol and resorcinol in their spatial structure. The first benzenediol is with two vicinal hydroxy-groups, while resorcinol is its meta-isomer.

Caffeic and gallic acids could be considered as phenolic compounds derived from catechol and pyrogallol, respect-ively. Their structural similarities with dihydroxyl and trihydroxyl aromatic compounds, as well as the fact that they are the usual constituents of polyphenolic complex in various natural products, motivated further interest in probing the voltammetric behavior of the produced biosensors in the presence of the two phenolic acids. On the CVs recorded in the absence of either phenolic compound (Fig. 2, black lines), a reductive wave was recorded with potentials more negative than -0.3 V that resulted from the electrochemical reduction of the dissolved molecular oxygen catalyzed by the immobilized enzyme, undoubtedly verifying that the two laccases were not only electrochemically, but also catalytically active.

The CVs recorded in the presence of either gallic or caffeic acid (Fig. 2, red lines), showed a clearly expressed reductive wave starting much earlier below +0.1 V, which was due to the fact that the two phenolic acids mediated the electrochemical reduction of dissolved oxygen and therefore significantly decreased the overpotential of the oxygen reduction on laccase-bearing electrodes. As seen from the presented plots, the interactions of the two laccases with the two phenolic acids resembled the shapes of the voltammograms recorded in the presence of catechol and pyrogallol. It is noteworthy that the efficiency of the enzyme interaction with the two phenolic acids is different being much higher in the presence of caffeic acid as it could be deduced from the pronounced reductive waves (Fig. 2C, D). Reaction with lower intensity between either of the two laccases with gallic acid was possibly resulting from electrostatic repulsion of its anionic form generated at the operating pH 4.0 [35] and the electrode surface, which also bore negative charges due to the coverage with a Nafion film.

3.2. Differential pulse voltamperometric response of laccase-based bioelectrodes to gallic and caffeic acids using laccases from Trametes versicolor and Trametes pubescens

Differential pulse voltammograms (DPV) of same laccase electrodes are depicted in Figure 3 (Fig. 3A, B; dashed curves). No peaks were identified on the DPVs of laccase-bearing electrodes in background electrolyte and the sharp current decay at potentials more negative than -0.4 V confirmed that oxygen reduction reaction occurred on the bioelectrode’s surface. To investigate further the voltammetric behavior of immobilized laccase in the presence of the two phenolic acids – gallic and caffeic acids, the differential pulse voltammograms were recorded at varied substrates’ concentrations. The addition of gallic acid aliquots to the buffer followed by the record of resulting DPV (Fig. 3A) caused a significant increase in the current with a peak at -0.2 V on the voltammograms, the height of which increased with increasing gallic acid concentration.

No shift of the peak position was reported upon raising its concentration. Similarly, in the presence of caffeic acid (Fig. 3B), a clearly expressed peak at a more positive potential than the one for gallic acid was recorded, the height of which increased proportionally to substrate concentration. The difference in the behavior of the laccase biosensor in the presence of caffeic acid as an enzyme substrate was that the reductive peak occurred at a potential of +0.2 V and the peak potential slightly shifted positively with increasing substrate concentration. It is plausible that the penetration of gallic acid was hampered by the deprotonation of its carboxylic group at the working pH due to the electrostatic repulsion between the negatively charged Nafion membrane and the gallic acid anionic form, resulting in a significant shift of the reduction potential to more negative values than those of caffeic acid. The latter was not deprotonated at the operating pH of the media [36] and hence its molecules penetrated the membrane easier. A similar behavior of the second laccase from T. pubescens was seen under equivalent experimental conditions.

A Michaelis type dependence between the DPV peak height and caffeic acid concentration was observed over a range from 0.01 up to 1 mM (Fig. 4A). Enzyme inhibition by the substrate of T. pubescens laccase became obvious at concentrations exceeding 0.5 mM, while enzyme isolated from T. versicolor seemed unaffected by substrate inhibition even at 1 mM concentration. Under equivalent experimental conditions, the dependence of the DPV peak current on gallic acid concentration (Fig. 4B) was based on Michaelis type kinetics only for T. versicolor laccase, while differential response of the biosensor based on the enzyme from T. pubescens decayed sharply in the presence of trihydroxy aromatic compound (Fig. 4B, red).

Despite the substantial differences between the DP voltammograms and the large peak separations recorded in the presence of either gallic or caffeic acid, DPV studies performed in combinations of the two phenolic acids did not allow discrimination between di- and triphenols. On DPVs recorded in 1:1 mixture of gallic and caffeic acids (Fig. S2, Supplementary information), the peak at -0.2 V appearing in the presence of gallic acid alone merged with the one typical for caffeic acid and the peak potential shifted negatively. With increasing the gallic acid quota up to 10 times, the two peaks broadened and turned into humps, the position and height of which varied irregularly with increasing the concentration of the mixture.

 Information from DPV studies clearly demonstrated that this electrochemical technique could hardly be used for analysis of mixtures of the studied di- and triphenolic compounds, which motivated further investigating an alternative electrochemical approach that could potentially be further useful for the analysis of complex mixtures as amperometric detection.

3.3. Amperometric detection of gallic and caffeic acids with laccase-based biosensors using laccases from Trametes versicolor and Trametes pubescens

In Fig. 5 are depicted the dependencies of the electrode response on the concentration of gallic and caffeic acids assessed with the two types of laccase-based electrodes under the working conditions selected as optimal: working potential of -0.2 V at pH 4.0 [29]. Similarities in the shapes of the curves were substantial. The two types of electrodes showed hyperbolic trends of electrode response as a function of substrate concentration. However, the apparent kinetic constants for the two immobilized laccases, determined from non-linear regression analysis of experimental data, showed significant differences. The apparent Michaelis constants for immobilized T. versicolor laccase (Fig. 5, A) with respect to caffeic and gallic acids have been found to be very similar (Table 1), while the corresponding apparent maximum rates of the enzyme catalyzed reaction differed significantly with the  for the caffeic acid being almost 5 times higher than that for gallic acid.

Unlike these, for the immobilized laccase from T. pubescens (Fig. 5B) with respect to caffeic and gallic acids differed more than 3 times (0.19 mM ±0.03 against 0.06 mM ±0.04, respectively, Table 1) whereas the apparent maximum reaction rates diverged more than those estimated for T. versicolor laccase. These differences in the apparent kinetic constants of the two identically immobilized enzymes suggested that despite biochemical similarities of the two laccases, significant differences were reported in enzyme affinity to dihydroxy and trihydroxy aromatic compounds. Since the reaction rate was equal to the electrode response (current, in amperes [A]), significant differences in  for the two laccases (Table 1) indicated that T. pubescens laccase respectively oxidized caffeic and gallic acids 5 and 4 times as fast as that from T. versicolor. Moreover, these differences in the heterogeneous enzymatic activities were observed regardless of equivalent units of the immobilized laccases.

In all cases except one, significant inhibition effects by the substrate were seen at concentrations exceeding 0.1 mM with inhibition constants calculated by non-linear regression (Table 1). Only T. versicolor laccase showed no inhibition upon addition of gallic acid at concentrations exceeding 0.15 mM.

From the non-linear regression analysis of the kinetic curves, it was found that the electrode response obeyed the Eq.1:

                                                                                                                      Eq.1

Where,  is the apparent maximum rate of the enzyme-catalyzed reaction, A;

 are the apparent Michaelis constant and inhibition constant, respectively, M;

C is the concentration of the respective phenolic acid, M;

I is the electrode response directly proportional to the rate of enzyme-catalyzed reaction, A.

All electrochemical measurements were done in triplicate with a RSD not exceeding 3.5 %.

3.4. Cyclic voltammetry analysis of phenolic acids in herbal extracts using laccases from Trametes versicolor and Trametes pubescens

The available three types of herbal extracts were tested for their redox behavior by performing cyclic voltammetry with the produced two types of laccase-based enzyme electrodes in the operating buffer; to which, equal volumes of each extract were added. Shapes of the resulting CVs (Figs. S3 and S4, Supplementary information) suggested that in the extract of type PM1, triphenols predominated while the other two types of herbal extracts contained a combination of di- and trihydroxy aromatic compounds. It is noticeable that the biosensor based on laccase from T. pubescens was responding ca. three times more intensely than the analogous one based on laccase from T. versicolor.

Quantitative analysis of herbal extracts has been carried out by means of constant potential amperometry (amperometric detection) using standard addition method. Method of standard additions (MSA) is typically carried out by adding small volumes of concentrated solution of the analyte to the sample [37]. The major advantage of the standard addition method is the opportunity to practically eliminate effects of the complex matrix in the real samples. As an external standard, 10 mM solutions of gallic acid were chosen and the MSA was implemented with the two discussed types of laccase biosensors. The one produced based on the laccase from T. pubescens (Fig. 6) guaranteed at least ten times more intense biosensor response to the same amount of PM1 herbal extract, compared to the signal of the T. versicolor laccase-based biosensor (Fig. 7). The latter finding led to greater RSD and significantly overestimated levels of the analyzed compounds; from which, it could be concluded that the second type of biosensor was not appropriate for further analyses. Based on the latter finding, the content of phenolic acids of the other two types of herbal extracts was analyzed using T. pubescens laccase-based biosensor, method of standard addition and gallic acid as an external standard.

The outcomes from biosensor analysis were compared with those from HPLC analyses of the three types of extracts (Table 2). Quantities of the phenolic compounds, estimated from the MSA, were further multiplied by dilution factor and the resulted values were recalculated into gallic acid equivalents per grams of dry weight herbs (DW). Based on HPLC analysis of the phenolic acids, the studied herbal extracts included gallic, caffeic, chlorogenic, p-coumaric and trans-ferulic acids.

As concluded from the present results, the recovery percentage was good only for the type PM1 herbal extract while for the other two extracts biosensor method showed significant deviations from the satisfactory recovery percentages (95-105%). This might be due to the presence of significant quantities of m-benzenediols (e.g. resorcinol and its derivatives) and/or other types of polyphenols, which did not react with laccase.

The two electroanalytical techniques, DPV and constant potential amperometry, were used to find the most convenient approach for the assessment of phenolic content in model solutions to adopt it for the analysis of real samples comprising numerous interferents. However, the hypothesis that the two-model phenolic compounds, benzenediol caffeic acid and benzenetriol gallic acid, could be discriminated in their mixtures based on their DPV peaks that were not well-separated, was not verified due to the strong interferences between them when combined. The constant potential amperometry was shown as a better technique for the quantification of benzenediols and benzenetriols. Large differences in the rates of their electrochemical conversion did not provide means for their separate analysis as well.

1. Conclusion

The present results suggested that only enzymes with high homogeneous activity might be used for electroanalytical purposes. As previously discussed, low activity might lead to overestimated analyte levels (Figure 7). The decreased heterogeneous activity of T. versicolor laccase caused a stronger interference of a complex matrix that could not be eliminated even by the method of standard addition.

Indeed, HPLC analyses could provide not only quantitative, but also qualitative information regarding types of phenolic compounds such as benzenediols and benzenetriols. However, biosensing method provides two important advantages over the chromatographic analysis – it can be carried out rapidly with minimum sample pretreatment and equipment, which is much susceptible to miniaturization allows in-field analysis mostly.

1. Acknowledgements

This study was funded by the European Union-NextGenerationEU through the National Recovery and Resilience Plan of the Republic of Bulgaria; grant no. BG-RRP-2.004-0001-C01, DUEcoS. Authors gratefully acknowledge the generous gift of purified T. pubescens laccase from Prof. Roland Ludwig, BOKU, Vienna, Austria.

1. Conflict of Interest

The authors report no conflict of interest.

1. Authors Contributions

All authors designed and contributed to this study. Conceptualization, N.D. and I.I, methodology, M.S, software, T.C, validation, A.P, M.S. and T.C, formal analysis, M.S. and M.N, investigation, M.S, A.P. and M.N; resources, N.D, data curation, M.S. and M.N, writing—original draft preparation, M.S. and T.C, writing—review and editing, N.D, visualization, M.S, supervision, N.D. and I.I, project administration, I.I, funding acquisition, I.I. All authors have read and agreed to publish the final version of the manuscript.