Senussi, TO;
(2007)
Directed evolution to modify the substrate specificity of transketolase, a carbon-carbon bond-forming enzyme.
Doctoral thesis , UCL (University College London).
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Abstract
Transketolase (TK) (E.C. 2.2.1.1) has significant potential as a biocatalyst in the production of pharmaceuticals and fine chemicals, catalysing the irreversible and stereospecific transfer of a C2 (1,2-dihydroxyethyl) moiety from the ketol donor substrate P-hydroxypyruvate (p-HPA) to a wide range of aldehyde acceptor substrates, such as glycoaldehyde (GA). Commercial application of TK is restricted by the limited availability and expense of P- HPA as well as its limited activity towards novel substrates. This project describes efforts to generate and identify variants of E. coli TK capable of accepting novel ketol donors and/or aldehyde acceptors. Variants were prepared by saturation site directed mutagenesis (SSDM), and characterised, using novel high-throughput HPLC and TLC screens. The model P-HPA and GA reaction, as well as a range of novel acceptors, such as propionaldehyde, benzaldehyde and hydroxybenzaldehyde were examined. HPLC assays were also developed for all aldehyde substrates, for the detailed analysis of enzyme variants identified from libraries by rapid HPLC or TLC. During such analysis a variety'of buffers were tested for suitability in novel screens. Mops and Hepes were both found to be capable of substrate conversion in the absence of transketolase, hence the discovery of the first ever mimetic reaction for transketolase. Two techniques were used to identify residues to target for random mutagenesis: phylogenetic library design (10 sites determined by S. Costelloe) and structural library design (10 site library generated and screened in collaboration with E. Hibbert). HPLC and TLC analysis identified variants with improved reaction rates towards the model HPA and GA reaction (A29E, A29D, I189Y and 46IS), and also towards propionaldehyde (H26A, H26T, H26K, R358I, H461S, D469S, and D469T), but failed to definitively identify a mutant capable of activity towards benzaldehyde.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Directed evolution to modify the substrate specificity of transketolase, a carbon-carbon bond-forming enzyme |
| Identifier: | PQ ETD:593419 |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1446090 |
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