Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

AMB Express

Table 1 Effect of external NADH addition on the activity of lyophilized P450 whole-cell catalysts

Lyophilized E.coli C43 (DE3) harboring	Testosterone conversion [%]		Formation of 2βhydroxytestosterone [%]
Lyophilized E.coli C43 (DE3) harboring	− NADH	+ NADH	− NADH	+ NADH
pET22b-cyp105D + pCOLADuet-pdx-pdr	≤ 1	3	≤ 1	3
pET22b-cyp105D + pCOLADuet-pdx-pdr-adh	53 ± 6	72 ± 5	46 ± 4	61± 4

Reaction conditions: 10 mg/mL lyophilized cells in 0.5 mL Phosphate Sucrose EDTA (PSE)-buffer with 1 × nutrient solution, pH 7.5, in 2 mL reaction tubes, 1 mM testosterone 1 dissolved in 5% (v/v) propan-2-ol final concentration, 25 °C, 1100 rpm, 20 h reaction time. 0.25 mM NADH was added up to four times at 0 h, 2 h, 4 and 6 h incubation. For the cells co-expressing the adh, 0.5 mM NADH were added after 4 h. Experiments were performed in technical duplicates