The use of Diels Alder cycloaddition chemistries to access a diverse range of useful cyclic structures is well established throughout literature. However, the value of the product may be enhanced further still by linking this reaction with subsequent (biocatalytic) steps to create novel, structurally demanding, optically pure compounds. This project investigates the linking of Diels Alder (DA) chemistry to the enzyme, transketolase (TK) as a model integration pathway of a chemical syntheses and a biological transformation. The two-step process aims to provide a framework to synthesise small structurally diverse compounds with high enantiomeric excess. The demand for optically pure compounds is becoming a necessity due to the adverse affects frequently introduced by racemic compounds and the cost implications of the material possessing often only 50% active compound. Recombinant wild type Eschericha coli transketolase (EC 2.2.1.1) (WT-TK) was overexpressed in E. coli for the biocatalytic step of this two step synthesis. A substrate walking approach whereby a range of sequentially linked cyclic aldehydes, were applied to wild type transketolase and potential activity detected. Transketolase mutants, previously constructed based on information derived from the structural position within the active site of the dimeric enzyme were subsequently screened for activity with the cycloadduct of the Diels Alder reaction as aldehyde acceptor substrate for TK. Following identification, selection, culturing and sequencing of variants indicating enhanced activity towards the novel, bulky, hydrophobic, cyclic aldehyde the enantioselectivity and absolute stereochemistry of the product were determined. Activity displayed by wild type transketolase indicated an 18,000 fold activity improvement. Michaelis- Menten kinetic parameters were subsequently determined to have an apparent Km of 69.9 mM, kcat of 17.5 s-1 and a vmax of 0.07 mM.min-1 and preliminary process compatibility including product and substrate inhibition issues were highlighted.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:565763 |
| Date | January 2012 |
| Creators | Coward, L. G. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1370575/ |
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