Plete consumptionof caffeine contained 1 mM caffeine with an OD600 of 10, which made 886 14 7-methylxanthine (Fig. 2C and F). We also examined caffeine concentrations of 10mM, 25mM, and 50 mM having a total OD600 of 100, but we observed a decreasing activity with improved caffeine concentrations, suggesting that higher caffeine concentrations could possibly be inhibiting towards the reaction (information not shown). A total cell OD600 of 50 was chosen as the optimal cellular concentration for the production of 7-methylxanthine from caffeine. To achieve total consumption of caffeine, cells at the chosen density have been reacted with approximately two.five mM caffeine (Fig. three), in lieu of the previous 5 mM (Fig. 2A and D), resulting within the comprehensive conversion of 2,293 24 caffeine to 61 25 theobromine and two,233 26 7-methylxanthine soon after five hours. In the course of the reaction caffeine was completely consumed within 3 hours, nevertheless it took 5 hours to convert a lot of the theobromine to 7-methylxanthine.7methylxanthine purification and recoveryTable 1 Comparison of Concentrations of Caffeine Consumed and Compounds Created by Varied Cell Densities of pADP1 and pBDP1aOD600 (NdmA/ NdmB) 100:0 75:25 50:50 25:75 0:aCaffeine Consumed (M) 1022 19 961 64 643 25 62 Theobromine Developed (M) 988 24 24 11 -7Methylxanthine Produced (M) ten 545 315 493 667 19 22 481 Reported concentrations are averages of triplicate 1 mL reactions sampled in the conclusion of a 5-hour reaction initiated by the addition of 1 mM caffeineThe two,233 26 7-methylxanthine made in our small-scale mixed resting cell assay suggested that there would be adequate item for isolation offered a larger reaction volume. Consequently, we proceeded to scale up the reaction. The pADP1 and pBDP1 E. coli strains were every single grown in three two.8 L Fernbach flasks for a total of six 1 L cultures, resulting in adequate cells for a 560 mL mixed resting cell reaction containing a 50A:50B cell ratio (total OD600 of 50) and two.five mM caffeine. The cell-caffeine mixture was permitted to react for 5 hours to make sure maximum conversion before harvesting. At the conclusion from the large-scale reaction, caffeine was completely degraded, producing 2.14 mM 7-methylxanthine and 0.235 mM theobromine (Fig. S4). This resulted in an 85.six mol conversion of caffeine to 7-methylxanthine and also a 9.4 mol conversion to theobromine. The remaining 5 mol of consumed caffeine could be accounted for in two unidentified HPLC peaks at 3.44 and four.58 min not observed within a cell-only control [31]. Preparation with the reaction by 0.two m filtration and also the addition of methanol to a final concentration of five for HPLC purification resulted in 542.Piperonylic acid Protocol 85 mL supernatant, which was separated by preparatory-scale HPLC as described previously [31, 32].α-MSH Purity & Documentation Offered that the final concentration of 7-methylxanthine produced was 2.PMID:23880095 14 mM, the theoretical maximum volume of 7-methylxanthine that may very well be recovered from this procedure was 183.eight mg. The purification procedure was thriving in separating contaminants and undesirable compounds in the desired 7-methylxanthine with minimal loss of your solution, resulting inside a separation efficiency of 93.30 (Table S2). Following purification, the collectedMock and Summers Journal of Biological Engineering(2023) 17:Web page 5 ofFig. two Mixed-culture resting cell reactions consumed caffeine and generated 7-methylxanthine. Equal concentrations of pADP1 and pBDP1 cells have been reacted with all the following initial caffeine concentrations and in the follo.