Yu, Haoran;
(2019)
Protein Engineering of Transketolase for Industrial Applications.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
The stereospecifically controlled carbon-carbon bond forming ability of transketolase (TK) makes it promising as a biocatalyst in industry. However, E. coli TK suffers the limitation of low stability to elevated temperatures and limited scope of substrates, hampering its use in industrial processes. Flexible sites were first selected as the mutation targets for engineering thermostability. Forty-nine single variants were designed and characterised experimentally. The combination of A282P with H192P resulted in the best all-round variant with a 3-fold improved half-life at 60 °C. The double-mutant (H192P/A282P), and two single-mutant (I365L and G506A) variants were then recombined and evaluated for pairwise epistatic interactions between mutations. The quadruple variant (H192P/A282P/I365L/G506A) was the most stable, with a 21-fold increase in half-life at 60 oC compared to wild type. Molecular dynamics correlation was identified to mediate the short and long-range epistatic effects of mutations on various measures of protein stability. These effects were then exploited to counteract the activity-stability trade-off of the variant S385Y/D469T/R520Q in which simulations revealed that the active sites became considerably more flexible when engineered to gain new function. Six variants were constructed and characterised, of which four increased the thermostability, while their activities were generally similar or improved. Mutations distant from the active site were shown to re-stabilise the active site, acting via a correlated molecular dynamics network. The most stable variant (H192P/A282P/I365L/G506A) was finally used as a template for simultaneously optimizing the donor substrate and acceptor substrate specificity. A small but smart library was constructed and screened towards novel substrates. Several variants were identified to be more active towards propionaldehyde and sodium pyruvate than WT and the best one showed 9.2- fold improvement compared to wild type. Some of the variants were also found to have high catalytic efficiency towards 3-formylbenzoic acid and sodium pyruvate which was unprecedented for TKs.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Protein Engineering of Transketolase for Industrial Applications |
| 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 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/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 UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10071529 |
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