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Background   Trigonopsis variabilis   D -amino acid oxidase ( Tv DAO) 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  Tv DAO 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 Tv DAO whole-cell biocatalyst.    Results  As compared to the native  T. variabilis  host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in  P. pastoris  through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 × 10 3  U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of ≥ 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of  P. pastoris  using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used  Tv DAO expressing  Escherichia coli  strain) under conditions of  D -methionine conversion using vigorous aeration.    Conclusions  Stepwise optimization using a multi-level engineering approach has delivered a new  P. pastoris  whole cell  Tv DAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this  P. pastoris  catalyst has been evaluated for industrial purposes.

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