Alana Dreßen Characterization of amino acid ammonia lyases & aminomutases for the production of chiral α- and β-amino acids

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Inhaltsangabe zu „Characterization of amino acid ammonia lyases & aminomutases for the production of chiral α- and β-amino acids“ von Alana Dreßen

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)-β-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 Arabidopsis thaliana (AtPAL2), an enzyme which was not characterized in detail yet, performed best, compared to the enzymes from Petroselinum cripsum and 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. To improve the reusability of the AtPAL2 an immobilization strategy using cellulose binding modules (CBMs) from C. fimi was investigated. AtPAL2 fused with a C-terminal CBM immobilized on Avicel showed 81.5 % remaining activity in the amination of 2-Cl-CA after eleven 1 h batch reaction cycles. Furthermore AtPAL2-CBM was immobilized on Avicel in a plug-flow reactor by simply pumping the E. coli crude cell extract containing the enzyme through a column filled with Avicel, which was afterwards directly used for biotransformation by pumping the substrate through the column. The STY reached 248.6 g/L*d and the productivity was 18.5 g/gDCW ...

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