Laminar Flow-Based Electrochemical Microreactor for Efficient Regeneration of Nicotinamide Cofactors for Biocatalysis

Abstract

One of the long-standing challenges in biocatalysis is the search for methods to continuously regenerate essential cofactors such as NADH that would enable a wide range of enzymes to be used in the more environmentally friendly synthesis of chiral fine chemicals including pharmaceuticals, cosmetics, and food additives. This communication reports a microreactor-based cofactor regeneration method that exploits the microfluidic phenomenon of laminar flow: a reactant stream and a buffer stream are introduced in a microchannel and continue to flow side by side without turbulent mixing between two electrodes that cover opposing channel walls. Adjustment of the flow rate ratio of the two streams in laminar flow enables focusing of the reactant stream close to the cathode, thereby reversing a normally unfavorable reaction equilibrium essential for cofactor regeneration. The absence of a bulk phase in these microreactors prevents the undesired reverse reaction to take place, which has prevented the use of electrochemical cofactor regeneration in macroscale processes. Here, we demonstrate the regeneration of NADH with conversion efficiencies as high as 31%. We also show the subsequent in situ conversion of an achiral substrate, pyruvate, into a chiral product, l-lactate, within this microreactor.

Electrochemical study in both classical cell and microreactors of flavin adenine dinucleotide as a redox mediator for NADH regeneration

“…The first of these was a previously designed one microfluidic Y-shaped channel [5]. The Y-shaped channel with integrated electrodes was sandwiched between gasket layers of PDMS and the dimensions of the microchannel are as follows: length = 3 cm; height = width = 1000 m. Both anolyte and catholyte flowed along the same channel.…”

“…Previous works carried out on gold [5,6] indicate that, in the cathodic potential range from −0.1 to −1 V of W (or Pt) wire, only the flavinic compound is reducible; the other species required for indirect pyruvate reduction to the chiral l-lactate molecule (formate dehydrogenase, lactate dehydrogenase, NAD + , NADH) are not electroactive. Solvent reduction takes place at potentials below approximately −1 V/Pt wire.…”

“…NAD + /NADH (nicotinamide adenine dinucleotide, oxidized and reduced forms) is a coenzyme that is involved as a cofactor in several [1][2][3][4][5][6] dehydrogenation reactions (catalyzed by enzymes such as dehydrogenases). Because of the high cost of the pyridinic cofactor (NADH), as well as for its very interesting reactivity, continuous regeneration represents an important industrial challenge.…”

“…In our previous works [3][4][5][6]19], the feasibility of continuous electrogeneration of NADH in a microreactor, using FAD/FADH 2 as a redox mediator, was experimentally demonstrated; in addition, mass balance experiments (and theoretical simulation) were carried out in order to use electrogenerated NADH for the syntheses of chiral l-lactate from achiral pyruvate. The aim of this study is to focus more specifically on the electrochemical behavior of the flavin on a gold electrode in phosphate buffer media in order to get a better understanding of the reversibility/irreversibility of this system, to access these physicochemical parameters and finally, to define the limiting step of its electrochemical reduction.…”

Enzymatic Processing in Microfluidic Reactors

“…Regeneration of coenzyme is the most difficult point in an enzymecatalyzed process. Regeneration of NADH was performed in a Y-shape microreactor, which possesses an electrode within the microchannel (Yoon et al, 2005). Another example has been reported by Koch et al (2008).…”