Figure 1.

Steroid bioconversions by whole cells (WC) and cell-free extract (CFE) of E. coli C43(DE3) (pIT2cyp154C5) (pACYCcamAB) employing each 3 μM CYP154C5. Results are given as conversions (%) and total turnover numbers (TTN, μmol of substrate consumed per μmol of CYP154C5 present in the reaction mixture) obtained in biotransformations of pregnenolone (1), progesterone (3) and testosterone (5) using different initial substrate concentrations. All reactions were carried out in 50 mM potassium phosphate buffer pH 7.4 at 30°C for 20 h. Substrates were added from stock solutions in 36% w/v hydroxypropyl-β-cyclodextrin in water. For cofactor regeneration in reactions using CFE, 0.5 U/mL formate dehydrogenase from Candida boidinii and 150 mM sodium formate were employed. In reactions using whole cells cofactor regeneration was achieved by addition of 30 mM glucose.