Philpott, Helena Kate; (2022) Bionanotechnology platforms for biocatalysis. Doctoral thesis (Ph.D), UCL (University College London).

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Text Helena Kate Philpott (17054526) - PhD Thesis Corrected.pdf - Accepted Version Download (14MB) | Preview

Abstract

Biocatalysis, the use of enzymes to perform chemical reactions, is widely accepted as a greener, more sustainable alternative to traditional chemical synthesis. Enzymes provide exquisite regio and stereo-selectivity and can open synthetic routes to compounds which are inaccessible by other chemical means. Enzymes have been extensively investigated and exploited for these properties, and due to the recent advances in bioinformatics, high-throughput screening and gene synthesis, we now have an almost infinite library of potential enzymes. Despite this library of enzymes being available there are still only a few commercially large scale processes which use enzymes. The use of enzymes in industrial processes pose a number of problems including low yields due to low substrate loading, slow reaction rates, cross reactivities and issues with downstream processing. In order to tackle these problems several methods, including various immobilisation and encapsulation techniques, have been developed. This thesis investigated two such methods for immobilisation of enzymes. These methods, based upon cholesterol membrane insertion and complementary DNA binding, were designed and synthesised to tether enzymes to a lipid bilayer membrane. The tethering of individual enzymes to a surface can improve enzyme stability, activity, selectivity, specificity and reduce inhibition. These methods built upon this, extending to an enzymatic cascade with a design to eliminate three-dimensional diffusion, increase reaction rates and yields, and improve flux. Testing of these methods was divided into a proof of concept, utilising fluorescent proteins to examine the efficacy of immobilisation through confocal microscopy; and testing with individual and cascaded enzymes in order to determine the benefits of the immobilisation techniques. A novel cascade was selected which would utilise redox and functional group conversion reactions and involve three enzyme classes: alcohol dehydrogenases, ene-reductases and transaminases. It is expected that these techniques will help to overcome the problems faced by biocatalysis and provide new bionanotechnology reaction platforms.

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