Applied Microbiology and Biotechnology

, Volume 77, Issue 3, pp 613–624 | Cite as

Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

  • C. MartinEmail author
  • M. Pecyna
  • H. Kellner
  • N. Jehmlich
  • C. Junghanns
  • D. Benndorf
  • M. von Bergen
  • D. Schlosser
Biotechnologically Relevant Enzymes and Proteins


Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO4 and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k cat/K m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s−1 mM−1, respectively, with K m values of 4.2, 67.8, and 104.9 μM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15°C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.


Ascomycete Aquatic fungi Laccase Characterization Gene Expression 



We want to thank the German Research Foundation (DFG) for funding of C. Martin within the DFG graduate college 416.


  1. Abdel-Raheem A, Shearer CA (2002) Extracellular enzyme production by freshwater ascomycetes. Fungal divers 11:1–9Google Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped blast and psi-blast: A new generation of protein database search programs. Nucl Acids Res 25:3389–3402CrossRefGoogle Scholar
  3. Arst HN, MacDonald DW (1975) A gene cluster in Aspergillus nidulans with an internally located cis-acting regulatory region. Nature 254:26–31CrossRefGoogle Scholar
  4. Augustin T, Schlosser D, Baumbach R, Schmidt J, Grancharov K, Krauss G, Krauss G-J (2006) Biotransformation of 1-naphthol by a strictly aquatic fungus. Curr Microbiol 52:216–220CrossRefGoogle Scholar
  5. Baldrian P (2006) Fungal laccases—occurrence and properties. FEMS Microbiol Rev 30:215–242CrossRefGoogle Scholar
  6. Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18:298–305CrossRefGoogle Scholar
  7. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795CrossRefGoogle Scholar
  8. Benndorf D, Thiersch M, Loffhagen N, Kunath C, Harms H (2006) Pseudomonas putida KT2440 responds specifically to chlorophenoxy herbicides and their initial metabolites. Proteomics 6:3319–3329CrossRefGoogle Scholar
  9. Berg JM, Tymoczky JL, Stryer L, Clarke ND (2002) Biochemistry. Freeman, New YorkGoogle Scholar
  10. Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546CrossRefGoogle Scholar
  11. Bollag J-M, Liu S-Y, Minard RD (1982) Enzymatic oligomerization of vanillic acid. Soil Biol Biochem 14:157–163CrossRefGoogle Scholar
  12. Bucher VVC, Pointing SB, Hyde KD, Reddy CA (2004) Production of wood decay enzymes, loss of mass, and lignin solubilization in wood by diverse tropical freshwater fungi. Microb Ecol 48:331–337CrossRefGoogle Scholar
  13. Call HP, Mücke I (1997) History, overview and applications of mediated lignolytic systems, especially laccase-mediator-systems (Lignozym®-process). J Biotechnol 53:163–202CrossRefGoogle Scholar
  14. Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl Environ Microbiol 64:3175–3179Google Scholar
  15. Duran N, Rosa MA, D’Annibale, A, Gianfreda L (2002) Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: A review. Enzyme Microb Tech 31:907–931CrossRefGoogle Scholar
  16. Faraco V, Giardina P, Sannia G (2003) Metal-responsive elements in Pleurotus ostreatus laccase gene promoters. Microbiology 149:2155–2162CrossRefGoogle Scholar
  17. 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 148:2159–2169Google Scholar
  18. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) The proteomics protocols handbook. Humana, Totowa, NJ, USAGoogle Scholar
  19. Gianfreda L, Xu F, Bollag JM (1999) Laccases: A useful group of oxidoreductive enzymes. Bioremed J 3:1–26CrossRefGoogle Scholar
  20. Hall TA (1999) Bioedit: A user-friendly biological sequence alignment editor and analysis program for windows 95/98NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  21. Hodkinson M, Dalton SA (1973) Interactions between DDT and river fungi. II. Influence of culture conditions on the compatibility of fungi and p,p'-DDT. Bull Environ Contam Toxicol 10:356–359CrossRefGoogle Scholar
  22. Hoegger PJ, Kilaru S, James TY, Thacker JR, Kües U (2006) Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences. FEBS J 273:2308–2326CrossRefGoogle Scholar
  23. Huang W-Y, Sheu S-J (2006) Separation and identification of the organic acids in Agelicae radix and Ligustici rhizoma by HPLC and CE. J Sep Sci 29:2616–2624CrossRefGoogle Scholar
  24. Johannes C, Majcherczyk A (2000) Laccase activity tests and laccase inhibitors. J Biotechnol 78:193–199CrossRefGoogle Scholar
  25. Junghanns C, Moeder M, Krauss G, Martin C, Schlosser D (2005) Degradation of the xenoestrogen nonylphenol by aquatic fungi and their laccases. Microbiology 151:45–57CrossRefGoogle Scholar
  26. Junghanns C, Krauss G, Schlosser D (2007) Potential of aquatic fungi derived from diverse freshwater environments to decolourise synthetic azo and anthraquinone dyes. Bioresource Technol (in press)Google Scholar
  27. Kellner H, Louis P, Buscot F (2007) Diversity of laccase-like multicopper oxidases genes in Morchellaceae: Identification of genes potentially involved in extracellular activities related to plant litter decay. FEMS Microb Ecol 61:153–163CrossRefGoogle Scholar
  28. Kiiskinen LL, Saloheimo M (2004) Molecular cloning and expression in Saccharomyces cerevisiae of laccase gene from the ascomycete Melanocarpus albomyces. Appl Environ Microbiol 70:137–144CrossRefGoogle Scholar
  29. Kiiskinen LL, Viikari L, Kruus K (2002) Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces. Applied Microbiology and Biotechnology 59:198CrossRefGoogle Scholar
  30. Kiiskinen L-L, Kruus K, Bailey M, Ylosmaki E, Siika-aho M, Saloheimo M (2004) Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme. Microbiology 150:3065–3074CrossRefGoogle Scholar
  31. Kirk PM, Cannon PF, David JC, Staplers JA (eds) (2001) Ainsworth and Bisby’s dictionary of the fungi. 9th edn. CABI Publishing, Wallingford, UKGoogle Scholar
  32. Kumar S, Tamura K, Nei M (2004) Mega3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163CrossRefGoogle Scholar
  33. Larrondo LF, Avila M, Salas L, Cullen D, Vicuna R (2003) Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns. Microbiology 149:1177–1782CrossRefGoogle Scholar
  34. Leonowicz A, Cho NS, Luterek J, Wilkołazka A, Wojtas-Wasilewska M, Matuszewska A, Hofrichter M, Wesenberg D, Rogalski J (2001) Fungal laccase: Properties and activity on lignin. J Basic Microbiol 41:185–227CrossRefGoogle Scholar
  35. Liers C, Ullrich R, Pecyna M, Schlosser D, Hofrichter M (2007) Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha. Enzyme Microb Tech 41:785–793CrossRefGoogle Scholar
  36. Litvintseva AP, Henson JM (2002) Cloning, characterization, and transcription of three laccase genes from Gaeumannomyces graminis var. tritici, the take-all fungus. Appl Environ Microbiol 68:1305–1311CrossRefGoogle Scholar
  37. Martin C, Moeder M, Daniel X, Krauss G, Schlosser D (2007) Biotransformation of the polycyclic musks HHCB and AHTN and metabolite formation by fungi occurring in freshwater environments. Environ Sci Technol 41:5395–5402CrossRefGoogle Scholar
  38. Matys V, Fricke E, Geffers R, Gossling E, Haubrock M, Hehl R, Hornischer K, Karas D, Kel AE, Kel-Margoulis OV, Kloos DU, Land S, Lewicki-Potapov B, Michael H, Münch R, Reuter I, Rotert S, Saxel H, Scheer M, Thiele S, Wingender E (2003) TransfacÒ: Transcriptional regulation, from patterns to profiles. Nucl Acids Res 31:374–378CrossRefGoogle Scholar
  39. Mayer AM, Staples RC (2002) Laccase: New functions for an old enzyme. Phytochemistry 60:551–565CrossRefGoogle Scholar
  40. Neuhoff V, Stamm R, Eibl H (1985) Clear background and highly sensitive protein staining with coomassie blue dyes in polyacrylamide gels: A systematic analysis. Electrophoresis 6:427–448CrossRefGoogle Scholar
  41. Nikolcheva LG, Bärlocher F (2002) Phylogeny of Tetracladium based on 18S rDNA. Czech Mycol 53:285–295Google Scholar
  42. Ravelet C, Grosset C, Krivobok S, Montuelle B, Alary J (2001) Pyrene degradation by two fungi in a freshwater sediment and evaluation of fungal biomass by ergosterol content. Appl Microbiol Biotechnol 56:803–808CrossRefGoogle Scholar
  43. Saito T, Hong P, Kato K, Okazaki M, Inagaki H, Maeda S, Yokogawa Y (2003) Purification and characterization of an extracellular laccase of a fungus (family Chaetomiaceae) isolated from soil. Enzyme and Microb Tech 33:520–526CrossRefGoogle Scholar
  44. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual. Press Cold Spring Harbor, New YorkGoogle Scholar
  45. Saparrat MCN, Guillen F, Arambarri AM, Martinez AT, Martinez MJ (2002) Induction, isolation and characterisation of two laccases from the white rot basidiomycete coriolopsis rigida. Appl Environ Microbiol 68:1534–1540CrossRefGoogle Scholar
  46. 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 68:3514–3521CrossRefGoogle Scholar
  47. Schlosser D, Grey R, Fritsche W (1997) Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood. Appl Microb Biotechnol 47:412–418CrossRefGoogle Scholar
  48. Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: Sequence statistics and functional implications in gene expression. Nucl Acids Res 15:7155–7174CrossRefGoogle Scholar
  49. Shearer CA, Descals E, Kohlmeyer B, Kohlmeyer J, Marvanova L, Padgett D, Porter D, Raja HA, Schmit JP, Thorton HA, Voglymayr H (2007) Fungal biodiversity in aquatic habitats. Biodivers Conserv 16:49–67CrossRefGoogle Scholar
  50. Shevchenko A, Jensen ON, Podtelejnikov AV, Sagliocco F, Wilm M, Vorm O, Mortensen P, Shevchenko A, Boucherie H, Mann M (1996) Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci U S A 93:14440–14445CrossRefGoogle Scholar
  51. Siqueira JO, Nair MG, Hammerschmidt R, Safir GR (1991) Significance of phenolic compounds in plant-soil microbial systems. Crit Rev Plant Sci 10:63–121Google Scholar
  52. Soden DM, Dobson ADW (2001) Differential regulation of laccase gene expression in Pleurotus sajor-caju. Microbiology 147:1755–1763Google Scholar
  53. Soden DM, O’Callaghan J, Dobson ADW (2002) Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host. Microbiology 148:4003–4014Google Scholar
  54. Stapleton PC, Dobson ADW (2003) Carbon repression of cellobiose dehydrogenase production in the white rot fungus Trametes versicolor is mediated at the level of gene transcription. FEMS Microbiol Lett 221:167–172CrossRefGoogle Scholar
  55. Tetsch L, Bend J, Hölker U (2006) Molecular and enzymatic characterisation of extra- and intracellular laccases from the acidophilic ascomycete Hortaea acidophila. Antonie van Leeuwenhoek 90:183–194CrossRefGoogle Scholar
  56. Thiele DJ (1992) Metal-regulated transcription in eukaryotes. Nucl Acids Res 20:1183–1191CrossRefGoogle Scholar
  57. Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26CrossRefGoogle Scholar
  58. Wessagowit V, Nalla VK, Rogan PK, McGrath JA (2005) Normal and abnormal mechanisms of gene splicing and relevance to inherited skin diseases. J Dermatol Sci 40:73–84CrossRefGoogle Scholar
  59. Yoshida H (1883) Chemistry of lacquer (urushi). J Chem Soc 43:472–486Google Scholar
  60. Yoshitake A, Katayama Y, Nakamura M, Limura Y, Kawai S, Morohoshi N (1993) N-linked carbohydrate chains protect laccase-iii from proteolysis in Coriolus versicolor. J Gen Microbiol 139:179–185Google Scholar
  61. Zhao J, Hyman L, Moore C (1999) Formation of mRNA 3 ′ ends in eukaryotes: Mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol Mol Biol R 63:405–445Google Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • C. Martin
    • 1
    Email author
  • M. Pecyna
    • 1
  • H. Kellner
    • 3
  • N. Jehmlich
    • 2
  • C. Junghanns
    • 1
  • D. Benndorf
    • 2
  • M. von Bergen
    • 2
  • D. Schlosser
    • 1
  1. 1.UFZ, Department of Environmental MicrobiologyHelmholtz Centre for Environmental Research – UFZLeipzigGermany
  2. 2.UFZ, Department of ProteomicsHelmholtz Centre for Environmental Research – UFZLeipzigGermany
  3. 3.Institute of Botany, Terrestrial EcologyUniversity of LeipzigLeipzigGermany

Personalised recommendations