Process issues in redox biocatalysis: Cyclohexanone monooxygenase catalysed chiral lactone syntheses

This thesis investigates the Baeyer-Villiger oxidation of cyclic ketones to optically enriched lactones by the enzyme cyclohexanone monooxygenase (CHMO), cloned into Escherichia coli JM107 pQR210. Two model substrates were selected (2-hexyl cyclopentanone and 4-methyl cyclohexanone) to conduct investigations with. A major constraint found was that whole cell catalysis produced low reaction rates and poor enzyme stability. Isolated enzyme was stabilised effectively by using elevated levels of the cofactor NADPH. Recycle of the expensive NADPH was investigated by detailed studies of thermostable glucose and alcohol dehydrogenases. These were characterised by marked product inhibition. Alcohol dehydrogenase from Thermoanaerobacter brockii (TBADH) was chosen for the ease of removal of the acetone product from the system and the high affinity for NADPH. The interaction between CHMO and TBADH was modeled by simultaneous numerical integration of their rate equations leading to an understanding of the effect of different enzyme ratios on system performance. This model also predicts the conditions necessary to maximise cofactor stability and re-usability. Quantification of a range of processing strategies was performed, fed-batch operation was found to be 2.5 times more productive than batch. Multi-gram syntheses of lactones were performed at 2L scale with both free and immobilised enzymes. NADPH recycle was effective at producing over 700 reaction cycles. Immobilised CHMO was found to be significantly more stable than free enzyme under process conditions, a catalyst with retained activity of 12% and specific activity of 1.2Ug-1 was produced. TBADH produced 42% retained and 13.6Ug-1 specific activity. Co-immobilisation of both enzymes on the same support produced a catalyst with an activity of 0.6Ug-1