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Title: Evaluation of a novel dual resin substrate feed-product removal (SFPR) strategy applied to an oxidative bioconversion
Author: Raja, N. Y.
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 2013
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A novel dual resin based, substrate feed and product removal (SFPR) strategy has been investigated to overcome the substrate and product inhibition in an industrially important Baeyer-Villiger monooxygenase catalysed bioconversion in order to enhance the productivity of the bioconversion process. The bioconversion of the ketone substrate, bicyclo[3.2.0]hept-2-en-6-one, to the lactone products, (1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one, catalysed by a recombinant whole cell biocatalyst, Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus, was used as the model reaction to prove the feasibility of the novel dual resin SFPR concept. Before the application of the dual resin SFPR strategy to the Baeyer-Villiger bioconversion, adsorption of the ketone and lactone onto non-specific resins was investigated. Several resins were initially characterised at the bench scale by determining adsorption isotherms for the ketone and lactone compounds. Thereafter adsorption isotherms were generated via a high throughput resin screening (HTRS) method using both 96 wells and 24 wells microplate platforms. Comparison of the adsorption isotherm data between the bench scale and the two HTRS platforms, together with results of resin mixing in wells of the 96 wells and 24 wells microplate platforms, as investigated by high speed imaging experiments, shows that the 24 well microplate platform was the most suitable to investigate adsorption kinetics of the ketone and lactone on the resins. Based on the adsorption studies, resins Dowex® Optipore L493 and Amberlite® XAD7 were chosen to be separately used for substrate feeding in the dual resin SFPR strategy. Dowex® Optipore L493 was chosen for its high capacity of 0.21 g/gadsorbent for ketone, whereas Amberlite® XAD7 was chosen for its high selectivity of ketone over lactone compared to any other resin. Amberlite® IRC50 was chosen for lactone removal in the dual resin SFPR strategy because of its high selectivity of lactone over ketone than any other resin. To demonstrate the feasibility of the dual resin SFPR strategy, the Baeyer-Villiger bioconversion was performed in shake flasks and compared to bioconversions without the use of resins and with the use of a single resin based SFPR strategy. At an initial ketone concentration of 3g/l, both resin based strategies performed significantly better than the bioconversion performed without resins. The dual resin SFPR strategy, carried out with both types of resins free in suspension without spatial separation, also showed improvement compared to results obtained with the single resin SFPR strategy. The dual resin SFPR strategy was also performed with the spatial separation of the two resins by housing one of the resins in a porous bag. This allowed observation of the majority of lactone product adsorbed onto the Amberlite® IRC50 resin as expected based on adsorption studies. Carrying out the Baeyer-Villiger bioconversion with the implementation of the dual resin SFPR strategy in shake flasks saw an increase of productivity compared to the Baeyer-Villiger bioconversions carried out without resins by as much as 132% and in comparison with the single resin SFPR strategy by as much 10%, thus demonstrating a ‘proof of concept’ of the novel dual resin SFPR bioconversion strategy. After demonstrating a ‘proof of concept’ for the dual resin SFPR strategy, its application in a miniature stirred tank bioreactor was investigated to open the way for scale up studies. Two configurations were investigated, namely the conventional reactor system where both resins were added directly into the bioreactor, and the recycle reactor system where a column housed one of the two types of resins. Using resins with low adsorption capacities and the need of an extra resin type in a dual resin SFPR strategy, makes the recycle reactor configuration a more attractive system, however it was the conventional reactor configuration that performed better than the recycle reactor system. The L493-IRC50 combination in the conventional reactor configuration achieves a 21% greater productivity than in the recycle reactor. The dual resin SFPR strategy using the L493-IRC50 combination performed better than any other resin based SFPR strategy when carried out with both resins in the reactor. It reached a productivity of 0.85g/l/h after 2.5 hours of reaction, 5% higher than the productivity achieved with the single resin strategy in the conventional reactor configuration. The novel dual resin based SFPR strategy and the HTRS method developed in this work has the potential to be applied in any bioconversion that needs to overcome substrate and product inhibition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available