Biodegradation of synthetic dye using partially purified and characterized laccase and its proposed mechanism

  • Z. Ghobadi Nejad
  • S. M. BorgheiEmail author
  • S. Yaghmaei
Original Paper


The supernatant obtained from the extracellular laccase produced by Phanerochaete chrysosporium was used as the enzyme source to conduct a partial purification, characterization and dye decolorization study. The partially purified enzyme was stable in the pH range of 3–5 and showed an optimum activity at pH 4.0, using guaiacol as a substrate. Laccase stability of pH was determined and discovered to retain 100% of its activity at a pH of 4.0 after 2 h. The maximum enzyme activity was obtained between 30 and 50 °C. The maximum velocity and Michaelis constant were calculated as 3.171 µM−1·min and 1628.23 µM, respectively. The enzyme was activated by Fe2+, Zn2+, Ca2+ and Cu2+, while Hg2+, Mn2+, Co2+, Mg2+, Cd2+, Ni2+ reduced the laccase activity. The partially purified enzyme was strongly inhibited by 1 mM of NaN3 and sodium thioglycolate. Among the eight different dyes (Malachite green, Safranin, Crystal violet, Methylene blue, Eriochrome black T, Methyl red, Methyl orange, Rhodamine B and Nigrosin), the enzyme showed highly efficient decolorizing activity (99%) toward Malachite green after treatment for 24 h at 30 °C. Antibacterial results showed that the product obtained by treating the dye with the enzyme is completely non-toxic to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. High-performance liquid chromatography and mass spectroscopy analysis of the extracted product confirmed the complete biodegradation of Malachite green and Leucomalachite green. Di-benzyl methane and 4-(dimethylamino) benzaldehyde were the ultimate products identified in this research.


Antibacterial activity Decolorization Detoxification Dye Laccase 



This work was supported by the Biotechnology Development Council of the Islamic Republic of Iran (Grant No: 960402). This research was also supported by the Iran National Science Foundation (INSF) (Grant No: 95833882). The authors would like to acknowledge Biochemical and Bioenvironmental Research Center, Sharif University of Technology, for providing valuable help to accomplish the research work. They also thank the Graduate School of the Environment and Energy, Science and Research Branch, Islamic Azad University.

Compliance with ethical standards

Conflict of interest

The authors have no financial conflicts of interest to declare.

Supplementary material

13762_2019_2226_MOESM1_ESM.docx (31 kb)
Supplementary material 1 (DOCX 30 kb)


  1. Afreen S, Shamsi TN, Baig MA et al (2017) A novel multicopper oxidase (laccase) from cyanobacteria: purification, characterization with potential in the decolorization of anthraquinonic dye. PLoS ONE 12:e0175144CrossRefGoogle Scholar
  2. Asgher M, Iqbal HMN, Asad MJ (2012) Kinetic characterization of purified laccase produced from Trametes versicolor IBL-04 in solid state bio-processing of corncobs. BioResources 7:1171–1188Google Scholar
  3. Attéké C, Mounguengui S, Tchinda J-BS et al (2013) Biodegradation of reactive blue 4 and orange G by Pycnoporus sanguineus strain isolated in Gabon. J Bioremediat Biodegrad 4:1000206Google Scholar
  4. Barapatre A, Aadil KR, Jha H (2017) Biodegradation of malachite green by the ligninolytic fungus Aspergillus flavus. CLEAN Soil Air Water 45:1600045CrossRefGoogle Scholar
  5. Baumer JD, Valério A, de Souza SMGU et al (2018) Toxicity of enzymatically decolored textile dyes solution by horseradish peroxidase. J Hazard Mater 360:82–88CrossRefGoogle Scholar
  6. Chakroun H, Mechichi T, Martinez MJ et al (2010) Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: application on bioremediation of phenolic compounds. Process Biochem 45:507–513CrossRefGoogle Scholar
  7. Cockerill FR (2011) Performance standards for antimicrobial susceptibility testing: twenty-first informational supplement. Clinical and Laboratory Standards Institute (CLSI)Google Scholar
  8. Couto SR, Herrera JLT (2006) Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 24:500–513CrossRefGoogle Scholar
  9. Crini G, Badot P-M (2008) Application of chitosan, a natural aminopolysaccharide, for dye removal from aqueous solutions by adsorption processes using batch studies: a review of recent literature. Prog Polym Sci 33:399–447CrossRefGoogle Scholar
  10. Cristóvão RO, Tavares APM, Ferreira LA et al (2009) Modeling the discoloration of a mixture of reactive textile dyes by commercial laccase. Bioresour Technol 100:1094–1099CrossRefGoogle Scholar
  11. Desai SS, Tennali GB, Channur N et al (2011) Isolation of laccase producing fungi and partial characterization of laccase. Biotechnol Bioinform Bioeng 1:543–549Google Scholar
  12. Du L-N, Wang S, Li G et al (2011) Biodegradation of malachite green by Pseudomonas sp. strain DY1 under aerobic condition: characteristics, degradation products, enzyme analysis and phytotoxicity. Ecotoxicology 20:438–446CrossRefGoogle Scholar
  13. D’Souza DT, Tiwari R, Sah AK, Raghukumar C (2006) Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes. Enzym Microb Technol 38:504–511CrossRefGoogle Scholar
  14. Ghobadi Nejad Z, Yaghmaei S, Hosseini RH (2009) Production of extracellular protease and determination of optimal condition by Bacillus licheniformis bbrc 100053 (research note). Int J Eng B Appl 22:221Google Scholar
  15. Ghobadi Nejad Z, Borghei M, Yaghmaei S (2018a) Effect of polyethylene glycol and triton X-100 on the enzymatic treatment of bisphenol A. Int J Eng Trans B Appl 31:1816–1823Google Scholar
  16. Ghobadi Nejad Z, Borghei SM, Yaghmaei S (2018b) Kinetic studies of Bisphenol A in aqueous solutions by enzymatic treatment. Int J Environ Sci Technol. Scholar
  17. Gomori G (1955) [16] Preparation of buffers for use in enzyme studies. Methods Enzymol 1:138–146CrossRefGoogle Scholar
  18. Grassi E, Scodeller P, Filiel N et al (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 65:635–643CrossRefGoogle Scholar
  19. Hadibarata T, Syafiuddin A, Al-Dhabaan FA, Elshikh MS (2018) Biodegradation of Mordant orange-1 using newly isolated strain Trichoderma harzianum RY44 and its metabolite appraisal. Bioprocess Biosyst Eng 41(5):621–632CrossRefGoogle Scholar
  20. Jain K, Shah V, Chapla D, Madamwar D (2012) Decolorization and degradation of azo dye–Reactive Violet 5R by an acclimatized indigenous bacterial mixed cultures-SB4 isolated from anthropogenic dye contaminated soil. J Hazard Mater 213:378–386CrossRefGoogle Scholar
  21. Jeon SJ, Lim SJ (2017) Purification and characterization of the laccase involved in dye-decolorization by the white rot fungus Marasmius scorodonius. J Microbiol Biotechnol 27:1120–1127Google Scholar
  22. Jeon JR, Baldrian P, Murugesan K, Chang Ys (2012) Laccase-catalysed oxidations of naturally occurring phenols: from in vivo biosynthetic pathways to green synthetic applications. Microb Biotechnol 5:318–332CrossRefGoogle Scholar
  23. Jiang M, Ten Z, Ding S (2013) Decolorization of synthetic dyes by crude and purified laccases from Coprinus comatus grown under different cultures: the role of major isoenzyme in dyes decolorization. Appl Biochem Biotechnol 169:660–672CrossRefGoogle Scholar
  24. Jin X, Ning Y (2013) Laccase production optimization by response surface methodology with Aspergillus fumigatus AF1 in unique inexpensive medium and decolorization of different dyes with the crude enzyme or fungal pellets. J Hazard Mater 262:870–877CrossRefGoogle Scholar
  25. Juárez-Gómez J, Rosas-Tate ES, Roa-Morales G et al (2018) Laccase inhibition by mercury: kinetics, inhibition mechanism, and preliminary application in the spectrophotometric quantification of mercury ions. J Chem 2018:7462697. CrossRefGoogle Scholar
  26. Kumar R, Ahmad R (2011) Biosorption of hazardous crystal violet dye from aqueous solution onto treated ginger waste (TGW). Desalination 265:112–118CrossRefGoogle Scholar
  27. Lam YW, Wang HX, Ng TB (2000) A robust cysteine-deficient chitinase-like antifungal protein from inner shoots of the edible chive Allium tuberosum. Biochem Biophys Res Commun 279:74–80CrossRefGoogle Scholar
  28. Lin J, Zhang X, Li Z, Lei L (2010) Biodegradation of Reactive blue 13 in a two-stage anaerobic/aerobic fluidized beds system with a Pseudomonas sp. isolate. Bioresour Technol 101:34–40CrossRefGoogle Scholar
  29. Liu L, Lin Z, Zheng T et al (2009) Fermentation optimization and characterization of the laccase from Pleurotus ostreatus strain 10969. Enzym Microb Technol 44:426–433CrossRefGoogle Scholar
  30. Lomascolo A, Record E, Herpoël-Gimbert I et al (2003) Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer. J Appl Microbiol 94:618–624CrossRefGoogle Scholar
  31. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  32. Lu L, Zhao M, Zhang B-B et al (2007) Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme. Appl Microbiol Biotechnol 74:1232–1239CrossRefGoogle Scholar
  33. Maalej-Kammoun M, Zouari-Mechichi H, Belbahri L et al (2009) Malachite green decolourization and detoxification by the laccase from a newly isolated strain of Trametes sp. Int Biodeterior Biodegrad 63:600–606CrossRefGoogle Scholar
  34. Mathur G, Nigam R, Jaiswal A, Kumar C (2013) Bioprocess parameter optimization for laccase production in solid state fermentation. J Biotechnol Bioeng Res 4:521–530Google Scholar
  35. Méndez-Hernández JE, Ramírez-Vives F, Solís-Oba M et al (2013) Detoxification and mineralization of Acid Blue 74: study of an alternative secondary treatment to improve the enzymatic decolourization. World J Microbiol Biotechnol 29:805–814CrossRefGoogle Scholar
  36. Mukherjee T, Das M (2014) Degradation of malachite green by enterobacter asburiae strain XJUHX-4TM. CLEAN Soil Air Water 42:849–856CrossRefGoogle Scholar
  37. Nagai M, Sato T, Watanabe H et al (2002) Purification and characterization of an extracellular laccase from the edible mushroom Lentinula edodes, and decolorization of chemically different dyes. Appl Microbiol Biotechnol 60:327–335CrossRefGoogle Scholar
  38. Niladevi KN, Jacob N, Prema P (2008) Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: purification and characterization. Process Biochem 43:654–660CrossRefGoogle Scholar
  39. Pandey A, Singh P, Iyengar L (2007) Bacterial decolorization and degradation of azo dyes. Int Biodeterior Biodegrad 59:73–84CrossRefGoogle Scholar
  40. Patel H, Gupte A, Gupte S (2009) Effect of different culture conditions and inducers on production of laccase by a basidiomycete fungal isolate Pleurotus ostreatus HP-1 under solid state fermentation. BioResources 4:268–284Google Scholar
  41. Pearce CI, Lloyd JR, Guthrie JT (2003) The removal of colour from textile wastewater using whole bacterial cells: a review. Dye Pigment 58:179–196CrossRefGoogle Scholar
  42. Revankar MS, Lele SS (2007) Synthetic dye decolorization by white rot fungus, Ganoderma sp. WR-1. Bioresour Technol 98:775–780CrossRefGoogle Scholar
  43. Sadhasivam S, Savitha S, Swaminathan K, Lin F-H (2008) Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1. Process Biochem 43:736–742CrossRefGoogle Scholar
  44. Shanmugam S, Ulaganathan P, Swaminathan K et al (2017) Enhanced biodegradation and detoxification of malachite green by Trichoderma asperellum laccase: degradation pathway and product analysis. Int Biodeterior Biodegrad 125:258–268CrossRefGoogle Scholar
  45. Stoilova I, Krastanov A, Stanchev V (2010) Properties of crude laccase from Trametes versicolor produced by solid-substrate fermentation. Adv Biosci Biotechnol 1:208–215CrossRefGoogle Scholar
  46. Taguchi T, Ebihara K, Yanagisaki C et al (2018) Decolorization of recalcitrant dyes by a multicopper oxidase produced by Iodidimonas sp. Q-1 with iodide as a novel inorganic natural redox mediator. Sci Rep 8:6717. CrossRefGoogle Scholar
  47. Wu Y-R, Luo Z-H, Chow RK-K, Vrijmoed LLP (2010) Purification and characterization of an extracellular laccase from the anthracene-degrading fungus Fusarium solani MAS2. Bioresour Technol 101:9772–9777CrossRefGoogle Scholar
  48. Yang XQ, Zhao XX, Liu CY et al (2009) Decolorization of azo, triphenylmethane and anthraquinone dyes by a newly isolated Trametes sp. SQ01 and its laccase. Process Biochem 44:1185–1189CrossRefGoogle Scholar
  49. Yesilada O, Birhanli E, Geckil H (2018) Bioremediation and decolorization of textile dyes by white rot fungi and laccase enzymes. In: Prasad R (ed) Mycoremediation and environmental sustainability. Springer, Cham, pp 121–153CrossRefGoogle Scholar
  50. Yuan X, Tian G, Zhao Y et al (2016) Degradation of dyes using crude extract and a thermostable and pH-stable laccase isolated from Pleurotus nebrodensis. Biosci Rep 36:e00365CrossRefGoogle Scholar

Copyright information

© Islamic Azad University (IAU) 2019

Authors and Affiliations

  • Z. Ghobadi Nejad
    • 1
  • S. M. Borghei
    • 2
    • 3
    Email author
  • S. Yaghmaei
    • 3
  1. 1.Department of Natural Resources and Environment, Science and Research BranchIslamic Azad University, TehranTehranIran
  2. 2.Biochemical and Bioenvironmental Research CentreSharif University of TechnologyTehranIran
  3. 3.Department of Chemical and Petroleum EngineeringSharif University of TechnologyTehranIran

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