Hernández López, Roberto Icken;
(2021)
Development of a high-throughput screening method for transketolase and protein engineering for biotechnology applications.
Doctoral thesis (Ph.D), UCL (University College London).
Preview |
Text
PhD Thesis Roberto Icken Hernández López.pdf - Accepted Version Download (2MB) | Preview |
Abstract
Abstract Transketolase (TK) is an interesting enzyme for the biotechnology industry because it can catalyse the formation of specific carbon-carbon bonds with high stereospecificity and selectivity. These characteristics make TK interesting for the formation of high-value chemicals and pharmaceutical intermediates such as those used to synthesise antibiotics and others according to the substrates on the bioconversion. However, its application within large-scale processes is currently limited by low activity on new reactions, and poor stability at the high temperatures often used during industrial processes. One route to overcome these limitations is to use site-directed mutagenesis or directed evolution to improve the enzyme function and stability. The success of directed evolution relies upon designing a suitable screening method that can directly identify the best mutants from large numbers of variants, with the desired set of attributes. This thesis aims to develop an improved screening platform by adaptation of a previous screening method based upon colorimetric reactions. To assess and quantify TK activity towards the conversion of lithium hydroxypyruvate (Li-HPA) and propionaldehyde (PA) to (3S)-1,3-dihydroxypentan-2-one (HK), over a wide range of substrate concentrations. Moreover, several experiments were performed to establish the best conditions to grow E. coli for TK production, protein extraction methods and quantification of TK. In addition, PCR conditions were established for the development of mutagenic libraries using the MEGAWHOP. Finally, five different truncated TK variants were generated, all of them showed activity using Li-HPA, glycolaldehyde (GA) and PA as substrates. Results obtained in this project set up the basis to generate TK variants with better stability and activity, screen large numbers of variants using the high-throughput platform developed and finally it was shown that truncated versions of TK could keep its activity.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Development of a high-throughput screening method for transketolase and protein engineering for biotechnology applications |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/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. |
| Keywords: | biochemical engineering, directed evolution, transketolase, high throughput screening methods, PCR, mutagenic library |
| 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 Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10128371 |
Archive Staff Only
 |
View Item |
