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Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst.

Sandra Abad, Jozef Nahalka, Gabriele Bergler, S Alison Arnold, Robert Speight, Ian Fotheringham, Bernd Nidetzky and Anton Glieder.
Graz University of Technology
Microbial Cell Factories 2010, 9:24
Gene names: TvDAO. Host systems: Pichia pastoris[Yeast]. Gene species: Trigonopsis variabilis[Yeast]. Optimized: Yes. Protein activity: Yes.
Abstract: Trigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst.
Comments: TvDAO is an industrially important enzyme produced from Trigonopsis variabilis. Anton Glieders group at Graz technical University, Austria together with Ingenza (Roslin, UK) redesigned the TvDAO gene for Pichia pastoris expression using the DNA2.0 software tools and synthesis. The Pichia system produced >200g/L and the final enzyme was significantly more stable and more active.