Enantioseparation using a counter-current bioreactor

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University.The potential of countercurrent chromatography (CCC) as a small footprint bioreactor/separator for manufacture of enantiopure chiral molecules was explored, using as a model reaction the isolation of L-amino butyric acid (L-ABA) from a DL-ABA racemate and the enantioselectivity of D-amino acid oxidase (DAAO). Bioconversion of D-ABA to ketobutyric acid (KBA) by DAAO, immobilised by selective partitioning in the stationary phase of the CCC centrifuge, was accompanied by separation of unreacted L-ABA from KBA by the countercurrent action of the centrifuge. For effective bioreactor/separator action, a high partition of the biocatalyst to the stationary phase was required in order to retain the biocatalyst in the coil, with differing partitions of substrates and products between the stationary phase (SP) and mobile phase (MP) so that these could be separated. Aqueous two-phase systems (ATPS) were the major two-phase systems used to provide SP and MP, as these are well reported to be effective in preserving enzyme activity. The distribution ratios of DL-ABA, KBA and DAAO were measured in a range of phases with polyethylene glycols (PEGs) of different molecular weights, different salts, and different compositions of PEG and salt, using an automated robotic method, developed for the purpose. A system of 14% w/w PEG 1000/ 14% w/w potassium phosphate, pH 7.6, gave the best combination of distributions ratios (CPEG phase/Csalt phase = CSP/CMP) for ABA, KBA and biocatalyst (DAAO) of 0.6, 2.4 and 19.6 respectively. A limited number of aqueous-organic and ionic liquid two-phase systems were also reviewed, but found unsatisfactory. CCC operating conditions such as substrate concentration, biocatalyst concentration, the mobile phase flow rate (residence time in the CCC coil), temperature, rotational speed and operational modes (single flow and multiple-dual flow) and types of mixing (cascade and wave-like) were optimised to produce total conversion of D-ABA to KBA, which was then completely separated from unreacted, enantiomerically pure (>99% ee), LABA. Advantages of the CCC bioreactor over conventional technology include reduced equipment footprint, cheaper running costs, and faster purifications. However, in its current format the drawbacks, such as enzyme instability and excessive optimisation time, reduce its commercial appeal. Additional investigations into the use of whole cell preparations of biocatalyst in the CCC bioreactor showed potential to overcome the problem of enzyme instability and this may in the future give the CCC bioreactor a place in the enantioseparation field