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Article

Purification and Biochemical Characterization of Trametes hirsuta CS5 Laccases and Its Potential in Decolorizing Textile Dyes as Emerging Contaminants

by
Guadalupe Gutiérrez-Soto
1,*,
Carlos Eduardo Hernández-Luna
2,*,
Iosvany López-Sandin
1,
Roberto Parra-Saldívar
1,2 and
Joel Horacio Elizondo-Luevano
3
1
Laboratorio de Ciencias Naturales, Biomolecular Innovation Group, Facultad de Agronomía, Universidad Autónoma de Nuevo León, Francisco Villa s/n, Ex-Hacienda el Canadá, General Escobedo 66050, NL, Mexico
2
Magan Centre of Applied Mycology (MCAM), Faculty of Engineering and Applied Sciences Cranfield, Cranfield Univesity, Bedford MK43 0AL, UK
3
Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, Pedro de Alba S/N., Ciudad Universitaria, San Nicolás de los Garza 66455, NL, Mexico
*
Authors to whom correspondence should be addressed.
Environments 2025, 12(1), 16; https://doi.org/10.3390/environments12010016
Submission received: 3 November 2024 / Revised: 29 December 2024 / Accepted: 3 January 2025 / Published: 7 January 2025
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Abstract

:
This study explores the purification, characterization, and application of laccases from Trametes hirsuta CS5 for degrading synthetic dyes as models of emerging contaminants. Purification involved ion exchange chromatography, molecular exclusion, and chromatofocusing, identifying th ree laccase isoforms: ThIa, ThIb, and ThII. Characterization included determining pH and temperature stability, kinetic parameters (Km, Kcat), and inhibition constants (Ki) for inhibitors like NaN3, SDS, TGA, EDTA, and DMSO, using 2,6-DMP and guaiacol as substrates. ThII exhibited the highest catalytic efficiency, with the lowest Km and highest Kcat. Optimal activity was observed at pH 3.5 and 55 °C. Decolorization tests with nine dyes showed that ThII and ThIa were particularly effective against Acid Red 44, Orange II, Indigo Blue, Brilliant Blue R, and Remazol Brilliant Blue R. ThIb displayed higher activity towards Crystal Violet and Acid Green 27. Among substrates, guaiacol showed the highest Kcat, while 2,6-DMP was preferred overall. Inhibitor studies revealed NaN3 as the most potent inhibitor. These results demonstrate the significant potential of T. hirsuta CS5 laccases, especially ThIa and ThII, as biocatalysts for degrading synthetic dyes and other xenobiotics. Their efficiency and stability under acidic and moderate temperature conditions position them as promising tools for sustainable wastewater treatment and environmental remediation.

1. Introduction

Emerging pollutants (EPs) are a diverse group of chemical and biological compounds recently identified in the environment, often at low concentrations but with significant adverse effects on ecosystems and human health [1]. Their management remains underregulated, making them a growing environmental concern [2]. The environmental and economic relevance of EPs spans water quality, ecosystem health, and the financial burden associated with their remediation [3]. Notably, EPs encompass a wide range of chemical compounds, including pharmaceuticals, personal care products, endocrine-disrupting substances, microplastics, and persistent organic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) [1]. These pollutants primarily originate from various anthropogenic activities, including industrial processes, urbanization, agriculture, and the extensive use of pharmaceuticals and personal care products [2,4]. Within the agricultural sector, synthetic dyes represent an increasingly critical issue, alongside other EPs [5]. When introduced into irrigation water, dyes exert deleterious effects on soil microbial communities, disrupting nutrient cycles and adversely affecting plan germination and growth [6]. This is exacerbated by the presence of toxic compounds frequently associated with synthetic dye effluents, such as chlorine, formaldehyde, solvents, aromatic amines, xenobiotics, pigments, heavy metals (e.g., lead, chromium, mercury), and other organic and inorganic contaminants [5,7]. These pollutants can infiltrate the food chain via plant absorption, resulting in soil toxicity and bioaccumulation. Recalcitrant textile dyes further impair photosynthesis, inhibit plant growth, and accumulate in tissues, and when they enter the food chain, they increase mutagenicity, carcinogenicity, and toxicity [8]. Additionally, dye-laden effluents disrupt biogeochemical cycles of nutrients in soil ecosystems, exacerbating soil contamination and undermining agricultural productivity.
Microorganism-based biotechnologies have emerged as sustainable and efficient solutions for mitigating emerging pollutants (EPs), addressing the limitations of conventional remediation methods. Among these, white-rot fungi stand is particularly notable for their ability to degrade recalcitrant compounds through ligninolytic enzymes, such as laccases, manganese peroxidases, and lignin peroxidases [5,6,7]. These enzymes can transform complex xenobiotics and persistent organic pollutants, providing a versatile and environmentally friendly biotechnological approach [8,9]. Within this group, Trametes hirsuta stands out due to the high catalytic efficiency of its laccases. These enzymes, characterized by their high redox potential, can oxidize a broad spectrum of phenolic and non-phenolic compounds, making them valuable for diverse applications such as dye decolorization, wastewater treatment, and bioremediation [10]. These characteristics have led to its use in applications such as dye decolorization, wastewater treatment, and bioremediation [11]. However, factors such as pH, temperature, and the intrinsic properties of dyes significantly affect the activity of these enzymes, underlining the need to identify new strains with enhanced enzymatic properties [12]. The phenotypic and genomic diversity of basidiomycetes presents significant potential for discovering strains with superior capabilities for EP remediation [13]. Trametes hirsuta CS5, a native strain from Northeastern Mexico, was previously reported to be capable of producing high redox potential laccases. Thus, the present work focuses on the purification and biochemical characterization of laccases produced by T. hirsuta CS5, specially evaluating their catalytic efficiency in decolorizing nine textile dyes representative of EPs. By expanding the understanding of laccase enzymatic diversity under real-world conditions, this research underscores the importance of exploring native fungal strains to develop innovative strategies in environmental biotechnology. The findings position T. hirsuta CS5 as a promising biotechnological resource for addressing the environmental challenges posed by EPs, contributing to the advancement of sustainable remediation technologies.

2. Materials and Methods

2.1. Chemicals

The matrices used for the different chromatographic steps were DEAE-Sephacel, Biogel P-100 and Poly Buffer Exchanger 94, as well as the substrates and dyes used (2,6-Dimethoxyphenol (Methoxyphenol), Guaiac, 2,2′–azino-bis (3-ethylbenzothiazolin-6-sulfonic)-diammonium salt (ABTS), syringaldazine (3,5-dimethoxy-4-hydroxybenzaldeidazine), o-anisidine, 2,4-dichlorophenol (2,4-DCP), 4-aminoantipyrine (4-AAP), 3-dimethylaminobenzoic acid (DMAB), 3-methyl-2-benzothiazoline hydrazone (MBTH), Poly R-478, Red Acid 44 (AR 44), Remazol Bright Blue Reagent (BBRR), Crystal Violet (CV), Indigo Carmine, Bright Blue R-250, Acid Green, Black 5 R, Orange II, Acrylamide, bis-N,N′-methylene-bis-acrylamide, sodium lauryl sulfate (SDS), Ammonium Persulfate, N,N,N′,N′-Tetramethylethylenediamine (TEMED), 2-mercaptoethanol, Trizma-base, and glycine) in kinetic assays, decolorization, and electrophoretic analysis were obtained from the commercial house Sigma-Aldrich (Estado de México, Mexico). The components of the culture media glucose, yeast extract, malt extract, and bacteriological agar were acquired from BD Difco (New Jersey, USA). The other reagents, acids, and inorganic salts used in this study were reagent-grade from CTR Scientific (Monterrey, Mexico). All reagents, chemicals, dyes, and substrates used were of analytical grade.

2.2. Microorganism

Strain Trametes hirsuta CS5 was isolated from the mountains of the Sierra Madre Oriental, Mexico, and maintained at 4 °C on YMGA (4 g yeast extract, 10 g malt extract, 4 g glucose, 15 g agar per liter).

2.3. Culture Medium

The strains were grown in YMGA medium (glucose 0.4%, malt extract 1.0%, malt extract 0.4%, and bacteriological agar 1.5%) for 5 days, after which the liquid media were inoculated. The production medium consisted of bran flakes (2%) in a 60 mM potassium phosphate buffer, adjusted to pH 6.0. This medium was inoculated with a homogenate of mycelium grown in YMGA (2 mL per 100 mL of the medium) under sterile conditions. The cultures were then incubated at 28 °C and 150 rpm for seven days.

2.4. Obtaining Crude Extracts

After 18 days of culture, the biomass was separated from the supernatant using a cotton–polyester sieve (50:50). The final volume was measured and frozen at −4 °C for 48 h to remove polysaccharides. The extracts were then thawed at room temperature and filtered using Schleicher & Schuell 520 B filter paper. The extracts were concentrated to approximately 200 mL using a Millipore Cartridge Prep/Scale TFF (Darmstadt, Germany). The final concentration of 15 mL was achieved with an AMICON ultrafiltration system using a YM10 membrane at a pressure of 40 psi (Darmstadt, Germany). The entire concentration process was performed in an ice bath.

2.5. Laccase Purification and Electrophoretic Analysis

The first purification step was performed using ion exchange chromatography with a DEAE-Sephacel column (bed volume: 196.3 cm3), equilibrated with a 20 mM potassium phosphate buffer, adjusted to pH 6.0. The samples were eluted with a linear potassium phosphate gradient of 20 to 150 mM. Fractions of 8 mL were collected, and absorbance was measured at 280 nm to define the protein peaks at 610 nm for the type I copper center, and 468 nm for laccase activity (via DMP oxidation) using a Shimadzu UV-VIS 1800 spectrophotometer (Kyoto, Japan).
The method used to determine laccase activity is based on the oxidation of the chromogenic substrate 2,6-Dimethoxyphenol (DMOP) by the enzyme, generating an orange color detectable at 468 nm [14]. The reaction mixture consisted of 2.8 mL of a 200 mM sodium acetate buffer (pH 4.5), 0.1 mL of 60 mM DMOP, and 0.01 mL of the enzymatic extract, giving a total volume of 3 mL. The reaction was initiated by adding the enzyme extract (or enzyme preparation) and monitored for a change in optical density at 468 nm. The units of laccase activity are expressed in μmol/min (U) using a molar extinction coefficient (ε) of 49,600 M−1 cm−1. After identifying the peak of laccase activity, the active fractions were collected and concentrated using an AMICON Ultrafiltration system with a YM10 membrane. A 100 μL aliquot of the concentrate was taken for protein analysis and activity determination (at a 1:100 dilution) and subsequently frozen at −4 °C.
The second purification step involved gel filtration chromatography using a P-100 Biogel column (bed volume: 319 cm3) equilibrated with a 20 mM potassium phosphate buffer and adjusted to pH 6.0. Then, 8 ml fractions were collected, and absorbance was measured at 280, 610, and 468 nm to identify protein peaks and determine the fractions with laccase activity. Once the active fraction was defined, it was also concentrated using the AMICON system. A 100 μL sample was taken for purity and yield analysis.
The final purification step was performed using a Poly Buffer Exchanger 94 column (bed volume: 6.36 cm3), equilibrated with 25 mM Tris-HCl, and adjusted to pH 9.4. An ampholyte solution (Poly Buffer 94 Sigma), adjusted to pH 3.5 with 1N HCl, was used for sample elution. Fractions of 2 mL were collected, measured at 280, 610, and 468 nm (as described above), and frozen in 0.5 mL aliquots until use.
Electrophoretic analysis was performed using native and discontinuous SDS-PAGE gels [15] to determine laccase purity. Each well was loaded with 24 μg of protein from each of the purification steps. For SDS-PAGE gel staining, 0.1% Coomassie Blue in 10% acetic acid and 40% methanol solution was used for one hour. The gels were washed with the same solution without dye. The zymogram was revealed with DMP.

2.6. Determination of Operational and Kinetic Parameters

To determine the optimal pH, a 100:50 mM potassium citrate-phosphate buffer in the pH range from 2.5 to 6.0, 2,6-DMP (2 mM) as substrate, and 0.01 mL of the enzyme (0.5 U) was used. As described above, the optical deviation change was measured at 468 nm. Then, 5 U of the laccase was incubated in 1 mL of the citrate-phosphate buffer to assess pH stability. Aliquots of 10 μL were taken to measure activity at times 0, 1, 2, 3, and 6 h, using the reaction mixture with 2,6-DMPO in the same buffer adjusted to 3.5.
The optimal temperature was determined by oxidizing 2,6-DMP using the reaction mixture described in the previous section, incubated at 25 to 85 °C. The reaction was initiated with the addition of 0.5 U of the enzyme. Thermostability was calculated at 55, 65, and 75 °C, determining the activity at 0, 1, 2, and 3 h on 2,6-DMOP in sodium acetate buffer at pH 3.5. All trials were performed in triplicate, with a less than 5% deviation.
The kinetic parameters were estimated using Lineweaver–Burk plots by performing time courses on the substrates 2,6-DMOP and guayacol at different concentrations: 0.12, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 mM. The reaction mixtures were in a 200 mM sodium acetate buffer, pH 3.5. The wavelength for 2,6-DMPO (ε = 49,600 M−1 cm−1) is 468 nm, and for guaiac, 436 nm (ε = 6400 M−1 cm−1). Measurements for the change in optical deviation were performed for one minute and in triplicate. Once the laccases’ kinetic parameters were determined, the effect of some inhibitors on their activity was evaluated. Reaction mixtures with 2,6-DMP were tested in the presence of concentrations of 0.1, 0.5, 1.0, 5.0 10, and 25 mM of sodium azide, SDS, EDTA, thioglycolic acid, and dimethyl sulfoxide (DMSO) as inhibitors.

2.7. Decolorization of Emerging Dye Contaminants Assay

The following emerging dyes were used in this study: Acid Red 44, Orange II, Reactive Black 5, Indigo Blue, Brilliant Blue R-250, Crystal Violet, Remazol Brilliant Blue R, and Acid Green 27. The reaction mixtures were prepared in a 200 mM sodium acetate buffer (pH 3.5) containing 100 ppm of each dye, 0.05 U of each pure native enzyme, and 5 U of Coriolopsis gallica (Cg) laccase. Absorbance readings were taken at 0, 1, 2, and 3 h across the 200–700 nm range, in triplicate, using a 96-well microplate in a Thermo Scientific® UV-VIS microplate spectrophotometer (Waltham, MA, USA). Table S1 summarizes the chemical groups and applications of the dyes. Acid Red 44 and Orange II were selected as representative azo dyes, while Reactive Black 5 was used as a diazo dye model. Moreover, Indigo Blue represented the indigo group, Poly R-478 was used as a polymeric dye model, Brilliant Blue R-250 and Crystal Violet served as triaryl methane dye models, and Remazol Brilliant Blue R and Acid Green 27 represented anthraquinone dyes due to their practical applications and economic significance. All tests were performed in triplicate and at room temperature.

2.8. Experimental Design

For the statistical analysis of experimental data, an analysis of variance (ANOVA) was applied using Tukey’s test, at 5% significance, using InfoStat v.2020 software, which allowed us to achieve more reliable results in the kinetic parameters, inhibition constants, and decolorization percentages.

3. Results

In this study, laccases from Trametes hirsuta CS5 were purified to evaluate their ability to degrade synthetic dyes, which are recognized as emerging pollutants due to their recalcitrance and harmful effects on aquatic ecosystems [8]. The results demonstrated that both laccases exhibit significant potential for degrading a variety of industrial dyes, highlighting their applicability in bioremediation processes. A detailed characterization of their physicochemical and operational properties, including thermal stability and pH profiles, was conducted, confirming their viability for industrial applications under demanding conditions.

3.1. 5 Laccase Purification of the Trametes hirsuta CS

Table 1 shows the Trametes hirsuta CS 5 results, which produce three laccase isoforms. The separation of ThIa and ThIb required three separation steps (ion exchange, molecular exclusion, and chromatofocusing column), while ThII only needed two (ion exchange, molecular exclusion). The latter had the highest yield (12%), while ThIa and ThIb isoforms had the highest specific activity with 191.7 and 171.37 U mg−1, respectively, although with low yields. Concerning the purification factor, ThI isoforms showed values close to 6, while the ThII isoform was less than 6.
Figure S1 shows the electrophoretic analysis of the fractions obtained from ion exchange and molecular exclusion. In contrast, Figure 1 shows the SDS-PAGE analysis used to determine the purity of the isoforms after the chromatofocusing column and calculating their molecular weight. The molecular masses for ThIa, ThIb, and ThII isoforms were 68, 65, and 60 kDa, respectively. On the other hand, the pI values were 6.28, 4.46, and 4.18 for ThIa, ThIb, and ThII, respectively (Figure S2). It is worth mentioning that all three isoforms showed the typical absorption spectrum of blue laccases, with the peak at 610 nm associated with the type I copper center and the peak at 330 nm corresponding to the binuclear type III copper center.

3.2. Operational and Kinetic Parameters of the Trametes hirsuta CS

pH and temperature are two factors that play an essential role in activity, i.e., they can either favor maximal enzyme activity or inhibit it. To evaluate the effect of pH on enzyme activity, 2,6-DMPO in a citrate-phosphate buffer was used. Under these conditions, all Trametes hirsuta CS5 laccase studied here showed intense activity in the acidic range, with optimum pH at 3.5 (Figure 2). Although, from 4.5 onwards, the activity started to decrease, ThIa was more susceptible to the increase in pH by decreasing 40% of the activity, while ThIb and ThII only decreased by 20%. This behavior was more evident at pH 5.0.
In analyzing pH stability for six hours, the three isoforms retained 100% of their activity in the range of 3.5 to 8.5 (Figure 3). However, at pH 2.5, the ThIa laccase loses about 30% of its activity, while ThIb and ThII lose 4 and 12%, respectively. Suggesting that ThIa is more susceptible to acidic pH.
Temperature is one of the parameters that influence enzyme activity, either by increasing activity (optimum temperature) or causing protein denaturation by prolonged exposure to a specific temperature (thermostability). For this reason, as part of the characterization, assays were performed with 2,6-DMOP to determine the effect of pH on the activity of the laccase isoforms. All three laccase isoforms showed temperature optima at 55 °C. Although ThIa and ThII were thermotolerant at 75 °C, retaining approximately 80% of their activity, ThIa stood out at 85 °C, maintaining 60%. This is contrary to what was observed with ThIb laccase, which, at 65 °C, lost more than 40% of its activity (Figure 4).
When the thermostability of the enzymes was evaluated at 55 °C, the Trametes hirsuta CS5 isoforms retained 100% of their activity for three hours. At 65 °C, only ThIb retained 100% of its activity after three hours of incubation, while ThIa and ThII retained approximately 80 and 30%, respectively. However, at 75 °C, none of the laccases showed activity after one hour of incubation. Although ThIb retained 7% of the activity, suggesting that it is the isoform with the highest thermostability, contrary to what was observed with ThII, which retained less than 1%, demonstrating greater susceptibility to temperature since it was the same behavior observed at 65 °C (Figure 5).
The operational characterization included the determination of the kinetic parameters Km and Kcat. These constants show how much substrate is being transformed and at what time, and the ratio between them (Kcat/Km) indicates the catalytic efficiency (Table 2). However, variations in these parameters were reported for laccases depending on the buffer and substrate being used. In the present work, we used a 200 mM sodium acetate buffer pH of 3.5, with 2,6-DMOP and Guaiacol as substrates. The graphical model used for the calculation of these parameters was the Lineweaver–Burk model. In general, the isoforms of Tramtes hirsuta CS5 laccases showed higher affinity for 2,6-DMP, ThII being the isoform with the lowest Km value at 74 mM. However, on guaiacol, the isoform with the highest affinity was ThIb at 390 mM. Concerning Vmax, ThIa and ThIb isoforms showed higher values on guaiacol (7.47 × 10−5 and 3.30 × 10−5 M·min, respectively) and thus a higher turnover number (Kcat) with 15.11 and 3.46 s−1, respectively. ThII was the isoform with the highest Vmax value (4.02 × 10−5 μM) over DMP but the lowest value over guaiacol (1.04 × 10−4 μM). For both substrates, the laccase isoform showed a higher catalytic efficiency (Kcat/Km). Table S1 shows other substrates on which the isoforms of Trametes hirsuta CS5 laccases showed activity, which include substrates typical of laccases and others such as veratryl alcohol and the pairs 4-AA: 4-Aminoantipyrine + 2,4-DCP: 2,4-Dichlorophenol, MTBH: 3-methyl-2-benzothiazoline hydrazone + DMBA: 3-dimethylamino benzoic acid reported for peroxidases.
Figure 6 shows the percentages of inhibition of laccase activity. Sodium azide (NaN3) was the compound that, at the lowest concentration (0.1 mM), inhibited 100% of the activity. This suggests that NaN3 may interact with the active site of laccase irreversibly, blocking its catalytic activity. SDS had a higher inhibitory effect on ThIb and ThII isoforms with values close to 90%, whereas ThIa lost only 50% of the activity. In the case of thioglycolic acid, ThIb laccase showed a higher percentage of inhibition (97%) at lower concentrations (10 mM) than the other isoforms. EDTA also shows high inhibition, especially on ThIb and ThII isoforms, with more than 95% activity inhibition at a concentration of 25 mM, while ThIa loses less than 20% of activity. Finally, DMSO presented lower inhibition values, with ThIa being the most susceptible, with a loss of activity of about 40%, while ThIb and ThII only lost about 40 % of their activity.
Table 3 shows the inhibition constants (Ki) for the laccase isoforms of Trametes hirsuta CS5. According to the observed inhibition percentages, NaN3 showed the lowest Ki, with ThIa being susceptible to this inhibitor (7.11 × 10−4 mM). This behavior was similar in TGC and DMSO. On the other hand, ThIb was the laccase isoform with the highest Ki for EDTA (0.417 mM) and ThII for SDS (0.088 mM).

3.3. Decolorization of Emerging Dye Contaminants by Laccases Trametes hirsuta CS5

Table 4 shows the results of the decolorization percentages of the isoforms of the CS5 laccase from Trametes hirsuta and their comparison with the Coriolopsis gallica (Cg) laccase. AR44 was the monoazo with the highest decolorizing activity (p < 0.05), mainly due to the Cg (93.6) and ThII (91.4) laccases. While in the mixtures with OII, ThII was the enzyme that showed the highest activity (76%). In the case of the results with RB5, despite presenting lower decolorization values (<40%) concerning the monoazos, all four showed similar activity on this one. In Indigo Blue, the Cg laccase presented the highest decolorizing activity (64.3%), followed by ThII and ThIa (40.4 and 66.6%). This same activity pattern was observed in the mixtures with Poly R-478 but with values lower than 45%. On the triaryl methane dye BBR, the laccases ThII, ThIa, and Cg decolorized more than 50%, the first being the most active (77.8%). In the case of the triarylmethane (CV) dye, the lowest percentages of decolorization were observed (<25%), with Cg and ThIb standing out as the most active laccases. On the other hand, the results of the anthraquinone dyes showed a similar behavior to that of the monoazos, where one of the dyes was more susceptible to enzymatic action. In this case, RBBR presented the highest percentages of discoloration (mainly in the treatments with the laccases ThII and Cg with 82 and 88.7%, respectively). At the same time, AG 27 was more susceptible to the isoform Th Ib and Cg, although with lower discoloration values (71.3 and 69.7%, respectively).
Figure 7 presents the decolorization kinetics of the tested dyes, highlighting distinct behaviors among the laccases. For AR44 and RB5, ThII and Cg exhibited similar decolorization patterns, whereas this trend was not observed for OII. In the case of BI, all laccases displayed a consistent linear reduction in color intensity over time, indicating uniform degradation kinetics. The degradation of Poly R-478 and CV showed a gradual decline without abrupt changes during the first 2 h, followed by a more pronounced decrease in remaining color at the 3 h mark. However, the decolorization percentages for CV were notably lower than those for the polymeric dye Poly R-478. Additionally, the laccases from Trametes hirsuta CS5 exhibited similar decolorization patterns for RRB, RBBR, and AG27, suggesting shared mechanisms or structural preferences in their interactions with these dyes. Figure S3 shows the AR44, RRBR, and CV bleaching spectra, where changes in the dye spectra can be observed with each Trametes hirsuta CS5 isoform. AR44 (like the azo dye degrading model) had the absorption main peak at 510 nm, on which ThIa and ThII isoforms showed higher decolorization activity. On the other hand, RBBR (anthraquinone dye model) showed at 598 nm as the main absorption peak. In this case, all isoforms had activity. Meanwhile, CV (triaryl methane dye) degradation was measured at 588 nm and showed a similar behavior to that of RBBR.

4. Discussion

4.1. Laccase Purification and Electrophoretic Analysis

Trametes hirsuta CS5 is a strain native to Nuevo León, Mexico, and was demonstrated to produce laccases with high redox potential [16]. Under the conditions outlined in this study, three isoforms of laccases (ThIa, ThIb, and ThII) were produced. While most studies report two isoforms for this strain [17,18], up to seven genes associated with the production of the isoforms were identified [19]. The specific activity of the primary isoform, ThI, was comparable to that of Lac 1 from T. hirsuta MX2 [17]. However, ThI required one more step for the separation of the ThI1a and ThIb isoforms, which presented high values of specific activity (>170 U mg−1 for both isoforms) compared to the Lac 1 laccase of T. hirsuta MX2 (<30 U mg−1). Similar observations were made for the ThII laccase when compared to the specific activity of the Lac 1 laccase of T. hirsuta MX2, which also showed a purification factor (9-fold) than the one reported in this work. When comparing with other similar purification protocols [20], based on precipitation with salts, ion exchange chromatography, and molecular exclusion, it can be observed that the levels of specific activity and the purification factor are similar. However, including chromatofocusing (in the case of ThI) increases the specific activity. Thus, the purification factor was decreased even though the SDS-PAGE analysis did not reveal other protein bands present in the fractions with laccase activity (Figure 1). Thus, the variation in laccase purification efficiency, including specific activity, yield, and purification factor, can be attributed to several factors related to the enzyme’s source, the purification methods employed, and the conditions under which the enzyme is produced and purified. These factors influence the efficiency and effectiveness of the purification process, leading to different outcomes. Molecular weights ranged between 60 and 68 kDa, demonstrating proteins of higher weight compared to what was reported for other Trametes hirsuta laccase such as TthLaccc-S [20] and the laccase of the MTCC 11397 strain [21], whose reported weights were 57 and 45 kDa, respectively, although like laccases produced by other species of Trametes have lower weights [22]. Regarding the isoelectric point, the three isoforms showed values from 4.0 to 6.3, lower than the Trametes sp. HS-03 isoforms from 3.5 to 7.3 [23]. Likewise, the UV/visible spectrum of the isoforms presented the typical trace with a peak of 610 nm corresponding to the type I copper center and the 330 nm peak to the binuclear copper scepter type III of the blue laccases seen in other strains of T. hirsuta [24]. The T1 site provides them with a light blue color in solution. It is characterized by a pronounced band of optical absorption at the wavelength of 600 nm (ε = 5000 M−1·cm−1) and by a weak parallel superfine division in the EPR spectra [19].

4.2. Determination of Operational and Kinetic Parameters

Concerning the optimal pH, the three isoforms had the exact value of 3.5, like that reported for other laccases produced by the genus Trametes over 2,6-DMP [20,21,25]. This behavior of isoforms with the same optimal pH value differed from that reported for the three laccases produced by Trametes sp. HS-03, which had optimal pH values of 2.5, 3.5, and 4.0 [23]. However, this is similar to the optimal pH of 3.0 reported for the house produced by Trametes hirsuta SYBC-L19 [26]. The optimal pH of basidiomycete laccases varies due to several factors, including genetic mutations, structural modifications, and environmental adaptations [27]. These enzymes, crucial for lignin degradation and other industrial applications, exhibit different pH optima to suit specific substrates and conditions [28]. The diversity in optimal pH is primarily driven by the need to enhance catalytic efficiency and stability under varying industrial and environmental conditions. In the case of stability, the three isoforms showed the typical behavior of blue laccases [29]. The three laccases exhibit optimal activity within a pH range of 3.5 to 7.5, with the ThIb laccase demonstrating stability even at pH 2.5, where it retained 96% of its activity for six hours. This stability can be attributed to the isoforms’ molecular structures and sequence identities. For example, the Lac3 isoform from Pleurotus nebrodensis retained 100% of its activity within a pH range of 5 to 8 [30], although for a shorter duration than that assessed in this study (1 h vs. 6 h). These differences in pH stability are influenced by various factors, including molecular structure, environmental conditions, and potential modifications or immobilizations [30,31]. The decline in laccase activity with increasing pH is primarily attributed to inhibition by hydroxyl ions (OH−), rather than enzyme denaturation. Laccases, as multi-copper oxidases, exhibit optimal functionality under acidic conditions where their copper centers, particularly T2 and T3, effectively facilitating electron transfer from the T1 site, a critical component of their catalytic mechanism. At neutral or alkaline pH, hydroxyl ions bind to the T2/T3 copper centers, obstructing electron transfer and thereby reducing enzymatic activity [32]. This inhibition mechanism is not exclusive to hydroxyl ions; other anions, such as chloride ions, also contribute to reduced activity through competitive inhibition [33]. On the other hand, the laccases from Trametes hirsuta CS5 exhibited an optimal temperature like other laccases of the same genus, which are characterized by their thermostability, as previously reported [25]. These enzymes demonstrated remarkable activity retention (between 100 and 80%) over a broad temperature range of 55–75 °C, suggesting their ability to maintain functionality under heat conditions. Comparatively, their thermostability at 55 °C for 3 h was more significant than that observed in the rLcc9 laccase from Coprinopsis cinerea, which was genetically modified to enhance its thermal stability [27]. This underscores the robustness of T. hirsuta laccases, even without genetic modifications, compared to the engineered version of rLcc9. Moreover, the laccase isoform ThI maintained activity above 65 °C, further supporting its performance in industrial applications requiring high-temperature conditions. Nevertheless, laccases are sensitive to temperature changes, which can alter their conformation and thus their catalytic activity. In some cases, a thermoactivation phenomenon is observed, where enzyme activity increases with temperature until it reaches an optimum point before declining. For example, Lac 37 II from Trametes trogii reaches its peak performance at 60 °C, but its activity declines rapidly beyond this limit due to the loss of structural stability [34]. Molecular dynamics studies have shown that temperature increases generate fluctuations in structural regions such as loops and helices, as observed in rPOXA laccase 1B, which explains the loss of activity at extreme temperatures [35]. Therefore, the observed fluctuations are due to a balance between thermoactivation, which temporarily increases activity at moderate ranges, and structural destabilization at higher temperatures.
The kinetic results obtained for the laccase isoforms of Trametes hirsuta CS5 using 2,6-DMP and guaiacol as substrates show significant variations in Km, Vmax, Kcat, and the Kcat/Km ratio, allowing for the evaluation of each isoform’s catalytic efficiency. For 2,6-DMP, the three isoforms (ThIa, ThIb, and ThII) exhibit Km values ranging from 74 to 81 µM, with relatively high catalytic efficiency for ThIa and ThII, indicating greater affinity and reaction rate compared to ThIb, which has the lowest efficiency. Overall, the affinity of the Trametes hirsuta CS5 laccase isoforms was higher than that reported for other laccases, such as Ganoderma australe (188.977 µM) [36], or for high redox potential fungal laccase (HRPL) variants, whose design reduced affinity for low-redox potential compounds like ABTS and DMP [27] but improved for high redox potential K4[Mo(CN)8], reflecting the high redox potential at the T1Cu site. In this context, the laccases of Trametes hirsuta CS5 have also been reported as high redox potential enzymes [16], being capable of oxidizing the 4-amino antipyrine + 2-dichlorophenol pair, as previously described for LiP determination [37], as well as the coupling of 3-methyl-2-benzothiazol inone hydrazone (MBTH) and 3-(dimethylamino)benzoic acid (DMAB), used for MnP [38]. Regarding the Kcat for DMP, all three isoforms displayed values lower than those of the Ganoderma australe laccase (237 s−1) [36], although the reported enzyme had a lower Kcat/Km ratio. In contrast, the two ThI isoforms exhibited Kcat/Km values similar to those reported for HRPL variants [27], while the ThII laccase showed a higher ratio than previously reported. These results suggest an opposite behavior to that of the laccases, as these native isoforms exhibit greater affinity but a low turnover number, although with a high Kcat/Km ratio. Suggesting that the low turnover number is compensated by high affinity, which may be associated with the primary structure. For instance, the presence of specific amino acids in the active site can influence substrate binding and turnover rates, such as the role of Asp-206 and His-458 in substrate recognition within the active site of Trametes versicolor [39], which could similarly affect the isoforms of Trametes hirsuta CS5. The high Kcat/Km ratio observed in the ThII laccase isoform suggests a strong affinity for the substrate, often resulting from optimal hydrophobic interactions and electronic properties that facilitate efficient substrate binding [40]. The kinetic results obtained with guaiac for the laccases of Trametes hirsuta CS5 indicate a different behavior than that observed with 2,6-DMP. Although a low affinity (high value of Km) and a relatively low exchange number (Kcat) were observed, the catalytic efficiency (Kcat/Km ratio) was higher than that reported for the T. trogii YDHSD laccase [25]. This relative efficiency suggests that T. hirsuta CS5 laccases, despite their low initial affinity for guaiac, may be particularly effective in applications where high catalytic activity is required in the presence of low to moderate substrate concentrations. Compared to other strains of Trametes hirsuta [41], catalytic efficiency stands out for substrates such as guaiacol, which could be attributed to structural differences in the active site that facilitates the accommodation and oxidation of specific phenolic substrates. Regarding the effect of inhibitors, the three laccases showed typical behavior at low concentrations of NaN3 [25], which acts as a strong nucleophile, potentially interacting with the catalytic sites of enzymes, such as laccase, by binding to specific amino acid residues [42]. Like what was reported for the T. trogii YDHSD laccase [25], the laccases of Trametes hirsuta CS5 require a similar percentage of inhibition with SDS and EDTA, only at higher concentrations. These differences can generally be explained by the gene families producing laccases [43].

4.3. Decolorization of Emerging Dye Contaminants

The decolorization results with the Trametes hirsuta CS5 laccase showed that the ThII isoform was the native laccase with the highest range of dyes of the azos, diazos, triarylmethane, and anthraquinone groups, which can be explained by being the isoform with the highest catalytic efficiency over 2,6-DMP, while it was the one with the lowest efficiency over guaiacol (Table 2). This behavior was like that observed in azo dyes, where AR44 had a discoloration greater than 90%, while OII was only bleached in 76%. These percentages were higher than those reported for the Trametes pavonia laccase on monoazo dyes (50.7%), such as Methyl Red [44]. Compared to other laccases, similar discoloration values (>80%) were reported for azo dyes (such as Congo red), although at longer times (>72 h) [45] than those reported in the present research. In the case of discoloration values for RB5, for the Trametes trogii laccase, discolorations greater than 90% were reported. Although these are higher than those presented, they were obtained in the presence of the mediator 1-hydroxybenzotriazole and higher enzyme concentrations [46]. On the other hand, the use of mediators is questioned due to the increase in costs in the process and the environmental impact they generate. On the other hand, for the Bjerkandera asterna TMF1 laccase, an efficiency of 60% was reported in the presence of other monoazo dyes [47]. Therefore, the results of ThII on AR44, OII, and RB5 make it a candidate to be tested in mixtures of dyes that increase its decolorizing potential. For indigo dyes, basidiomycetes are considered a great alternative, reporting discoloration rates above 90%, although over time higher [48], or with higher temperature ranges [49] than those used in the Trametes hirsuta CS5 laccase. Triarylmethane dyes are recognized for the limitations that represent their bioremediation, including their complex chemical structure and their marked negative impact on ecosystems [50]. For this reason, the efficiency of basidiomycete laccases will depend on the dye structure, the strain, and the reaction conditions (time, pH, and temperature) [45]. Therefore, prospective studies such as those reported in this research are of great relevance. Of the three house isoforms produced, ThIb was the one that showed the highest bleaching activity against CV. However, ThII preferentially bleached to BBR and with a higher percentage of discoloration (78%), which was like that reported to the bleaching laccase of Ceriporiopsis subvermispora over Bromocresol Purple (71.6%) and Methyl Violet (68.1%) [51]. With the same behavior observed in the triarylmeta bond dyes, the ThIb and ThII isoforms showed preference over AG27 (71%) and RBBR (82%), respectively. In both cases, the values were similar to those reported for RBBR, treated with the Trametes polyzona H18 laccase [52], and the Cerrena sp. laccase. BMD. TA.1 [45], although at a lower concentration (100 ppm) than that used in the present research (200 ppm). This highlights the importance of understanding the interaction between laccase isoforms and dye structures for efficient bioremediation. The variability in the results of the isoforms of laccases produced by the same strain can be explained by several factors, among the main ones being the presence of multiple laccase isoenzymes, a phenomenon widely documented in different basidiomycetes, as well as the catalytic and structural properties that influence the specificity of the substrate and performance under different environmental conditions [52]. For example, Coprinus comatus produces six laccase isoenzymes, with LacA being efficient in dye discoloration when used with a redox mediator [53]. In this sense, studies in Pleurotus ostreatus have shown that variability in laccase activity may be related to the source of inoculum and genetic variations between crops [54]. Therefore, the production of multiple isoforms allows basidiomycetes to adapt to specific conditions, translating into differentiated results in applications such as dye decolorization [55]. Likewise, the Ping-Pong bi-bi kinetic model of the laccase plays a fundamental role in the degradation of dyes. They catalyze oxidation reactions through a monoelectronic mechanism, in which they transfer electrons from various substrates, such as phenols and aromatic compounds, to molecular oxygen, reducing it to water. During this process, a root intermediate is formed that plays a crucial role in the breakdown of complex substrates, such as synthetic dyes and organic pollutants. These radicals can trigger secondary reactions, promoting the fragmentation of molecules into simpler and less toxic structures, highlighting the importance of laccases in bioremediation applications and environmental treatments [56]. Studies show that fungal laccases such as Cerrena sp. and Pycnoporus sanguineus have significant degradation rates for anthraquinone dyes, achieving more than 80% discoloration in short incubation periods [45,49]. In contrast, triaryl methane and azo dyes typically have more stable structures that resist the action of the laccase, resulting in a lower degradation efficiency [45]. Azo dyes require additional reduction processes for effective degradation, complicating their treatment compared to anthraquinone dyes [57]. Despite this, laccases offer an ecological approach by operating in non-severe temperature and pH conditions, eliminating the need for aggressive chemicals and energy-intensive processes, significantly reducing environmental impacts [58]. In addition, enzymatic immobilization in supports such as ZnFe2O4 nanoparticles [59] or recently magnetite nanocomposites (Fe3O4/C) [60] are a strategy to improve their stability and reuse, reducing long-term costs.
Finally, it is important to mention that laccase-mediated dye degradation is an environmentally friendly process that reduces the formation of toxic by-products compared to chemical treatments. Laccases not only decolorization dyes but also detoxify effluents, reducing their toxicity to aquatic ecosystems and minimizing risks such as the generation of multi-drug-resistant microorganisms [61,62]. Furthermore, from an economic point of view, another advantage of using laccases compared to physicochemical treatments is its low production cost, since they can be obtained from agro-industrial waste and fruit waste peels [63]. For this reason, the characterization of new laccases with robust properties and a wide range of substrates is of scientific relevance since this biocompatible and safe approach solves problems associated with chemical treatments that can harm human and environmental health [64]. Therefore, future research will characterize the degradation products and toxicity of emerging contaminants treated with the Trametes hirsuta CS5 laccase.

5. Conclusions

Trametes hirsuta CS5 is a native strain producer of three laccases: ThIa, ThIb, and ThII. ThIa and ThIb required separation via chromatofocus chromatography, being the isoforms with lower yields. Molecular weight, optimal pH, and temperature were similar at typical blue laccases, although ThIa showed higher thermostability. Concerning kinetics parameters, all isoforms had major affinity on 2,6-DMP, highlighting Km and Kcat values of ThII. All three isoforms had typical inhibitory behavior, although ThIb had the lowest Ki values for NaN3 and SDS. In contrast, ThII retained the highest activity in respiration of ATG, EDTA, and DMSO, although all three retained more than 60% of the activity in the presence of 25 mM DMSO. About the decolorizing capacity of the Trametes hirsuta CS5 laccases, ThIa and ThII had similar behaviors with a wide range of dyes of different chemical groups. At the same time, ThIb only showed relevant activity on CV and AG27. These results allow us to conclude that the T. hirsuta CS5 laccases, especially ThIa and ThII, possess characteristics such as thermostability, tolerance to inhibitors, and a wide range of catalytic activity that make them ideal for biodegradation processes of emerging pollutants and other xenobiotic compounds. Thus, in future research, the degradation products are characterized to identify the kinetic mechanism of each isoform and their applications in the degradation of emerging pollutants present in real effluents.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/environments12010016/s1. Figure S1: Polyacrylamide gel electrophoresis analysis of CS5; Figure S2: Chromatofocusing separation, Figure S3: Decolorization spectrum of dyes models, Table S1: Dyes used, Table S2: Substrate specificity.

Author Contributions

Conceptualization, G.G.-S. and C.E.H.-L.; methodology, G.G.-S. and C.E.H.-L.; software, I.L.-S.; validation, G.G.-S., C.E.H.-L. and R.P.-S.; formal analysis, G.G.-S. and C.E.H.-L.; investigation, G.G.-S. and I.L.-S.; resources, G.G.-S.; data curation, G.G.-S. and C.E.H.-L.; writing—original draft preparation, G.G.-S. and J.H.E.-L.; writing—review and editing, G.G.-S. and I.L.-S.; visualization, R.P.-S. and J.H.E.-L.; supervision, G.G.-S. and C.E.H.-L.; project administration, G.G.-S. and C.E.H.-L.; funding acquisition, C.E.H.-L. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

All databases are available in the Natural Science Laboratory of Facultad de Agronomía UANL.

Acknowledgments

The authors acknowledge the support received from the Universidad Autónoma de Nuevo León. We thank M.C. Carlos Alberto Hernandez Martinez, Director of the Faculty of Agronomy, for all the facilities provided.

Conflicts of Interest

The authors declare no conflicts of interest.

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Environments 12 00016 g001
Figure 1. SDS-PAGE analysis. Lane 1 corresponds to molecular weight markers, 2 corresponds to ThIa laccase, 3 to ThIb, and 4 to ThII isoform.
Figure 1. SDS-PAGE analysis. Lane 1 corresponds to molecular weight markers, 2 corresponds to ThIa laccase, 3 to ThIb, and 4 to ThII isoform.
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Figure 2. Optimum pH determination.
Figure 2. Optimum pH determination.
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Figure 3. pH stability. (A) corresponds to ThIa isoform, (B) to ThIb laccase, and (C) to ThII isoform.
Figure 3. pH stability. (A) corresponds to ThIa isoform, (B) to ThIb laccase, and (C) to ThII isoform.
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Figure 4. Optimal temperature determination.
Figure 4. Optimal temperature determination.
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Figure 5. Temperature stability.
Figure 5. Temperature stability.
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Figure 6. Effect of some inhibitors. (A) corresponds to sodium azide (NaN3), (B) to Sodium Dodecyl Sulfate (SDS), (C) to thioglycolic acid (TGA), (D) to ethylenediamine tetraacetic acid (EDTA), and (E) to dimethyl sulfoxide (DMSO).
Figure 6. Effect of some inhibitors. (A) corresponds to sodium azide (NaN3), (B) to Sodium Dodecyl Sulfate (SDS), (C) to thioglycolic acid (TGA), (D) to ethylenediamine tetraacetic acid (EDTA), and (E) to dimethyl sulfoxide (DMSO).
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Figure 7. Decolorization kinetics graphs of laccase of Trametes hirsuta CS5 on nine synthetic dyes. (A) corresponds to Acid Red 44, (B) Orange II, (C) Reactive Black 5, (D) Blue Indigo, (E) Poly R-478, (F) Remazol Black B, (G) Violet Crystal, (H) Remazol Brilliant Blue R, and (I) Acid Green 27.
Figure 7. Decolorization kinetics graphs of laccase of Trametes hirsuta CS5 on nine synthetic dyes. (A) corresponds to Acid Red 44, (B) Orange II, (C) Reactive Black 5, (D) Blue Indigo, (E) Poly R-478, (F) Remazol Black B, (G) Violet Crystal, (H) Remazol Brilliant Blue R, and (I) Acid Green 27.
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Table 1. Purification table of Trametes hirsuta CS5 laccases.
Table 1. Purification table of Trametes hirsuta CS5 laccases.
StepTotal Protein
(mg)		Total Units
(U)		Specific Activity
(U mg−1)		Purification
(Fold)		Yield
(%)
Culture supernatant79.672429.5530.491.00100.00
Concentrate YM1077.251835.8123.040.7675.6
DEAE-Sephacel ThI4.74284.2760.031.9711.7
Biogel P-100 ThI2.9989.6830.050.993.7
* Chromatofocus ThIa0.0814.43191.706.290.6
* ChromatofocusThIb0.046.17171.375.620.3
DEAE-Sephacel ThII8.09113.5114.030.464.7
Biogel P-100 ThII3.77291.2077.182.5312.0
* Polybuffer Exchanger 94.
Table 2. Kinetic constants of Trametes hirsuta CS5 laccases.
Table 2. Kinetic constants of Trametes hirsuta CS5 laccases.
Isoform2,6-DMPGuaiacol
Km
(μM)		Vmax
(M·min)		Kcat
(s−1)		Kcat/Km
(M·s−1)		km
(μM)		Vmax
(M·min)		Kcat
(s−1)		Kcat/Km
(s−1)
ThIa81 a3.52 × 10−5 b7.13 a8.8 × 104 b548 a7.47 × 10−5 c15.11 a2.7 × 104 b
ThIb77 a1.75 × 10−5 c2.0 c2.3 × 104 c390 c3.30 × 10−5 b3.46 b8.8 × 103 a
ThII74 b4.02 × 10−5 a6 b8.9 × 105 a466 b1.04 × 10−4 a1.7 × 105 c3.7 × 108 c
The assays were performed with 0.05 U of the enzymes. The data corresponds to a representative experiment in triplicate. The standard deviation was less than 5%. a–c Different literals indicate statistically significant differences in the column.
Table 3. Inhibition constants of Trametes hirsuta CS5 laccases.
Table 3. Inhibition constants of Trametes hirsuta CS5 laccases.
IsoformNaN3
Ki *		SDS
Ki		ATG
Ki		EDTA
Ki		DMSO
Ki
ThIa7.11 × 10−4 c0.207 a0.325 c0.450 b0.051 b
ThIb4.17 × 10−2 a0.222 a0.855 b0.417 b0.645 a
ThII1.38 × 10−3 a0.088 b1.156 a0.616 a0.669 a
* The assays were performed with 0.05 U of the enzymes. The data corresponds to a representative experiment in triplicate. The standard deviation was less than 5%. a–c Different literals indicate statistically significant differences in the column.
Table 4. Percentage of discoloration.
Table 4. Percentage of discoloration.
DyeDicoloration (%)
CgThIaThIbThII
AR 44 (Monoazo)93.6 a87.8 b33.7 c91.4 a
OII (Monoazo)64.6 b62.9 b36.9 c76.0 a
RB5 (Diazo)32.3 b32.9 b31.8 b39.1 a
BI (Indigo)64.3 a36.6 b20.8 c40.4 b
Poly R-478 (Polymeric dye)44.7 a24.0 c33.2 b19.7 c
BBR (Triarylmethane)69.6 b54.5 c27.2 d77.8 a
CV (Triarylmethane)25.0 a14.3 b24.3 a12.7 b
RBBR (Anthraquinone)88.7 a77.2 c53.0 d82.0 b
AG 27 (Anthraquinone)69.7 a50.1 b71.3 a54.1 b
AR 44 = Acid Red 44; OII = Orange II; RB5 = Reactive Black 5; BI = Blue Indigo; BBR = Brilliant Blue R-250; CV = Crystal Violet; RBBR = Remazol Brilliant Blue R; AG27 = Acid Green 27. Incubation for 3 h with 0.005 U of the enzymes and 250 ppm of the dyes. As a positive control, a pure laccase (1 U) from Coriolopsis gallica (Cg) was used. The data corresponds to a representative experiment in triplicate. The standard deviation was less than 5%. a–d Different literals indicate statistically significant differences between rows.
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MDPI and ACS Style

Gutiérrez-Soto, G.; Hernández-Luna, C.E.; López-Sandin, I.; Parra-Saldívar, R.; Elizondo-Luevano, J.H. Purification and Biochemical Characterization of Trametes hirsuta CS5 Laccases and Its Potential in Decolorizing Textile Dyes as Emerging Contaminants. Environments 2025, 12, 16. https://doi.org/10.3390/environments12010016

AMA Style

Gutiérrez-Soto G, Hernández-Luna CE, López-Sandin I, Parra-Saldívar R, Elizondo-Luevano JH. Purification and Biochemical Characterization of Trametes hirsuta CS5 Laccases and Its Potential in Decolorizing Textile Dyes as Emerging Contaminants. Environments. 2025; 12(1):16. https://doi.org/10.3390/environments12010016

Chicago/Turabian Style

Gutiérrez-Soto, Guadalupe, Carlos Eduardo Hernández-Luna, Iosvany López-Sandin, Roberto Parra-Saldívar, and Joel Horacio Elizondo-Luevano. 2025. "Purification and Biochemical Characterization of Trametes hirsuta CS5 Laccases and Its Potential in Decolorizing Textile Dyes as Emerging Contaminants" Environments 12, no. 1: 16. https://doi.org/10.3390/environments12010016

APA Style

Gutiérrez-Soto, G., Hernández-Luna, C. E., López-Sandin, I., Parra-Saldívar, R., & Elizondo-Luevano, J. H. (2025). Purification and Biochemical Characterization of Trametes hirsuta CS5 Laccases and Its Potential in Decolorizing Textile Dyes as Emerging Contaminants. Environments, 12(1), 16. https://doi.org/10.3390/environments12010016

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