Electrochemical Cofactor Regeneration Research Articles

Enhancing Efficiency and Selectivity of Electrochemical Regeneration of NADH By Dynamic Operation

The demand for the reduced nicotinamide adenine dinucleotide (1,4-NADH) and reduced nicotinamide adenine dinucleotide phosphate 1,4-NAD(P)H in industrial processes, particularly in the pharmaceutical sector, necessitates their continuous regeneration to mitigate cost implications [1]. In this respect, direct electrochemical cofactor regeneration is a promising alternative, but challenges in achieving optimal selectivity have hindered its widespread industrial integration.This study focuses on advancing the efficiency and selectivity of electrochemical NADH regeneration through dynamic potential inputs, inspired by recent findings demonstrating improved selectivity using dynamic potential inputs instead of steady-state conditions [2]. Various dynamic, pulsed potential profiles are tested and compared with steady-state conditions, revealing distinct advantages in selectivity. Nicotinamide adenine dinucleotide reduction reaction is then coupled with the enzymatic conversion of a substrate (cyclohexenone) to a product (cyclohexanone). In this electroenzymatic biotransformation, 1,4-NADH formed electrochemically is utilized as a second substrate of enzyme enoate reductase. The success of this coupling is evident in the observed formation of the desired product. This research not only contributes to the fundamental understanding of electrochemical co-factor regeneration but also demonstrates its practical application. By combining the advantages of electrochemical and enzymatic reactions, we pave the way for more sustainable and economically viable industrial processes. The presented results mark a significant step towards harnessing the full potential of electrochemical regeneration in facilitating enzymatic transformations with enhanced efficiency and selectivity through a pulsed dynamic operation.

Construction of a bioelectrocatalytic system with bacterial surface displayed enzyme-nanomaterial hybrids

To take advantage of the high specificity of enzymatic catalysis along with the high efficiency of electrochemical cofactor regeneration, a bacterial surface displayed enzyme-nanomaterial hybrid bioelectrocatalytic system is herein developed. A cofactor-dependent xylose reductase, capable of reducing xylose to xylitol, is displayed on the surface of Bacillus subtilis, followed by the attachment of copper nanomaterials via the binding of His-tagged enzyme with the nickel ion. This hybrid system can regenerate NADPH with a highest efficiency of 71.6% in 4 h without the usage of extra electron mediators, and 2.35 mM of xylitol can be synthesized after a series of optimization processes. This work opens up new possibilities for the construction and application of bioelectrocatalytic systems with enzyme-nanomaterial hybrids.

Renewable hydrogen production and storage via enzymatic interconversion of CO2 and formate with electrochemical cofactor regeneration.

The urgent need to reduce CO2 emissions has motivated the development of CO2 capture and utilization technologies. An emerging application is CO2 transformation into storage chemicals for clean energy carriers. Formic acid (FA), a valuable product of CO2 reduction, is an excellent hydrogen carrier. CO2 conversion to FA, followed by H2 release from FA, are conventionally chemically catalyzed. Biocatalysts offer a highly specific and less energy intensive alternative. CO2 conversion to formate is catalyzed by formate dehydrogenase (FDH), which usually requires a cofactor to function. Several FDHs have been incorporated in bioelectrochemical systems where formate is produced by the biocathode and the cofactor is electrochemically regenerated. H2 production from formate is also catalyzed by several microorganisms possessing either formate hydrogenlyase or hydrogen-dependent CO2 reductase complexes. Combination of these two processes can lead to a CO2-recycling cycle for H2 production, storage, and release with potentially lower environmental impact than conventional methods.

Enzymatic Electrosynthesis of Glycine from CO2 and NH3

AbstractEnzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2. However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value‐added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L−1 h−1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Enzymatic Electrosynthesis of Glycine from CO2 and NH3.

Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2 . However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L-1 h-1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.

Covalent Immobilization of Dehydrogenases on Carbon Felt for Reusable Anodes with Effective Electrochemical Cofactor Regeneration.

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon-felt-based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD+ ), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD+ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD+ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches.

Wastewater-powered high-value chemical synthesis in a hybrid bioelectrochemical system

SummaryA microbial electrochemical system could potentially be applied as a biosynthesis platform by extracting wastewater energy while converting it to value-added chemicals. However, the unfavorable thermodynamics and sluggish kinetics of in vivo whole-cell cathodic catalysis largely limit product diversity and value. Herein, we convert the in vivo cathodic reaction to in vitro enzymatic catalysis and develop a microbe-enzyme hybrid bioelectrochemical system (BES), where microbes release the electricity from wastewater (anode) to power enzymatic catalysis (cathode). Three representative examples for the synthesis of pharmaceutically relevant compounds, including halofunctionalized oleic acid based on a cascade reaction, (4-chlorophenyl)-(pyridin-2-yl)-methanol based on electrochemical cofactor regeneration, and l-3,4-dihydroxyphenylalanine based on electrochemical reduction, were demonstrated. According to the techno-economic analysis, this system could deliver high system profit, opening an avenue to a potentially viable wastewater-to-profit process while shedding scientific light on hybrid BES mechanisms toward a sustainable reuse of wastewater.

Improved soluble expression and use of recombinant human renalase.

Electrochemical bioreactor systems have enjoyed significant attention in the past few decades, particularly because of their applications to biobatteries, artificial photosynthetic systems, and microbial electrosynthesis. A key opportunity with electrochemical bioreactors is the ability to employ cofactor regeneration strategies critical in oxidative and reductive enzymatic and cell-based biotransformations. Electrochemical cofactor regeneration presents several advantages over other current cofactor regeneration systems, such as chemoenzymatic multi-enzyme reactions, because there is no need for a sacrificial substrate and a recycling enzyme. Additionally, process monitoring is simpler and downstream processing is less costly. However, the direct electrochemical reduction of NAD(P)+ on a cathode may produce adventitious side products, including isomers of NAD(P)H that can act as potent competitive inhibitors to NAD(P)H-requiring enzymes such as dehydrogenases. To overcome this limitation, we examined how nature addresses the adventitious formation of isomers of NAD(P)H. Specifically, renalases are enzymes that catalyze the oxidation of 1,2- and 1,6-NAD(P)H to NAD(P)+, yielding an effective recycling of unproductive NAD(P)H isomers. We designed several mutants of recombinant human renalase isoform 1 (rhRen1), expressed them in E. coli BL21(DE3) to enhance protein solubility, and evaluated the activity profiles of the renalase variants against NAD(P)H isomers. The potential for rhRen1 to be employed in engineering applications was then assessed in view of the enzyme’s stability upon immobilization. Finally, comparative modeling was performed to assess the underlying reasons for the enhanced solubility and activity of the mutant enzymes.

An l-glucitol oxidizing dehydrogenase from Bradyrhizobium japonicum USDA 110 for production of d-sorbose with enzymatic or electrochemical cofactor regeneration

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an L-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with D-glucitol oxidation to D-fructose but also converted L-glucitol to D-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K m value for L-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a D-sorbitol dehydrogenase (EC 1.1.1.14).

Development of a high performance electrochemical cofactor regeneration module and its application to the continuous reduction of FAD

Abstract Technical reactor limitations and low productivities have been shown to limit the implementation of electroenzymatic syntheses beyond lab-scale. One possible solution is a continuous flow-through reactor based on electrochemical plate and 6 frame filter press cells, as proposed in this study. With the aim of maximizing electroenzymatic productivities, the developed reactor set-up allows high electrochemical cofactor regeneration rates using porous, three-dimensional reticulated vitreous carbon electrodes with exceptionally large surface areas up to 19,685 m2 m−3. This system provides increased mass transfer rates and flavin adeninedinucleotide (FAD) was reduced at rates up to 93 mM h−1. The electrochemical FAD reduction was coupled to the styrene monooxygenase (StyA) catalyzed (S)-epoxidation of styrene. Electroenzymatic productivities increased with FAD reduction rates up to 1.3 mM h−1. This set-up now set the stage for efficient in vitro FAD regeneration and allows a broad electrochemical application of flavin dependent enzymes as biocatalysts.

Electrochemical Screening of Redox Mediators for Electrochemical Regeneration of NADH

We report the electrochemical cofactor regeneration for electroenzymatic reduction as a promising alternative to the enzymatic recycling due to the avoidance of couple products. In the mediated electron transfer reaction appropriate redox mediators for the cofactor reduction are necessary. We have synthesized different redox mediators-based on rhodium complexes-and have screened their electrochemical performance at different pH values and concentrations. Suitable mediators have been tested in the presence of the cofactor NAD+ and then additionally in the presence of D-sorbitol dehydrogenase (DSDH) from Rhodobacter sphaeroides. The electroenzymatically generated product was analyzed by HPLC. As result of the screening some mediator species were identified which enable an efficient electrochemical cofactor reduction.

Electroenzymatic Oxidation and Reduction of Directed Immobilized Dehydrogenases for Electrochemical Cofactor Regeneration

not Available.

Directed Immobilisation of Modificated Galactitol-Dehydrogenase on Gold Electrodes for Electrochemical Cofactor Regeneration

A supplemental cysteine-tag modification of the enzyme galactitol dehydrogenase (GatDH), expressed in Escherichia coli has enabled a directed and covalent immobilisation of the protein on gold surfaces without secondary linkers. The successful enzyme immobilisation was verified by SPR measurements, and the activity of the immobilized GatDH was demonstrated by cyclic voltammetry (CV) while NAD+ and the redox mediator 4-carboxy-2,5,7-trinitrofluorenyliden-malonnitrile (CTFM) were both kept in solution. The results represent a proof of concept for the development of electrochemical reactors for the generation of enantiopure fine chemicals as building blocks for pharmaceutical products.

Synthesis of α-ketoglutarate using glutamate dehydrogenase combined with electrochemical cofactor regeneration system

Synthesis of α-ketoglutarate using glutamate dehydrogenase combined with electrochemical cofactor regeneration system

Productive Asymmetric Styrene Epoxidation Based on a Next Generation Electroenzymatic Methodology

AbstractWe have established a novel and scalable methodology for the productive coupling of redox enzymes to reductive electrochemical cofactor regeneration relying on efficient mass transfer of the cofactor to the electron‐delivering cathode. Proof of concept is provided by styrene monooxygenase (StyA) catalyzing the asymmetric (S)‐epoxidation of styrene with high enantiomeric excess, space‐time yields, and current efficiencies. Highly porous reticulated vitreous carbon electrodes, maximized in volumetric surface area, were employed in a flow‐through mode to rapidly regenerate the consumed FADH2 cofactor required for StyA activity. A systematic investigation of the parameters determining cofactor mass transfer revealed that low FAD concentrations and high flow rates enabled the continuous synthesis of the product (S)‐styrene oxide at high rates, while at the same time the accumulation of the side‐products acetophenone and phenylacetaldehyde was minimized. At 10 μM FAD and a flow rate of 150 mL min−1, an average space‐time yield of 0.35 g L−1 h−1 could be achieved during 2 h with a final (S)‐styrene oxide yield of 75.2%. At two‐fold lower aeration rates, the electroenzymatic reaction could be sustained for 12 h, albeit at the expense of lower (59%) overall space‐time yields. Under these conditions, as much as 20.5% of the utilized current could be channeled into (S)‐styrene oxide formation. In comparison with state‐of‐the‐art electroenzymatic methodologies for the same conversion, (S)‐styrene oxide synthesis could be improved up to 150‐fold with respect to both reaction time and space‐time yield. These productivities constitute the most efficient reaction reported for asymmetric in vitro epoxidations of styrene.

Directed Immobilization of Modificated Galactitol-Dehydrogenase on Gold Electrodes for Electrochemical Cofactor Regeneration

not Available.

Regeneration of the nicotinamide cofactor using a mediator-free electrochemical method with a tin oxide electrode

Tin (IV) oxide was made using an anodization and annealing method and was used as a working electrode in an electrochemical cofactor regeneration reaction. This material was formed with a large surface area, and by changing the preparation conditions, it was possible to control the morphology. Tin oxide has redox properties similar to those of frequently used mediators required for electron transfer between cofactors and an electrode. Therefore, by using tin oxide as a novel electrode, mediator-free electrochemical cofactor regeneration may be possible. Oxidation and reduction of the nicotinamide cofactors, NAD(P)H and NAD(P) +, were carried out under various reaction conditions. The results showed a high efficiency for oxidizing NADH over a broad range of pH and temperatures. The oxidation tendency of NADPH was also observed, and it demonstrated a similar reaction tendency as NADH. When using a tin oxide electrode, NAD + was readily reduced to NADH, though the efficiency of this reaction was lower than for NADH oxidation. Oxidation of 2-propanol to acetone was used as a model system using alcohol dehydrogenase and the cofactor regeneration system suggested in this study. The electroenzymatic reaction showed efficient regeneration of NADP + without a mediator.

Synthesis, Characterization and Application of New Rhodium Complexes for Indirect Electrochemical Cofactor Regeneration

AbstractElectroenzymatic synthesis often suffers from electrochemical reaction steps which proceed slower than the coupled enzyme reaction. For indirect electrochemical cofactor regeneration, we here report two new mediators with superior properties compared to the established rhodium complex (2,2′‐bipyridyl)(pentamethylcyclopentadienyl)rhodium [Cp*Rh(2,2′‐bipyridine)]. After constructing a robotic system for fast and reliable cyclic voltammetry measurements, we screened twelve rhodium complexes with substituted 2,2′‐bipyridine ligands for their reduction potentials and catalytic activity towards the reduction of NADP. Promising complexes were investigated in more detail by cyclic voltammetry and under batch electrolysis conditions. The new complexes Cp*Rh(5,5′‐methyl‐2,2′‐bipyridine) and Cp*Rh(4,4′‐methoxy‐2,2′‐bipyridine) reduced NADP to NADPH three times faster than the established mediator, resulting in volumetric productivities of up to 136 mmol L−1 d−1 and turnover frequencies of up to 113 h−1. This increased reaction rate of these new mediators makes indirect electrochemical approach significantly more competitive to other methods of cofactor regeneration.Abbreviations: ADH=alcohol dehydrogenase; Ag|AgCl=silver|silver chloride reference electrode; bpy=2,2′‐bipyridine; ci=current increase; Cp*=pentamethylcyclopentadienyl; CV=cyclic voltammetry; Ep=peak potential; equiv=equivalent; NADP/NADPH=nicotinamide adenine dinucleotide phosphate oxidised/reduced form.

Electroenzymatic synthesis of chiral alcohols in an aqueous–organic two-phase system

The reduction of acetophenone to ( R)-phenylethanol catalysed by the alcohol dehydrogenase from Lactobacillus brevis in combination with electrochemical regeneration of NADPH mediated by a rhodium complex is reported. The reaction in buffer solution was optimised with regard to high productivity (up to 14 gL −1d −1) and enantioselectivity (>99.9%). Enzyme stability under the reaction conditions was increased either by addition of bovine serum albumin as a sacrificial protein or by immobilisation, leading to full conversion and enzyme ttn’s of up to 75,000. To improve the utilisation of cofactor and mediator as well as to broaden the substrate spectrum to more hydrophobic substrates, we introduced an organic phase of methyl tert-butyl ether. This is the first reported two-phase approach for electrochemical cofactor regeneration, which yielded mediator and cofactor ttn’s twice as high as in the one-phase approach. Furthermore, a concentrated product solution of 180 mM enantiopure ( R)-phenylethanol was obtained, facilitating product work-up.

Productivity of Selective Electroenzymatic Reduction and Oxidation Reactions: Theoretical and Practical Considerations

AbstractThe volumetric productivities, final product concentration and total process times, of electroenzymatic processes comprising reductive electrochemical cofactor regeneration coupled to dehydrogenase or oxygenase catalyzed redox reactions determine process performance. Exemplified for the production of fine chemicals, operational windows were defined to consider these parameters. This theoretical approach allows the identification of limiting process parameters and promising process developments. Several biocatalytic processes for syntheses of specialty chemicals are already in a performance range of technical relevance. Electroenzymology thus evolved to a strong additional technology in the toolbox of biocatalysis and organic chemistry.