Enzymatic Bioelectrocatalysis Research Articles

(Invited) Using Molecular Dynamics Simulations to Design Mediated Bioelectrochemical Interfaces

Cell-free bioelectrocatalysis has drawn significant research attention as a Green approach for producing commodity and fine chemicals. Enzymatic bioelectrocatalysis utilizes electrodes to drive challenging enzymatic redox reactions, such as CO2 reduction and selective oxidation of lignocellulosic biomass to generate value-added products. For many oxidoreductases, the redox cofactor is buried within the insulating protein matrix and, thus, inaccessible to the heterogeneous electrode surface. In such cases, electrochemically active small molecules, called redox mediators, have been proven effective in enabling efficient electron transfer by acting as electron shuttles between the electrode and enzyme cofactor. To this end, redox-active hydrogels have become a critical component of many bioelectrocatalysis applications as a way to create an efficient enzyme/electrode interface. Polymers containing covalently attached redox mediators are cross-linked in the presence of a desired oxidoreductase onto the surface of an electrode to form a bioelectrocatalytic hydrogel. This strategy simultaneously immobilizes enzymes at the electrode interface – thereby simplifying product purification and mitigating mass transfer limitations – and improving electron transfer kinetics necessary for electrochemically regenerating the redox cofactor. Despite the success and broad application of redox polymers in enzymatic bioelectrocatalysis, critical interactions between polymer-bound mediators and redox enzymes remain poorly understood, thereby adding significant complications to the design of novel redox polymers. Moreover, there are notable (and seemingly contradictory) discrepancies between the activity of commonly used enzymes, such as glucose oxidase, with freely solvated mediators compared to their polymer-bound counterparts. We have employed a combination of electroanalytical techniques with an array of computational tools (including density function theory (DFT), static docking simulations, and molecular dynamics simulations) to provide new insights into the role that both protein structure and redox polymer composition play in designing efficient bioelectrochemical interfaces. Specifically, we compare the interactions of a commonly used naphthoquinone-modified linear poly(ethylenimine) hydrogel with two structurally analogous enzymes, GOx and FAD-dependent glucose dehydrogenase, that lead to divergent electrocatalytic activity despite both being equally active towards a comparable freely diffusing mediator.

Bioengineering of Cyanobacteria for Electrosynthesis

Market analysis has shown that one of the challenges with using enzymes for electrosynthesis is the need for cofactor regeneration of non-redox cofactors (i.e. ATP, GTP, coenzyme A, etc.). Moving from enzymatic bioelectrocatalysis to microbial bioelectrocatalysis is one way to address this problem, but many microorganizations are not designed for the synthesis chemistry your desire and extracellular electron transport is slow. This paper will discuss the use of bioengineering techniques to overexpress synthetic oxidoreductase enzymes in microorganisms simulataneously with outer membrane cytochromes to improve extracellular electron transport. The example system shown will be for nitrogen reduction to ammonia with cyanobacteria.

Unusual Long-Term Stability of Enzymatic Bioelectrocatalysis in Organic Solvents

Organic solvent tolerant enzymes are of tremendous importance for the pharmaceutical industry in search of high added-value chemicals. However, enzymes exhibit usually weak long-term activity and stability in various media. Here, we report a general strategy allowing stabilizing and keeping enzymes active in biphasic systems upon immobilization on an electrode surface. It consists in the combination of an engineered solvent-tolerant enzyme, combined with fine-tuned osmium-based redox polymers where the concentration of osmium complex has been specifically tuned to minimize its deswelling associated with the use of porous gold electrodes. This approach is validated with bilirubin oxidase as a model system. This copper enzyme is able to oxidize a wide range of substrates, combined with the reduction of O2 to water. While all other enzymatic systems irreversibly lose their activity and stability in the presence of 7.5 M methanol or below, this optimized enzymatic system stays functional in 12.5 M methanol with no loss in current density. It exhibits a half-life of more than 8 days, which is unprecedented in the literature. We show that this electrode can also operate in DMSO, dioxane, acetonitrile and acetone, thus opening up a very wide variety of applications in the field of bioelectrocatalysis and bioelectrosynthesis. Figure: Example of a long-term stability experiment for a mediated bilirubin oxidase biocathode in 12.5 M methanol. Black curve: immobilized on a bare gold electrode; red curve: immobilized on a 11/2 layers porous gold electrode. Experiments performed at +0.1V vs. Ag/AgCl. The total loading was fixed at 600 µg.cm-2 for both experiments. Figure 1

Exploiting CO2 laser to boost graphite inks electron transfer for fructose biosensing in biological fluids

The possibility to print electronics by means of office tools has remarkedly increased the possibility to design affordable and robust point-of-care/need devices. However, conductive inks suffer from low electrochemical and rheological performances limiting their applicability in biosensors. Herein, a fast CO2 laser approach to activate printed carbon inks towards direct enzymatic bioelectrocatalysis (3rd generation) is proposed and exploited to build biosensors for D-fructose analysis in biological fluids.The CO2 laser treatment was compared with two lab-grade printed transducers fabricated with solvent (SB) and water (WB) based carbon inks. The use of the laser revealed significant morpho-chemical variations on the printed inks and was investigated towards enzymatic direct catalysis, using Fructose dehydrogenase (FDH) integrated into entirely lab-produced biosensors. The laser-driven activation of the inks unveils the inks' direct electron transfer (DET) ability between FDH and the electrode surface. Sub-micromolar limits of detection (SB-ink LOD = 0.47 μM; WB-ink LOD = 0.24 μM) and good linear ranges (SB-ink: 5–100 μM; WB-ink: 1–50 μM) were obtained, together with high selectivity due to use of the enzyme and the low applied overpotential (0.15 V vs. pseudo-Ag/AgCl). The laser-activated biosensors were successfully used for D-fructose determination in complex synthetic and real biological fluids (recoveries: 93–112%; RSD ≤8.0%, n = 3); in addition, the biosensor ability for continuous measurement (1.5h) was also demonstrated simulating physiological D-fructose fluctuations in cerebrospinal fluid.

Enzymatic Bioelectrocatalysis for Organic Electrosynthesis

Organic electrosynthesis has become a popular research area in the last decade due to a desire for more sustainable and greener organic synthesis methods. However, electrosynthesis frequently has challenges with selectivity. This talk will detail the design of bioelectrocatalytic systems for organic electrosynthesis with a focus on improving the selectivity and efficiency of electrosynthesis systems. Specifically, the talk will describe bioelectrocatalytic systems for C-H activation and chiral synthesis. It will discuss both materials design of electrodes and enzyme design for electrochemistry, but also discuss strategies for improving performance of bioelectroatalytic systems using bi-phasic electrochemical systems.

Unusual long-term stability of enzymatic bioelectrocatalysis in organic solvents

Organic solvent tolerant enzymes are of tremendous importance for the pharmaceutical industry in search of high added-value chemicals. However, enzymes exhibit usually weak long-term activity and stability in various media. Here, we report a general strategy allowing stabilizing and keeping enzymes active in mixed-solvent systems upon immobilization on an electrode surface. It consists in the combination of an engineered solvent-tolerant enzyme, combined with fine-tuned osmium-based redox polymers where the concentration of osmium complex has been specifically tuned to minimize its deswelling, associated with the use of porous gold electrodes. This approach is validated with bilirubin oxidase as a model system. This copper enzyme is able to oxidize a wide range of substrates, combined with the reduction of O2 to water. While all other enzymatic systems irreversibly lose their activity and stability in the presence of 7.5 M methanol or below, this optimized enzymatic system stays functional in 12.5 M methanol with no loss in current density. It exhibits a half-life of more than 8 days, which is unprecedented in the literature. We show that this electrode can also operate in DMSO, dioxane, acetonitrile and acetone, thus opening up a very wide variety of applications in the field of bioelectrocatalysis and bioelectrosynthesis.

(Invited) Use of Redox Mediators and Polymers to Provide Additional Control of Bioelectrocatalytic Systems

Mediated electron transfer is a critical strategy for "wiring" biomolecules to electrode interfaces in bioelectrochemical systems. Specifically in enzymatic bioelectrocatalysis, soluble redox mediators and redox polymers have been critical for both improving the catalytic performance, but also improving the selectivity of the bioelectrodes. This paper will discuss the design and implementation of redox mediators and redox polymers in bioelectrocatalytic systems. Application of these concepts to the fields of electrosynthesis, bioanalysis supercapacitors, and redox flow biobatteries will be discussed.

Designing Enzymatic Redox Mediators: Quantifying the Impact of Molecular Structure on Biocatalytic Activity

Enzymatic bioelectrocatalysis involves the application of enzymes to facilitate the conversion of chemical to electrical energy. This approach has been widely applied in the fields of biosensing, biofuel cells, and to a lesser degree the synthesis of fine chemicals. While direct electrochemical communication between the enzymes and electrodes is generally preferable, it is often the case that efficient electron transfer rates can only be achieved when redox mediators are employed to shuttle electrons reversibly from the redox site of the enzyme to the electrode interface. Unfortunately, there still exist many oxidoreductases for which no effective exogenous mediator is known. This may be due to insufficient understanding of the role that molecular structure of the mediator plays in determining its ability to facilitate electron transfer to a given enzyme. Consequently, selection and/or design of novel redox mediators remains a challenge that is often accomplished using a “guess and check” approach to maximize electron transfer rates. Developing a rapid, and convenient method of predicting the role played by other structural and chemical features beyond redox potential of a mediator is an important goal in advancing mediated bioelectrocatalysis. This talk will describe our recent efforts in identifying structure - function relationships of redox mediators that control efficient electron transfer rates. I will highlight results from stopped-flow spectrophotometry experiments which review the nature of electron transfer events between quinone redox couples in altering bioelectrocatalytic activity.

(Invited) Using Enzymatic Bioelectrocatalysis for Nitrogen Reduction to Ammonia and Chiral Amines

Enzymatic biocatalysts have been proposed for a variety of chemical manufacturing applications due to their high selectivity, but they can be utilized as electrocatalysts. This talk will discuss the "wiring" of nitrogenase (the only enzyme known to catalyze reduction of nitrogen to ammonia) to electrode surfaces for electrosynthesis. The talk will start with a discussion of nitrogenase bioelectrodes for ammonia production followed by the use of nitrogenase bioelectrodes for the selective production of chiral amines and chiral amino acids.

Enzymatic Bioelectrocatalysis

Enzymatic bioelectrocatalysis relies on immobilizing oxidoreductases on electrode surfaces, leading to different applications, such as biosensors [...]

Fundamental insight into redox enzyme-based bioelectrocatalysis.

Redox enzymes can work as efficient electrocatalysts. The coupling of redox enzymatic reactions with electrode reactions is called enzymatic bioelectrocatalysis, which imparts high reaction specificity to electrode reactions with nonspecific characteristics. The key factors required for bioelectrocatalysis are hydride ion/electron transfer characteristics and low specificity for either substrate in redox enzymes. Several theoretical features of steady-state responses are introduced to understand bioelectrocatalysis and to extend the performance of bioelectrocatalytic systems. Applications of the coupling concept to bioelectrochemical devices are also summarized with emphasis on the achievements recorded in the research group of the author.

Rational Surface Modification of Carbon Nanomaterials for Improved Direct Electron Transfer-Type Bioelectrocatalysis of Redox Enzymes

Interfacial electron transfer between redox enzymes and electrodes is a key step for enzymatic bioelectrocatalysis in various bioelectrochemical devices. Although the use of carbon nanomaterials enables an increasing number of redox enzymes to carry out bioelectrocatalysis involving direct electron transfer (DET), the role of carbon nanomaterials in interfacial electron transfer remains unclear. Based on the recent progress reported in the literature, in this mini review, the significance of carbon nanomaterials on DET-type bioelectrocatalysis is discussed. Strategies for the oriented immobilization of redox enzymes in rationally modified carbon nanomaterials are also summarized and discussed. Furthermore, techniques to probe redox enzymes in carbon nanomaterials are introduced.

Recent Progress in Applications of Enzymatic Bioelectrocatalysis

Bioelectrocatalysis has become one of the most important research fields in electrochemistry and provided a firm base for the application of important technology in various bioelectrochemical devices, such as biosensors, biofuel cells, and biosupercapacitors. The understanding and technology of bioelectrocatalysis have greatly improved with the introduction of nanostructured electrode materials and protein-engineering methods over the last few decades. Recently, the electroenzymatic production of renewable energy resources and useful organic compounds (bioelectrosynthesis) has attracted worldwide attention. In this review, we summarize recent progress in the applications of enzymatic bioelectrocatalysis.

Rational design of electroactive redox enzyme nanocapsules for high-performance biosensors and enzymatic biofuel cell

The potential application of biodevices based on enzymatic bioelectrocatalysis are limited by poor stability and electrochemical performance. To solve the limitation, modifying enzyme with functional polymer to tailor enzyme function is highly desirable. Herein, glucose oxidase (GOx) was chosen as a model enzyme, and according to the chemical structure of GOx cofactor (flavin adenine dinucleotide, FAD), we customize a biomimetic cofactor containing vinyl group (SFAD) for GOx, and prepared an GOx nanocapsule via in-situ polymerization. The characterization of particle size distribution, TEM, fluorescence and electrochemical performance indicated the successful formation of electroactive GOx nanocapsule with SFAD-containing polymeric network (n (GOx-SFAD-PAM)). The network can act as an electronic “highway” to link the active site with electrode, with capability to accelerate electron transfer as well as enhanced GOx stability. Further investigation of bioelectrocatalysis shows that n (GOx-SFAD-PAM)-based biosensor has low detection potential (−0.4 vs. Ag/AgCl), high sensitivity (64.97 μAmM−1cm−2), good anti-interference performance, quick response (3⁓5s) and excellent stability, and that n (GOx-SFAD-PAM)-based enzymatic biofuel cell (EBFC) has the high maximum power density (1011.21 μWcm−2), which is a 385-fold increase over that of native GOx-based EBFC (2.62 μWcm−2). This study suggests that novel enzyme nanocapsule with electroactive polymeric shell might provide a prospective solution for the performance improvement of enzymatic bioelectrocatalysis-based biodevices.

Enzymatic Bioelectrocatalysis for Enzymology Applications

AbstractEnzymatic biosensors are commonly used to evaluate the concentration of individual analytes in samples (e. g., glucose in blood, lactate in sweat, etc.), where the working electrode utilizes an enzyme (a “bioelectrode”) as the chemically selective agent for electrochemical analyses. However, those same enzymatic bioelectrodes can be used for the analytical characterization of enzymes as a new biophysical characterization tool for enzymologists. This Minireview will detail recent work focused on utilizing enzymatic bioelectrocataysis for enzymology applications. Specifically, it will talk about the use of enzymatic bioelectrodes for studying kinetics, inhibition, transport, and thermodynamics. It will also compare and contrast electrochemical techniques with spectrophotometric techniques for enzymology.

Recent advances in surface chemistry of electrodes to promote direct enzymatic bioelectrocatalysis

Abstract Redox enzymes catalyze major reactions in microorganisms to supply energy for life. Their use in electrochemical biodevices requires their integration on electrodes, while maintaining their activity and optimizing their stability. In return, such applicative development puts forward the knowledge on involved catalytic mechanisms, providing a direct electrode connection of the enzyme is fulfilled. Enzymes being large molecules with active site embedded in an insulating moiety, direct bioelectrocatalysis supposes strategies for specific orientation of the enzyme to be developed. In this review, we summarize recent advances during the past 3 years in the chemical modification of electrodes favoring direct electrocatalysis. We present the different methodologies used according to the electrode materials, including metals, carbon-based electrodes, or porous structures and discuss the gained insights into bioelectrocatalysis. We especially focus on enzyme engineering, which appears as an emerging strategy for enzyme anchoring. Remaining challenges will be discussed with regard to these later findings.

Direct Electrochemical Investigation into the Mechanism and Thermodynamics of Mofe-Dependent Nitrogenase

Enzymatic bioelectrocatalysis often requires an artificial redox mediator to observe significant electron transfer rates. The use of such mediators can add a substantial overpotential and obfuscate the protein’s native kinetics, which limits the voltage of a biofuel cell and alters the analytical performance of biosensors. We have recently developed a material for facilitating direct electrochemical communication with redox proteins based on a novel pyrene-modified linear poly(ethyleneimine). By immobilizing the catalytic subunit of nitrogenase (MoFe protein), to demonstrate the ATP-independent direct electroenzymatic reduction of N2 to NH3. In addition, this novel hydrogel material has enabled us to study the electron transfer kinetics of each electroactive cofactor for the MoFe protein (P-cluster and FeMoco) independently, and has been combined with stop-flow IR spectroscopy to study changes in global conformational changes of the protein during catalysis.

(Invited) Redox Active Metallic and Glyco-Based Nanoparticles for Enzymatic Bioelectrocatalysis

The clean production of electrical energy from renewable resources has received recently a growing attention from the research community. A way to achieve this goal is the use of biological catalysts to catalyze the oxidation of renewable fuels. One of the streamlines on the research dealing with green electrical energy production is represented by oxidoreductase enzymes as the biocatalysts. Thanks to their specificity towards their respective substrates and activity in relatively mild conditions (pH, temperature, etc…), they are ideal candidates to replace noble metals regularly found in fuel cells. However, due to low power densities achieved utilizing these redox proteins and their limited stability; enzymatic fuel cells (EFCs) are so far only envisioned to power small and disposable electronic devices. In most literature reports, the redox enzymes are essentially immobilized on chemically modified electrode surfaces. There, they harvest and convert the chemical energy stored in fuels and turn it into electrical energy. The growing field in bioelectrocatalysis is beneficial not only for the biofuel cell technology but also for the design of efficient biosensing or bioreactor devices. In this presentation, we will focus on two different approaches for the design of EFCs currently studied in our laboratory. The rationalization of the electrode surface requirement to achieve efficient bioelectrocatalysis (mediated or direct electron transfer) [1] allowed for the synthesis of redox active gold nanoparticles (AuNPs) bearing electroactivable nitro groups and negatively charged groups. The obtained hydroxylamine-modified AuNPs are catalytically active for nicotinamide adenine dinucleotide (NADH) oxidation and for NAD+ regeneration in bioelectrocatalysis (with NAD-dependent glucose dehydrogenase). In contrast, the same particles possessing also negatively charged carboxylic groups had been shown to favorably orientate some multi-copper oxidases having their T1 copper center facing the metallic particles and consequently enhancing oxygen electroreduction. On the other hand, we will also present our recent strategy to design solubilized enzymatic fuel cell (SEFC). The entrapment of different redox mediator is accomplished by host-guest interaction with β-cyclodextrin-coated glyconanoparticles for the mediated glucose and oxygen conversion [2,3]. Hence, the cell was designed with permselective membranes to enable substrate and proton diffusion while trapping the enzymes and redox glyconanoparticles in separate compartments. The SEFC suffered a limited power loss of about 25% after 7 days of continuous charge-discharge cycling at 50 µA [3].

Direct enzymatic bioelectrocatalysis: differentiating between myth and reality.

Enzymatic bioelectrocatalysis is being increasingly exploited to better understand oxidoreductase enzymes, to develop minimalistic yet specific biosensor platforms, and to develop alternative energy conversion devices and bioelectrosynthetic devices for the production of energy and/or important chemical commodities. In some cases, these enzymes are able to electronically communicate with an appropriately designed electrode surface without the requirement of an electron mediator to shuttle electrons between the enzyme and electrode. This phenomenon has been termed direct electron transfer or direct bioelectrocatalysis. While many thorough studies have extensively investigated this fascinating feat, it is sometimes difficult to differentiate desirable enzymatic bioelectrocatalysis from electrocatalysis deriving from inactivated enzyme that may have also released its catalytic cofactor. This article will review direct bioelectrocatalysis of several oxidoreductases, with an emphasis on experiments that provide support for direct bioelectrocatalysis versus denatured enzyme or dissociated cofactor. Finally, this review will conclude with a series of proposed control experiments that could be adopted to discern successful direct electronic communication of an enzyme from its denatured counterpart.

DNA Redox Hydrogels: Improving Mediated Enzymatic Bioelectrocatalysis

We present the preparation of a redox DNA hydrogel for mediated bioelectrocatalysis of oxidoreductase enzymes for biosensor and biofuel cell applications. The noncovalent functionalization of DNA with redox molecules is achieved by intercalation of aromatic redox probes into the DNA double helix or electrostatic binding of redox-active tetraalkylammonium ions to phosphate groups on DNA. Prepared DNA redox hydrogels demonstrate the capability of mediating bioelectrocatalytic glucose oxidation by oxidoreductase enzymes. This is the first evidence that redox DNA hydrogels can replace redox polymer hydrogels for self-exchange-based mediation for bioelectrocatalytic applications. This study contributes toward advances in the use of DNA, an emerging biomaterial, in enzymatic bioelectrocatalysis-based applications.