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Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells

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Journal of Industrial Microbiology & Biotechnology

Abstract

It has been demonstrated that Agaricus bisporus tyrosinase is able to oxidize various phenolic compounds, thus being an enzyme of great importance for a number of biotechnological applications. The tyrosinase-coding PPO2 gene was isolated by reverse-transcription polymerase chain reaction (RT-PCR) using total RNA extracted from the mushroom fruit bodies as template. The gene was sequenced and cloned into pYES2 plasmid, and the resulting pY-PPO2 recombinant vector was then used to transform Saccharomyces cerevisiae cells. Native polyacrylamide gel electrophoresis followed by enzymatic activity staining with l-3,4-dihydroxyphenylalanine (l-DOPA) indicated that the recombinant tyrosinase is biologically active. The recombinant enzyme was overexpressed and biochemically characterized, showing that the catalytic constants of the recombinant tyrosinase were higher than those obtained when a commercial tyrosinase was used, for all the tested substrates. The present study describes the recombinant production of A. bisporus tyrosinase in active form. The produced enzyme has similar properties to the one produced in the native A. bisporus host, and its expression in S. cerevisiae provides good potential for protein engineering and functional studies of this important enzyme.

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Acknowledgments

We thank the native speaker Prof. H. Caffery for proofreading and providing valuable linguistic advice.

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Authors and Affiliations

  1. Consiglio Nazionale delle Ricerche (CNR), Institute of Sciences of Food Production (ISPA), Operative Unit of Lecce, via Provinciale Lecce-Monteroni, 73100, Lecce, Italy

    Gianluca Bleve, Stefano Spagnolo & Francesco Grieco

  2. Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, via Prov. Lecce-Monteroni, 73100, Lecce, Italy

    Chiara Lezzi & Carla Perrotta

Authors
  1. Chiara Lezzi
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  2. Gianluca Bleve
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  3. Stefano Spagnolo
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  4. Carla Perrotta
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  5. Francesco Grieco
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Correspondence to Francesco Grieco.

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Lezzi, C., Bleve, G., Spagnolo, S. et al. Production of recombinant Agaricus bisporus tyrosinase in Saccharomyces cerevisiae cells. J Ind Microbiol Biotechnol 39, 1875–1880 (2012). https://doi.org/10.1007/s10295-012-1192-z

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  • DOI: https://doi.org/10.1007/s10295-012-1192-z

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