3 Biotech

, 8:505 | Cite as

Decolorization and detoxification of textile wastewaters by recombinant Myceliophthora thermophila and Trametes trogii laccases

  • Klára Herkommerová
  • Jiří Dostál
  • Iva PichováEmail author
Original Article


Laccases are multi-copper oxidoreductases with broad biotechnological applications. Here, we report detailed biochemical characterization of purified recombinant laccases originating from Myceliophthora thermophila (MtL) and Trametes trogii (TtL). We identified optimal conditions for decolorization of commercial dyes and textile wastewater samples. We also tested the toxicity of decolorized wastewater samples using human peripheral blood mononuclear cells. MtL and TtL were expressed in Saccharomyces cerevisiae, and secreted enzymes were purified by consecutive hydrophobic and gel chromatography. The molecular masses of TtL (~ 65 kDa) and MtL (> 100 kDa) suggested glycosylation of the recombinant enzymes. Deglycosylation of MtL and TtL led to 25% and 10% decreases in activity, respectively. In a thermal stability assay, TtL retained 61% and MtL 86% of the initial activity at 40 °C. While TtL retained roughly 50% activity at 60 °C, MtL lost stability at temperatures higher than 40 °C. MtL and TtL preferred syringaldazine as a substrate, and the catalytic efficiencies for ABTS oxidation were 7.5 times lower than for syringaldazine oxidation. In the presence of the mediator HBT, purified TtL almost completely decolorized dyes within 30 min and substantially decolorized wastewater samples from a textile factory (up to 74%) within 20 h. However, products of TtL-catalyzed decolorization were more toxic than MtL-decolorized products, which were almost completely detoxified.


Laccases Textile dyes Wastewater Decolorization Toxicity PBMCs 



This work was supported by Grant NPU LO 1302 from the Ministry of Education, Youth and Sports of the Czech Republic. The authors thank Zdeněk Voburka for N-terminal protein sequencing, Martin Hubálek for performing the peptide mass fingerprinting analysis, Miroslav Hájek for the toxicity study, Petr Beier for a consultation in dye chemistry, Zuzana Antošová for a consultation of decolorization experiments and Radek Stloukal (Lentikat’s a.s., Straž pod Ralskem, Czech Republic) for providing samples of textile industry wastewater originating from CNM textile a.s. (Baška, Czech Republic.)

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

13205_2018_1525_MOESM1_ESM.pdf (825 kb)
Supplementary material 1 (PDF 825 KB)


  1. Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gubitz GM (2000) Decolorization and detoxification of textile dyes with a laccase from Trametes hirsuta. Appl Environ Microbiol 66:3357–3362CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ai MQ, Wang FF, Huang F (2015) Purification and characterization of a thermostable laccase from Trametes trogii and its ability in modification of kraft lignin. J Microbiol Biotechnol 25:1361–1370. CrossRefPubMedGoogle Scholar
  3. Ali N, Hameed A, Ahmed S (2009) Physicochemical characterization and Bioremediation perspective of textile effluent, dyes and metals by indigenous Bacteria. J Hazard Mater 164:322–328. CrossRefPubMedGoogle Scholar
  4. Antosova Z, Sychrova H (2016) Yeast hosts for the production of recombinant laccases: a review. Mol Biotechnol 58:93–116. CrossRefPubMedGoogle Scholar
  5. Antosova Z, Herkommerova K, Pichova I, Sychrova H (2018) Efficient secretion of three fungal laccases from Saccharomyces cerevisiae and their potential for decolorization of textile industry effluent—a comparative study. Biotechnol Prog 34:69–80. CrossRefPubMedGoogle Scholar
  6. Arca-Ramos A et al (2016) Assessing the use of nanoimmobilized laccases to remove micropollutants from wastewater. Environ Sci Pollut Res 23:3217–3228. CrossRefGoogle Scholar
  7. Archna LKN, Kiran S R. R (2012) Biological methods of dye removal from textile effluents—a review. J Biochem Tech 3:177–180Google Scholar
  8. Barrios-Estrada C, de Jesús Rostro-Alanis M, Muñoz-Gutiérrez BD, Iqbal HMN, Kannan S, Parra-Saldívar R (2018) Emergent contaminants: Endocrine disruptors and their laccase-assisted degradation—a review. Sci Total Environ 612:1516–1531. CrossRefPubMedGoogle Scholar
  9. Bello-Gil D et al (2018) An enzymatic system for decolorization of wastewater dyes using immobilized CueO laccase-like multicopper oxidase on poly-3-hydroxybutyrate. Microbial Biotechnol. CrossRefGoogle Scholar
  10. Benzina O et al (2013) Decolorization and detoxification of two textile industry effluents by the laccase/1-hydroxybenzotriazole system. Environ Sci Pollut Res 20:5177–5187. CrossRefGoogle Scholar
  11. Bilal M, Asgher M, Parra-Saldivar R, Hu HB, Wang W, Zhang XH, Iqbal HMN (2017) Immobilized ligninolytic enzymes: An innovative and environmental responsive technology to tackle dye-based industrial pollutants—a review. Sci Total Environ 576:646–659. CrossRefPubMedGoogle Scholar
  12. Brinch DS, Pedersen PB (2002) Toxicological studies on laccase from Myceliophthora thermophila expressed in Aspergillus oryzae. Regul Toxicol Pharmacol 35:296–307. CrossRefPubMedGoogle Scholar
  13. Bulter T, Alcalde M, Sieber V, Meinhold P, Schlachtbauer C, Arnold FH (2003) Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl Environ Microbiol 69:987–995. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Cannatelli MD, Ragauskas AJ (2017) Two decades of laccases: advancing sustainability in the chemical industry. Chem Rec 17:122–140. CrossRefPubMedGoogle Scholar
  15. Carneiro PA, Umbuzeiro GA, Oliveira DP, Zanoni MVB (2010) Assessment of water contamination caused by a mutagenic textile effluent/dyehouse effluent bearing disperse dyes. J Hazard Mater 174:694–699. CrossRefPubMedGoogle Scholar
  16. Champagne PP, Nesheim ME, Ramsay JA (2013) A mechanism for NaCl inhibition of Reactive Blue 19 decolorization and ABTS oxidation by laccase. Appl Microbiol Biotechnol 97:6263–6269. CrossRefPubMedGoogle Scholar
  17. Chatha SAS, Asgher M, Iqbal HMN (2017) Enzyme-based solutions for textile processing and dye contaminant biodegradation—a review. Environ Sci Pollut Res 24:14005–14018. CrossRefGoogle Scholar
  18. Chen SC et al (2012) Biochemical characterization of a novel laccase from the basidiomycete fungus Cerrena sp. WR1. Protein Eng Des Sel 25:761–769. CrossRefPubMedGoogle Scholar
  19. Chhabra M, Mishra S, Sreekrishnan TR (2015) Immobilized laccase mediated dye decolorization and transformation pathway of azo dye acid red 27. J Environ Health Sci Eng 13:38. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Colao MC, Lupino S, Garzillo AM, Buonocore V, Ruzzi M (2006) Heterologous expression of lcc 1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microb Cell Fact 5:31. CrossRefPubMedPubMedCentralGoogle Scholar
  21. Daâssi D, Zouari-Mechichi H, Frikha F, Martinez MJ, Nasri M, Mechichi T (2013) Decolorization of the azo dye Acid Orange 51 by laccase produced in solid culture of a newly isolated Trametes trogii strain. 3 Biotech 3:115–125. CrossRefPubMedGoogle Scholar
  22. Dellai A, Dridi D, Lemorvan V, Robert J, Cherif A, Mosrati R, Mansour HB (2013) Decolorization does not always mean detoxification: case study of a newly isolated Pseudomonas peli for decolorization of textile wastewater. Environ Sci Pollut Res 20:5790–5796. CrossRefGoogle Scholar
  23. Forgacs E, Cserháti T, Oros G (2004) Removal of synthetic dyes from wastewaters: a review. Environ Int 30:953–971. CrossRefPubMedGoogle Scholar
  24. Grandclément C et al (2017) From the conventional biological wastewater treatment to hybrid processes, the evaluation of organic micropollutant removal: a review. Water Res 111:297–317. CrossRefPubMedGoogle Scholar
  25. Grassi E, Scodeller P, Filiel N, Carballo R, Levin L (2011) Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators. Int Biodeterior Biodegrad 65:635–643. CrossRefGoogle Scholar
  26. Guan ZB, Shui Y, Song CM, Zhang N, Cai YJ, Liao XR (2015) Efficient secretory production of CotA-laccase and its application in the decolorization and detoxification of industrial textile wastewater. Environ Sci Pollut Res 22:9515–9523. CrossRefGoogle Scholar
  27. Herkommerova K, Zemancikova J, Sychrova H, Antosova Z (2018) Immobilization in polyvinyl alcohol hydrogel enhances yeast storage stability and reusability of recombinant laccase-producing S. cerevisiae. Biotechnol Lett 40:405–411. CrossRefPubMedGoogle Scholar
  28. Holkar CR, Jadhav AJ, Pinjari DV, Mahamuni NM, Pandit AB (2016) A critical review on textile wastewater treatments: possible approaches. J Environ Manage 182:351–366. CrossRefPubMedGoogle Scholar
  29. Hollmann F, Gumulya Y, Tölle C, Liese A, Thum O (2008) Evaluation of the laccase from Myceliophthora thermophila as industrial biocatalyst for polymerization reactions. Macromolecules 41:8520–8524. CrossRefGoogle Scholar
  30. Kadri T, Rouissi T, Brar SK, Cledon M, Sarma S, Verma M (2017) Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review. J Environ Sci 51:52–74. CrossRefGoogle Scholar
  31. Kepp KP (2015) Halide binding and inhibition of laccase copper clusters: The role of reorganization energy. Inorg Chem 54:476–483. CrossRefPubMedGoogle Scholar
  32. Khlifi R, Belbahri L, Woodward S, Ellouz M, Dhouib A, Sayadi S, Mechichi T (2010) Decolourization and detoxification of textile industry wastewater by the laccase-mediator system. J Hazard Mater 175:802–808. CrossRefPubMedGoogle Scholar
  33. Legerská B, Chmelová D, Ondrejovič M (2016) Degradation of synthetic dyes by laccases—a mini-review. Nova Biotechnol Chim 15:90–106. CrossRefGoogle Scholar
  34. Luo Q, Chen Y, Xia J, Wang K-Q, Cai Y-J, Liao X-R, Guan Z-B (2018) Functional expression enhancement of Bacillus pumilus CotA-laccase mutant WLF through site-directed mutagenesis. Enzyme Microbial Technol 109:11–19. CrossRefGoogle Scholar
  35. Maestre-Reyna M et al (2015) Structural and functional roles of glycosylation in fungal laccase from Lentinus sp. PLoS One 10:e0120601. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Mahmood S, Khalid A, Arshad M, Mahmood T, Crowley DE (2016) Detoxification of azo dyes by bacterial oxidoreductase enzymes. Crit Rev Biotechnol 36:639–651. CrossRefPubMedGoogle Scholar
  37. Mate DM, Alcalde M (2015) Laccase engineering: From rational design to directed evolution. Biotechnol Adv 33:25–40. CrossRefPubMedGoogle Scholar
  38. Moilanen U, Kellock M, Várnai A, Andberg M, Viikari L (2014) Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce. Biotechnol Biofuels 7:177. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Naghdi M, Taheran M, Brar SK, Kermanshahi-pour A, Verma M, Surampalli RY (2018) Removal of pharmaceutical compounds in water and wastewater using fungal oxidoreductase enzymes. Environ Pollut 234:190–213. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Nyanhongo GS, Gomes J, Guebitz G, Zvauya R, Read J, Steiner W (2002) Decolorization of textile dyes by laccases from a newly isolated strain of Trametes modesta. Water Res 36:1449–1456. CrossRefPubMedGoogle Scholar
  41. Osma JF, Toca-Herrera JL, Rodríguez-Couto S (2010) Uses of Laccases in the food industry. Enzyme Res. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Patel H, Gupte A (2016) Optimization of different culture conditions for enhanced laccase production and its purification from Tricholoma giganteum AGHP. Bioresour Bioprocess 3:11. CrossRefGoogle Scholar
  43. Pezzella C, Guarino L, Piscitelli A (2015) How to enjoy laccases. Cell Mol Life Sci 72:923–940. CrossRefPubMedGoogle Scholar
  44. Piscitelli A, Giardina P, Mazzoni C, Sannia G (2005) Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae. Appl Microbiol Biotechnol 69:428–439. CrossRefPubMedGoogle Scholar
  45. Ranieri D, Colao MC, Ruzzi M, Romagnoli G, Bianchi MM (2009) Optimization of recombinant fungal laccase production with strains of the yeast Kluyveromyces lactis from the pyruvate decarboxylase promoter. FEMS Yeast Res 9:892–902. CrossRefPubMedGoogle Scholar
  46. Raseda N, Hong S, Kwon OY, Ryu K (2014) Kinetic evidence for the interactive inhibition of laccase from Trametes versicolor by pH and chloride. J Microbiol Biotechnol 24:1673–1678CrossRefPubMedGoogle Scholar
  47. Rezaei S, Tahmasbi H, Mogharabi M, Ameri A, Forootanfar H, Khoshayand MR, Faramarzi MA (2015) Laccase-catalyzed decolorization and detoxification of Acid Blue 92: statistical optimization, microtoxicity, kinetics, and energetics. J Environ Health Sci Eng 13:31. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226. CrossRefPubMedGoogle Scholar
  49. Robinson T, McMullan G, Marchant R, Nigam P (2001) Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresour Technol 77:247–255. CrossRefPubMedGoogle Scholar
  50. Sen SK, Raut S, Bandyopadhyay P, Raut S (2016) Fungal decolouration and degradation of azo dyes: a review. Fungal Biol Rev 30:112–133. CrossRefGoogle Scholar
  51. Senthivelan T, Kanagaraj J, Panda RC (2016) Recent trends in fungal laccase for various industrial applications: an eco-friendly approach—a review. Biotechnol Bioprocess Eng 21:19–38. CrossRefGoogle Scholar
  52. Shanmugam S, Ulaganathan P, Swaminathan K, Sadhasivam S, Wu Y-R (2017) Enhanced biodegradation and detoxification of malachite green by Trichoderma asperellum laccase: degradation pathway and product analysis. Int Biodeterior Biodegrad 125:258–268. CrossRefGoogle Scholar
  53. Sharma B, Dangi AK, Shukla P (2018) Contemporary enzyme based technologies for bioremediation: a review. J Environ Manage 210:10–22. CrossRefPubMedGoogle Scholar
  54. Singh K, Arora S (2011) Removal of synthetic textile dyes from wastewaters: a critical review on present treatment technologies. Crit Rev Environ Sci Technol 41:807–878. CrossRefGoogle Scholar
  55. Singh RL, Singh PK, Singh RP (2015) Enzymatic decolorization and degradation of azo dyes—a review. Int Biodeterior Biodegrad 104:21–31. CrossRefGoogle Scholar
  56. Sondhi S, Kaur R, Kaur S, Kaur PS (2018) Immobilization of laccase-ABTS system for the development of a continuous flow packed bed bioreactor for decolorization of textile effluent. Int J Biol Macromol 117:1093–1100. CrossRefPubMedGoogle Scholar
  57. Tauber MM, Gubitz GM, Rehorek A (2008) Degradation of azo dyes by oxidative processes—laccase and ultrasound treatment. Bioresour Technol 99:4213–4220. CrossRefPubMedGoogle Scholar
  58. Vanhulle S et al (2008) Cytotoxicity and genotoxicity evolution during decolorization of dyes by White Rot Fungi. World J Microbiol Biotechnol 24:337–344. CrossRefGoogle Scholar
  59. Vite-Vallejo O, Palomares LA, Dantán-González E, Ayala-Castro HG, Martínez-Anaya C, Valderrama B, Folch-Mallol J (2009) The role of N-glycosylation on the enzymatic activity of a Pycnoporus sanguineus laccase. Enzyme Microb Technol 45:233–239. CrossRefGoogle Scholar
  60. Wang J, Lu L, Feng F (2017) Improving the Indigo Carmine decolorization ability of a Bacillus amyloliquefaciens laccase by site-directed mutagenesis. Catalysts 7:275. CrossRefGoogle Scholar
  61. Widsten P, Kandelbauer A (2008) Laccase applications in the forest products industry: a review. Enzyme Microb Technol 42:293–307. CrossRefGoogle Scholar
  62. Xu F (1997) Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J Biol Chem 272:924–928. CrossRefPubMedGoogle Scholar
  63. Yang J, Yang XD, Lin YH, Ng TB, Lin J, Ye XY (2015) Laccase-catalyzed decolorization of Malachite Green: Performance optimization and degradation mechanism. PLoS One 10:14. CrossRefGoogle Scholar
  64. Zeng X, Cai Y, Liao X, Zeng X, Luo S, Zhang D (2012) Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase. Process Biochem 47:160–163. CrossRefGoogle Scholar
  65. Zhu C, Bao G, Huang S (2016) Optimization of laccase production in the white-rot fungus Pleurotus ostreatus (ACCC 52857) induced through yeast extract and copper. Biotechnol Biotechnol Equip 30:270–276. CrossRefGoogle Scholar
  66. Zilly A, da Silva Coelho-Moreira J, Bracht A, Marques de Souza CG, Carvajal AE, Koehnlein EA, Peralta RM (2011) Influence of NaCl and Na2SO4 on the kinetics and dye decolorization ability of crude laccase from Ganoderma lucidum. Int Biodeterior Biodegrad 65:340–344. CrossRefGoogle Scholar
  67. Zouari-Mechichi H, Mechichi T, Dhouib A, Sayadi S, Martínez AT, Martínez MJ (2006) Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme. Enzyme Microb Technol 39:141–148. CrossRefGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2018

Authors and Affiliations

  1. 1.Laboratory of Viral and Microbial ProteinsInstitute of Organic Chemistry and Biochemistry of the Czech Academy of SciencesPragueCzech Republic
  2. 2.Department of Membrane TransportInstitute of Physiology of the Czech Academy of SciencePragueCzech Republic

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