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High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

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Abstract

By combining gene design and heterologous over-expression of Rhodotorula gracilis d-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.

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Acknowledgments

The authors would like to thank Thorsten Bachler and Margaretha Schiller for their excellent technical support. The Österreichische Forschungsförderungsgesellschaft mbH (FFG), the provinces of Vienna, Styria and Tyrol as well as the Steirische Wirtschaftsförderungsgesellschaft mbH (SFG) and Ingenza Ltd. are acknowledged for financial support. BN and JN acknowledge the Slovak-Austrian travel project SK-AT-0024-08.

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Author notes

  1. Jozef Nahalka

    Present address: Institute of Chemistry, Center of Glycomics, Slovak Academy of Sciences, Dúbravska cesta 9, 84538, Bratislava, Slovak Republic

Authors and Affiliations

  1. Austrian Centre of Industrial Biotechnology, Petersgasse 14, Graz, 8010, Austria

    Sandra Abad, Jozef Nahalka, Margit Winkler, Gabriele Bergler, Anton Glieder & Bernd Nidetzky

  2. Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, Graz, 8010, Austria

    Jozef Nahalka & Bernd Nidetzky

  3. Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, Graz, 8010, Austria

    Anton Glieder

  4. Ingenza Ltd, Wallace Building, Roslin BioCentre, Roslin, EH25 9PP, UK

    Robert Speight

Authors
  1. Sandra Abad
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  2. Jozef Nahalka
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  3. Margit Winkler
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  4. Gabriele Bergler
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  5. Robert Speight
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  6. Anton Glieder
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  7. Bernd Nidetzky
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Corresponding author

Correspondence to Bernd Nidetzky.

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Abad, S., Nahalka, J., Winkler, M. et al. High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris . Biotechnol Lett 33, 557–563 (2011). https://doi.org/10.1007/s10529-010-0456-9

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  • DOI: https://doi.org/10.1007/s10529-010-0456-9

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