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A thermostable L-aspartate oxidase: a new tool for biotechnological applications

  • Biotechnologically relevant enzymes and proteins
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Abstract

L-Amino acid oxidases (LAAOs) are homodimeric flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the stereospecific oxidative deamination of L-amino acids to α-keto acids, ammonia, and hydrogen peroxide. Unlike the D-selective counterpart, the biotechnological application of LAAOs has not been thoroughly advanced because of the difficulties in their expression as recombinant protein in prokaryotic hosts. In this work, L-aspartate oxidase from the thermophilic archea Sulfolobus tokodaii (StLASPO, specific for L-aspartate and L-asparagine only) was efficiently produced as recombinant protein in E. coli in the active form as holoenzyme. This recombinant flavoenzyme shows the classical properties of FAD-containing oxidases. Indeed, StLASPO shows distinctive features that makes it attractive for biotechnological applications: high thermal stability (it is fully stable up to 80 °C) and high temperature optimum, stable activity in a broad range of pH (7.0–10.0), weak inhibition by the product oxaloacetate and by D-aspartate, and tight binding of the FAD cofactor. This latter property significantly distinguishes StLASPO from the E. coli counterpart. StLASPO represents an appropriate novel biocatalyst for the production of D-aspartate and a well-suited protein scaffold to evolve a LAAO activity by protein engineering.

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Acknowledgments

This work was supported by grants from Fondo di Ateneo per la Ricerca to L. Pollegioni and G. Molla. We are grateful for the support of Consorzio Interuniversitario per le Biotecnologie. D. Bifulco is a student of the Ph.D. program in Biotechnology, School in Biological and Medical Sciences, University of Insubria.

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Authors and Affiliations

  1. Dipartimento di Biotecnologie e Scienze della Vita, Università degli Studi dell’Insubria, via J.H. Dunant 3, Varese, Italy

    Davide Bifulco, Loredano Pollegioni & Gianluca Molla

  2. The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano, ICRM–CNR Milano, and Università degli Studi dell’Insubria, via Mancinelli 7, Milano, Italy

    Loredano Pollegioni, Davide Tessaro, Stefano Servi & Gianluca Molla

  3. Dipartimento di Chimica, Materiali, Ingegneria Chimica G. Natta, Politecnico di Milano, via Mancinelli 7, Milano, Italy

    Davide Tessaro & Stefano Servi

Authors
  1. Davide Bifulco
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  2. Loredano Pollegioni
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  3. Davide Tessaro
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  4. Stefano Servi
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  5. Gianluca Molla
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Correspondence to Loredano Pollegioni.

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Bifulco, D., Pollegioni, L., Tessaro, D. et al. A thermostable L-aspartate oxidase: a new tool for biotechnological applications. Appl Microbiol Biotechnol 97, 7285–7295 (2013). https://doi.org/10.1007/s00253-013-4688-1

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  • DOI: https://doi.org/10.1007/s00253-013-4688-1

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