Enzymatic Synthesis via Electrochemical Regeneration of NADH at an Enzyme-Viologen Modified Electrode

Cross-Linked LDH Crystals for Lactate Synthesis Coupled to Electroenzymatic Regeneration of NADH

“…The pH optima for LiDH and LDH in solution differ by one unit (6.5 and 7.5, respectively). In previous experiments, , the pH of the electrolysis solution was buffered at 7.0, a compromise betwwen the optimal pH of each enzyme. As shown in Figure , the activity of cross-linked lactate dehydrogenase in 0.1 M phosphorous buffer is relatively constant throughout the pH range tested (4.0−9.0), which would allow the LDH crystals to be used over a much wider range of conditions than are normally possible with lactate dehydrogenase.…”

“…Immobilized LiDH Activity Assay. In a batch reactor 6,7 equipped with magnetic stirrer were mixed 15 mL of 0.05 M sodium phosphate buffer, pH 6.5; 0.45 mL of EDTA disodium salt (50 mM aqueous solution containing 2% bovine serum albumin); 0.45 mL of β−NADH (7.0 mM in 0.05 M phosphate buffer, pH 6.5); 0.45 mL of NAD + (20 mM in 0.05 M phosphate buffer, pH 6.5); 0.45 mL of thioctic amide (28 mM; 20 mg/2 mL of absolute ethanol and 1.5 mL of 0.05 M phosphate buffer, pH 6.5), and LiDH immobilized on a reticulated vitreous carbon (RVC) electrode under a Nafion membrane. , The contents of the reactor were circulated to a spectrophotometer flow microcell using an ISCO Model 2300 HPLC pump, and the change of absorbance at 340 nm was recorded.…”

“…Synthesis of l - Lactate by Preparative Electrolysis. We have described in detail the procedure used for these electrolyses in our previous publications. ,,, However, at the request of a reviewer of the paper, we repeat some of the details here. A 2 mg sample of LiDH is dissolved in 2 mL of 0.6 mM FAD + /2 mM NAD(H) solution, and the entire solution is evaporated onto the electrode by alternate syringe injection and evaporation by air drying.…”

“…A problem encountered in all such electroenzymatic reactions is the instability of the enzymes in solution. We and other research groups have shown that the first (NADH-regenerating) enzyme can be stabilized by immobilizing it on the electrode surface. ,− However, the instability of the second enzyme remains a problem. For this reason, it is generally necessary to add fresh enzyme to the electrolysis solution periodically during the course of an extended electrolysis. − , It is this problem which we wish to address in the present paper.…”

“…In our previously reported experiments, , the electroenzymatic regeneration of NADH was coupled with electroenzymatic syntheses of several organic acids. Lactic, malic, and glutamic acids were prepared successfully utilizing the appropriate substrates and redox enzymes.…”