Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye


The lack of a commercially available robust and inexpensive laccase is a major barrier to the widespread application of this enzyme in various industrial sectors. By using an efficient system developed in Streptomyces lividans, we have produced by homologous expression 350 mg L−1 of a bacterial laccase with a high purity and without any extensive purification. This is the highest production yield reported in the literature for a bacterial laccase. The secreted enzyme achieved oxidation under a wide pH range depending on the substrate: 4.0 for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) and 9.0 for 2,6-dimethoxyphenol. Furthermore, this bacterial laccase was found to be quite resistant under various conditions. It withstands pH from 3.0 to 9.0, shows a great thermostability at 70°C and was highly resistant toward conventional inhibitors. For instance, while the laccase of Trametes versicolor was completely inhibited by 1 mM NaN3, the laccase of Streptomyces coelicolor was fully active under the same conditions. To assess application potential of this laccase, we have investigated its ability to decolourise Indigo carmine. This enzyme was able to rapidly decolourise the dye in the presence of syringaldehyde as a redox mediator.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. Alexandre G, Zhulin IB (2000) Laccases are widespread in bacteria. Trends Biotech 18:41–42

  2. Alves A, Record E, Lomascolo A, Scholtmeijer K, Asther M, Wessels JGH, Wosten HAB (2004) Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus. Appl Environ Microbiol 70:6379–6384

  3. Bertrand T, Jolivalt C, Briozzo P, Caminade E, Joly N, Madzak C, Mougin C (2002) Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics. Biochemistry 41:7325–7333

  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

  5. Burton SG (2003) Laccases and phenol oxidases in organic synthesis—a review. Curr Org Chem 7:1317–1331

  6. Camarero S, Ibarra D, Martinez MJ, Martinez AT (2005) Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes. Appl Environ Microbiol 71:1775–1784

  7. Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179

  8. Childs RE, Bardsley WG (1975) The steady-state kinetics of peroxidase with 2,2’’-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) as chromogen. Biochem J 145:93–103

  9. Claus H (2004) Laccases: structure, reactions, distribution. Micron 35:93–96

  10. Endo K, Hayashi Y, Hibi T, Hosono K, Beppu T, Ueda K (2003) Enzymological characterization of EpoA, a laccase-like phenol oxidase produced by Streptomyces griseus. J Biochem (Tokyo) 133:671–677

  11. Enguita FJ, Martins LO, Henriques AO, Carrondo MA (2003) Crystal structure of a bacterial endospore coat component. A laccase with enhanced thermostability properties. J Biol Chem 278:19416–19425

  12. Galhaup C, Goller S, Peterbauer CK, Strauss J, Haltrich D (2002) Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148:2159–2169

  13. Hakulinen N, Kiiskinen LL, Kruus K, Saloheimo M, Paananen A, Koivula A, Rouvinen J (2002) Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nat Struct Biol 9:601–605

  14. Hullo MF, Moszer I, Danchin A, Martin-Verstraete I (2001) CotA of Bacillus subtilis is a copper-dependent laccase. J Bacteriol 183:5426–5430

  15. Hurtubise Y, Shareck F, Kluepfel D, Morosoli R (1995) A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins. Mol Microbiol 17:367–377

  16. Jaouani A, Guillen F, Penninckx MJ, Martinez AT, Martinez MJ (2005) Role of Pycnoporus coccineus laccase in the degradation of aromatic compounds in olive oil mill wastewater. Enzyme Microb Technol 36:478–486

  17. Kenealy WR, Jeffries TW (2003) Enzyme processes for pulp and paper: a review of recent developments. In: Goodell B, Nicholas DD, Schultz TP (eds) Wood deterioration and preservation: advances in our changing world, ACS Symposium Series 845, Washington, pp 210–239

  18. Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA (2000) Practical Streptomyces genetics. John Innes Foundation, Norwich

  19. Kluepfel D, Vats-Mehta S, Aumont F, Shareck F, Morosoli R (1990) Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. Biochem J 267:45–50

  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

  21. Leonowicz A, Cho NS, Luterek J, Wilkolazka A, Wojtas-Wasilewska M, Matuszewska A, Hofrichter M, Wesenberg D, Rogalski J (2001) Fungal laccase: properties and activity on lignin. J Basic Microbiol 41:185–227

  22. Lomascolo A, Record E, Herpoel-Gimbert I, Delattre M, Robert JL, Georis J, Dauvrin T, Sigoillot JC, Asther M (2003) Overproduction of laccase by a monokaryotic strain of Pycnoporus cinnabarinus using ethanol as inducer. J Appl Microbiol 94:618–624

  23. Machczynski MC, Vijgenboom E, Samyn B, Canters GW (2004) Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity. Protein Sci 13:2388–2397

  24. Madzak C, Mimmi MC, Caminade E, Brault A, Baumberger S, Briozzo P, Mougin C, Jolivalt C (2006) Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis. Protein Eng Des Sel 19:77–84

  25. Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277:18849–18859

  26. Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60:551–565

  27. Minussi RC, Pastore GM, Duran N (2007) Laccase induction in fungi and laccase/N–OH mediator systems applied in paper mill effluent. Bioresour Technol 98:158–164

  28. Moldes D, Sanroman MA (2006) Amelioration of the ability to decolorize dyes by laccase: relationship between redox mediators and laccase isoenzymes in Trametes versicolor. World J Microbiol Biotechnol 22:1197–1204

  29. Muñoz C, Guillén F, Martínez AT, Martínez MJ (1997) Induction and characterization of laccase in the ligninolytic fungus Pleurotus eryngii. Curr Microbiol 34:1–5

  30. Perie FH, Reddy GVB, Blackburn NJ, Gold MH (1998) Purification and characterization of laccases from the white-rot basidiomycete Dichomitus squalens. Arch Biochem Biophys 353:349–355

  31. Piontek K, Antorini M, Choinowski T (2002) Crystal structure of a laccase from the fungus Trametes versicolor at 1.90-A resolution containing a full complement of coppers. J Biol Chem 277:37663–37669

  32. Rodriguez Couto S, Toca Herrera JL (2006) Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 24:500–513

  33. Sharma P, Goel R, Capalash N (2007) Bacterial laccases. World J Microbiol Biotechnol 23:823–832

  34. Slomczynski D, Nakas JP, Tanenbaum SW (1995) Production and characterization of laccase from Botrytis cinerea 61-34. Appl Environ Microbiol 61:907–912

  35. Suzuki T, Endo K, Ito M, Tsujibo H, Miyamoto K, Inamori Y (2003) A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression. Biosci Biotechnol Biochem 67:2167–2175

  36. Ward AC (1992) Rapid analysis of yeast transformants using colony-PCR. BioTechniques 13:350

  37. Yoshida H (1883) Chemistry of lacquer (urushi). J Chem Soc Trans 43:472–486

Download references


This work was supported by the Canada research chair on value-added papers from the Centre Intégré en Pâtes et Papiers (Trois-Rivières, Canada), AgroTerra Biotech (Trois-Rivières, Canada), the Institut National de la Recherche Scientifique—Institut Armand-Frappier (Laval, Canada) and the Natural Sciences and Engineering Research Council of Canada.

Author information

Correspondence to Marc Beauregard.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Dubé, E., Shareck, F., Hurtubise, Y. et al. Homologous cloning, expression, and characterisation of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye. Appl Microbiol Biotechnol 79, 597–603 (2008).

Download citation


  • Laccase
  • Streptomyces coelicolor
  • Homologous cloning
  • Expression
  • Characterisation
  • Dye decolourisation
  • Textile