Investigation of process stability of a whole-cell biocatalyst with Baeyer–Villiger monooxygenase activity in continuous bioreactors

Abstract

The stability of a whole-cell biocatalyst based on genetically modified Escherichia coli cells harboring cyclohexanone monooxygenase was investigated in a free and immobilized form in continuous bioreactors. The biotransformation of a bicyclic ketone to a racemic mixture of the corresponding lactones by an enzymatic cascade was performed in a membrane bioreactor (MBR) for free cells and in a fluidized-bed bioreactor (FBBR) for immobilized cells. Both reactors were operated at the residence time of 6.25 h with the biocatalyst concentration of 1 % w/w. Different oxygen mass transfer rates were set via the impeller speed in MBR or air flow rate in FBBR, respectively. The potential of continuous bioreactors to achieve a steady state was utilized for the analysis of different factors affecting the biocatalyst stability. An apparent steady state could be maintained in these bioreactors up to 40 h. The operation at high conversions or high oxygenation rates allowed to exclude poisoning by substrate or oxygen as causes of biocatalyst activity loss. Further experiments showed that the cells did not lose viability or plasmid. The problem in biocatalyst stability was not in the stability of cyclohexanone monooxygenase but in insufficient regeneration of cofactors. The only partially effective measure was the addition of catalase with the feed concentration of 0.025 g/L that decomposed hydrogen peroxide formed in the enzymatic cascade. However, both free and immobilized biocatalysts lost their activity completely in about a week.