Laccase Isoenzymes Research Articles

The roles of Pleurotus ostreatus HAUCC 162 laccase isoenzymes in decolorization of synthetic dyes and the transformation pathways

Fungal laccases have shown great potential in industrial and environmental applications. They are generally produced as laccase isoenzymes. Thus, to further study the properties of different laccase isoenzymes and their performance in bio-remediation is essential for a deep understanding of laccase function and application. In this study, three Pleurotus ostreatus HAUCC 162 laccase isoenzymes were heterologously expressed, and the effects of different inhibitors, metal ions, and organic solvents on the activity of recombinant laccases were evaluated. In the dye decolorization test, LACC6 showed the highest ability to remove Malachite green (MG), Remazol Brilliant Blue R (RBBR), Bromophenol blue (BB), and Methyl orange (MO) among the three recombinant laccases. Removal rates within 24 h were 91.5%, 84.9%, 79.1%, and 73.1% for MG (100 mg/L), RBBR (100 mg/L), BB (100 mg/L), and MO (100 mg/L), respectively. The MG and RBBR transformation pathways were proposed by using High Performance Liquid Chromatography-Mass Spectrometry (LC–MS) analysis. Based on the results of this work, the production of recombinant LACC6 or improving the portion of LACC6 in the crude extracellular laccase may advance synthetic dye removal.

Laccase Isoenzymes of Trametes hirsuta LE-BIN072: Degradation of Industrial Dyes and Secretion under the Different Induction Conditions

It is well-known that various oxidative enzymes of wood-degrading fungi are widely demanded in various fields of biotechnology. Laccases attract particular attention, since they have different substrate specificities and can be used not only in pulp and paper industry, but also for the degradation of various xenobiotics, including decolorization of dyes. In this work, a comparative analysis of the native major (LacA) and the recombinant minor (rLacC, rLacD, rLacF) Trametes hirsuta 072 laccase isoenzymes applicability for bleaching of four widely used recalcitrant dyes was conducted. It was shown that all isoenzymes decolorized dyes to different extent, and the minor isoenzymes rLacD and rLacF are the most promising for decolorization of congo red and phenol red dyes. Moreover, we provide detailed exoproteome analysis of T. hirsuta 072 growing on the media supplemented with compounds that were previously shown to increase transcription levels of the minor laccase isoenzymes.

Enhancing laccase production by white-rot fungus Funalia floccosa LPSC 232 in co-culture with Penicillium commune GHAIE86.

To obtain enzymatic preparations with higher laccase activity levels from Funalia floccosa LPSC 232, available for use in several applications, co-cultures with six filamentous microfungi were tested. A laccase non-producing soil fungus, identified as Penicillium commune GHAIE86, showed an outstanding ability to increase laccase activity (3-fold as compared to that for monoculture) when inoculated in 6-day-old F. floccosa cultures. Maximum laccase production with the F. floccosa and P. commune co-culture reached 60U/mL, or twice that induced by chemical treatments alone. Our study demonstrated that co-culture with soil fungi might be a promising method for improving laccase production in F. floccosa. Although the enhancement of laccase activity was a function of P. commune inoculation time, two laccase isoenzymes produced by F. floccosa remained unchanged when strains were co-cultured. These data are compatible with the potential of F. floccosa in agricultural applications in soil, whose enzyme machinery could be activated by soil fungi such as P. commune.

Purification and biochemical characterization of two isolated laccase isoforms from Agaricus bisporus CU13 and their potency in dye decolorization

Agaricus bisporus CU13 laccase was purified using ammonium sulfate precipitation (40–80%), Sephadex G100, and DEAE Sephadex A50 anion exchange column chromatography, respectively. Two laccase isoenzymes (Lacc1 & Lacc2) with purification folds of 1.40 and 5.81 respectively, were obtained from DEAE Sephadex A50 column. Optimal temperature and pH were recorded at 55 °C and pH 5.0 for both laccase isoenzymes using ABTS as substrate. Lacc1 was more thermostable than Lacc2 with residual activity of 95, 80 and 6%, while Lacc2 only retained 72, 25 and 0.4% of its activity after incubation for 90 min. at 50, 60 and 70 °C, respectively. Lacc2 retained about 93 and 86% of the initial activity at pH 9.0 and 7.0, whereas Lacc1 was stable at pH 7.0 and 5.0 followed by pH 9.0 and retained about 87, 76, and 36% of its activity respectively, after 4 h of incubation. Lacc1 was activated by 40% in the presence of Cu2+ (10 mM). Km and Vmax values found to be 0.394 and 0.158 μM, and 0.1351 and 0.4755 μmol min−1 for Lacc1 and Lacc2, respectively. The efficiency of both isoenzymes to decolorize Acid blue dye, make the enzyme seems to be a prospective for further biotechnological applications.

Heterologous expression and characterization of three laccases obtained from Pleurotus ostreatus HAUCC 162 for removal of environmental pollutants

Chlorophenols (CPs), nitrophenols (NPs), and sulfonamide antibiotics (SAs) are three types of environmental pollutants that are of great concern because of their prevalence and toxicity. In this study, three laccase isoenzymes obtained from Pleurotus ostreatus HAUCC 162 were heterologously expressed and characterized with respect to their ability to degrade CPs, NPs, and, SAs. The three recombinant laccases can efficiently degrade the three types of considered pollutants using a laccase-mediator system (LMS). Their specific efficiencies for the removal of 2NP, 3NP, 4NP, 4CP, 2,4-dichlorophenol (DCP), 2,6-DCP, sulfadiazine (SDZ), sulfamethazine (SMZ), and sulfamethoxazole (SMX) over 60min were 59.21%, 47.91%, 60.24%, 74.9%, 28.9%, 35.1%, 98.1%, 97.5%, and 97.8%, respectively. Based on the analysis of the oxidation products of the CPs, NPs, and SAs, pollutant removal pathways are proposed, namely, the production of 3-nitromuconate and 3-chloromuconate as the key intermediates of 4-NP and 2, 4-DCP; and oxidative coupling for the transformation of SDZ by LMS. The results of present work indicated the laccases could efficiently remove NPs, CPs, and SAs in LMS, which offers an opportunity to apply P. ostreatus HAUCC 162 laccase in the field of environmental biotechnology.

Degradation of dyes using crude extract and a thermostable and pH-stable laccase isolated from Pleurotus nebrodensis.

Three laccase isoenzymes (Lac1, Lac2 and Lac3) have been purified to homogeneity from Pleurotus nebrodensis in our previous study. Lac2 was shown to be the dominant isoform, capable of oxidizing the majority of laccase substrates and manifesting good thermostability and pH stability. Hence, Lac2 was selected to decolourize structurally different dyes and the colour removal efficiencies of Lac2 and the crude extract of P. nebrodensis were compared. By monitoring the λmax of the reaction system during the course of biotransformation, clear hypsochromic shifts were observed for most of the dyes examined, illustrating that at least one peak disappeared as a result of laccase treatment. In general, Lac2 was more efficient within a short time (1 h) and the crude extract, in general, could achieve similar or even higher efficiency when the duration of treatment was extended to 24 h. Malachite green (MG) was chosen to study the detoxifying potential of Lac2, because of the relatively simple structure and high toxicity of the dye towards microorganisms. The toxicity of MG towards both bacteria (Bacillus subtilis, Bacillus licheniformis, Pseudomonas fluorescens and Escherichia coli) and fungi (Fusarium graminearum and Trichoderma harzianum) was dramatically decreased and the potential mechanism was estimated by GC–MS as to remove four methyl groups firstly and the two newly formed amine groups would be degraded or polymerized further. The present study facilitates an understanding of the application of P. nebrodensis laccases and furnishes evidence for the safety of their utilization in the treatment of wastewater emanating from textile industries.

Morphological and enzymatic response of the thermotolerant fungus Fomes sp. EUM1 in solid state fermentation under thermal stress.

Thermotolerance of the fungus Fomes sp. EUM1 was evaluated in solid state fermentation (SSF). This thermotolerant strain improved both hyphal invasiveness (38%) and length (17%) in adverse thermal conditions exceeding 30°C and to a maximum of 40°C. In contrast, hyphal branching decreased by 46% at 45°C. The production of cellulases over corn stover increased 1.6-fold in 30°C culture conditions, xylanases increased 2.8-fold at 40°C, while laccase production improved 2.7-fold at 35°C. Maximum production of lignocellulolytic enzymes was obtained at elevated temperatures in shorter fermentation times (8-6 days), although the proteases appeared as a thermal stress response associated with a drop in lignocellulolytic activities. Novel and multiple isoenzymes of xylanase (four bands) and cellulase (six bands) were secreted in the range of 20-150 kDa during growth in adverse temperature conditions. However, only a single laccase isoenzyme (46 kDa) was detected. This is the first report describing the advantages of a thermotolerant white-rot fungus in SSF. These results have important implications for large-scale SSF, where effects of metabolic heat are detrimental to growth and enzyme production, which are severely affected by the formation of high temperature gradients.

Selection of High Laccase-Producing Coriolopsis gallica Strain T906: Mutation Breeding, Strain Characterization, and Features of the Extracellular Laccases.

Commercial application of laccase is often hampered by insufficient enzyme stocks, with very low yields obtained from natural sources. This study aimed to improve laccase production by mutation of a Coriolopsis gallica strain and to determine the biological properties of the mutant. The high-yield laccase strain C. gallica TCK was treated with N-methyl-N-nitro-N-nitrosoguanidine and ultraviolet light. Among the mutants isolated, T906 was found to be a high-production strain of laccases. The mutant strain T906 was stabilized via dozens of passages, and the selected ones were further processed for optimization of metallic ion, inducers, and nutritional requirements, which resulted in the optimized liquid fermentation medium MF9. The incubation temperature and pH were optimized to be 30°C and 4.5, respectively. The mutant strain T906 showed 3-times higher laccase activity than the original strain TCK under optimized conditions, and the maximum laccase production (303 U/ml) was accomplished after 13 days. The extracellular laccase isoenzyme 1 was purified and characterized from the two strains, respectively, and their cDNA sequence was determined. Of note, the laccase isoenzyme 1 transcription levels were overtly increased in T906 mycelia compared with values obtained for strain TCK. These findings provide a basis for C. gallica modification for the production of high laccase amounts.

English

Laccases are enzymes that have a great potential for use in breaking down toxic compounds. Fungal laccases show high enzymatic activity, especially those produced by basidiomycetes. Depending on the culture conditions and the strain used, a variety of isoenzymes and/or enzymatic activities can be obtained. In this study, extracellular laccase enzymes produced by Pleurotus ostreatus was identified in a submerged culture (SmF), with and without copper sulphate as a chemical inducer, and in a solid-state culture (SSF), using wheat straw as natural inducer. This study was conducted to observe the expression of the enzymes produced under the culture conditions tested and their persistence during the culture, as well as the extracellular activity produced and the correspondence that the isoenzymes presented between the intracellular and extracellular media. A positive effect of the inducers on the specific laccase activity was observed either in SmF with copper sulphate or SSF (41.11 and 40.43 UI/mg protein, respectively), compared with that obtained in SmF without copper sulphate (2.87 UI/mg protein). This effect was different only at the time when the highest activity appeared (360 and 120 h, respectively), showing advantages in SSF. The same three isoenzymes were observed in the three kinds of cultures. The main differences among the laccase profiles reside in the time when they appeared in each culture and only an additional form of lower molecular weight was observed in SSF. The laccase enzymes in the intracellular extracts were equal to those in the extracellular ones. The laccase isoenzymes profiles suggest that the presence of inducers helps in maintaining the activity through the culture time. Key words: Phenol oxidases, basidiomycete, enzymatic activity, copper, wheat straw, solid-state culture (SSF), submerged culture (SMF).&nbsp

Heterologous production, characterization and dye decolorization ability of a novel thermostable laccase isoenzyme from Trametes trogii BAFC 463

Laccases are multicopper polyphenol oxidases that are able to catalyze the oxidation of a wide range of phenolic compounds with the simultaneous reduction of O2 to H2O. Despite their promising industrial uses, feasible incorporation of laccases in harsh processes requires the bioprospecting and/or engineering of enzymes to be stable and active in acidic or alkaline pHs, high temperatures, oxidative conditions and tolerant to high salinity and/or organic solvents. Here we used a PCR-based screening to clone two novel laccase coding sequences from the white-rot basidiomycete Trametes trogii. Recombinant expression of lcc3 gene in Komagataella (=Pichia) pastoris showed that it encodes a thermo active and thermostable laccase with an optimum temperature of 50°C and with a half-life of 45min at 70°C and a stability higher than 3h at 60°C. Furthermore, recombinant LCC3 was capable of decolorizing between 50% and 100% of indigoid, triarylmethane, azoic and anthraquinonic synthetic dyes in the presence of the natural redox mediator acetosyringone within 2h of incubation at pH 6 and 70°C.

A novel ethanol-tolerant laccase, Tvlac, from Trametes versicolor.

To produce and characterize novel laccases with ethanol tolerance from Trametes versicolor using agriculture by-products as energy source. Trametes versicolor 1017 produces two laccase isoenzymes with a total activity of 10 Uml(-1) within 8 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A novel isoenzyme, named Tvlac, was identified, purified and characterized. Its optimum pH and temperature were from 4.5 to 5 and 55 to 60°C, respectively. Its activity was stimulated by ethanol at 10% (v/v) which increased the V 0. The biochemical properties of Tvlac substantiate the potential of this enzyme for applications under an aqueous ethanol mixture environment.

Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions

Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unclear. Multiple laccase isoforms produced by the same organism imply a greater flexibility and/or adaptation to constantly changing environments. In this study, we explore and discuss the laccase isoform diversity of Trametes versicolor under submerged culture conditions. Oak sawdust was used as a natural, abundant and cheap source of laccase inducers. Two laccase bands (lcc1 and lcc2) were detected by SDS-PAGE zymography. Lcc2 exhibited an apparent molecular mass of 100kDa, and was purified to its characteristic blue color for biochemical characterization. Lcc2 displayed high affinities toward all the substrates used in this study (0.01±0.0043, 0.5±0.053, 2.23±0.53 and 3.77±0.85mM, for ABTS, DMP, GUA and SYR, respectively). Isoelectric focusing analysis of this band revealed the presence of 5 distinct laccase isoforms. The suggested existence of different laccase heterodimers in lcc2 may have had an influence on its catalyzing capacity. Laccase isoform diversity was corroborated by molecular characterization of 5 allelic mRNA sequences. Multiple alignment analysis, 3-dimensional mapping and theoretical sequence-structure-function assumptions suggests that the newly characterized alleles would most likely display diversity in their biochemical behavior if functionally expressed.

Synergistic action of laccases from Trametes hirsuta Bm2 improves decolourization of indigo carmine

Laccase isoenzymes (LacI,II,III) produced on wheat bran from Trametes hirsuta were partially purified through anion exchange chromatography. The three isoenzymes had the same MW of 65kDa, and their main physico-chemical properties were studied. As single isoenzymes, laccases were unable to decolourize dye. Among several mediators evaluated, syringaldehyde was the most effective in dye decolourization (100%). A remarkable increase in dye decolourization was observed when LacI, II, III in mixture or crude enzyme were added to the reaction system, indicating that the laccases acted synergistically. Laccases have a great potential of application in bioremediation processes. White rot fungi produces several laccase isoenzymes and many of them have been purified and characterized. However, the additive or synergic action between laccase isoenzymes in dye decolourization has not yet been described. Such studies will help to better understand their action and to improve the process with isoenzymes mixtures. This study showed synergistic action between isoenzymes laccases produced by Trametes hirsuta Bm2 during decolourization of indigo carmine.

Molecular characterization and expression profiling of novel laccase isoenzyme genes from Fusarium solani X701, a wood-rotting ascomycete

Fusarium solani X701 is a wood-rotting ascomycete. Four laccase isoenzyme genes, lcc1, lcc2, lcc3 and lcc4, were cloned from this fungus, expression of these genes was profiled, and their relationship with other laccases was explored. Expression profiling of the isoenzymes in different media clearly shown that lcc4 mRNA was significantly induced by lignin, suggesting that lcc4 be the responsible gene for catabolizing lignin. Expression of lcc1 was triggered by the switch from the presence to the absence of glucose, while lcc2 was significantly induced in the glucose medium. Expression levels of lcc3 were not affected by different carbon sources in the primary growth phase, but a short induction by lignin was observed in early secondary growth phase. Bioinformatical analyses revealed unique features in our isoenzymes in comparison with other laccase isoenzymes from ascomycete.

Identification of a laccase Glac15 from Ganoderma lucidum 77002 and its application in bioethanol production.

BackgroundLaccases have potential applications in detoxification of lignocellulosic biomass after thermochemical pretreatment and production of value-added products or biofuels from renewable biomass. However, their application in large-scale industrial and environmental processes has been severely thwarted by the high cost of commercial laccases. Therefore, it is necessary to identify new laccases with lower cost but higher activity to detoxify lignocellulosic hydrolysates and better efficiency to produce biofuels such as bioethanol. Laccases from Ganoderma lucidum represent proper candidates in processing of lignocellulosic biomass.ResultsG. lucidum 77002 produces three laccase isoenzymes with a total laccase activity of 141.1 U/mL within 6 days when using wheat bran and peanut powder as energy sources in liquid culture medium. A new isoenzyme named Glac15 was identified, purified, and characterized. Glac15 possesses an optimum pH of 4.5 to 5.0 and a temperature range of 45°C to 55°C for the substrates tested. It was stable at pH values ranging from 5.0 to 7.0 and temperatures lower than 55°C, with more than 80% activity retained after incubation for 2 h. When used in bioethanol production process, 0.05 U/mL Glac15 removed 84% of the phenolic compounds in prehydrolysate, and the yeast biomass reached 11.81 (optimal density at 600 nm (OD600)), compared to no growth in the untreated one. Addition of Glac15 before cellulase hydrolysis had no significant effect on glucose recovery. However, ethanol yield were improved in samples treated with laccases compared to that in control samples. The final ethanol concentration of 9.74, 10.05, 10.11, and 10.81 g/L were obtained from samples containing only solid content, solid content treated with Glac15, solid content containing 50% prehydrolysate, and solid content containing 50% prehydrolysate treated with Glac15, respectively.ConclusionsThe G. lucidum laccase Glac15 has potentials in bioethanol production industry.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0235-x) contains supplementary material, which is available to authorized users.

Characterization of laccase isoenzymes from the white-rot fungusGanodermasp.En3 and synergistic action of isoenzymes for dye decolorization

BACKGROUND Laccase is an important and valuable enzyme with great potential for biotechnological applications. Laccase is produced mainly by white-rot fungi and often secreted extracellularly as different isoenzymes. Although there have been some reports about the biochemical characteristics and dye decolorization capability of laccase isoenzymes, few studies have been performed to evaluate the synergistic action of different laccase isoenzymes for decolorizing different types of dyes. RESULTS Three laccase isoenzymes designated as Lac-2, Lac-3 and Lac-4 were purified from the white-rot fungus Ganoderma sp.En3. Lac-2 showed stronger pH stability and thermostability than other two isoenzymes. Lac-2 also had a stronger ability to tolerate metal ions and organic solvents compared with other two isoenzymes. Different laccase isoenzymes from Ganoderma sp.En3 had different and selective decolorization capability. The synergistic action of different laccase isoenzymes for the enzymatic decolorization of dyes was further evaluated. There existed a positive synergistic effect of different laccase isoenzymes on the decolorization capability. Combination of Lac-3 with other two isoenzymes Lac-2 or Lac-4 could efficiently promote the decolorization of azo dyes. CONCLUSION This research will contribute to the deep understanding of functions of different laccase isoenzymes and better application of laccase in the biodegradation of environmental pollutants. © 2014 Society of Chemical Industry]

Essential role of the N- and C-terminals of laccase from Pleurotus florida on the laccase activity and stability.

POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c△13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cΔ13-R5V, combined the R5V and △13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cΔ13-R5V. POXA1cΔ13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.

Reactive dyes and textile effluent decolorization by a mediator system of salt-tolerant laccase from Peniophora cinerea

Peniophora cinerea is a white-rot fungus with ability to decolorize dyes in saline textile effluent. After cultivation in a medium composed by sucrose, corn steep liquor, copper, and other minor components, P. cinerea produced 1000U/L of laccase, but no lignin peroxidase or manganese peroxidase. The produced laccase was purified by precipitation with ammonium sulfate (80%) and anion exchange chromatography. Isoelectric focusing revealed a total of eight salt tolerant laccase isoenzymes with pI between 3 and 6, and molecular weights in the range of 26.2 and 72.6kDa. Five compounds were then tested and compared for their ability to act as mediators in the decolorization of the reactive blue 19 dye by these laccases of P. cinerea. Syringaldehyde was the best mediator since it increased in almost 3-fold the dye decolorization when compared to the decolorization by using laccase alone. Addition of Mn2+ and oxalate to this system increased in 4.8-fold the initial decolorization rate (178μmol/Lmin). When applied for real effluent decolorization, this system promoted the highest decolorization after 72h at 50°C and pH 4. This laccase-mediator system was considered promissory for application on the treatment of industrial dye effluents.

Overproduction of Laccase by the White-Rot Fungus Pleurotus ostreatus Using Apple Pomace as Inducer

Laccase activity of Pleurotus ostreatus is significantly increased by the addition of apple pomace. Among various conditions, the best concentration of apple pomace and cultivation time for the production of laccase by P. ostreatus was 2.5% and 9 days, respectively. Reverse transcription polymerase chain reaction analyses of laccase isoenzyme genes, including pox1, pox3, pox4, poxc, poxa3, and poxa1b, revealed a clear effect of apple pomace on transcription induction. Our findings reveal that the use of apple pomace can be a model for the valuable addition of similar wastes and for the development of a solid-state fermenter and commercial production of oyster mushroom P. ostreatus.

Comparison of Two Laccases from Trametes versicolor for Application in the Decolorization of Dyes

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and (NH4)2SO4 precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and 55°C with 2,2'-azino-bis-[3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and 60°C. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a Vmax value of 51.28 U/mg, and the Km and Vmax values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.