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

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Abstract

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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Unit of Bioengineering (GEBI), University of Louvain, Place Croix du Sud 2/19, 1348, Louvain-la-Neuve, Belgium

    C. Junghanns

  2. Department of Environmental Biotechnology, International Graduate School (IHI) Zittau, Markt 23, 02763, Zittau, Germany

    M. J. Pecyna & M. Hofrichter

  3. Vita34 AG, Deutscher Platz 1a, 04103, Leipzig, Germany

    D. Böhm

  4. UFZ, Department of Proteomics, Helmholtz Centre for Environmental Research–UFZ, Permoserstrasse 15, 04318, Leipzig, Germany

    N. Jehmlich & M. von Bergen

  5. AVIOR Systems GmbH, Weissenfelser Strasse 67, 04229, Leipzig, Germany

    C. Martin

  6. Institute of Microbiology, University of Greifswald, Friedrich-Ludwig-Jahn-Strasse 15, 17487, Greifswald, Germany

    F. Schauer

  7. UFZ, Department of Environmental Microbiology, Helmholtz Centre for Environmental Research–UFZ, Permoserstrasse 15, 04318, Leipzig, Germany

    D. Schlosser

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  1. C. Junghanns
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Correspondence to D. Schlosser.

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Junghanns, C., Pecyna, M.J., Böhm, D. et al. Biochemical and molecular genetic characterisation of a novel laccase produced by the aquatic ascomycete Phoma sp. UHH 5-1-03. Appl Microbiol Biotechnol 84, 1095–1105 (2009). https://doi.org/10.1007/s00253-009-2028-2

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