The production and use of transketolase as a catalyst for carbon-carbon bond formation

This thesis describes the advances made towards the development of a complete pilot scale process for an enzyme catalysed carbon-carbon bond forming reaction. Transketolase is such an enzyme that has high synthetic potential as a stereospecific carbon-carbon bond forming reagent. Transketolase from Escherichia coli has been produced from recombinant forms of E. coli over-expressing the enzyme to a level of 25%–40% of the total protein. This level of expression has been achieved in high productivity fermentations up to 1000 L in scale. Transketolase was determined using a linked, multi-enzymic assay which was shown to be accurate and reliable using commercially available transketolase. The enzyme has also been purified to homogeneity and subsequently crystallised. An initial investigation into the substrate specificity of the enzyme illustrated the wide range of substrates accepted by this form of transketolase and led to the synthesis of two novel compounds. Further synthesis suggested that E. coli transketolase dictates the stereochemistry of two new chiral centres in the reaction product. Using the transketolase catalysed reaction between hydroxypyruvate and glycolaldehyde to form erythrulose as a model example, biotransformation scale-up conditions were mimicked to investigate the optimum conditions for reaction and productivity. Substrate concentrations of 0.5 M were used and the reaction was found not to be inhibited by high substrate concentrations but it was inhibited by the accumulation of product. Conversions of >95% were achieved based on the loss of glycolaldehyde, but the reaction only proceeded to -60% completion. The information derived from this research, and that of other members of the transketolase group, will help to provide more widely applicable guidelines for biotransformation process design, operation and scale-up