Characterization of amino acid ammonia lyases & mutases for the production of chiral $\alpha$- and $\beta$-amino acids

Enantiopure non-natural amino acids are valuable building blocks for the production of chemicals and pharmaceuticals or are themselves pharmacologically active. Examples are 2-chloro-phenylalanine, a precursor for the production of a hypertension pharmaceutical and L-Dopa, which is used for the treatment of Parkinson desease or (R)-$\beta$-phenylalanine, which is part of the side chain of the antitumor drug Taxol. To get access to this class of compounds, a toolbox of MIO-dependent ammonia lyases and aminomutases was generated including four wild-type phenylalanine and tyrosine ammonia lyases (PAL/TAL), one variant with altered substrate specificity, and three wild-type phenylalanine and tyrosine aminomutases (PAM/TAM) from plants, yeast, and bacteria. All ammonia lyases were comparatively characterized with respect to kinetic parameters, pH- and T-optima for the deamination reaction of their natural substrates. Scince the focus of this thesis was on the stereoselective amination of cinnamic acid (CA) derivatives to yield enantiopure α- and β-amino acids, the investigation of the substrate ranges and reaction parameters was the main goal. Among the tested ammonia lyases in the toolbox PAL from $\textit{Arabidopsis thaliana}$ (AtPAL2), an enzyme which was not characterized in detail yet, performed best, compared to the enzymes from $\textit{Petroselinum cripsum}$ and $\textit{Rhodosporidium toruloides}$, which are well studied and industrially applied. AtPAL2 shows significant faster conversion of 3-F-CA (91.2 %), 4-F-CA (84.7 %) and 2-Cl-CA (96.4 %) in batch reactions. Due to the importance of 2-Cl-L-Phe as key intermediate for the production of hypertension drugs, this reaction was further investigated in larger scale using different reactor types and reaction modes. With a continuous enzyme membrane reactor the space-time-yield (STY) and productivity per catalyst (cell dry weight: CDW) could be enhanced from 11.8 g/L*d and 10.8 g/gDCW in batch to 67.8 g/L*d and 46.8 g/gDCW. The product 2-Cl-L-Phe could be isolated by simple volume reduction and crystallization with a yield of 57 % and high purity. [...