Enzymes are highly specialized catalysts, used by all living organisms to catalyze chemical reactions necessary to sustain life. Although enzymes in the course of evolution became highly specialized catalysts towards one specific chemical reaction, many enzymes are capable of catalyzing one or more unnatural reactions. These promiscuous reactions can be highly interesting, especially when the product of a promiscuous reaction forms a facile building block for the synthesis of particular pharmaceuticals. The focus of this thesis is the enzyme 4-oxalocrotonate tautomerase, which is capable of catalyzing several highly interesting carbon-carbon bond-forming reactions. The products of these reactions can be used to synthesize gamma-aminobutyric-acid analogues (GABA analogues), a highly important class of pharmaceuticals. Via extensive mutagenesis and by means of a novel color-based screenings method, enzyme variants were identified that were able to catalyze these reactions much quicker and moreover would almost exclusively synthesize the desired enantiomer. These improved enzyme variants were combined with other enzymes and a chemical catalyst in one pot, showing that all steps in this novel synthesis route could be performed without intermediate purification. Although this synthesis route needs to be further improved before it can be applied industrially, it highlights the exciting opportunities available to search for new enzyme variants and develop new sustainable catalysts that could applied in the synthesis of pharmaceuticals.
|Qualification||Doctor of Philosophy|
|Place of Publication||[Groningen]|
|Publication status||Published - 2020|