One-Pot Bioelectrocatalytic Conversion of Chemically Inert Hydrocarbons to Imines.

Petroleum hydrocarbons are our major energy source and an important feedstock for the chemical industry. With the exception of combustion, the deep conversion of chemically inert hydrocarbons to more valuable chemicals is of considerable interest. However, two challenges hinder this conversion. One is the regioselective activation of inert carbon-hydrogen (C-H) bonds. The other is designing a pathway to realize this complicated conversion. In response to the two challenges, a multistep bioelectrocatalytic system was developed to realize the one-pot deep conversion from heptane to N-heptylhepan-1-imine under mild conditions. First, in this enzymatic cascade, a bioelectrocatalytic C-H bond oxyfunctionalization step based on alkane hydroxylase (alkB) was applied to regioselectively convert heptane to 1-heptanol. By integrating subsequent alcohol oxidation and bioelectrocatalytic reductive amination steps based on an engineered choline oxidase (AcCO6) and a reductive aminase (NfRedAm), the generated 1-heptanol was successfully converted to N-heptylhepan-1-imine. The electrochemical architecture provided sufficient electrons to drive the bioelectrocatalytic C-H bond oxyfunctionalization and reductive amination steps with neutral red (NR) as electron mediator. The highest concentration of N-heptylhepan-1-imine achieved was 0.67 mM with a Faradaic efficiency of 45% for C-H bond oxyfunctionalization and 70% for reductive amination. Hexane, octane, and ethylbenzene were also successfully converted to the corresponding imines. Via regioselective C-H bond oxyfunctionalization, intermediate oxidation, and reductive amination, the bioelectrocatalytic hydrocarbon deep conversion system successfully realized the challenging conversion from inert hydrocarbons to imines that would have been impossible by using organic synthesis methods and provided a new methodology for the comprehensive conversion and utilization of inert hydrocarbons.

Advances in electrochemical cofactor regeneration: enzymatic and non-enzymatic approaches.

Nicotinamide adenine dinucleotide(NAD(P)H) is a metabolically interconnected redox cofactor serving as a hydride source for the majority of oxidoreductases, and consequently constituting a significant cost factor for bioprocessing. Much research has been devoted to the development of efficient, affordable, and sustainable methods for the regeneration of these cofactors through chemical, electrochemical, and photochemical approaches. However, the enzymatic approach using formate dehydrogenase is still the most abundantly employed in industrial applications, even though it suffers from system complexity and product purity issues. In this review, we summarize non-enzymatic and enzymatic electrochemical approaches for cofactor regeneration, then discuss recent developments to solve major issues. Issues discussed include Rh-catalyst mediated enzyme mutual inactivation, electron-transfer rates, catalyst sustainability, product selectivity and simplifying product purification. Recently reported remedies are discussed, such as heterogeneous metal catalysts generating H+ as the sole byproduct or high activity and stability redox-polymer immobilized enzymatic systems for sustainable organic synthesis.

Substrate Channeling by a Rationally Designed Fusion Protein in a Biocatalytic Cascade.

Substrate channeling, where an intermediate in a multistep reaction is directed toward a reaction center rather than freely diffusing, offers several advantages when employed in catalytic cascades. Here we present a fusion enzyme comprised of an alcohol and aldehyde dehydrogenase, that is computationally designed to facilitate electrostatic substrate channeling using a cationic linker bridging the two structures. Rosetta protein folding software was utilized to determine an optimal linker placement, added to the truncated termini of the proteins, which is as close as possible to the active sites of the enzymes without disrupting critical catalytic residues. With improvements in stability, product selectivity (90%), and catalyst turnover frequency, representing 500-fold increased activity compared to the unbound enzymes and nearly 140-fold for a neutral-linked fusion enzyme, this design strategy holds promise for making other multistep catalytic processes more sustainable and efficient.

Rapid Entrapment of Phenazine Ethosulfate within a Polyelectrolyte Complex on Electrodes for Efficient NAD+ Regeneration in Mediated NAD+-Dependent Bioelectrocatalysis.

Over the past two decades, the designs of redox polymers have become critical to the field of mediated bioelectrocatalysis and are used in commercial glucose biosensors, as well as other bioelectrochemical applications (e.g., energy harvesting). These polymers are specifically used to immobilize redox mediators on electrode surfaces, allowing for self-exchange-based conduction of electrons from enzymes far from the electrode to the electrode surface. However, the synthesis of redox polymers is challenging and results in large batch-to-batch variability. Herein, we report a rapid entrapment of mediators for NAD+-dependent bioelectrocatalysis within reverse ionically condensed polyelectrolytes. A high ionic strength aqueous solution of oppositely charged polyelectrolytes, composed of cationic polyguanidinium (PG) chloride and anionic sodium hexametaphosphate (P6), undergoes phase inversion into a solid microporous polyelectrolyte complex (PEC) when introduced into a low ionic strength aqueous solution. The ionic strength-triggered phase inversion of PGP6 solutions was investigated as a means to entrap mediators on the surface of electrodes for mediated bioelectrocatalysis. Compared to the traditional cross-linked immobilizations using redox polymers, this phase inversion takes place within seconds and requires up to 60 min for complete stabilization. In this work, redox mediator phenazine ethosulfate (PES) was entrapped within PGP6 on electrode surfaces for nicotinamide adenine dinucleotide (NAD+)-dependent bioelectrocatalysis. In the bulk solution, NAD+-dependent dehydrogenase enzymes catalyze the oxidation of the substrate while reducing NAD to reduced nicotinamide adenine dinucleotide (NADH). The resulting NADH is reoxidized to NAD+ by the entrapped PES that gets reduced on the electrode, completing the NAD+-regeneration-based bioelectrocatalysis. To show the use of these new materials in an application, biofuel cells were evaluated using four different anodic enzyme systems (alcohol dehydrogenase, lactate hydrogenase, glycerol dehydrogenase, and glucose dehydrogenase).

Cascaded Biocatalysis and Bioelectrocatalysis: Overview and Recent Advances.

Enzyme cascades are plentiful in nature, but they also have potential in artificial applications due to the possibility of using the target substrate in biofuel cells, electrosynthesis, and biosensors. Cascade reactions from enzymes or hybrid bioorganic catalyst systems exhibit extended substrate range, reaction depth, and increased overall performance. This review addresses the strategies of cascade biocatalysis and bioelectrocatalysis for (a) CO2 fixation, (b) high value-added product formation, (c) sustainable energy sources via deep oxidation, and (d) cascaded electrochemical enzymatic biosensors. These recent updates in the field provide fundamental concepts, designs of artificial electrocatalytic oxidation-reduction pathways (using a flexible setup involving organic catalysts and engineered enzymes), and advances in hybrid cascaded sensors for sensitive analyte detection.

Fundamentals, Applications, and Future Directions of Bioelectrocatalysis.

Bioelectrocatalysis is an interdisciplinary research field combining biocatalysis and electrocatalysis via the utilization of materials derived from biological systems as catalysts to catalyze the redox reactions occurring at an electrode. Bioelectrocatalysis synergistically couples the merits of both biocatalysis and electrocatalysis. The advantages of biocatalysis include high activity, high selectivity, wide substrate scope, and mild reaction conditions. The advantages of electrocatalysis include the possible utilization of renewable electricity as an electron source and high energy conversion efficiency. These properties are integrated to achieve selective biosensing, efficient energy conversion, and the production of diverse products. This review seeks to systematically and comprehensively detail the fundamentals, analyze the existing problems, summarize the development status and applications, and look toward the future development directions of bioelectrocatalysis. First, the structure, function, and modification of bioelectrocatalysts are discussed. Second, the essentials of bioelectrocatalytic systems, including electron transfer mechanisms, electrode materials, and reaction medium, are described. Third, the application of bioelectrocatalysis in the fields of biosensors, fuel cells, solar cells, catalytic mechanism studies, and bioelectrosyntheses of high-value chemicals are systematically summarized. Finally, future developments and a perspective on bioelectrocatalysis are suggested.

Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide.

In the present work, direct electron transfer (DET) based biosensing system for the determination of glucose has been fabricated by utilizing gold binding peptide (GBP) fused flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Burkholderia cepacia. The GBP fused FAD-GDH was immobilized on the working electrode surface of screen-printed electrode (SPE) which consists of gold working electrode, a silver pseudo-reference electrode and a platinum counter electrode, to develop the biosensing system with compact design and favorable sensing ability. The bioelectrochemical and mechanical properties of GBP fused FAD-GDH (GDH-GBP) immobilized SPE (GDH-GBP/Au) were investigated. Here, the binding affinity of GDH-GBP on Au surface, was highly increased after fusion of gold binding peptide and its uniform monolayer was formed on Au surface. In the cyclic voltammetry (CV), GDH-GBP/Au displayed significantly high oxidative peak currents corresponding to glucose oxidation which is almost c.a. 10-fold enhanced value compared with that from native GDH immobilized SPE (GDH/Au). As well, GDH-GBP/Au has shown 92.37% of current retention after successive potential scans. In the chronoamperometry, its steady-state catalytic current was monitored in various conditions. The dynamic range of GDH-GBP/Au was shown to be 3-30 mM at 30 °C and exhibits high selectivity toward glucose in whole human blood. Additionally, temperature dependency of GDH-GBP/Au on DET capability was also investigated at 30-70 °C. Considering this efficient and stable glucose sensing with simple and easy sensor fabrication, GDH-GBP based sensing platform can provide new insight for future biosensor in research fields that rely on DET.

Electroenzymatic Nitrogen Fixation Using a MoFe Protein System Immobilized in an Organic Redox Polymer.

We report an organic redox-polymer-based electroenzymatic nitrogen fixation system using a metal-free redox polymer, namely neutral-red-modified poly(glycidyl methacrylate-co-methylmethacrylate-co-poly(ethyleneglycol)methacrylate) with a low redox potential of -0.58 V vs. SCE. The stable and efficient electric wiring of nitrogenase within the redox polymer matrix enables mediated bioelectrocatalysis of N3- , NO2- and N2 to NH3 catalyzed by the MoFe protein via the polymer-bound redox moieties distributed in the polymer matrix in the absence of the Fe protein. Bulk bioelectrosynthetic experiments produced 209±30 nmol NH3 nmol MoFe-1 h-1 from N2 reduction. 15 N2 labeling experiments and NMR analysis were performed to confirm biosynthetic N2 reduction to NH3 .

Dissolved carbon monoxide concentration monitoring platform based on direct electrical connection of CO dehydrogenase with electrically accessible surface structure.

CO dehydrogenase (CODH) employed in a dissolved CO biosensor development study harbors a solvent-exposed cofactor capable of DET to electrode. Here, CODH was immobilized on arrays of AuNPs of various dimensions to determine the effect of the size and shape of the electrode surface on the direct electrical connection between CODH and electrode surface. The results showed the degree of proximity between the CODH cofactor and electrode surface, which varied with AuNP size and caused significant changes to the electrical connection at the interface as well as to the substrate accessibility. Consequently, a high-density nanoscale SRS was fabricated on electrode to further facilitate direct electrical connection as well as to enable distribution of CODH into monolayer or near-monolayer for lowering the barrier of CO diffusion toward enzyme. The findings show the feasibility of controlling the direct electrical connection between CODH and the electrode as well as controlling the substrate accessibility.

Significant enhancement of direct electric communication across enzyme-electrode interface via nano-patterning of synthetic glucose dehydrogenase on spatially tunable gold nanoparticle (AuNP)-modified electrode.

In this study, the effect of inter-enzyme steric hindrance that occurs during enzyme immobilization on the electrode, on direct electrical communications of enzyme with electrode was investigated via nano-patterning of enzymes on the electrode. Here, the nano-patterning of enzymes was achieved through the combination of DET-capable enzyme that was produced via fusion of site-specific gold binding peptide (GBP) to catalytic subunit of enzyme and gold nanoparticle (AuNP) array with highly tunable dimensions of AuNPs, resulting in spatially controllable enzyme-electrode. The nano-scale spatial control between immobilized enzymes on the highly tuned AuNPs shows different DET efficiency across the enzyme-electrode interface, showing 18.47% of maximum electron recovery which is 3.2-fold enhanced electron recovery efficiency compared to spatially non-controlled enzymes on the electrode where showed 5.7% of electron recovery. The result affirms that inter-enzyme interaction is a significant parameter that decides the enzyme-electrode performance.

Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer.

Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold-binding peptide at its amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine combinations of recombinant GDH proteins on the electrode show significantly different direct electron-transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme-electrode interface even in the absence of the electron-transfer subunit (i.e., ß subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (-)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH2 redox wave and a stabilized bioelectrocatalytic current of more than 100 µA, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 µA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron-transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific "sticky-ended" proteins without loss of catalytic activity could be applied to other redox enzymes having a buried active site.

Development of anode zone using dual-anode system to reduce organic matter crossover in membraneless microbial fuel cells.

To prevent the occurrence of the organic crossover in membraneless microbial fuel cells (ML-MFCs), dual-anode MFC (DA-MFC) was designed from multi-anode concept to ensure anode zone. The anode zone addressed increase the utilization of organic matter in ML-MFCs, as the result, the organic crossover was prevented and performance of MFCs were enhanced. The maximum power of the DA-MFC was 0.46mW, which is about 1.56 times higher than the ML-MFC (0.29mW). Furthermore, the DA-MFC had advantage in correlation of organic substance concentration and dissolved oxygen concentration, and even electric over-potential. In addition, in terms of cathode fouling, the DA-MFC showed clearer surface. Hence, the anode zone should be considered in the advanced ML-MFC for practically use in wastewater treatment process, and also for scale-up of MFCs.

Increased Power in Sediment Microbial Fuel Cell: Facilitated Mass Transfer via a Water-Layer Anode Embedded in Sediment.

We report a methodology for enhancing the mass transfer at the anode electrode of sediment microbial fuel cells (SMFCs), by employing a fabric baffle to create a separate water-layer for installing the anode electrode in sediment. The maximum power in an SMFC with the anode installed in the separate water-layer (SMFC-wFB) was improved by factor of 6.6 compared to an SMFC having the anode embedded in the sediment (SMFC-woFB). The maximum current density in the SMFC-wFB was also 3.9 times higher (220.46 mA/m2) than for the SMFC-woFB. We found that the increased performance in the SMFC-wFB was due to the improved mass transfer rate of organic matter obtained by employing the water-layer during anode installation in the sediment layer. Acetate injection tests revealed that the SMFC-wFB could be applied to natural water bodies in which there is frequent organic contamination, based on the acetate flux from the cathode to the anode.