Stabilization of microbial cytochrome P-450 activity by creation of stationary-phase conditions in a continuously operated immobilized-cell reactor

Abstract

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygenase activity (referred to herein as Cyt P-450 meg) catalyzing 15β-steroid hydroxylation. This activity belongs to the widespread ferredoxin reductase- ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The level of Cyt P-450 meg activity reaches its maximum in the cells during the stationary phase of the growth curve and is not affected by Cyt P-450 inducers. Here we present the development of an approach for stabilizing the Cyt P-450 meg system so that it performs continuous steroid hydroxylation and will be a model system for Cyt P-450- based detoxification. It is based on cell immobilization and simulation of stationary-phase conditions in a continuously operated fluidized-bed bioreactor. The combination of an appropriate immobilization technique, operational conditions, and medium composition provided a stabilized cell environment resulting in 'freezing' of a physiological steady-state analog under stationary-phase conditions, allowing stable performance of continuous hydroxylation for several weeks. It is suggested that this approach may be extended for use with other environmentally induced enzymatic activities.