Skip to main content
SpringerLink
Log in

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

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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 via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  • 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

    CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

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

    Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

Authors and Affiliations

  1. Département de chimie-biologie, Université du Québec à Trois-Rivières, 3351 Des Forges, C.P. 500, Trois-Rivières, Quebec, Canada, G9A 5H7

    Etienne Dubé, Yves Hurtubise, Claude Daneault & Marc Beauregard

  2. INRS-Institut Armand-Frappier, Université du Québec, 531 boul. des Prairies, Ville de Laval, Quebec, Canada, H7V 1B7

    François Shareck

Authors
  1. Etienne Dubé
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. François Shareck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yves Hurtubise
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claude Daneault
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marc Beauregard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

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). https://doi.org/10.1007/s00253-008-1475-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1475-5

Keywords

Search

Navigation