STABILIZATION OF MICROBIAL CYTOCHROME-P-450 ACTIVITY BY CREATION OF STATIONARY-PHASE CONDITIONS IN A CONTINUOUSLY OPERATED IMMOBILIZED-CELLREACTOR

Bacillus megaterium (ATCC 13368) exhibits cytochrome P-450 monooxygena se activity (referred to herein as Cyt P-450 meg) catalyzing 15 beta-s teroid hydroxylation. This activity belongs to the widespread ferredox in reductase-ferredoxin-Cyt P-450 type of monooxygenases, providing a representative model system for this type of activity. The lever of Cy t P-450 meg activity reaches its maximum in the cells during the stati onary phase of the growth curve and is not affected by Cyt P-450 induc ers. Here,ve present the development of an approach for stabilizing th e Cyt P-450 meg system so that it performs continuous steroid hydroxyl ation and will be a model system for Cyt P-450-based detoxification. I t is based on cell immobilization and simulation of stationary-phase c onditions in a continuously operated fluidized-bed bioreactor. The com bination of an appropriate immobilization technique, operational condi tions, and medium composition provided a stabilized cell environment r esulting 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 m ay be extended for use with other environmentally induced enzymatic ac tivities.