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

The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties

Abstract

Cerrena unicolor secreted two laccase isoforms with different characteristics during the growth in liquid media. In a synthetic low-nutrient nitrogen glucose medium (Kirk medium), high amounts of laccase (4,000 U l−1) were produced in response to Cu2+. Highest laccase levels (19,000 U l−1) were obtained in a complex tomato juice medium. The isoforms (Lacc I, Lacc II) were purified to homogeneity with an overall yield of 22%. Purification involved ultrafiltration and Mono Q separation. Lacc I and II had Mw of 64 and 57 kDa and pI of 3.6 and 3.7, respectively. Both isoforms had an absorption maximum at 608 nm but different pH optima and thermal stability. Optimum pH ranged from 2.5 to 5.5 depending on the substrate. The pH optima of Lacc II were always higher than those of Lacc I. Both laccases were stable at pH 7 and 10 but rapidly lost activity at pH 3. Their temperature optimum was around 60°C, and at 5°C they still reached 30% of the maximum activity. Lacc II was the more thermostable isoform that did not lose any activity during 6 months storage at 4°C. Kinetic constants (Km, kcat) were determined for 2,2′-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol and syringaldazine.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Abadulla E, Tzanov T, Costa S, Robra KH, Cavaco-Paulo A, Gübitz GM (2000) Decolorization and detoxification of texile dyes with laccase from Trametes hirsuta. Appl Environ Microbiol 66:3357–3362

  2. Al-Adhami AJH, Bryjak J, Greb-Markiewicz B, Peczyńska-Czoch W (2002) Immobilization of wood-rotting fungi laccases on modified cellulose and acrylic carriers. Proc Biochem 37:1387–1394

  3. Ander P, Messner K (1998) Oxidation of 1-hydroxybenzotriazole by laccase and lignin peroxidase. Biotechnol Tech 12:191–195

  4. Bourbonnais R, Paice MG, Freiermuth B, Bodie E, Borneman S (1997) Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl Environ Microbiol 63:4627–4632

  5. de Jong E, de Vries FP, Field JA, Van der Zwan RP, de Bont JAM (1992) Isolation and screening of basidiomycetes with high peroxidative activity. Mycol Res 96:1098–1104

  6. Dedeyan B, Klonowska A, Tagger S, Tron T, Iacazio G, Gil G, Petit J (2000) Biochemical and molecular characterization of laccase from Marasmius quercophilus. Appl Environ Microbiol 66:925–929

  7. Eggert C, Temp U, Eriksson KE (1996) The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62:1151–1158

  8. Elisashvili V, Kachlishvili E, Bakradze M (2002) Dependence of activities of polysaccharide hydrolases and oxidases from Cerrena unicolor on the source of carbon and aromatic acids in culture medium. Appl Biochem Microbiol 38:210–213

  9. Enebak SA, Blanchette RA (1989) Canker formation and decay in sugar maple and paper birch infected by Cerrena unicolor. Can J For Res 19:225–231

  10. Filazzola MT, Sannino F, Rao MA, Gianfreda L (1999) Effect of various pollutants and soil-like constituents on laccase from Cerrena unicolor. J Environ Qual 28:1929–1938

  11. Fukushima Y, Kirk TK (1995) Laccase component of the Ceriporiopsis subvermispora lignin-degrading system. Appl Environ Microbiol 61:872–876

  12. Galhaup C, Haltrich D (2001) Enchanced formation of laccase activity by the white-rot fungus Trametes pubescens in the presence of copper. Appl Microbiol Biotechnol 56:225–232

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

  14. Gianfreda L, Sannino F, Filazzola MT, Leonowicz A (1998) Catalytic behavior and detoxifying ability of a laccase from the fungal strain Cerrena unicolor. J Mol Catal, B Enzym 4:13–23

  15. Hatakka A (2001) Biodegradation of lignin. In: Hofrichter M, Steinbüchel A (eds) Lignin, humic substances and coal. Biopolymers 1:129–180

  16. Kim Y, Cho NS, Eom TJ, Shin W (2002) Purification and characterization of laccase from Cerrena unicolor and its reactivity in lignin degradation. Bull Korean Chem Soc 23:985–989

  17. Kirk TK, Schulz E, Connors WJ, Lorenz LF, Zeikus JG (1978) Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch Microbiol 117:277–285

  18. Ko KS, Jung HS (1999) Phylogenetic re-evaluation of Trametes consors based on mitochondrial small subunit ribosomal DANN sequences. FEMS Microbiol Lett 170:181–186

  19. Krieglsteiner GJ (2000) Die Grosspilze Baden—Württernbergs. Verlag Eugen Ulmer 1:501–502

  20. Kuhad RC, Singh A, Eriksson KE (1997) Microorganisms and enzymes involved in the degradation of plant fiber cell walls. In: Eriksson (ed) Advances in biochemical engineering biotechnology. Springer-Verlag, Germany, pp 46–125

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

  22. Leonowicz A, Grzywnowicz K (1981) Quantitative estimation of laccase forms in some white-rot fungi using syringaldazine as substrate. Enzyme Microb Technol 3:55–58

  23. Leonowicz A, Matuszewska A, Luterek J, Ziegenhagen D, Wojtas-Wasilewska M, Cho NS, Hofrichter M, Rogalski J (1999) Biodegradation of lignin by white rot fungi. Fungal Genet Biol 27:175–185

  24. Li K, Xu F, Eriksson KEL (1999) Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl Environ Microbiol 65:2654–2660

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

  26. Muñoz C, Guillén F, Martínez AT, Martínez MJ (1997) Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation. Appl Environ Microbiol 63:2166–2174

  27. Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso A, Sannia G (1997) A novel white laccase from Pleurotus ostreatus. J Biol Chem 272:31301–31307

  28. Papinuzzi VL, Forchiassin F (2003) Optimization of manganese peroxidase and laccase production in the South American fungus Fomes sclerodermeus (Löv.) Cke. J Ind Microbiol Biotech 30:536–541

  29. Périé FH, Reddy GJB, Blackburn NJ, Gold MH (1998) Purification and characterization of laccase from the white-rot basidiomycete Dichomitus squalens. Arch Biochem Biophys 353:349–355

  30. Robene-Soustrade I, Lung-Escarmant B, Bono JJ, Taris B (1992) Identification and partial characterization of an extracellular manganese-dependent peroxidase in Armillaria ostoyae and Armillaria mellea. Eur J For Pathol 22:227–236

  31. Rogalski J, Dawidowicz A, Jóźwik E, Leonowicz A (1999) Immobilization of laccase from Cerrena unicolor on controlled porosity glass. J Mol Catal, B Enzym 6:29–39

  32. Saparrat MCN, Guillén F, Arambarri AM, Martínez AT, Martínez MJ (2002) Induction, isolation and characterization of two laccases from the white rot Basidiomycete Coriolopsis rigida. Appl Environ Microbiol 68:1534–1540

  33. Schlosser D, Höfer C (2002) Laccase-catalyzed oxidation of Mn2+ in the presence of natural Mn3+ chelators as a novel source of extracellular H2O2 production and its impact on manganese peroxidase. Appl Environ Microbiol 68:3514–3521

  34. Stepanova EV, Pegasova TV, Gavrilova VP, Landesman EO, Koroleva OV (2002) Extracellular laccases from Cerrena unicolor 059, Cerrena unicolor 0784, and Pleurotus oastreatus 0432: a comparative assay. Appl Biochem Microbiol 39:375–381

  35. Thurston CF (1994) The structure and function of fungal laccase. Microbiology 140:19–26

  36. Ullrich R, Huong LM, Dung NL, Hofrichter M (2005) Laccase from the medicinal mushroom Agaricus blazei: production, purification and characterization. Appl Microbiol Biotechnol

  37. Wesenberg D, Kyriakides I, Agathos SN (2003) White-rot fungi and their enzymes for the treatment of industrial dye effluents. Biotechnol Adv 22:161–187

Download references

Acknowledgements

This research was supported by the German Environmental Foundation (Deutsche Bundesstiftung Umwelt, grant number AZ 21000/227), the Nowicky Foundation (Poland) and the administration of the International Graduate School Zittau (RD' in Dr. R. Konschak). We thank U. Schneider for excellent technical assistance and C. Liers for useful discussions.

Author information

Correspondence to Anna Michniewicz.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Michniewicz, A., Ullrich, R., Ledakowicz, S. et al. The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties. Appl Microbiol Biotechnol 69, 682–688 (2006). https://doi.org/10.1007/s00253-005-0015-9

Download citation

Keywords

  • Laccase Activity
  • Laccase Production
  • Veratryl Alcohol
  • Tomato Juice
  • Syringaldazine