Bacon, Sarah Louise; (2003) Recombinant transketolases with increased synthetic potential. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the progress made towards the development of recombinant enzymes for use in the catalysis of a carbon-carbon bond-forming reaction. Transketolase (TK) is one such enzyme and has considerable synthetic potential as a stereospecific C-C bond-forming reagent. Recombinant forms of TK from Escherichia coli have been produced, over-expressing to levels of 15-25% of the total cellular protein. The native enzyme is stereospecific for D-enantiomeric, linear sugar aldehyde acceptors. It also dictates the stereochemistry of the two new chiral centres in the reaction product. Furthermore, it has been shown to have activity with a range of simple α-unsubstituted aldehydes, so demonstrating a low specificity for the aldehyde component. Combined with the high stereoselectivity for the C-C bond-forming step, this makes TK an attractive catalyst for asymmetric synthesis. Using the enzyme crystal structure as a model for their design, a selection of TK mutants were generated with assorted combinations of amino acid changes located within the active site funnel. The synthetic potential of these modified TKs was analysed combinatorially with a range of aromatic aldehydes, using β-hydroxypyruvic acid as the ketol donor. Relaxation or reversal of the TK requirement for a D- α-hydroxyl group would provide even greater scope for its syntheic applications. Variants tailored for these needs were constructed and characterised using D-glyceraldehyde and DL-glyceraldehyde as aldehyde acceptors. In addition, an ion-exchange chromatographic method of purification was developed for a poly-lysine tagged version of TK, affording a 4.5-fold purification of the enzyme. The TK thus produced is available for subsequent investigations into its specific reactivity with a range of substrates.

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