As the demand for single enantiomer compounds continues to increase so must the supply, and biocatalysts are increasingly being used because of their superior stereo-, regio-, and enantioselectivity over alternative methods. Chiral carboxylic acids are vital components of a wide range of drugs and pharmaceuticals, and a number of methods for their production are in use today. The work that follows details investigations towards novel biocatalysts for the production of chiral carboxylic acids.Firstly, the development of an arylmalonate decarboxylase (AMDase) from Bordetella bronchiseptica is described. Screening the AMDase with a range of prochiral malonates revealed than an alkenylmalonate substrate was decarboxylated by the enzyme, and the implications of this finding on the AMDase mechanism are discussed. A library of alkenylmalonates were subsequently synthesised and successfully tested with the AMDase before the enzyme was subjected to mutagenesis, generating a library of mutants which exhibited up to 50-fold improvements in activity compared to the wild-type enzyme. However, attempts to reengineer the AMDase to create an enantiocomplementary decarboxylase were unsuccessful. An alternative approach to producing enantiocomplementary AMDases was attempted, starting from uncharacterised proteins thought to be related Asp/Glu racemases based on sequence alignments. A number of enzymes were selected, and overproduced in E. coli BL21(DE3). Investigations to reveal the native functions of the enzymes were carried out, and using both a dehydrogenase assay and a novel D-amino acid oxidase screen, racemase activity was exhibited with many of the enzymes. Four proteins exhibited promiscuous decarboxylase activity to some extent with 2-phenylmalonate, but crucially, not with prochiral malonates. The most active protein proved to be extremely unstable, and so subsequent mutagenesis was carried out on the next best candidate, a glutamate racemase from Aquifex pyrophilus. Ultimately, although the mutants produced were significantly more active than the wild-type enzyme, and furthermore became the first examples of Asp/Glu racemases, capable of decarboxylating prochiral malonates, the products of decarboxylation were racemic.Finally an N-acylamino acid racemase from Geobacillus kaustophilus was obtained and overproduced in E. coli BL21(DE3). The optimal conditions for enzyme-catalysed racemisation of N-acylamino acids were determined, and subsequent screening with a small number of non-proteinogenic amino acids tested the substrate scope of the enzyme. Although the HPLC assay developed was indicative of racemase activity with several substrates, chiral standards were unavailable for comparison. The promiscuity of the enzyme was also investigated, but no additional activities were observed.
|Date of Award||1 Aug 2012|
- The University of Manchester
|Supervisor||Jason Micklefield (Supervisor)|