Figure 3
Time course of dNS production under optimized reaction conditions. The initial 10.5-ml reaction mixture contained 600 mM glucose, 250 mM acetaldehyde, 30 mM adenine, 25 mM MgSO4·7H2O, 20 mM potassium phosphate buffer (pH 7.0), 1.0 mM MnCl2·4H2O, 0.1 mM glucose 1,6-diphosphate, 0.4% (v/v) polyoxyethylenelaurylamine, 1.0% (v/v) xylene, 10 mM adenosine, 4% (w/v) acetone-dried yeast, 15% (w/v) wet E. coli BL21/pACDR-pTS17 cells, and 30 U/ml commercial PNPase. The reaction was performed in pH 6.8 (without adjusting), 10°C for 30 h with shaking (120 rpm). During the dNS-forming reaction, 10 N NaOH was used as the alkali solution to adjust the pH to approximately pH 7.5 at 5 h and 18 h. In addition, 0.8% (w/v) acetone-dried yeast, 5 mM adenosine, 25 mM adenine, and 90 mM acetaldehyde were added at 5 h and 18 h. The arrowheads indicate these feeding points. Three separate experiments were performed. Open circles depict the average dI production. Closed circles with the dotted line depict the averages of the sum of the residual nucleosides (adenosine and inosine) and nucleobase (adenine and hypoxanthine). Error bars depict the standard deviations.