Redox-mediated decolorization of recalcitrant textile dyes by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> WL1 laccase

The efficiency of crude and partially purified Trichoderma harzianum WL1 laccase for the decolorization of synthetic dyes (Rhodamine 6G, Erioglaucine and Trypan blue) with complex aromatic structures were evaluated. Selection of dyes was based on their extensive usage in local dyeing and textile industries around the study area. Studies on the role of redox potential of laccases on dye decolorization are rarely discussed and hence, for the first time we have shown the redox mediated dye decolorizing efficiency of T. harzianum WL1 laccase with the commonly employed redox mediator 1-hydroxybenzotriazole (HBT). The process parameters such as initial dye concentration, enzyme load and HBT concentration were studied and found that they had a great influence on dye removal process. When the dyes were treated with increased concentration of enzyme, it showed a greater percentage of decolorization. Compared to the crude laccase, partially purified laccase accounts for maximum decolorization of all the dyes studied. In addition, the rate of dye decolorization was considerably enhanced in presence of 4 mM HBT. Maximum and minimum decolorization were recorded for Rhodamine 6G and Trypan blue, respectively. The results of this study further confirmed that, T. harzianum laccase was found to be suitable with HBT and this laccase-mediator system (LMS) could be applied for the decolorization of various classes of dyes.     

Decolorization of reactive dyes by a thermostable laccase produced by Ganoderma lucidum in solid state culture

Dye decolorizing potential of the white rot fungus Ganoderma lucidum KMK2 was demonstrated for recalcitrant textile dyes. G. lucidum produced laccase as the dominant lignolytic enzyme during solid state fermentation (SSF) of wheat bran (WB), a natural lignocellulosic substrate. Crude enzyme shows excellent decolorization activity to anthraquinone dye Remazol Brilliant Blue R (RBBR) without redox mediator whereas diazo dye Remazol Black-5 (RB-5) requires a redox mediator. Polyacrylamide gel electrophoresis (PAGE) of crude enzyme confirms that the laccase enzyme was the major enzyme involved in decolorization of either dyes. Native and SDS-PAGE indicates that the presence of single laccase with molecular weight of 43 kDa. N-Hydroxybenzotriazole (HBT) at a concentration of 1 mM was found as the best redox mediator. RB-5 (50 mg l^−l) was decolorized by 62% and 77.4% within 1 and 2 h, respectively by the crude laccase (25 U ml⁻¹). RBBR (50 mg l^−l) was decolorized by 90% within 20 h, however, it was more efficient in presence of HBT showing 92% decolorization within 2 h. Crude laccase showed high thermostability and maximum decolorization activity at 60 °C and pH 4.0. The decolorization was completely inhibited by the laccase inhibitor sodium azide (0.5 mM). Enzyme inactivation method is a good method which averts the undesirable color formation in the reaction mixture after decolorization. High thermostability and efficient decolorization suggest that this crude enzyme could be effectively used to decolorize the synthetic dyes from effluents.     

Decolorization of textile dyes by whole cultures of Ischnoderma resinosum and by purified laccase and Mn-peroxidase

Ischnoderma resinosum produced extracellular ligninolytic enzymes laccase and MnP. The activity of laccase achieved the maximum on day 10 (29.4 U L⁻¹), the MnP on day 14 (34.5 U L⁻¹). Laccase and Mn-peroxidase were purified from the culture liquid using gel permeation and ion-exchange chromatographies. Purified Mn-peroxidase performed decolorization of all textile dyes tested (Reactive Black 5, Reactive Blue 19, Reactive Red 22 and Reactive Yellow 15). Laccase was inactive with Reactive Black 5 and Reactive Red 22, while all dyes were decolorized after addition of the redox mediators violuric acid (VA) and hydroxybenzotriazole (HBT). The culture liquid from I. resinosum cultures was also able to decolorize all dyes as well as the synthetic dyebaths in the presence of VA and HBT. The highest decolorization rates were detected in acidic pH (3–4).     

Decolorization of textile dyes by enzymatic extract and submerged cultures of <Emphasis Type="Italic">Pleurotus sajor-caju</Emphasis>

Pleurotus sajor-caju PS2001 was screened in Petri dish plates to assess the dye-decolorizing ability of industrial textile dyes. P. sajor-caju PS2001 was also cultivated in solid-state fermentation containing sawdust of Pinus sp. and wheat bran to obtain the enzymatic extract, showing laccase and manganese-peroxidase activity, which was used to test the capacity to degrade the textile dyes. Additional tests of decolorization were performed in liquid cultures. Anthraquinone-type textile dyes proved to be substrates for the enzymatic system of P. sajor-caju PS2001. Cultures in Petri dish plates showed that the anthraquinone dye Reactive Blue 220 can act as a redox mediator for the enzymatic reactions involved in the decolorization process, and enables the azo dye degradation. Reactive Blue 220 and Acid Blue 280 were completely decolorized in 30 min and 60 min, respectively, during the tests with precipitated enzymatic extract, while the azo dyes showed resistance to degradation. Additionally, in submerged cultures with dyes, veratryl alcohol oxidases and lignin peroxidase activities were observed. These results suggest that the strain P. sajor-caju PS2001 has great potential for use in the bioremediation technology of recalcitrant pollutant such as textile effluents.     

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.     

Overexpression of a Novel Thermostable and Chloride-Tolerant Laccase from Thermus thermophilus SG0.5JP17-16 in Pichia pastoris and Its Application in Synthetic Dye Decolorization

Laccases have been used for the decolorization and detoxification of synthetic dyes due to their ability to oxidize a wide variety of dyes with water as the sole byproduct. A putative laccase gene (LacTT) from Thermus thermophilus SG0.5JP17-16 was screened using the genome mining approach, and it was highly expressed in Pichia pastoris, yielding a high laccase activity of 6130 U/L in a 10-L fermentor. The LacTT open reading frame encoded a protein of 466 amino acid residues with four putative Cu-binding regions. The optimal pH of the recombinant LacTT was 4.5, 6.0, 7.5 and 8.0 with 2,2''-azino-bis(3-ethylbenzothazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), guaiacol, and 2,6-dimethoxyphenol (2,6-DMP) as the substrate, respectively. The optimal temperature of LacTT was 90°C with guaiacol as the substrate. LacTT was highly stable at pH 4.0–11.0 and thermostable at 40°C–90°C, confirming that it is a pH-stable and thermostable laccase. Furthermore, LacTT also exhibited high tolerance to halides such as NaCl, NaBr and NaF, and decolorized 100%, 94%, 94% and 73% of Congo Red, Reactive Black B and Reactive Black WNN, and Remazol Brilliant Blue R, respectively. Interestingly, addition of high concentration of NaCl increased the RBBR decolorization efficiency of LacTT. These results suggest that LacTT is a good candidate for industrial applications such as dyestuff processing and degradation of dyes in textile wastewaters.     

Decolorization of aqueous solutions of disperse textile dyes by oxidoreductases

The potential of three oxidoreductases, a laccase preparation of Pleurotus sajor-caju PS-2001, horseradish peroxidase (HRP) and a microbial peroxidase (MP) was evaluated for the decolorization of disperse textile dyes (CI Disperse Red 343, CI Disperse Red 167 and CI Disperse Blue 148) used in polyester dyeing. Decolorization was studied in aqueous solutions varying in dye concentration, pH, temperature, enzyme concentration and the addition of mediators HBT and syringaldazine. The best conditions found for Disperse Red 343 with laccase, HRP and MP were: 15 mg L^?1 dye concentration, 50°C, pH 3.0 for laccase and pH 5.0 for peroxidases. Without mediator, the highest decolorizaton results (38.5% and 58.6%) were achieved with the highest tested concentrations of laccase (10 U mL^?1) and HRP (89.7 U mL^?1), respectively, but no significant difference in decolorization was found for the tested MP concentrations (29.9–89.7 U mL^‐1). HBT or syringaldazine increased decolorization with peroxidases significantly, but no effect was observed for the laccase. Decolorization of Disperse Red 167 (up to 15%) and Disperse Blue 148 (up to 25%) was much lower than of Disperse Red 343. With respect to enzyme concentration, the use of mediator and under the selected test conditions the laccase of P. sajor-caju PS-2001 turned out to be more efficient in disperse dye decolorization, than peroxidases HRP and MP.     

Study of dye decolorization in an immobilized laccase enzyme-reactor using online spectroscopy

Decolorization of textile dyes by a laccase from Trametes modesta immobilized on gamma-aluminum oxide pellets was studied. An enzyme reactor was equipped with various UV/Vis spectroscopic sensors allowing the continuous online monitoring of the decolorization reactions. Decolorization of the dye solutions was followed via an immersion transmission probe. Adsorption processes were observed using diffuse reflectance measurements of the solid carrier material. Generally, immobilization of the laccase does not seem to sterically affect dye decolorization. A range of commercial textile dyes was screened for decolorization and it was found that the application of this enzymatic remediation system is not limited to a certain structural group of dyes. Anthrachinonic dyes (Lanaset Blue 2R, Terasil Pink 2GLA), some azo dyes, Indigo Carmine, and the triphenylmethane dye Crystal Violet were efficiently decolorized. However, the laccase displayed pronounced substrate specificities when a range of structurally related model azodyes was subjected to the biotransformation. Azodyes containing hydroxy groups in ortho or para position relative to the azo bond were preferentially oxidized. The reactor performance was studied more closely using Indigo Carmine.     

Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes

The aim of this study was to evaluate the enzymatic action of partially purified bitter gourd peroxidase for the degradation/decolorization of complex aromatic structures. Twenty-one dyes, with a wide spectrum of chemical groups, currently being used by the textile and other important industries have been selected for the study. Here, for the first time we have shown peroxidases from Momordica charantia (300 EU/gm of vegetable) to be highly effective in decolorizing industrially important dyes. Dye solutions, containing 50-200 mg dye/l, were used for the treatment with bitter gourd peroxidase (specific activity of 99.0 EU/mg protein). M. charantia peroxidases were able to decolorize most of the textile dyes by forming insoluble precipitate. When the textile dyes were treated with increasing concentration of enzyme, it was observed that greater fraction of the color was removed but four out of eight reactive dyes were recalcitrant to decolorization by bitter gourd peroxidase. Step-wise addition of enzyme to the decolorizing reaction mixture at the interval of 1h further enhanced the dye decolorization. The rate of decolorization was enhanced when the dyes were incubated with fixed quantity of enzyme for increasing times. Decolorization of non-textile dyes resulted in the degradation and removal of dyes from the solution without any precipitate formation. Decolorization rate was drastically increased when the textile and other industrially important non-textile dyes were treated with bitter gourd peroxidase in presence of 1.0 mM 1-hydroxybenzotriazole. Complex mixtures of dyes were prepared by taking three to four reactive textile and non-textile dyes in equal proportions. Each mixture was decolorized by more than 80% when treated with the enzyme in presence of 1.0 mM 1-hydroxybenzotriazole. Our data suggest that the peroxidase/mediator system is an effective biocatalyst for the treatment of effluents containing recalcitrant dyes from textile, dye manufacturing, dyeing and printing industries.     

Implication of mycelium-associated laccase from Irpex lacteus in the decolorization of synthetic dyes

The white rot fungus Irpex lacteus is able to decolorize such synthetic dyes as Reactive Orange 16 and Remazol Brilliant Blue R. Here, we demonstrate that this type of dye decolorization is mainly related to a laccase-like enzyme activity associated with fungal mycelium. In its bound form, the enzyme detected showed a pH optimum of 3.0 for the oxidation of ABTS, DMP and guaiacol, and a pH of 7.0 for syringaldazine. The highest enzymatic activity was obtained with ABTS as substrate. Enzyme activity was fully inhibited with 50mM NaN(3). Depending on the chemical structure of dyes, redox mediators had a positive effect on the dye decolorization by fungal mycelium. Enzyme isolated from fungal mycelium was able to decolorize synthetic dyes in vitro.     

Molecular cloning and characterization of a novel metagenome-derived multicopper oxidase with alkaline laccase activity and highly soluble expression

Lac591, a gene encoding a novel multicopper oxidase with laccase activity, was identified through activity-based functional screening of a metagenomic library from mangrove soil. Sequence analysis revealed that lac591 encodes a protein of 500 amino acids with a predicted molecular mass of 57.4 kDa. Lac591 was overexpressed heterologously as soluble active enzyme in Escherichia coli and purified, giving rise to 380 mg of purified enzyme from 1 l induced culture, which is the highest expression report for bacterial laccase genes so far. Furthermore, the recombinant enzyme demonstrated activity toward classical laccase substrates syringaldazine (SGZ), guaiacol, and 2, 6-dimethoxyphenol (2, 6-DMP). The purified Lac591 exhibited maximal activity at 55°C and pH 7.5 with guaiacol as substrate and was found to be stable in the pH range of 7.0–10.0. The substrate specificity on different substrates was studied with the purified enzyme, and the optimal substrates were in the order of 2, 6-DMP > catechol > α-naphthol > guaiacol > SGZ > 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). The alkaline activity and highly soluble expression of Lac591 make it a good candidate of laccases in industrial applications for which classical laccases are unsuitable, such as biobleaching of paper pulp and dyestuffs processing.     

Cloning,expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization

A thermostable and pH-stable laccase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli. The recombinant laccase (rLac) achieved a specific activity of 7.12 U/mg after purification by Ni-affinity chromatography. Optimal enzyme activity was observed at pH 4.0 and 35 °C for 2,2′-azino-bis (3-ethylbenzthiazoline sulfonic acid) (ABTS) oxidization and pH 8.0 and 70 °C for 2,6-dimethoxyphenol (2,6-DMP) oxidization. Thermostability and pH stability studies showed that the rLac was stable over the range of 30–70 °C and pH 5.0–9.0 using 2,6-DMP as substrate. Circular dichroism analysis suggested that the secondary structure of the rLac mainly consisted of α-helix that played a vital role in maintaining laccase activity and revealed the potential mechanisms for the changes in laccase activity under varying pHs (3.0–11.0) and temperatures (20–90 °C). Finally, the rLac could decolorize the tested dyes with high decolorization efficiency.     

Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus

Two new laccase genes, named lac1 and lac2, were cloned from the edible basidiomycete Coprinus comatus. Comparison of the deduced amino acid sequences revealed two laccases showed 66.12 % identity and clustered with lac2 and lac3 from Coprinopsis cinerea in same phylogenetic group. Lac1 and lac2 encode proteins of 517 and 523 amino acids preceded by 18 and 21-residue signal peptides, respectively. Lac1 was functionally expressed in Pichia pastoris. The optimum pHs of recombinant Lac1 were 3.0, 6.0, 5.5 and 6.0 and the optimum temperatures were 65, 55, 70 and 50 °C for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The Km values of Lac1 were 34, 4,317, 7,611 and 14 μM, and the corresponding kcat values were 465.79, 7.67, 1.15 and 0.60 (s^?1 mM), for ABTS, guaiacol, 2,6-dimethylphenol and syringaldazine, respectively. The enzyme activity was completely inhibited by sodium azide (NaN₃) and 1,4-dithiothreitol (DTT) at the concentration of 5 mM. Laccase activity was also inhibited by several metal ions, especially Fe²⁺, while K⁺ and NH₄ ⁺ slightly enhanced laccase activity. Twelve synthetic dyes belonging to anthraquinone, azo and triphenylmethane dyes were decolorized by the recombinant Lac1 at different extents. The recombinant Lac1 decolorized azo dye Reactive Dark Blue KR up to 90 % without any mediator and increasing to 96 % with mediator, indicating its potential in the treatment of industrial effluent containing some recalcitrant synthetic dyes.     

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.     

Hydroxybenzotriazole increases the range of textile dyes decolorized by immobilized laccase

Laccase from Coriolopsis gallica UAMH8260 was immobilized on activated agarose and tested for repeated decolorization of industrial dyes. Immobilized enzyme retained 85% of the initial activity after 10 cycles, and 70% after 3 months of intermittant use in the decolorization of Reactive Blue 198 dye. Free laccase decolorized 13 of 38 industrial dyes tested but, in the presence of 1 mM 1-hydroxybenzotriazole as a free radical mediator, the enzyme decolorized 26 of the 38 dyes increasing both the range and rate of decolorization. Immobilized laccase showed a higher thermal stability at 70 °C than free enzyme but no increased resistance to organic solvents.     

Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes,and decolorization of chemically different dyes

A laccase (EC 1.10.3.2) was isolated from the culture filtrate of Lentinula edodes. The enzyme was purified to a homogeneous preparation using hydrophobic, anion-exchange, and size-exclusion chromatographies. SDS-PAGE analysis showed the purified laccase, Lcc 1, to be a monomeric protein of 72.2 kDa. The enzyme had an isoelectric point of around pH 3.0. The optimum pH for enzyme activity was around 4.0, and it was most active at 40 degrees C and stable up to 35 degrees C. The enzyme contained 23.8% carbohydrate and some copper atoms. The enzyme oxidized 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, p-phenylendiamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol, and ferulic acid, but not veratryl alcohol, tyrosine, and beta-(3,4-dihydroxyphenyl) alanine. The N-terminal amino acid sequence of Lcc 1 showed close homology to the N-terminal sequences determined for laccases from Phlebia radiata, Trametes villosa, and Trametes versicolor, but only low similarity was observed to a previously reported laccase from L. edodes. Lcc 1 was effective in the decolorization of chemically different dyes - Remazole Brilliant Blue R, Bromophenol Blue, methyl red, and Naphtol Blue Black - without any mediators, but the decolorization of two dyes - red poly(vinylamine)sulfonate-anthrapyridone dye and Reactive Orange 16 - did require some redox mediators.     

Bioinformatics aided microbial approach for bioremediation of wastewater containing textile dyes

Four textile azo dyes, Joyfix Red, Remazol Red, Reactive Red and Reactive Yellow, were studied for decolorization. Of nineteen soil bacterial isolates, two novel strains were found to highly decolorize Joyfix Red and were identified as Lysinibacillus sphaericus (KF032717) and Aeromonas hydrophila (KF032718) through 16S rDNA analysis. Laccase and Azoreductase enzyme modeling and enzyme–dye interaction performed using Schrödinger Suite imitated decolorization percentage. Results based on cumulative Glide score (Dry laboratory) and decolorization percentage of the other three dyes based on ultraviolet–visible (UV–vis) spectroscopy (Wet laboratory) were reliable. Biodegradation of Joyfix Red was confirmed by high-performance liquid chromatography (HPTLC) elution profile which showed four peaks at 1.522, 1.800, 3.068 and 3.804 min with that of parent dye which showed single peak at 1.472 min. Fourier transform infrared spectroscopy (FT-IR) analysis supported the biotransformation of Joyfix Red. Gas chromatography–mass spectroscopy (GC–MS) analysis showed sodium (3E,5Z)-4-amino-6-hydroxyhexa-13,5-triene-2-sulfonate was formed as end product during biodegradation. From these findings, it can be inferred that enzyme and dye interaction studies can assist in examining decolorization efficiency of bacteria and its enzyme, thereby enhancing the bioremediation process by reducing preliminary lengthy wet laboratory screening. This is the first report of a combinatorial in silico cum in vitro approach and its validation for the bioremediation of wastewater containing these textile azo dyes.     

Influence of structure on dye degradation with laccase mediator systems

A new laccase was purified from Trametes hirsuta IMA2002. The laccase had a molecular mass of 62 kDa and an isoelectric point of pH 7. It had an optimum pH of 3.0 and an optimum temperature of 55°C. The laccase was quite stable at 30°C and pH 4.0 with a half-life of more than 100 hours. On ABTS, syringaldazide, and DMP the laccase showed K_M and K_cat values of 75, 12 and 37 μM and 64, 83 and 54 s^-1, respectively. The structurally diverse commercial dyes Indigo Carmine, Lanaset Blue 2R, Diamond Black PV 200 and Diamond Fast Brown were oxidized by the laccase. While the rate and extent of decolorization of the latter dye was significantly enhanced by the presence of different types of mediators, the structurally similar azo-dye Tartrazine was not oxidized. Lanaset Blue 2R, a commercial textile dye containing an anthrachinoid structural fragment acted similarly to anthrachinone sulfonic acid by strongly enhancing the rate of the decolorization reaction. Twenty two model azo-dyes based on the molecular framework of 2,7-dihydroxy-1-phenylazonaphtalene-3,6-disulfonic acid were synthesized and the kinetics of their laccase-catalyzed decolorization was studied. Hydroxy-substituted dyes were the most susceptible to enzyme/mediator action. All reactions were well described by Michaelis-Menten-like kinetics and the Hammett free energy linear relationship could be successfully applied to describe the influence of dye structure (substituents on the aromatic ring) on decolorization. Strongly electron withdrawing substituents such as a nitro-group in the meta-position (+0.7) resulted in positive σ-constants whereas electron donating groups such as para-methyl (-0.3) resulted in negative values for σ-constants.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.