CONTINUOUS STEREOSPECIFIC DELTA-4-3-KETO-STEROID REDUCTION BY PAAH BEAD-ENTRAPPED CLOSTRIDIUM-PARAPUTRIFICUM CELLS

The development of a continuous anaerobic process for stereospecific Δ4-3-keto-steroid reduction by immobilized Clostridium paraputrificum cells cells is described. Following a study on conditions for cell growth and sporulation, spores of C. paraputrificum were aseptically immobilized in PAAH beads. Conditions for cell growth and induction in the immobilized state were determined, as well as the medium composition required to maintain a stabilized immobilized cell population. The effect of the concentration of ethylene glycol added as selected cosolvent on reaction kinetics, substrate solubility, specific activity, and cell growth, was investigated. A 10% (v/v) cosolvent input provided maximal activity along with enhanced solubility of the steroidal substrate. It was shown that cell growth was enhanced in the presence of the added cosolvent in addition to its effect on substrate solubility and enzymic activity. The immobilized cells readily performed Δ4, as well as 3-keto steroid reduction of several steroids, including ADD, AD, 16-dehydroprogesterone, progesterone, and hydrocortisone. It was shown that repeated batch-wise reduction cycle-in the presence of the cosolvent-resulted in rapid loss of activity, while the continuous uninterrupted process permitted the attaining of full bioconversion level, maintained stable for at least the period of 5 days of continuous operation tested. © 1990.