Restriction lift date: 2029-12-31
Enhancing the potential of hydrolases in enantioselective synthesis
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Date
2024
Authors
Torley, Rachel
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
This interdisciplinary research project focuses on the utilisation of a novel hydrolase of marine origin as a biocatalyst in asymmetric synthesis, bringing together synthetic organic chemistry and molecular biology. The primary objective of this research was to investigate the effect of histidine tags on the activity and selectivity of this biocatalyst.
Chapter 1 summarises literature reports of the applications of biocatalysts in asymmetric processes and, specifically, the reported effects of histidine tags on enzyme activity.
Chapter 2 describes the synthesis and use of ester substrates for biocatalysis screening against three variants of the novel marine esterase, esterase 26D. The enzyme variants included the wild-type, non-histidine tagged strain, and two histidine tagged enzymes (C-terminal and N-terminal tagged). Overall, the investigation showed that the histidine tag had no detectable effect on the selectivity or activity of the esterase 26D. An important outcome of the work was development of a standard procedure to be undertaken at the outset of future investigations of novel biocatalysts to definitively establish whether the histidine tag impacts on the enzyme activity.
Chapter 3 details the expression of the novel esterase, esterase 26D, variants. The non-histidine tag variant was cloned into E. coli where it was subsequently expressed alongside the readily available histidine tagged counterparts. Additionally, variants of other enzymes, esterase 32C and 51C, were cloned and expressed to provide another set of enzymes that could be screened for activity in the future.
Lastly, Chapter 4 contains the full experimental details and spectroscopic characterisation of the compounds synthesised in the work, the biocatalysis studies, along with the general methods employed for generating the desired enzymes.
Description
Keywords
Biocatalysis , Asymetric chemistry , Hydrolases
Citation
Torley, R. L. 2024. Enhancing the potential of hydrolases in enantioselective synthesis. MRes Thesis, University College Cork.