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The Biocatalytic Application of Novel Enzymes from Metagenomic Sources

Leipold, Leona; (2019) The Biocatalytic Application of Novel Enzymes from Metagenomic Sources. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Biocatalysis is defined as the use of biological molecules to catalyse chemical reactions. Biocatalysts have been used unwittingly for thousands of years in food and drink manufacture and have since become an integral part of the food, agriculture, and pharmaceutical industry. Their integration into pharmaceutical processes is due to both their intrinsic chemo-, regio- and stereospecificity as well as their alignment with the principles of green chemistry. Chemical manufacturing has been identified as one of the top ten polluting industries, therefore processes that are more sustainable and environmentally friendly are vital going forward. The work presented here uses a metagenomics approach to identify novel enzymes for biocatalysis. A drain metagenome database was queried for a number of enzyme classes and their retrieval and characterisation has been detailed here. Using this method, 29 novel transaminases from a household drain metagenome were cloned and overexpressed. The most promising enzymes were fully characterised and the effects of pH, temperature, amine donor concentration and co-solvent determined. Several enzymes demonstrated good substrate tolerance as well as an unprecedented robustness for a wild-type transaminase. One enzyme in particular readily accepted isopropylamine as an amine donor, giving the same conversion with 2-50 equivalents, as well as being tolerant to a number of co-solvents, and operational in up to 50% DMSO. Two novel enzymes belonging to the limonene epoxide hydrolase family were also cloned and characterised, and their function compared to the previously studied Re-LEH. The substrate scope was investigated, and the enzymes used in a preparative scale reaction to demonstrate their industrial application. The feasibility of using the two enzymes in a reaction cascade have also been described, as well as the development of a spectrophotometric assay for the identification of novel transaminases that accept bulky substrates.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The Biocatalytic Application of Novel Enzymes from Metagenomic Sources
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10067792
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