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Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae

  • Applied Genetics and Molecular Biotechnology
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Abstract

Heterologous expression of Pleurotus ostreatus POXC and POXA1b laccases in two yeasts, Kluyveromyces lactis and Saccharomyces cerevisiae, was performed. Both transformed hosts secreted recombinant active laccases, although K. lactis was much more effective than S. cerevisiae. rPOXA1b transformants always had higher secreted activity than rPOXC transformants did. The lower tendency of K. lactis with respect to S. cerevisiae to hyperglycosylate recombinant proteins was confirmed. Recombinant laccases from K. lactis were purified and characterised. Specific activities of native and recombinant POXA1b are similar. On the other hand, rPOXC specific activity is much lower than that of the native protein, perhaps due to incomplete or incorrect folding. Both recombinant laccase signal peptides were correctly cleaved, with rPOXA1b protein having two C-terminal amino acids removed. The availability of the established recombinant expression system provides better understanding of laccase structure–function relationships and allows the development of new oxidative catalysts through molecular evolution techniques.

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Acknowledgements

This work was supported by grants from the Ministero dell'Università e della Ricerca Scientifica (Progetti di Rilevante Interesse Nazionale, PRIN 2002 and 2004), INTAS (International Association for the promotion of cooperation with the scientists from the New Independent States of the former Soviet Union, ref. no. 03-51-5889), and Centro Regionale di Competenza BioTekNet. A. Piscitelli was the recipient of a French/Italian University fellowship. The authors thank Prof. Claudio Falcone for kindly making available K. lactis strain and plasmid, Prof. Piero Pucci for helpful discussions, and Dr. Thierry Tron for the critical reading of the manuscript and for kindly making available S. cerevisiae strain and plasmids.

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Authors and Affiliations

  1. Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, via Cinthia, 80126, Naples, Italy

    Alessandra Piscitelli, Paola Giardina & Giovanni Sannia

  2. Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Roma La Sapienza, Piazzale Aldo Moro, 5, 00185, Rome, Italy

    Cristina Mazzoni

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  1. Alessandra Piscitelli
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  2. Paola Giardina
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  3. Cristina Mazzoni
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  4. Giovanni Sannia
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Correspondence to Giovanni Sannia.

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Piscitelli, A., Giardina, P., Mazzoni, C. et al. Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae . Appl Microbiol Biotechnol 69, 428–439 (2005). https://doi.org/10.1007/s00253-005-0004-z

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  • DOI: https://doi.org/10.1007/s00253-005-0004-z

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