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Natural and recombinant fungal laccases for paper pulp bleaching

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Abstract

Three laccases, a natural form and two recombinant forms obtained from two different expression hosts, were characterized and compared for paper pulp bleaching. Laccase from Pycnoporus cinnabarinus, a well known lignolytic fungus, was selected as a reference for this study. The corresponding recombinant laccases were produced in Aspergillus oryzae and A. niger hosts using the lacI gene from P. cinnabarinus to develop a production process without using the expensive laccase inducers required by the native source. In flasks, production of recombinant enzymes by Aspergilli strains gave yields close to 80 mg l−1. Each protein was purified to homogeneity and characterized, demonstrating that the three hosts produced proteins with similar physico-chemical properties, including electron paramagnetic resonance spectra and N-terminal sequences. However, the recombinant laccases have higher Michaelian (K m) constants, suggesting a decrease in substrate/enzyme affinity in comparison with the natural enzyme. Moreover, the natural laccase exhibited a higher redox potential (around 810 mV), compared with A. niger (760 mV) and A. oryzae (735 mV). Treatment of wheat straw Kraft pulp using laccases expressed in P. cinnabarinus or A. niger with 1-hydroxybenzotriazole as redox mediator achieved a delignification close to 75%, whereas the recombinant laccase from A. oryzae was not able to delignify pulp. These results were confirmed by thioacidolysis. Kinetic and redox potential data and pulp bleaching results were consistent, suggesting that the three enzymes are different and each fungal strain introduces differences during protein processing (folding and/or glycosylation).

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Acknowledgements

We thank P. Mansuelle (UMR 6560, IFR Jean Roche, Marseille, France) for the N-terminal sequence analysis. This research was supported by the EU Quality of Living Resources programme (Project QLK3-99-590, Fungal metalloenzymes oxidizing aromatic compound of industrial interest) and GIS-EBL (Région Provence Alpes Côte d’Azur and Conseil Général des Bouches-du-Rhône, France). C.S. is grateful to the Conseil Régional Provence-Alpes-Côte d’Azur, France, the Institut National de la Recherche Agronomique and Tembec-Tarascon S.A. for a PhD scholarship.

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Authors and Affiliations

  1. UMR 1163 INRA/Université de Provence de Biotechnologie des Champignons Filamenteux, IFR 86 BAIM, Universités de Provence et de la Méditerranée, ESIL, 163 avenue de Luminy, C.P. 925, 13288, Marseille Cedex 09, France

    C. Sigoillot, E. Record, J. L. Robert, A. Levasseur, J. C. Sigoillot & M. Asther

  2. UPR 9036 de Bioénergétique et Ingénierie des Protéines–CNRS 31, Chemin Joseph Aiguier, 13402, Marseille Cedex 20, France

    V. Belle & A. Fournel

  3. Department of Applied Microbiology and Gene Technology, TNO Nutrition and Food Research Institute, Utrechtseweg 48, P.O. Box 360, 3700 AJ, Zeist, The Netherlands

    P. J. Punt & C. A. M. J. J. van den Hondel

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  1. C. Sigoillot
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  2. E. Record
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  3. V. Belle
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  4. J. L. Robert
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  5. A. Levasseur
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  6. P. J. Punt
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  7. C. A. M. J. J. van den Hondel
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  8. A. Fournel
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  9. J. C. Sigoillot
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  10. M. Asther
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Correspondence to C. Sigoillot.

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Sigoillot, C., Record, E., Belle, V. et al. Natural and recombinant fungal laccases for paper pulp bleaching. Appl Microbiol Biotechnol 64, 346–352 (2004). https://doi.org/10.1007/s00253-003-1468-3

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  • DOI: https://doi.org/10.1007/s00253-003-1468-3

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