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A Semi-Rational Approach to Engineering Laccase Enzymes

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Abstract

In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t 1/2 at pH 5 = 35 days), neutral (t 1/2 at pH 7 = 38 days) and alkaline (t 1/2 at pH 10 = 62 days) pH values.

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References

  1. Thurston, F. (1994). The structure and function of fungal laccases. Microbiology, 140, 19–26.

    Article  CAS  Google Scholar 

  2. Ullah, M. A., Bedford, C. T., & Evans, C. S. (2002). Reactions of pentachlorophenol with laccase from Coriolus versicolor. Applied Microbiology and Biotechnology, 53, 230–234.

    Article  Google Scholar 

  3. Bourbonnais, R., & Paice, M. G. (1990). Oxidation of nonphenolic substrates—an expanded role for laccase in lignin biodegradation. FEBS Letters, 267, 99–102.

    Article  CAS  Google Scholar 

  4. Xu, F. (2005). Applications of oxidoreductases: Recent progress. Industrial Biotechnology, 1, 38–50.

    Article  CAS  Google Scholar 

  5. Riva, S. (2006). Laccases: Blue enzyme for green chemistry. Trends in Biotechnology, 24, 219–226.

    Article  CAS  Google Scholar 

  6. Alcalde, M., Ferrer, M., Plou, F. J., & Ballesteros, A. (2006). Environmental biocatalysis: From remediation with enzymes to novel green processes. Trends in Biotechnology, 24, 281–287.

    Article  CAS  Google Scholar 

  7. Pezzella, C., Autore, F., Giardina, P., Piscitelli, A., Sannia, G., & Faraco, V. (2009). The Pleurotus ostreatus laccase multi-gene family: Isolation and heterologous expression of new family members. Current Genetics, 55(1), 45–57.

    Article  CAS  Google Scholar 

  8. Palmieri, G., Cennamo, G., & Sannia, G. (2005). Remazol Brilliant Blue R decolourisation by the fungus Pleurotus ostreatus and its oxidative enzymatic system. Enzyme and Microbial Technology, 36, 17–24.

    Article  CAS  Google Scholar 

  9. Palmieri, G., Giardina, P., & Sannia, G. (2005). Laccase-mediated Remazol Brilliant Blue R decolourization in a fixed-bed bioreactor. Biotechnology Progress, 21, 1436–1441.

    Article  CAS  Google Scholar 

  10. Faraco, V., Pezzella, C., Miele, A., Giardina, P., & Sannia, G. (2009). Bio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes. Biodegradation, 20(2), 209–220.

    Article  CAS  Google Scholar 

  11. Faraco, V., Pezzella, C., Giardina, P., Piscitelli, A., Vanhulle, S., & Sannia, G. (2009). Decolourization of textile dyes by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus. Journal of Chemical Technology and Biotechnology, 84, 414–419.

    Article  CAS  Google Scholar 

  12. Giardina, P., Palmieri, G., Scaloni, A., Fontanella, B., Faraco, V., Cennamo, G., et al. (1999). Protein and gene structure of a blue laccase from Pleurotus ostreatus. Biochemical Journal, 341, 655–663.

    Article  CAS  Google Scholar 

  13. Miele, A., Giardina, P., Sannia, G., & Faraco, V. (2009). Random mutants of a Pleurotus ostreatus laccase as new biocatalysts for industrial effluents bioremediation. Journal of Applied Microbiology. doi:10.1111/j.1365-2672.2009.04505.

  14. Festa, G., Autore, F., Fraternali, F., Giardina, P., & Sannia, G. (2007). Development of new laccases by directed evolution: Functional and computational analyses. Proteins, 72, 25–34.

    Article  Google Scholar 

  15. Piscitelli, A., Giardina, P., Mazzoni, C., & Sannia, G. (2005). Recombinant expression of Pleurotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 69, 428–439.

    Article  CAS  Google Scholar 

  16. Gietz, D., St Jean, A., Woods, R. A., & Schiestl, R. H. (1992). Improved method for high efficiency transformation of intact yeast cells. Nucl Acids Research, 20, 1425.

    Article  CAS  Google Scholar 

  17. Faraco, V., Ercole, C., Festa, G., Giardina, P., Piscitelli, A., & Sannia, G. (2008). Heterologous expression of heterodimeric laccases from Pleurotus ostreatus in Kluyveromyces lactis. Applied Microbiology and Biotechnology, 77, 1329–1335.

    Article  CAS  Google Scholar 

  18. Autore, F., Del Vecchio, C., Fraternali, F., Giardina, P., Sannia, G., & Faraco, V. (2009). Molecular determinants of peculiar properties of a Pleurotus ostreatus laccase: Analysis by site-directed mutagenesis. Enzyme and Microbial Technology, 45, 507–513.

    Article  CAS  Google Scholar 

  19. Hakulinen, N., Kiiskinen, L. L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A., et al. (2002). Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site. Nature Structural Biology, 9, 601–605.

    CAS  Google Scholar 

  20. Gelo-Pujic, M., Kim, H. H., Butlin, N. G., & Palmore, G. T. (1999). Electrochemical studies of a truncated laccase produced in Pichia pastoris. Applied and Environmental Microbiology, 65, 5515–5521.

    CAS  Google Scholar 

  21. Bulter, T., Alcalde, M., Sieber, V., Meinhold, P., Schlachtbauer, C., & Arnold, F. H. (2003). Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Applied and Environmental Microbiology, 69, 987–995.

    Article  CAS  Google Scholar 

  22. Zumarraga, M., Camarero, S., Shleev, S., Martinez-Arias, A., Ballesteros, A., Plou, F. J., et al. (2008). Altering the laccase functionality by in vivo assembly of mutant libraries with different mutational spectra. Proteins, 71(1), 250–260.

    Article  CAS  Google Scholar 

  23. Bloom, J. D., Labthavikul, S. T., Otey, C. R., & Arnold, F. H. (2006). Protein stability promotes evolvability. Proceedings of the National Academy of Sciences of the United States of America, 103, 5869–5874.

    Article  CAS  Google Scholar 

  24. Bertrand, T., Jolivalt, C., Briozzo, P., Caminade, E., Joly, N., Madzak, C., et al. (2002). Crystal structure of a four-copper laccase complexed with an arylamine: Insights into substrate recognition and correlation with kinetics. Biochemistry, 41, 7325–7333.

    Article  CAS  Google Scholar 

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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), and from the Ministero Degli Affari Esteri di Intesa con il Ministero dell’Università e della Ricerca (Progetti di ricerca di base e tecnologica approvati nei protocolli di cooperazione scientifica e tecnologica bilaterale come previsto dal protocollo bilaterale tra Italia e Turchia).

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Authors and Affiliations

  1. Department of Organic Chemistry and Biochemistry, University of Naples “Federico II”, Complesso Universitario Monte S. Angelo, via Cintia, 4, 80126, Naples, Italy

    Annalisa Miele, Paola Giardina, Alessandra Piscitelli, Giovanni Sannia & Vincenza Faraco

  2. School of Biotechnological Sciences, University of Naples “Federico II”, Naples, Italy

    Annalisa Miele, Eugenio Notomista & Vincenza Faraco

  3. Department of Structural and Functional Biology, University of Naples “Federico II”, Naples, Italy

    Eugenio Notomista

Authors
  1. Annalisa Miele
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  2. Paola Giardina
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  3. Eugenio Notomista
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  4. Alessandra Piscitelli
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  5. Giovanni Sannia
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  6. Vincenza Faraco
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Correspondence to Vincenza Faraco.

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Miele, A., Giardina, P., Notomista, E. et al. A Semi-Rational Approach to Engineering Laccase Enzymes. Mol Biotechnol 46, 149–156 (2010). https://doi.org/10.1007/s12033-010-9289-y

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