Electrochemical Regeneration Of NADH 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.

Functionalized Metal-Organic Framework for NADH Regeneration by Hydrogen in a Redox Flow Bioreactor.

The electrochemical regeneration of reduced nicotinamide adenine dinucleotide (NADH) using [Rh(Cp*)(bpy)Cl]+ holds significant promise for the industrial synthesis of chiral chemicals. However, challenges persist due to the high consumption of NADH and the limited efficiency of its cyclic regeneration, which currently hinder widespread application. To address these obstacles, based on in-situ growth of 3D ordered metal-organic framework (NU-1000) on the surface of graphite felt, [Rh(Cp*)(bpy)Cl]+ were immobilized on the Zr6 nodes of NU-1000 by solvent-assisted ligand incorporation (SALI), and applied in a flow bioreactor. Moreover, we employ a gas diffusion electrode (GDE) to oxidize H2, providing a clean proton source for the electrochemical regeneration of NADH. Consequently, highly efficient enzymatic electrocatalytic synthesis of L-lactate was achieved when coupled with L-lactate dehydrogenases (LDH) as a model reaction, and the total turnover number (TTN) reached 19600 and 1750 for [Rh(Cp*)(bpy)Cl]+ and NAD+ after 48 h, corresponding to a high turnover frequency (TOF) of 2350 h-1 and 210 h-1 for [Rh(Cp*)(bpy)Cl]+ and NAD+, respectively. This work provides new insights for the construction of efficient enzymatic electrosynthesis systems in industrial production.

Electrochemical NADH Regeneration Mediated by Pyridine Amidate Iridium Complexes Interconverting 1,4- and 1,6-NADH

Electrochemical NADH Regeneration Mediated by Pyridine Amidate Iridium Complexes Interconverting 1,4- and 1,6-NADH

Direct Electrochemical Regeneration of NADH and Its Biomimetics

Enzyme-catalyzed redox transformations are an attractive approach for conversion of unactivated alkanes into value-added products under mild reaction conditions. This is especially true since advancements in the area of enzyme engineering has led to a dramatic expansion in the use of modified oxidoreductases for selective functionalization of commodity petrochemicals in non-aqueous media. Despite their appeal, many redox enzymes rely on stoichiometric quantities of expensive coenzymes, such as nicotinamide adenine dinucleotide (NADH), that are not easily regenerated. The use of direct electrochemistry to catalytically regenerate NADH has long been an aspiration of chemists as a convenient and straightforward method that would enable use of the expensive cofactor in enzyme-catalyzed synthesis on an industrial scale. Unfortunately, direct electrochemical reduction of the oxidized form of nicotinamide adenine dinucleotide (NAD+) results in unstable radical intermediates that rapidly form biologically inactive byproducts. Recent progress has been made towards the development of inexpensive artificial NADH biomimetics as replacements for the natural coenzyme, but the strategies to regenerate these biomimetic coenzymes is equally limiting. A primary focus of our research is to design synthetic analogues of NAD+ and NADH that are both enzymatically active and electrochemically regenerable. We will present our recent progress in understanding the structure-reactivity relationships that lead to dimerization over the preferred formation of 1,4-dihydronicotinamide in NAD+ and its biomimetics, and we will describe strategies to prevent dimerization of electrochemically reduced NAD+.

Bioelectrocatalysis with a palladium membrane reactor

Enzyme catalysis is used to generate approximately 50,000 tons of value-added chemical products per year. Nearly a quarter of this production requires a stoichiometric cofactor such as NAD+/NADH. Given that NADH is expensive, it would be beneficial to regenerate it in a way that does not interfere with the enzymatic reaction. Water electrolysis could provide the proton and electron equivalent necessary to electrocatalytically convert NAD+ to NADH. However, this form of electrocatalytic NADH regeneration is challenged by the formation of inactive NAD2 dimers, the use of high overpotentials or mediators, and the long-term electrochemical instability of the enzyme during electrolysis. Here, we show a means of overcoming these challenges by using a bioelectrocatalytic palladium membrane reactor for electrochemical NADH regeneration from NAD+. This achievement is possible because the membrane reactor regenerates NADH through reaction of hydride with NAD+ in a compartment separated from the electrolysis compartment by a hydrogen-permselective Pd membrane. This separation of the enzymatic and electrolytic processes bypasses radical-induced NAD+ degradation and enables the operator to optimize conditions for the enzymatic reaction independent of the water electrolysis. This architecture, which mechanistic studies reveal utilizes hydride sourced from water, provides an opportunity for enzyme catalysis to be driven by clean electricity where the major waste product is oxygen gas.

Metal-organic framework functionalized bipolar electrodes for bulk electroenzymatic synthesis

Electrochemical regeneration of the cofactor nicotinamide adenine dinucleotide (NADH) is crucial for the enzymatic synthesis of a variety of valuable chemicals. [Rh(Cp*)(bpy)Cl]+ has been reported as one of the most efficient non-enzymatic catalysts for electrochemical regeneration of NADH, but the efficiency of the existing systems is still far from satisfying. Here we propose to suspend metal–organic framework functionalized glassy carbon beads in a bipolar electrochemical cell under mechanical stirring for bulk electroenzymatic synthesis. The beads are modified with a metal–organic framework NU-1000 shell, in which [Rh(Cp*)(bpy)Cl]+ catalyst is incorporated via the solvent-assisted ligand incorporation approach. Enzymatically active 1,4-NADH is electrochemically regenerated, and finally bipolar electrochemical bulk conversion of pyruvate into L-lactate is performed in the presence of NADH dependent L-lactate dehydrogenase as a model system. The very high turnover frequency (TOF) of 3100 ± 106 h−1 opens up promising perspectives for employing this concept also for various other electroenzymatic bulk processes.

Electroenzymatic Reduction of Furfural to Furfuryl Alcohol by an Electron Mediator and Enzyme Orderly Assembled Biocathode.

The electroenzymatic valorization of biomass derivatives into valuable biochemicals has a promising outlook. However, bottlenecks including poor electron transfer between the electrode surface and oxidoreductase, inefficient regeneration of cofactors, and high cost of enzymes and electron mediators hindered the realistic applications of the technique. Herein, to address the above technical barriers, a novel bio-electrocatalytic system that integrates the electrochemical NADH regeneration and enzymatic reaction was constructed, using an orderly assembled composite bioelectrode consisting of an outer immobilized enzyme layer and a sandwiched redox mediator rhodium complex layer. The as-prepared composite bioelectrode was further applied for the highly selective hydrogenation of furfural into furfural alcohol. Results indicated that the enzyme activity was significantly improved, while the furfural valorization was promoted by effective interfacial electron transition and co-factor regeneration on the composite bioelectrode. Considerable high furfural conversion (96.4%) can be achieved accompanied by a furfural alcohol selectivity of 90.0% at -1.2 V (vs Ag/AgCl). The novel composite bioelectrode also showed good stability and reusability. Up to 85.1% of the original furfural alcohol selectivity can be preserved after 10 times of recycling. This work presents a promising green alternative for the valorization of furfural, which also shows great potential extending to the valorization of other biomass compounds.

Influence of electrode potential, pH and NAD+ concentration on the electrochemical NADH regeneration

Electrochemical NAD+ reduction is a promising method to regenerate NADH for enzymatic reactions. Many different electrocatalysts have been tested in the search for high yields of the 1,4-isomer of NADH, the active NADH, but aside from electrode material, other system parameters such as pH, electrode potential and educt concentration also play a role in NADH regeneration. The effect of these last three parameters and the mechanisms behind their influence on NADH regeneration was systematically studied and presented in this paper. With percentages of active NADH ranging from 10 to 70% and faradaic efficiencies between 1 and 30%, it is clear that all three system parameters drastically affect the reaction outcome. As a proof of principle, the NAD+ reduction in the presence of pyruvate and lactate dehydrogenase was performed. It could be shown that the electrochemical NADH regeneration can also be done successfully in parallel to enzymatically usage of the regenerated cofactor.

Advances of bioelectrocatalysis by oxidoreductases

<p indent=0mm>Bioelectrocatalysis attracts much attention with the advantages of high selectivity, high efficiency, no byproduct and environmental friendliness. Electrification of industrial biotechnology is challenging for the different operating conditions and catalysts of electrochemical and biochemical reactions. In this review, we present the state-of-the-art information on enzymatic electrosynthesis for the production of fine chemicals. Multidisciplinary strategies of electrode surface modification and enzyme immobilization have been integrated in electrocatalytic devices with various applications in biosensors and bioelectrocatalysis. However, the efficiency of bioelectrosynthesis is determined by the intramolecular electron transfer rate, and interfacial electron transfer rate between the oxidoreductases and electrode. This review aims to summarize recent progresses on the mechanism studies of intramolecular electron transferof redox proteins, including tunneling, hopping, proton-coupled electron transfer, etc. Modular origin of electron transfer chain was also discussed from the perspective of the evolution of spatial adjacency network. Novel and efficient oxidoreductases with enhanced intramolecular and interfacial electron transfer rate can be obtained by protein engineering. The strategies for facilitating intramolecular electron transfer are addressed, which includes the regulation of co-evolved electron transfer network, the designs of molecular switch and the assembly of conductive modules. Oxidoreductases are engineered to improve their biocatalytic performance by using the tools of molecular evolution, modeling, structure prediction, and mutation. Co-evolved molecular switches controll proton-coupled electron transfer and regulate electron transfer inside the multi-center redox proteins. Assembly of surface-binding conductive peptides to oxidoreductases facilitates electricity-driven catalysis. Moreover, the modifications of oxidoreductases allow their predictable immobilization on functionalized electrode surface with improved stability, controlled orientation, surface coverage and enhanced electron transfer. After that, electron transfer within a series of well-defined peptides with orientation-controlled and surface-confined enzymes was addressed. Strategies also developed to increase the biocompatible active surface of electrodes and to promote charge transfer reactions. Nano- and macroporous electrodes based on metal nanoparticles, nanocarbon tubes, graphene, and metallic inverse opals have been designed and fabricated with predictable surface functionalities and electrochemical properties. Electrode/mediator/cofactor/enzyme conjugates can enhance the <italic>in vitro</italic> bioelectricity generation of cofactor. Screening of redox mediators for electrochemical regeneration of NADH was also summarized. Multifunctional surfaces of nano or meso-porous electrodes, where oxidoreductases were bounded to structured electrodes with necessary components (e.g., mediators, cofactor, etc.) with tailored properties, were summarized. Optimizations of functional electrode materials and surfaces can improve the efficiency of cofactor regeneration in the electrochemical reactor modules. Key challenges and future research for better bioelectrocatalysis are discussed and expected briefly. Mining and engineering of novel and robust enzymes from genomic and metagenomic libraries would benefit the bioelectrocatalysis. Based on genome mining, discovery of novel oxidoreductases from extreme microorganism provide thermophilic, alkalophilic, halophilic and organic solvent tolerant oxidoreductases. Reactors for bioelectrocatalysis are optimized to provide a platform for the production of chiral chemicals, which brings great promises for biomanufacturing. Further study of bio-inspired multi-enzyme immobilization which mimics quick electron transfer in multi-enzyme complex is suggested. A variety of approaches for bioelectrocatalysis have been successfully applied for the synthesis of chiral chemicals which are intermediates of fine chemicals and pharmaceuticals. Bioelectrosynthesis processes could replace many chemically catalyzed routes in the future, and aims to construct a new platform for more efficient and green biomanufacturing.

Investigating the enzymatic CO2 reduction to formate with electrochemical NADH regeneration in batch and semi-continuous operations

The enzymatic production of formate from CO2 with the immobilized NADH-dependent formate dehydrogenase (FDH) on the activated electrospun polystyrene nanofibers (EPSNF) was investigated to develop a sustainable process for CO2 reduction. Direct electrochemical regeneration on a Cu foam electrode was employed to supply the reaction with the reduced cofactor (NADH). The formate production was studied in two modes of batch and semi-continuous operations with the cofactor recycle. Results indicated that the regenerated cofactor concentrations in both systems were nearly identical (0.5 mM) which ensured the desirable activity of the immobilized enzyme. This showed that the electrochemical regeneration system was effective even in the semi-continuous operation. Although the cumulative formate concentration in the batch operation was higher, the total amounts of the produced formate were higher for the semi-continuous mode for more than 42% which was justified by the fact that the lower formate concentration in the semi-continuous mode would be favorable to the progress of the enzymatic CO2 to formate conversion. Finally, it was concluded that the proposed semi-continuous process in this work could be considered as a promising process for the enzymatic CO2 conversion.

Enzymatic CO2 reduction to formate by formate dehydrogenase from Candida boidinii coupling with direct electrochemical regeneration of NADH

Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

Direct electrochemical regeneration of 1,4-NADH at the copper foam and bimetallic copper foam

Herein, Cu foam and bimetallic Cu foams are introduced as the cost efficient and easy to scale up electrode for efficient electrochemical regeneration of NADH. The porous copper foam was electrochemically deposited at the copper foil. Cu foam was used as a template for preparation of Ag and Pt coated Cu foam by galvanic replacement of Cu with silver and platinum. The nature and structure of the prepared foams were characterized by energy dispersive X-ray spectrometry and scanning electron microscopy. Electrochemical behavior of NAD+ was studied by cyclic voltammetry at the surface of prepared foams. Also regeneration of the enzymatic cofactor NADH in a batch electrochemical reactor using Cu foil, Cu foam, Ag coated and Pt coated Cu foam electrodes has been investigated. The effect of thickness of Cu foam and the reduction potential of NAD+ on the recovery percent of 1,4-NADH was investigated to achieve the most efficient conditions for electrochemical regeneration of NADH. The regeneration yields of enzymatically active NADH were about 80%, 70% and 75% using the Cu foam, Ag and Pt coated Cu foam electrodes, respectively which all of them were more than that observed at Cu foil electrode 54%.

Electrochemical NADH regeneration and electroenzymatic CO2 reduction on Cu nanorods/glassy carbon electrode prepared by cyclic deposition

Mimicking photosynthesis using NADH is a sustainable method to convert CO2 and mitigate global warming. Here, Cu nanorods with twin crystal structure on glassy carbon (GC) were prepared by a cyclic electrodeposition method with voltage from −0.2V to −1.0. The prepared CuGC electrodes were used for the electrochemical NADH regeneration and electroenzymatic CO2 reduction at −1.0V using formate dehydrogenase from Candida boidinii. The selective activation of NADH (1,4-NADH) approached 67% as the deposition cycle number increased. The electron mediator [Cp*Rh(bpy)Cl]Cl complex (Rh(III)) was used to obtain nearly 100% active NADH on the CuGC electrode. The electron transfer rate to Rh(III) is crucial for optimal NADH regeneration: Rh(III) should be reduced to RhH quickly as it has the capability to decompose NADH catalytically. This allows sufficiently high NAD+ conversion and NADH regeneration reaction rates for the electroenzymatic CO2 reduction to formate. The optimum concentrations of Rh(III) and NAD+ were estimated to be 0.25 and 1.00mM, respectively. For the CuGC electrode prepared with 300 deposition cycles, the formate formation rate was estimated to be (6.28±0.02)×10−3μmol/mgCbsFDH/min, a three-fold increase compared to previously reported results on Cu foil electrode.

An Electroenzymatic CO2 Conversion to Formate on Cu Electrode Using NADH Co-Enzyme

Mimicking photosynthesis using has been attempted to convet CO2. The reaction scheme consists of electrochemical NADH regeneration and enzymatic CO2 reduction. Carbon and metal electrodes were not suitable for electrochemical NADH regenreation because the inactive NADH formation by dimerization of NADH was not avoidable. Electron mediators were required to get nearly 100% active NADH by the electrochemical method. Rh carbene

The reductive amination of α-ketoisocaproic acid using a leucine dehydrogenase-modified gold electrode

ABSTRACTIn the present paper, the reductive amination of α-ketoisocaproic acid to L-leucine was monitored using a new modified electrode based on gold nanoparticles (AuNPs) and leucine dehydrogenase (LeuDH). The modified electrode was prepared by self-assembling of AuNPs with thiocytosine followed by the immobilization of the enzyme. The enzyme-modified electrode response was investigated by chronoamperometry for indirect quantification of ammonium ion () involved in the reaction. This reaction is catalyzed by LeuDH and takes place in the presence of ammonium chloride, as nitrogen source, and nicotinamide adenine dinucleotide (NADH), as cofactor. The enzyme-modified electrode was also used for in situ electrochemical NADH regeneration. After one step NADH regeneration the conversion of ammonium ion was improved. The modified electrode has good sensitivity (0.91 × 10−3 A/M) and reproducibility with an RSD of 8.4% (n = 4). The experimental determination of ammonium ion was confirmed by standard Berthelot spectrophotometric method.

Regeneration of the NADH Cofactor by a Rhodium Complex Immobilized on Multi-Walled Carbon Nanotubes

The regeneration of the enzymatic cofactor nicotinamide adenine dinucleotide (NADH) by rhodium-based catalysts such as [Rh(Cp*)(bpy)Cl]+ (Cp* = pentamethylcyclopentadienyl, bpy = 2,2′-bipyridine) and derivatives have previously been studied extensively in solution. In this work, we report a synthetic route of a rhodium complex with a pyrene-substituted phenanthroline ligand (pyr-Rh). The immobilization of the pyr-Rh complex was accomplished on multi-walled carbon nanotubes (MWCNTs) via π-π stacking to obtain effective and durable indirect electrochemical regeneration of NADH. Cyclic voltammetry and amperometry were used to demonstrate the electrochemical activity of the surface-confined pyr-Rh complex. The loading quantity of the pyr-Rh complex was found to be 47 ± 2 nmol/mg of MWCNTs. The reusability of the electrodes modified with the pyr-Rh complex was investigated and an average turnover frequency of 3.6 ± 0.1 s−1 over ten cycles in the presence of 2 mM nicotinamide adenine dinucleotide (NAD+) was observed. Lastly, malate dehydrogenase (MDH), a NADH-dependent enzyme, was evaluated in the presence of the immobilized pyr-Rh complex to confirm the catalyst's capability to regenerate biologically active NADH for biocatalysis.

Mechanistic Aspects of Multistep Bioelectrocatalytic Oxidation of Fuels

Quantitative models were developed for dihydroxyacetone (DHA) production from glycerol oxidation coupled with electrocatalysis. A two-step kinetic model was developed to describe the reactions in bioelectrocatalysis involving electrochemical NADH regeneration. Planar and porous bioelectronic interfaces are modeled to simulate, predict and evaluate kinetics and diffusion in the bioconversion (Figure 1). Key parameters involve diffusivity of cofactor/substrate, electrocatalytic activity and enzyme kinetics. Model-based analysis indicates that the bioreactor is controlled by glycerol dehydrogenase kinetics for enzyme loadings lower than 1 mM and shifts to mass transport and incipient NADH oxidation kinetic control at higher enzyme loadings.

Conversion of CO2 to formate in an electroenzymatic cell using Candida boidinii formate dehydrogenase

Electroenzymatic conversion of environmentally detrimental CO2 into useful chemicals using a NADH-dependent formate dehydrogenase has been of great interest, but the system remained at a conceptual level because of its complexity. In this study, we found that CO2 was a direct substrate for formate formation with Candida boidinii formate dehydrogenase (CbsFDH), but HCO3− was not. Enzymatic formate formation with CbsFDH was inhibited at NADH concentrations higher than 0.35 mM. Electrochemical NADH regeneration was performed at a Cu electrode using the [Cp*Rh(bpy)Cl]+ complex, which catalyzed the conversion of NAD+ into the active NADH with almost 100% selectivity. The electroenzymatic reaction for HCO2H formation was conducted at 1.0 V, 0.25 mM NADH and 0.25 mM Rh complex during electrochemical NADH regeneration. CO2 was reduced into formate by enzymatic catalysis under the NADH regeneration condition, where protons and electrons was continuously supplied into a cathode cell through Nafion® membrane from water splitting at a Pt anode. The interactions of the Cu electrode, Rh mediator, NADH, and CbsFDH were analyzed for the first time. The Rh(III) mediator was hydrolyzed and reduced reversibly into a Rh(I) intermediate (Mred1) as well as irreversibly into a Rh(I) hydride intermediate (Mred2) at the Cu electrode. Interestingly, the Rh(I) species showed activity toward the direct reduction of CO2 as well as NADH regeneration, although the primary CO2 reduction occurred through CbsFDH at 1.0 V.

Immobilization of flavin adenine dinucleotide (FAD) onto carbon cloth and its application as working electrode in an electroenzymatic bioreactor

A high porosity carbon cloth with immobilized FAD was employed as working electrode in electrochemical NADH-regeneration procedure. Carbon cloth was oxidized with hot acids to create surface carboxyl group and then coupled by adenine amino group of FAD with carbodiimide in the presence of N-hydroxysulfosuccinimide. The bioelectrocatalytic NADH-regeneration was coupled to the conversion of achiral substrate pyruvate into chiral product l-lactate by l-lactate dehydrogenase (l-LDH) within the same reactor. The conversion was completed at 96h in bioreactor with FAD-modified carbon cloth, resulting in about 6mM of l-lactate from 10mM of pyruvate. While with bare carbon cloth, the yield at 120h was around 5mM. Immobilized FAD on the surface of carbon cloth electrode facilitated it to carry electrons from electrode to electron transfer enzymes; thereby NADH-regeneration was accelerated to drive the enzymatic reaction efficiently.

Factors affecting the electrochemical regeneration of NADH by (2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complexes: Impact on their immobilization onto electrode surfaces

Complexes of the (2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium family ([Cp*Rh(bpy)Cl] +, which is actually hydrolyzed in the form of [Cp*Rh(bpy)H 2O] 2+ in aqueous medium) are suitable solution-phase mediators likely to regenerate nicotinamide cofactors associated to dehydrogenases involved in many biocatalytic applications. Their practical application as bioelectrocatalysts, e.g., in fine chemicals synthesis or biosensors, remains however restricted to their durable immobilization in an active form onto solid electrode surfaces. This paper reports some new observations on the electrocatalytic properties of this mediator towards NAD + reduction, notably the critical effect of pH and cofactor-to-mediator concentration ratio, and investigates the behavior of a series of ([Cp*Rh(bpy)Cl] +) derivatives bearing various substituents on the bipyridine ligand in view of their subsequent integration in electrochemical bioreactors. It will be shown that such compounds containing S- or N- moieties (i.e., often used as precursors to functionalize electrode surfaces) lead to inactivation of the electrocatalyst because their interaction with the Rh center prevents the formation of the active rhodium hydride complex. It was thus necessary to find another strategy of immobilization, and we found that adsorption of [Cp*Rh(bpy)Cl] + by π-stacking on single-walled carbon nanotubes is an effective mean to reach this goal, leading to efficient and stable catalytic responses for NAD + reduction. Preliminary electroenzymatic experiments in the presence of d-sorbitol dehydrogenase further point out the interest of this approach for bioelectrocatalysis purposes and provide the proof-of-concept for this immobilization strategy.