Applied Microbiology and Biotechnology

, Volume 84, Issue 6, pp 1095–1105 | Cite as

Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete Phoma sp. UHH 5-1-03

  • C. Junghanns
  • M. J. Pecyna
  • D. Böhm
  • N. Jehmlich
  • C. Martin
  • M. von Bergen
  • F. Schauer
  • M. Hofrichter
  • D. SchlosserEmail author
Biotechnologically Relevant Enzymes and Proteins


A laccase from the aquatic ascomycete Phoma sp. UHH 5-1-03 (DSM 22425) was purified upon hydrophobic interaction and size exclusion chromatography (SEC). Mass spectrometric analysis of the laccase monomer yielded a molecular mass of 75.6 kDa. The enzyme possesses an unusual alkaline isoelectric point above 8.3. The Phoma sp. laccase undergoes pH-dependent dimerisation, with the dimer (∼150 kDa, as assessed by SEC) predominating in a pH range of 5.0 to 8.0. The enzyme oxidises common laccase substrates still at pH 7.0 and 8.0 and is remarkably stable at these pH values. The laccase is active at high concentrations of various organic solvents, all together indicating a considerable biotechnological potential. One laccase gene (lac1) identified at the genomic DNA level and transcribed in laccase-producing cultures was completely sequenced. The deduced molecular mass of the hypothetical protein and the predicted isoelectric point of 8.1 well agree with experimentally determined data. Tryptic peptides of electrophoretically separated laccase bands were analysed by nano-liquid chromatography–tandem mass spectrometry. By using the nucleotide sequence of lac1 as a template, eight different peptides were identified and yielded an overall sequence coverage of about 18%, thus confirming the link between lac1 and the expressed laccase protein.


Alkaline isoelectric point Aquatic ascomycete Dimeric laccase Enzyme stability Laccase gene 



We gratefully acknowledge the funding of this study by the European Commission within the frame of the Integrated Project SOPHIED (NMP2-CT-2004-505899) of the 6th framework programme. Many thanks to A. Bazes, E. Enaud, M. Trovaslet, S. Vanhulle (all Université Catholique de Louvain, Louvain-la-Neuve, Belgium) and H. Kellner (University Gembloux, Belgium) for fruitful discussions. Furthermore, we want to thank R. Bötz (UFZ) for excellent technical assistance.


  1. 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. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  2. Baldrian P (2006) Fungal laccases—occurence and properties. FEMS Microbiol Rev 30:215–242CrossRefGoogle Scholar
  3. 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
  4. Beloqui A, Pita M, Polaina J, Martinez-Arias A, Golyshina OV, Zumarraga M, Yakimov MM, Garcia-Arellano H, Alcalde M, Fernandez VM, Elborough K, Andreu JM, Ballesteros A, Plou FJ, Timmis KN, Ferrer M, Golyshin PN (2006) Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships. J Biol Chem 281:22933–22942CrossRefGoogle Scholar
  5. 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
  6. Benndorf D, Balcke GU, Harms H, von Bergen M (2007) Functional metaproteome analysis of protein extracts from contaminated soil and groundwater. ISME J 1:224–234CrossRefGoogle Scholar
  7. Berka RM, Schneider P, Golightly EJ, Brown SH, Madden M, Brown KM, Halkier T, Mondorf K, Xu F (1997) Characterization of the gene encoding an extracellular laccase of Myceliophthora thermophila and analysis of the recombinant enzyme expressed in Aspergillus oryzae. Appl Environ Microbiol 63:3151–3157Google Scholar
  8. Esser K, Minuth W (1970) The phenoloxidases of the ascomycete Podospora anserina. Communication IV. Genetic regulation of the formation of laccase. Genetics 64:441–458Google Scholar
  9. Ettrich R, Kopecky V, Hofbauerova K, Baumruk V, Novak P, Pompach P, Man P, Plihal O, Kuty M, Kulik N, Sklenar J, Ryslava H, Kren V, Bezouska K (2007) Structure of the dimeric N-glycosylated form of fungal beta-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies. BMC Struct Biol 7:32–46CrossRefGoogle Scholar
  10. Faraco V, Ercole C, Festa G, Giardina P, Piscitelli A, Sannia G (2008) Heterologous expression of heterodimeric laccase from Pleurotus ostreatus in Kluyveromyces lactis. Appl Microbiol Biotechnol 77:1329–1335CrossRefGoogle Scholar
  11. Fernandez-Larrea J, Stahl U (1996) Isolation and characterization of a laccase gene from Podospora anserina. Mol Gen Genet 252:539–551Google Scholar
  12. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) The proteomics protocols handbook. Humana, TotowaGoogle Scholar
  13. Gianfreda L, Xu F, Bollag JM (1999) Laccases: a useful group of oxidoreductive enzymes. Bioremediat J 3:1–26CrossRefGoogle Scholar
  14. Giardina P, Palmieri G, Scaloni A, Fontanella B, Faraco V, Cennamo G, Sannia G (1999) Protein and gene structure of a blue laccase from Pleurotus ostreatus. Biochem J 341:655–663CrossRefGoogle Scholar
  15. 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
  16. 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
  17. Iyer G, Chattoo BB (2003) Purification and characterization of laccase from the rice blast fungus, Magnaporthe grisea. FEMS Microbiol Lett 227:121–126CrossRefGoogle Scholar
  18. Johannes C, Majcherczyk A (2000) Laccase activity tests and laccase inhibitors. J Biotechnol 78:193–199CrossRefGoogle Scholar
  19. Junghanns C, Krauss G, Schlosser D (2008a) Potential of aquatic fungi derived from diverse freshwater environments to decolourise synthetic azo and anthraquinone dyes. Bioresource Technol 99:1225–1235CrossRefGoogle Scholar
  20. Junghanns C, Parra R, Keshavarz T, Schlosser D (2008b) Towards higher laccase activities produced by aquatic ascomycetous fungi through combination of elicitors and an alternative substrate. Eng Life Sci 8:277–285CrossRefGoogle Scholar
  21. Kellner H, Jehmlich N, Benndorf D, Hoffmann R, Rühl M, Hoegger PJ, Majcherczyk A, Kües U, von Bergen M, Buscot F (2007a) Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry. Enzyme Microb Tech 41:694–701CrossRefGoogle Scholar
  22. Kellner H, Luis P, Buscot F (2007b) Diversity of laccase-like multicopper oxidase genes in Morchellaceae: identification of genes potentially involved in extracellular activities related to plant litter decay. FEMS Microbiol Ecol 61:153–163CrossRefGoogle Scholar
  23. 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
  24. Kiiskinen LL, Viikari L, Kruus K (2002) Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces. Appl Microbiol Biotechnol 59:198–204CrossRefGoogle Scholar
  25. 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
  26. Lin C-T, Lin M-T, Chen Y-T, Shaw J-F (1995) Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method. Plant Mol Biol 28:303–311CrossRefGoogle Scholar
  27. 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
  28. Martin C, Pecyna M, Kellner H, Jehmlich N, Junghanns C, Benndorf D, von Bergen M, Schlosser D (2007) 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. Appl Microbiol Biotechnol 77:613–624CrossRefGoogle Scholar
  29. 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
  30. Perry CR, Matcham SE, Wood DA, Thurston CF (1993) The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus. J Gen Microbiol 139:171–178Google Scholar
  31. Reid ID, Paice MG (1994) Biological bleaching of kraft pulps by white-rot fungi and their enzymes. FEMS Microbiol Rev 13:369–375CrossRefGoogle Scholar
  32. Reinhammar B (1970) Purification and properties of laccase and stellacyanin from Rhus vernicifera. BBA-Bioenergetics 205:35–47CrossRefGoogle Scholar
  33. Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226CrossRefGoogle Scholar
  34. Robles A, Lucas R, Martinez-Canamero M, Ben Omar N, Perez R, Galvez A (2002) Characterisation of laccase activity produced by the hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH32. Enzyme Microb Tech 31:516–522CrossRefGoogle Scholar
  35. Rogalski J, Dawidowicz A, Jozwik E, Leonowicz A (1999) Immobilization of laccase from Cerrena unicolor on controlled porosity glass. J Mol Catal B: Enzym 6:29–39CrossRefGoogle Scholar
  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  37. Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15:7155–7174CrossRefGoogle Scholar
  38. 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
  39. 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
  40. Trovaslet M, Enaud E, Guiavarc'h Y, Corbisier A-M, Vanhulle S (2007) Potential of a Pycnoporus sanguineus laccase in bioremediation of wastewater and kinetic activation in the presence of an anthraquinonic acid dye. Enzyme Microb Tech 41:368–376CrossRefGoogle Scholar
  41. Wahleithner JA, Xu F, Brown K, Brown S, Golightly E, Halkier T, Kauppinen S, Pederson A, Schneider P (1996) The identification and characterization of four laccases from the plant pathogenic fungus Rhizoctonia solani. Curr Genet 29:395–403CrossRefGoogle Scholar
  42. Wesenberg D, Kyriakides I, Agathos SN (2003) White-rot fungi and their enzymes for the treatment of industrial dye effluents. Biotechnol Adv 22:161–187CrossRefGoogle Scholar
  43. Xu F, Shin W, Brown SH, Wahleithner JA, Sundaram UM, Solomon EI (1996) A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity, and stability. BBA-Protein Struct M 1292:303–311CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • C. Junghanns
    • 1
  • M. J. Pecyna
    • 2
  • D. Böhm
    • 3
  • N. Jehmlich
    • 4
  • C. Martin
    • 5
  • M. von Bergen
    • 4
  • F. Schauer
    • 6
  • M. Hofrichter
    • 2
  • D. Schlosser
    • 7
    Email author
  1. 1.Unit of Bioengineering (GEBI)University of LouvainLouvain-la-NeuveBelgium
  2. 2.Department of Environmental BiotechnologyInternational Graduate School (IHI) ZittauZittauGermany
  3. 3.Vita34 AGLeipzigGermany
  4. 4.UFZ, Department of ProteomicsHelmholtz Centre for Environmental Research–UFZLeipzigGermany
  5. 5.AVIOR Systems GmbHLeipzigGermany
  6. 6.Institute of MicrobiologyUniversity of GreifswaldGreifswaldGermany
  7. 7.UFZ, Department of Environmental MicrobiologyHelmholtz Centre for Environmental Research–UFZLeipzigGermany

Personalised recommendations