Decolorization and Detoxification of Synthetic Dyes by Trametes versicolor Laccase Under Salt Stress Conditions

Abstract

Fungal laccases are promising oxidative enzymes for bioremediation applications, particularly in the degradation of synthetic dyes present in industrial effluents. Here, we evaluated the inhibitory effects of sodium chloride (NaCl) and sodium sulfate (Na₂SO₄) on the activity of Trametes versicolor laccase and its ability to decolorize Congo Red (CR), Malachite Green (MG), and Remazol Brilliant Blue R (RBBR). Enzyme assays revealed concentration-dependent inhibition, with IC₅₀ values of 0.22 ± 0.04 M for NaCl and 1.00 ± 0.09 M for Na₂SO₄, indicating stronger inhibition by chloride. Kinetic modeling showed mixed-type inhibition for both salts. Despite this effect, the enzyme maintained significant activity: after 12 h, decolorization efficiencies reached 95 ± 4.0% for MG, 88 ± 3.0% for RBBR, and 75 ± 3.0% for CR, even in the presence of 0.5 M salts. When applied to a mixture of the three dyes, decolorization decreased only slightly in saline medium (94.04 ± 4.0% to 83.43 ± 5.1%). FTIR spectra revealed minor structural changes, but toxicity assays confirmed marked detoxification, with radicle length in lettuce seeds increasing from 20–38 mm (untreated dyes) to 41–48 mm after enzymatic treatment. Fungal growth assays corroborated reduced toxicity of treated dyes. These findings demonstrate that T. versicolor laccase retains functional robustness under ionic stress, supporting its potential application in saline textile wastewater remediation.

1. Introduction

The textile industry is one of the most significant contributors to environmental pollution due to the large volumes of wastewater it generates during various stages of fabric processing. These effluents often contain high concentrations of synthetic dyes, many of which are resistant to conventional treatment methods and pose serious environmental and health risks. Among the diverse dyes used in textile manufacturing, Congo red (CAS Number 573-58-0), an azo dye, Malachite green (CAS Number 569-64-2), a triphenylmethane dye, and the anthraquinone-based dye Remazol brilliant blue R (CAS Number 2580-78-1) are of particular concern due to their toxicity, mutagenicity, and persistence in aquatic ecosystems [1,2,3,4].

The global production of synthetic dyes intended for the textile industry reaches 7 × 10⁵ tons, of which 10–15% are discarded into the environment [5,6]. The contamination of water by textile dyes affects the biological, physical, and chemical processes of these environments. It reduces, for example, photosynthesis, because the dyes block the passage of sunlight and the chemical substances can induce mutagenic effects [7,8]. Conventional methods for eliminating these effluents are mainly based on physical and chemical processes, such as adsorption, concentration, chemical transformation, and incineration; these processes are costly and can generate toxic byproducts, limiting their application [9]. Alternatively, biological methods such as the use of microorganisms and their ligninolytic enzymes have gained attention as treatment methods for dyes due to their efficiency, high performance at low concentrations, and the formation of non-toxic residues with low operational costs [4,10,11]. Integrated systems, such as the combination of advanced oxidation processes with biological treatment have already been evaluated with the aim of obtaining less harmful, cost-effective and environmentally sustainable treatment [12].

Ligninolytic fungi are a group of microorganisms with strong degradation capacity, relying on unique extracellular enzyme systems such as laccase, manganese peroxidase, and lignin peroxidase, which enable efficient lignin degradation [13]. They have also been shown to effectively degrade a variety of environmental pollutants, including synthetic dyes [14,15]. Enzymatic bioremediation is increasingly recognized as an effective strategy for treating dye-polluted effluents. Among the enzymes explored for this application, laccase, a multicopper oxidase with the ability to oxidize a wide variety of aromatic and phenolic compounds, has demonstrated high potential [16,17]. Laccases are predominantly produced by ligninolytic fungi, which play a crucial role in the natural degradation of lignin, a complex aromatic polymer found in plant cell walls. Among these fungi, Trametes versicolor, a white-rot basidiomycete, is one of the most studied and efficient producers of laccase. The laccases from T. versicolor exhibit high redox potential and broad substrate specificity, making them especially suitable for the oxidation of various recalcitrant pollutants, including synthetic dyes [18]. These enzymes catalyze one-electron oxidation reactions using molecular oxygen as the terminal electron acceptor, generating water as the only byproduct, which further enhances their environmental appeal. Due to their robustness, catalytic efficiency, and ability to operate under a wide range of pH and temperature conditions, T. versicolor laccases are widely investigated for applications in bioremediation, particularly for the treatment of industrial wastewater containing complex aromatic compounds such as textile dyes. However, a significant limitation to their practical application in real effluent conditions is the presence of high concentrations of salts, especially sodium chloride and sodium sulfate, commonly used in textile dyeing processes [19]. While these salts are critical for neutralizing the negative zeta potential of cotton fibers and thereby enhancing dye fixation [20], chloride ions are well-documented inhibitors of laccase activity. Their inhibitory effect is attributed to direct interactions with the enzyme’s active site or to conformational perturbations that compromise catalytic efficiency [21,22]. This salt-induced inhibition represents a significant challenge for implementing laccase-based treatments in saline textile effluents, underscoring the necessity of further investigation about the enzyme performance under such conditions. In this study, we evaluate the efficacy of T. versicolor laccase in degrading three representative textile dyes, Congo red (azo dye), Malachite green (triarylmethane dye), and Remazol Brilliant Blue R (anthraquinone dye), while also examining the impact of sodium chloride (NaCl) and sodium sulfate (Na₂SO₄) on the enzymatic activity. Although the inhibitory action of salts, particularly NaCl, on laccases has been extensively reported, most of these studies have relied on short-term assays with artificial substrates such as ABTS, providing only a limited understanding of enzyme behavior under realistic conditions. To date, little is known about how laccases perform over extended periods of time when challenged with structurally diverse and environmentally relevant dyes in saline environments. Our study addresses this gap by systematically evaluating the ability of Trametes versicolor laccase to decolorize and detoxify Congo Red, Malachite Green, and RBBR in the presence and absence of salts over long incubation periods. This approach not only advances current knowledge on laccase inhibition dynamics but also provides critical insights into their potential for practical dye bioremediation under saline stress. Understanding the influence of these salts is essential for assessing the feasibility of laccase-mediated bioremediation strategies in industrial wastewater treatment scenarios.

2. Materials and Methods

2.1. Materials

ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) as well as Congo red, Malachite green and Remazol Brilliant Blue R were obtained from Sigma-Aldrich Co. (Saint Louis, MO, USA). The analytical-grade chemicals were of the highest purity available.

2.2. Microorganism and Laccase Production

Trametes versicolor used in this study is part of the Basidiomycete Collection maintained by the Laboratory of Microorganism Biochemistry at the State University of Maringá (UEM), and is registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SISGEN) under the accession number A4E5EC1, as of 1 November 2018. For strain maintenance, the fungus was cultured on potato dextrose agar (PDA) plates. Laccase production was promoted under solid-state fermentation (SSF), using 5 g of wheat bran as the substrate in 250 mL Erlenmeyer flasks. The substrate moisture content was adjusted to 83% by the addition of a mineral solution [23]. Cultivation was continued for 7 days in the absence of light and at 28 °C. Crude enzyme extraction was performed by adding 15 mL of cold distilled water to each flask, followed by agitation at 10 °C for 30 min, filtration through gauze, and centrifugation at 1792× g for 10 min. A previously reported SDS-PAGE method was employed [24], with slight modifications. Specifically, β-mercaptoethanol was omitted and samples were not heated prior to electrophoresis, ensuring non-reducing conditions. Laccase activity was determined using the ABTS assay as described elsewhere [25]. The apparent molecular mass of laccase was estimated by comparing its electrophoretic mobility with that of protein standards (Bio-Rad, 10–250 kDa). Native sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis confirmed the production of a single laccase isoform with an estimated molecular mass of 45 kDa, consistent with findings previously reported by another research group [26]. The crude enzyme extract was subsequently lyophilized and stored at −20 °C until further use.

2.3. Laccase Activity Determination

Laccase activity was measured spectrophotometrically using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, 1.0 mM) as the substrate. The reaction mixture (final volume: 1.0 mL) contained 50 mM sodium acetate buffer (pH 5.0), ABTS, and an appropriate volume of enzyme solution [27]. Assays were carried out at 40 °C in a quartz cuvette, and the increase in absorbance was monitored at 420 nm for 5 min using a UV–Vis spectrophotometer. Laccase activity was calculated from the linear portion of the absorbance curve using the molar extinction coefficient of 36,000 M⁻¹·cm⁻¹. One unit of enzyme activity (U) was defined as the amount of enzyme required to oxidize 1 µmol of ABTS per minute under the assay conditions.

2.4. Effects of Na₂SO₄ and NaCl on Laccase Kinetic Parameters

The initial reaction rates were determined at varying concentrations of ABTS (0.1–1.0 mM) in 50 mM sodium acetate buffer (pH 5.0) at 40 °C, with a reaction time of 5 min in the absence and presence of variable concentrations of Na₂SO₄ and NaCl. To determine the best kinetic model that describes the data, Scientist^® software (version 2.0; MicroMath Scientific Software, Salt Lake City, UT, USA) was used. This software allows us to fit a single equation with two (2) independent variables to the data using an iterative non-linear least-squares procedure. The independent variables were the substrate (ABTS) concentration and the salt concentration (NaCl or Na₂SO₄). Selection of the best kinetic model (equation) was based on the model selection criterion (MSC), the standard deviations of the fitted parameters, and the sum of squared deviations between experimental and calculated reaction rates. The MSC is defined as [28]:

$$\mathrm{M}\mathrm{S}\mathrm{C}=\ln \left[{\displaystyle \frac{{\displaystyle \sum _{\mathrm{i}=1}^{\mathrm{n}}{\mathrm{w}}_{\mathrm{i}}{({\mathrm{Y}}_{\mathrm{o}\mathrm{b}{\mathrm{s}}_{\mathrm{i}}}-{\overline{\mathrm{Y}}}_{\mathrm{o}\mathrm{b}\mathrm{s}})}^2}}{{\displaystyle \sum _{\mathrm{i}=1}^{\mathrm{n}}{\mathrm{w}}_{\mathrm{i}}{({\mathrm{Y}}_{\mathrm{o}\mathrm{b}{\mathrm{s}}_{\mathrm{i}}}-{\mathrm{Y}}_{\mathrm{c}\mathrm{a}\mathrm{l}})}^2}}}\right]-{\displaystyle \frac{2\mathrm{p}}{\mathrm{n}}}$$

(1)

The variables are: Y_obs, measured reaction rates; $\overline{\mathrm{Y}}\mathrm{o}\mathrm{b}\mathrm{s}$, mean of all reaction rates; Y_cal, theoretical reaction rates; w, statistical weights. The calculations were performed with n experimental observations and p parameters to be determined.

2.5. Use of the Laccase for Decolorizing Synthetic Dyes in the Absence and Presence of Salts (Na₂SO₄ and NaCl)

In 10 mL test tubes, 4.5 mL of the following dyes were added: Congo Red (50 ppm), Malachite Green (40 ppm), and RBBR (100 ppm). A mixture of all three dyes was also used. Next, 500 µL of enzyme extract (5 U) was added to each tube. The mixtures were kept at 40 °C without stirring. Absorption spectra of each mixture were recorded in the visible light range (400 to 800 nm) at 0 h (negative control) and subsequently at different times of incubation. To evaluate the percentage of decolorization, the absorbance at the maximum absorption wavelength of each dye was used: 495 nm for Congo red, 620 nm for Malachite green, and 595 nm for RBBR. Decolorization of the dye mixture was assessed by determining the area under the curves using the program GraphPad Prism 10. To evaluate the effects of presence of Na₂SO₄ and NaCl on the capability of decolorization of dyes, the experiments were conducted in the same way but in the presence of different concentrations of NaCl or Na₂SO₄.

2.6. Infrared Spectroscopy

Fourier Transform Infrared Spectroscopy (FTIR) was used to analyze the molecular structure and functional groups of the synthetic dyes before and after enzymatic treatment with laccase. Samples were first dried at 40 °C until constant weight to remove residual moisture. For each measurement, 1 mg of dried dye sample was finely ground and homogenized with 200 mg of spectroscopic-grade potassium bromide (KBr, Sigma-Aldrich). The mixture was pressed into transparent pellets under a pressure of approximately 10 MPa using a manual hydraulic press. FTIR spectra were collected on a PerkinElmer ( Waltham, MA, USA) spectrometer in the wavenumber range of 4000–500 cm⁻¹, with a spectral resolution of 2 cm⁻¹. Each spectrum was obtained by averaging 128 scans, and all analyses were performed in triplicate. Baseline correction and normalization were applied before further interpretation of the spectra.

2.7. Ecotoxicity Analysis of the Dyes Before and After Laccase Treatment

Two complementary bioassays were employed to evaluate the ecotoxicity of the dyes: a phytotoxicity test using lettuce seeds (Lactuca sativa) and a fungal growth inhibition test using Aspergillus awamori. All dye solutions were prepared in distilled water at a final concentration of 100 ppm, either untreated or after enzymatic treatment with laccase.

2.7.1. Phytotoxicity Assay (Lactuca sativa)

Sterile 90 mm Petri dishes were lined with Whatman No. 1 (Maidstone, UK) filter paper and moistened with 2 mL of each dye solution (treated or untreated). Distilled water was used as a negative control. Twenty lettuce seeds were evenly distributed in each plate. The plates were incubated at 25 ± 1 °C in the dark for 4 days. After incubation, the length of the radicles was measured using a ruler. Each treatment was performed in triplicate (n = 60 seeds per condition). Results were expressed as mean radicle elongation (mm) ± standard deviation [29]. Statistical comparisons between treated and untreated dye solutions were performed using Student’s t-test (p < 0.05). Additionally, one-way ANOVA followed by Bonferroni post hoc testing was applied to compare radicle growth in the control group (distilled water only) with the growth observed in the presence of laccase-treated dyes.

2.7.2. Fungal Growth Inhibition Assay (Aspergillus awamori)

A. awamori spores (1 × 10⁵ spores/mL) were prepared in sterile distilled water and inoculated onto Potato Dextrose Agar (PDA) plates supplemented with either untreated or laccase-treated dyes (final concentration: 100 ppm). PDA plates without dye addition served as growth controls. Inoculation was performed by spotting 10 µL of the spore suspension in the center of each plate. The plates were incubated at 28 °C for 5 days, and fungal growth was monitored daily by measuring the colony diameter along two perpendicular axes. Each treatment was performed in triplicate.

3. Results

3.1. Evaluation of T. versicolor Laccase Activity in the Presence of Na₂SO₄ and NaCl

Figure 1 shows the impact of various concentrations of Na₂SO₄ and NaCl on the catalytic activity of the T. versicolor laccase, using ABTS as the substrate. Both salts exhibited inhibitory effects on the enzymatic activity; however, the inhibition caused by NaCl was significantly more pronounced than that observed for Na₂SO₄ along the same concentration range. The IC₅₀ (salt concentration causing 50% inhibition) was 0.22 ± 0.04 M for NaCl, and 1.00 ± 0.09 M for Na₂SO₄.

To get an insight into the inhibition mechanism, the substrate concentration dependence of the laccase was measured in the absence and presence of either NaCl or Na₂SO₄. The results are shown in Figure 2. The laccase showed the usual Michaelian behavior with near saturation being achieved at the ABTS concentration near 1 mM. Apparently neither NaCl nor Na₂SO₄ modified this Michaelian behavior, but inhibition is quite evident over the whole ABTS concentration range. A numerical analysis, however, should provide more detailed information about the mechanism of the inhibition and provide useful numerical data. Using the algorithm provided by the Scientist software (version 2), several equations, each one representing a specific mechanism, were fitted to the whole data set obtained with each salt. Equations describing competitive, uncompetitive, non-competitive (and the mixed type variant) inhibition were fitted to the data in the search for the most probable mechanism. The search included the variants linear (complete inhibition), hyperbolic and parabolic inhibition. For both salts, the mixed type variant of mixed non-competitive linear (complete) inhibition proved to be the most probable mechanism. This mechanism is described by the following equation [30,31]:

$$\mathrm{v}={\displaystyle \frac{{\mathrm{V}}_{\max }[\mathrm{S}]}{{\mathrm{K}}_{\mathrm{M}}\left(1+\frac{[\mathrm{I}]}{{\mathrm{K}}_{\mathrm{i}1}}\right)+[\mathrm{S}]\left(1+\frac{[\mathrm{I}]}{{\mathrm{K}}_{\mathrm{i}2}}\right)}}$$

(2)

V_max is the maximal reaction rate and K_M the Michaelis-Menten constant. Equation (2) predicts two enzyme-inhibitor complexes, EI and ESI, K_i1 and K_i2 being the corresponding dissociation constants. The continuous lines in Figure 2 allow a visual evaluation of how theory describes the experimental curves. It is apparent that the theoretical curves cover quite well the course of the experimental points without systematic deviations. Numerical values of the optimized values of V_max, K_M, K_i1 and K_i2, together with the corresponding standard errors, are listed in

Table 1. Optimized kinetic constants and corresponding statistical indicators of fit goodness obtained by fitting Equation (2) to the experimental data shown in Figure 2.

Kinetic Constants and Statistical Indicators	NaCl	Na2SO4
Vmax (µmol/min)	4.84 ± 0.05	4.85 ± 0.05
KM (mM)	0.0712 ± 0.0035	0.0686 ± 0.0037
Ki1 (M)	0.0364 ± 0.0036	0.788 ± 0.145
Ki2 (M)	0.234 ± 0.021	1.221 ± 0.077
Model selection criterion	5.858	4.939
Correlation (r)	0.998	0.997
Sum of squared deviations	0.133	0.207

. The latter also gives the values of MSC, correlation coefficient (r) and sum of squared deviations, which are indicators of the goodness of fit. The optimized V_max and K_M values obtained in the NaCl and Na₂SO₄ experiments were practically the same, a necessary outcome if one takes into account that they reflect intrinsic properties of the same enzyme.

The K_i1 and K_i2 values of NaCl and Na₂SO₄ differ substantially. They are both higher for Na₂SO₄, an outcome that reflects the different potencies of both salts as inhibitors, NaCl being more effective. For both salts, on the other hand, the EI complex is more easily formed than the ESI complex. More specifically, for NaCl the E·NaCl complex is 21.6 more stable than the E·S·NaCl complex; and, in the case of Na₂SO₄, the E· Na₂SO₄ complex forms 5.2 more easily than the E·S· Na₂SO₄ complex.

3.2. Decolorization of Synthetic Dyes by Trametes Versicolor Laccase: The Effects of Salts

To evaluate the effect of NaCl and Na₂SO₄ on the decolorization capacity of the Trametes versicolor laccase, experiments were conducted using three synthetic dyes: Congo red, Malachite green, and Remazol brilliant blue R (RBBR). The decolorization experiments were done in the absence of salts as well as in the presence of 0.5 M NaCl and 0.5 M Na₂SO₄. At zero time, and after 2 and 12 h of incubation under static conditions, spectral absorption curves were recorded in the range of 400–800 nm (Figure 3A–C). After 2 h of incubation, a slight reduction in decolorization of all three dyes was observed in the presence of salts. However, after 12 h, no significant difference in decolorization was observed for any of the dyes when compared to the control without salt: after 12 h, decolorization efficiencies reached 95 ± 4.0% for MG, 88 ± 3.0% for RBBR, and 75 ± 3.0% for CR, even in the presence of 0.5 M salts. Figure 4 presents the results of a similar experiment using a mixture of the three dyes. In this case, although decolorization was still observed after 12 h of incubation in the presence of 0.5 M NaCl, it is evident that the presence of salt caused a slight reduction (from 94.04 ± 4.0% to 83.43 ± 5.10%) in decolorization compared to the condition without salt.

Importantly, after 12 h the residual laccase activity did not vary by more than 10% compared to the initial activity, indicating good enzyme stability throughout the assays.

3.3. Fourier Transform Infrared Spectroscopy

The FTIR spectra of the synthetic dyes before and after treatment with laccase are shown in Figure 5. In Figure 5A,B, the spectrum of Congo Red (CR) exhibited peaks corresponding to −NH₂ and −OH groups at higher wavenumbers; weak bands from overtones and combination vibrations at 3030–2300 cm⁻¹; N=N and C=C stretching conjugated with the aromatic ring around 1600 cm⁻¹ and 1400 cm⁻¹; and aromatic amines at 1354–1180 cm⁻¹. The asymmetric stretching vibration of the S–O (SO₃H) group appears at 1196 cm⁻¹, and the band at 1336 cm⁻¹ corresponds to NO₂. Minor differences can be observed between the initial spectrum and the spectrum after 12 h treatment, which may suggest slight modifications in the dye structure. For Malachite Green (Figure 5C,D), the broad peak around 3400 cm⁻¹ indicates the presence of −OH and −NH groups. Peaks at 2910 and 1365 cm⁻¹ are associated with C–H stretching and bending in methyl groups, respectively. Absorbance changes in the 1415–1455 cm⁻¹ region may reflect variations in C–C stretching vibrations in aromatic rings. The peak at 1035 cm⁻¹ corresponds to C–N stretching vibrations and the presence of aliphatic amines. The RBBR spectrum (Figure 5E,F) shows a broad band around 3400 cm⁻¹ indicative of N–H and O–H stretching vibrations. Peaks at 1610 cm⁻¹ can be attributed to C=C, C=O, and C=N functional groups, while bands at 1241, 725, and 626 cm⁻¹ are associated with C–O, S–O, and C–CO–C stretching vibrations. Small differences observed after 12 h treatment may indicate slight structural changes in the dye molecules; however, further analyses (e.g., LC-MS or HPLC) would be required to confirm any fragmentation or formation of derivative compounds.

3.4. Toxicity Studies

Phytotoxicity was assessed using lettuce seeds whose germination was evaluated after 4 days of incubation in the presence of either untreated or laccase-treated dyes under dark conditions at 28 °C. As shown in Figure 6, significant phytotoxic effects were observed in media saturated with malachite green, Congo red, RBBR, and their mixture.

A reduction in phytotoxicity was observed for all samples following enzymatic treatment for 12 h. Seeds incubated with water (control) exhibited an average radicle length of 50.00 ± 4.00 mm. Seeds exposed to untreated and treated malachite green showed mean radicle lengths of 25.32 ± 3.00 mm and 43.00 ± 9.75 mm, respectively. For Congo red, the corresponding values were 35.00 ± 4.00 mm (untreated) and 47.00 ± 3.00 mm (treated). For RBBR, the corresponding values were 38.00 ± 4.00 mm (untreated) and 47.95 ± 4.00 mm (treated). Lastly, the dye mixture yielded radicle lengths of 20.42 ± 4.00 mm for the untreated solution and 41.00 ± 4.00 mm for the laccase-treated solution.

In order to investigate further the toxicity of the dyes before and after enzymatic treatment, a second type of method was employed: the toxicity of dyes on fungal cells. PDA plates containing both treated and untreated dyes were monitored over a period of five days. As shown in Figure 7, spore development was observed only in plates containing the laccase-treated dyes.

4. Discussion

Textile effluents are complex mixtures containing high concentrations of salts and structurally diverse synthetic dyes, such as azo, anthraquinone, and triphenylmethane derivatives. These compounds are recalcitrant, toxic, and often resistant to conventional biological treatment. White-rot fungi and their oxidative enzymes, especially laccases, have emerged as promising candidates for the bioremediation of these effluents due to their ability to oxidize a wide range of xenobiotic compounds. Laccases decolorize synthetic dyes through oxidative cleavage of chromophoric structures. The enzyme initiates a one-electron transfer, forming phenoxy radicals that disrupt the extended π-electron conjugation system responsible for visible color [32]. These radicals can undergo subsequent non-enzymatic reactions, promoting further breakdown of dye molecules into smaller, less complex structures [33]. This process not only removes color but also reduces toxicity by eliminating or transforming hazardous groups such as aromatic amines and sulfonated moieties [32,34,35,36,37,38,39]. This mechanism makes laccases valuable biocatalysts for environmentally friendly treatment of dye-polluted effluents. However, the high salt concentrations typical of textile wastewaters, especially NaCl and Na₂SO₄, can negatively impact enzyme performance [40].

In the present study, both NaCl and Na₂SO₄ inhibited T. versicolor laccase activity in a concentration-dependent manner, with NaCl showing a stronger inhibitory effect, in agreement with previous reports for Ganoderma lucidum and other fungal laccases [41,42,43]. The inhibition was more pronounced at acidic pH, consistent with mechanisms proposed for chloride ion binding affecting the type 1 copper site [44]. Kinetic analysis suggested that both salts act as mixed-type inhibitors, likely forming EI and ESI complexes that are largely inactive under the tested conditions. While some laccases, such as those from Pycnoporus sanguineus or Oudemansiella canarii, exhibit incomplete or even stimulatory responses to salts [44,45,46,47], the results here highlight species-specific and context-dependent variability in salt sensitivity, which should be considered when designing bioremediation strategies.

Although T. versicolor laccase effectively decolorized and detoxified the three dyes tested in this study, FTIR analysis revealed only minor spectral changes after treatment. This likely reflects the limited sensitivity of FTIR to detect subtle or localized molecular modifications. Laccase-mediated oxidation primarily targets chromophoric groups, generating reactive radicals that disrupt conjugated double-bond systems, while the main molecular backbone of the dye molecules often remains largely intact. Similar findings have been reported in other studies, where only small FTIR shifts were observed following enzymatic dye degradation (

Table 2. Expected FTIR changes after laccase treatment.

Dye	Affected Groups	Expected FTIR Changes	Reference
Congo red	–N=N –SO3H Aromatic C=C	Disappearance or decrease of N=N (~1580–1600); Reduced S=O (~1040–1180); new C=O (~1700)	[47,48,49,50]
Malachite green	–N(CH3)2 Aromatic C–H	Decrease in C–N (~1120–1250) and C–H (~2800–3000); new O–H (~3200–3600), C=O (~1700)	[26,51,52,53]
RBBR	–SO3−, C=O, Aromatic rings	Decrease in S=O (~1040–1190) and C=O (~1660–1700); loss of aromatic bands; new O–H, C=O	[54,55]

).

These results highlight the importance of employing complementary analytical techniques, such as LC-MS or HPLC, to identify specific degradation products and fully elucidate the transformation pathways [56,57]. Nevertheless, the marked reduction in ecotoxicity observed for all treated dyes strongly suggests that significant chemical changes occurred, leading to the formation of less harmful intermediates even without complete mineralization.

The most important finding and primary novelty of this study is that, despite similar previous reports pointing toward high inhibitory effects by salts on the T. versicolor laccase, the enzyme retained sufficient activity to promote substantial decolorization of Congo Red, Malachite Green, RBBR, and their mixture under saline conditions when incubated for extended periods. These results indicate that, even under ionic stress, the laccase activity can be partially preserved, allowing relevant levels of dye decolorization to occur. Notably, the long-term assays conducted herein, spanning several hours, go beyond the short-term ABTS-based tests commonly reported in the literature, offering a more realistic assessment of the laccase performance under prolonged salt exposure. Taken together, these results demonstrate that T. versicolor laccase can effectively decolorize and detoxify structurally diverse dyes under saline stress. By systematically evaluating both enzyme inhibition mechanisms and functional outcomes over extended periods, this study provides new insights into the robustness of fungal laccases and highlights their potential applicability in practical saline wastewater remediation scenarios. In

Table 3. Comparison of the results of the present work with some previous reports about the action of fungal laccases on decolorization and detoxification of synthetic dyes and the effects of salts.

Fungal Laccase	Synthetic Dyes/ Salts Tested	Decolorization Efficiency	Detoxification	Reference
Ganoderma lucidum (crude laccase)	RBBR/NaCl and Na2SO4	Complete decolorization with 1.0 M Na2SO4, ~50% with 0.1 M NaCl	ND	[40]
Trametes hirsuta (immobilized laccase)	Reactive Blue 19 (RB19), NaCl	80% decolorization.	Up to 84% detoxification for RB19	[35]
Trametes versicolor (purified laccase)	Reactive Blue 19/NaCl and Na2SO4	Detailed mechanism of Cl− inhibition provided.	ND	[21]
Trametes versicolor (crude laccase)	RBBR, Congo red (CR), Malachite green (MG)/NaCl and Na2SO4	95 ± 4.0% for MG, 88 ± 3.0% for RBBR, and 75 ± 3.0% for CR/0.5 M salts	Toxicity of all dyes significantly decreased	This work

ND = not determined.

the results obtained in this work were compared with similar previous works.

A question that has not been approached experimentally in the present work deserves a few comments due to its importance for the degradation of dyes and other contaminants. This question refers to the mineralization of the dyes, the process which would result in the almost complete elimination of these compounds from the environment. Complete mineralization of dyes is impossible to achieve using laccases alone. For this reason, association of these enzymes with other biological or physicochemical processes is essential, particularly in saline environments. A promising approach involves laccase pre-treatment, which breaks chromophores and increases the biodegradability of aromatic intermediates, followed by the action of halotolerant bacteria that can complete the degradation pathway, ultimately leading to CO₂ and H₂O. This sequential strategy has proven to be especially effective for real textile effluents containing both dyes and high salt concentrations. Another alternative is to combine laccases with advanced oxidation processes (AOPs). For instance, laccase coupled with ultrasound can synergistically accelerate the breakdown of complex dye molecules while reducing toxicity, whereas photocatalysis benefits from enzymatic pre-oxidation, which enhances the susceptibility of the dyes to light-driven degradation. Similarly, bioelectrochemical systems can further oxidize dye intermediates that have been initially modified by laccases, promoting electrochemical mineralization. For optimal performance under saline conditions, immobilized laccases should be used to enhance stability and allow multiple reuse cycles and, whenever possible, Na₂SO₄ should be favored over NaCl, as chloride ions cause stronger enzymatic inhibition. Finally, integrating biological treatments with complementary physicochemical methods, while carefully monitoring both toxicity assays (e.g., Microtox, Daphnia tests) and conventional parameters such as COD (Chemical oxygen demand) and TOC (Total organic carbon), is crucial to ensure effective detoxification, complete mineralization, and overall environmental safety.

Finally, some limitations should be mentioned. This study focused on the free form of T. versicolor laccase and a restricted set of salts and dyes, whereas industrial effluents usually present more complex matrices, including surfactants, heavy metals, and variable pH and temperature conditions. Further research could explore enzyme immobilization, the use of redox mediators, or protein engineering approaches to enhance laccase tolerance to saline stress and broaden its substrate range. Moreover, combining laccase treatment with other biological or physicochemical processes may lead to synergistic effects, improving efficiency in real wastewater systems. In summary, the present findings highlight the resilience of T. versicolor laccase under ionic stress and support its potential application in integrated, eco-friendly strategies for textile wastewater remediation.