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Applied Microbiology and Biotechnology

, Volume 101, Issue 7, pp 2853–2864 | Cite as

Recombinant expression and characterization of a l-amino acid oxidase from the fungus Rhizoctonia solani

  • Katharina Hahn
  • Katrin Neumeister
  • Andreas Mix
  • Tilman Kottke
  • Harald Gröger
  • Gabriele Fischer von Mollard
Biotechnologically relevant enzymes and proteins

Abstract

l-Amino acid oxidases (L-AAOs) catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. l-AAOs are homodimeric enzymes with FAD as a non-covalently bound cofactor. They are of potential interest for biotechnological applications. However, heterologous expression has not succeeded in producing large quantities of active recombinant l-AAOs with a broad substrate spectrum so far. Here, we report the heterologous expression of an active l-AAO from the fungus Rhizoctonia solani in Escherichia coli as a fusion protein with maltose-binding protein (MBP) as a solubility tag. After purification, it was possible to remove the MBP-tag proteolytically without influencing the enzyme activity. MBP-rsLAAO1 and 9His-rsLAAO1 converted basic and large hydrophobic l-amino acids as well as methyl esters of these l-amino acids. The progress of the conversion of l-phenylalanine and l-leucine into the corresponding α-keto acids was determined by HPLC and 1H-NMR analysis of reaction mixtures, respectively. Enzymatic activity was stimulated 50–100-fold by SDS treatment. K m values ranging from 0.9–10 mM and v max values from 3 to 10 U mg−1 were determined after SDS activation of 9His-rsLAAO1 for the best substrates. The enzyme displayed a broad pH optimum between pH 7.0 and 9.5. In summary, a successful overexpression of recombinant l-AAO in E. coli was established that results in a promising enzymatic activity and a broad substrate spectrum for biotechnological application.

Keywords

l-Amino acid oxidase Heterologous expression E. coli Maltose-binding protein Solubility tag 

Notes

Acknowledgements

We thank Denise Weinberg and Thomas Geisler for excellent technical assistance.

Compliance with ethical standards

Funding

This study was funded by Deutsche Forschungsgemeinschaft (grant number KO3580/4-1).

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Katharina Hahn
    • 1
  • Katrin Neumeister
    • 1
  • Andreas Mix
    • 2
  • Tilman Kottke
    • 3
  • Harald Gröger
    • 4
  • Gabriele Fischer von Mollard
    • 1
  1. 1.Biochemie III, Fakultät für ChemieUniversität BielefeldBielefeldGermany
  2. 2.Anorganische Chemie und Strukturchemie, Fakultät für ChemieUniversität BielefeldBielefeldGermany
  3. 3.Physikalische und Biophysikalische Chemie, Fakultät für ChemieUniversität BielefeldBielefeldGermany
  4. 4.Organische Chemie I, Fakultät für ChemieUniversität BielefeldBielefeldGermany

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