Dual lifetime referencing enables pH-control for oxidoreductions in hydrogel-stabilized biphasic reaction systems

pH-shifts are a serious challenge in cofactor dependent biocatalytic oxidoreductions. Therefore, a pH control strategy was developed for reaction systems, where the pH value is not directly measurable. Such a reaction system is the biphasic aqueous-organic reaction system, where the oxidoreduction of hydrophobic substrates in organic solvents is catalysed by hydrogel-immobilized enzymes, and enzyme-coupled cofactor regeneration is accomplished via formate dehydrogenase, leading to a pH-shift. Dual lifetime referencing (DLR), a fluorescence spectroscopic method, was applied for online-monitoring of the pH-value within the immobilizates during the reaction, allowing for a controlled dosage of formic acid. It could be shown that by applying trisodium 8-hydroxypyrene-1, 3, 6-trisulfonate as pH indicator and Ru(II) tris(4, 7-diphenyl-1, 10-phenantroline) (Ru[dpp]) as a reference luminophore the control of the pH-value in a macroscopic gel-bead-stabilized aqueous/organic two phase system in a range of pH 6.5 to 8.0 is possible. An experimental proof of concept could maintain a stable pH of 7.5 +/- 0.15 during the reaction for at least 105 h. With these results, it could be shown that DLR is a powerful tool for pH-control within reaction systems with no direct access for conventional pH-measurement.