Ferreria-Torres, C.; (2008) Microscale process characterisation of oxidative bioconversions. Doctoral thesis , University of London.

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Abstract

In this work, the potential of automated microscale process sequences for the evaluation of recombinant biocatalysts and the use of microwell data to inform larger scale operations is examined. As a model bioconversion, the cyclohexanone monooxygenase (CHMO) Baeyer-Villiger oxidation of a range of cyclic ketone substrates was examined cyclopentanone, cyclohexanone and bicyclo 3.2.0 hept-2-en-6-one. Three whole cell biocatalysts were evaluated which included two strains of E. coll. TOP10 pQR239 and JM107 and A. calcoaceticusNCIMB 9871. Initial studies to establish the microwell process used the well-characterised E. coli TOP 10 pQR239 biocatalyst that was previously prepared in our laboratory. It was shown that quantitative and reproducible data could be obtained for fermentation, enzyme induction and bioconversion operations carried out in 96 Deep Square Well (96 DSW) plate formats. It was possible to produce up to 5.8 gocw-l" of the E. coli biocatalyst and support growth rates up to 0.55 h". The biocatalyst had a specific CHMO activity of 11 U.gocw"1 and could catalyse the conversion of bicyclo 3.2.0 hept-2-en-6-one at an initial rate of product formation of 62 pmoLl min*1 with high yields of up to 92%. The microscale process sequence was then operated in an automated fashion using a Multiprobe II EX liquid handling robot (Packard Instrument Company, Meriden, Connecticut, US). The process performance of the three biocatalysts was then examined following a combinatorial approach in which biocatalyst was produced at initial glycerol concentrations of 10 and 20 in fermentation and, following induction (when required), bioconversion rates were measured for each of the three ketone substrates at initial concentrations of 0.5 g.l"1 and 1.0 g.l"1. The E. coli TOP10 pQR239 biocatalyst showed the best overall performance and the results obtained were comparable to those already reported in the literature. Finally, the ability of microwell results to be predictive of larger scale operations was examined. Given the demand for molecular oxygen at both, the fermentation and bioconversion stages, the oxygen mass transfer coefficient, kLa, was considered as a basis for scale-up. ki,a values in both shaken microwell plates and a 2 1 stirred bioreactor were determined over a range of operating conditions. Values in the range 15 - 188 h"1 could be obtained at both scales. Processes carried out at the two scales at matched a values showed excellent agreement both in terms of the quantitative values of the results obtained and the way in which the process performance varied with kia. The results obtained in this thesis further show the potential of automated microwell experimentation to support rapid and more cost effective bioprocess development.

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