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Title: Directed evolution of D-amino acid oxidase for the oxidation of non-proteinogenic amino acids
Author: Wilson, Yvonne Martha
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2009
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Random mutagenesis libraries were prepared by propagating the Trigonopsis variabilis D-amino acid oxidase (TvDAAO) gene in a mutator strain and by performing error-prone PCR (epPCR) on the Rhodotorula gracilis D-amino acid oxidase (RgDAAO) gene. These libraries were initially screened using a solid-phase for detection of activity towards β-amino acids. To overcome reproducibility problems, a high-throughput liquid-phase screening method in 96-well plate format was developed, evaluated and used to screen the RgDAAO epPCR library. In the absence of any detectable activity towards the selected β-amino acid substrates, an alternative strategy, which minimised the variant library size and increased the range of substrates, was applied. Single site saturation libraries were generated by saturation mutagenesis PCR at the RgDAAO active site residues F58, M213, Y238 and R285. The resulting variant libraries were screened in the liquid-phase for activity towards a range of proteinogenic and non-proteinogenic amino acids. Saturation mutagenesis at the conserved active site residues Y238 and R285 produced mainly deleterious exchanges. However, saturation at the active site residues F58 and M213 produced a range of beneficial and deleterious variants, allowing substrate profiles to be produced for each. Evaluation of these profiles permitted the identification of individual variants. Although no oxidase activity was detected towards β-amino acids, improved activity was observed in both the F58 and M213 saturation mutagenesis libraries towards several proteinogenic and non-proteinogenic substrates. The improved activity detected in the assay for the variants F58L and H58I towards (rac)-tetrahydroisoquinoline-3-carboxylic acid and for F58M towards (R)-pipecolonic acid was confirmed by performing whole cell biotransformations and analysing the progression of the reactions by HPLC.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available