NADH Regeneration Research Articles

Bioinspired Photocatalytic NADH Regeneration by Covalently Metalated Carbon Nitride for Enhanced CO2 Reduction.

Natural photosynthesis is a highly unified biocatalytic system, which coupled cofactor (NAD(P)H) regeneration and enzymatic CO2 reduction efficiently for solar energy conversion. Mimicking nature, a novel system with Rh complex covalently grafted onto NH2 -functionalized polymeric carbon nitride (NH2 -PCN) was constructed. The integrated connection of the light-harvesting and electron mediation modules as Rhm3 -N-PCN could promote the efficient NAD+ reduction to NADH. As a result, the integrated system exhibited a conversion of ∼66 % within 20 minutes. By further coupling in situ generated NADH with formate dehydrogenase (FDH), a photoenzymatic production of formic acid (HCOOH) from CO2 was accomplished. Moreover, by immobilizing FDH onto a hydrophobic membrane, an enhanced HCOOH production of ∼5.0 mM can be obtained due to the concentrated CO2 on the gas-liquid-solid three-phase interface. Our work herein provides an integrated strategy for coupling the anchored electron mediator with immobilized enzyme for enhanced artificial photosynthesis.

Nanoporous Graphitic Carbon Nitride as Catalysts for Cofactor Regeneration

An efficient nicotinamide adenine dinucleotide (NADH) regeneration system is of great significance for the application of oxidoreductase-catalyzed reactions. Photochemical regeneration has been a research hotspot in recent years. Graphite carbon nitride (g-C3N4) has important application prospects. However, bulk g-C3N4 (BCN) has some disadvantages that lead to a low catalytic efficiency. Herein, nanoporous g-C3N4 (PCN) was prepared using silica nanoparticles as templates, and the effects of the mass ratio of cyanamide to silica and the particle size of the templates on the structure, optical, and catalytic properties of PCN were investigated. Then, cyanamide was separately co-polymerized with two types of thiophene compounds and imidazole-, benzene-, and quinolone-based compounds, and the effects of the structure and number of aromatic rings introduced into PCN on the properties of catalysts were studied. The results show that PCN had much better catalytic performance than BCN, the initial reaction rate of NADH regeneration could be upgraded from 18.01 to 37.14 mmol/(g·min) after PCN was modified with thiophene compound, and the yield of NADH after 20 min increased from 56.8 to 82.7%. When other aromatic compounds were added in the PCN preparation process, a catalytic performance similar to that of thiophene-modified PCN was acquired.

Improving the Enzymatic Cascade of Reactions for the Reduction of CO2 to CH3OH in Water: From Enzymes Immobilization Strategies to Cofactor Regeneration and Cofactor Suppression

The need to decrease the concentration of CO2 in the atmosphere has led to the search for strategies to reuse such molecule as a building block for chemicals and materials or a source of carbon for fuels. The enzymatic cascade of reactions that produce the reduction of CO2 to methanol seems to be a very attractive way of reusing CO2; however, it is still far away from a potential industrial application. In this review, a summary was made of all the advances that have been made in research on such a process, particularly on two salient points: enzyme immobilization and cofactor regeneration. A brief overview of the process is initially given, with a focus on the enzymes and the cofactor, followed by a discussion of all the advances that have been made in research, on the two salient points reported above. In particular, the enzymatic regeneration of NADH is compared to the chemical, electrochemical, and photochemical conversion of NAD+ into NADH. The enzymatic regeneration, while being the most used, has several drawbacks in the cost and life of enzymes that suggest attempting alternative solutions. The reduction in the amount of NADH used (by converting CO2 electrochemically into formate) or even the substitution of NADH with less expensive mimetic molecules is discussed in the text. Such an approach is part of the attempt made to take stock of the situation and identify the points on which work still needs to be conducted to reach an exploitation level of the entire process.

Efficient biosynthesis of D-mannitol by coordinated expression of a two-enzyme cascade

D-mannitol is widely used in the pharmaceutical and medical industries as an important precursor of antitumor drugs and immune stimulants. However, the cost of the current enzymatic process for D-mannitol synthesis is high, thus not suitable for commercialization. To address this issue, an efficient mannitol dehydrogenase LpGDH used for the conversion and a glucose dehydrogenase BaGDH used for NADH regeneration were screened, respectively. These two enzymes were co-expressed in Escherichia coli BL21(DE3) to construct a two-enzyme cascade catalytic reaction for the efficient synthesis of d-mannitol, with a conversion rate of 59.7% from D-fructose achieved. The regeneration of cofactor NADH was enhanced by increasing the copy number of Bagdh, and a recombinant strain E. coli BL21/pETDuet-Lpmdh-Bagdh-Bagdh was constructed to address the imbalance between cofactor amount and key enzyme expression level in the two-enzyme cascade catalytic reaction. An optimized whole cell transformation process was conducted under 30 ℃, initial pH 6.5, cell mass (OD600) 30, 100 g/L D-fructose substrate and an equivalent molar concentration of glucose. The highest yield of D-mannitol was 81.9 g/L with a molar conversion rate of 81.9% in 5 L fermenter under the optimal conversion conditions. This study provides a green and efficient biotransformation method for future large-scale production of D-mannitol, which is also of great importance for the production of other sugar alcohols.

Topologically and chemically engineered conjugated polymer with synergistically intensified electron generation, transfer and utilization for photocatalytic nicotinamide cofactor regeneration

Electron generation, transfer and utilization in photocatalytic reduction reactions jointly govern the solar-to-chemical conversion efficiency. In natural photosynthesis, the ultrathin thylakoid membrane loads numerous pigments and proteins for light harvesting and electron transfer, which also integrates ferredoxin-NADP+ reductase bearing molecule hydrides for electron utilization in NADPH regeneration. Inspired by this, herein, conjugated polymer nanolayer coordinated with (Cp*RhCl2)2 (CPNL-Rh) is developed through a topological and chemical engineering strategy to synergistically intensify electron generation, transfer and utilization for NADH regeneration. Specifically, the triazine moieties well distributed in CPNL-Rh largely harvest visible light to generate electrons. The electrons are rapidly transferred to Rh cocatalyst through CPNL. Rh cocatalyst finally accepts electrons and protons in solution to form molecule hydrides for implementing NADH regeneration. The turnover frequency of CPNL-Rh for NADH regeneration reaches 44.8 h−1, among the highest value ever reported and ~346% higher than that of bulk CP with free Rh cocatalyst.

Hierarchically porous metal organic framework immobilized formate dehydrogenase for enzyme electrocatalytic CO2 reduction

The utilization of CO2 has become an important issue aiming to the achievement of carbon neutrality. The enzyme electrocatalytic CO2 reduction to produce carbonaceous fuels has been increasingly appealing. While the low CO2 aqueous solubility severely limits the CO2 reduction efficiency, and the expensive cofactor restricts the application potential. In this study, microporous UiO-66-NH2 was firstly converted into hierarchically porous structure (HP-UiO-66-NH2) containing both micropores and mesopores to simultaneously enhance the enrichment of CO2 and the immobilization of formate dehydrogenase by optimizing the pore structure. The relative contents of the protein secondary structures were similar after immobilization. The mechanism of enzyme electrocatalytic CO2 reduction was proposed. The enzyme stability was highly enhanced after immobilization and the CO2 reduction was enhanced by the enzyme electrocatalytic system through the combination of CO2 enrichment and electrocatalytic NADH regeneration. The optimized catalytic system can obtain formate yield of 1.826 mM in 3 h (5.57 times higher than the free enzyme system) with a generation rate of 6086.7 μmol gcat‑1h‑1.

Efficient enzymatic synthesis of (S)-1-(3′-bromo-2′-methoxyphenyl)ethanol, the key building block of lusutrombopag

(S)-1-(3′-Bromo-2′-methoxyphenyl)ethanol ((S)-1b) is the key precursor for the synthesis of Lusutrombopag. The bioreduction of 1-(3′-bromo-2′-methoxyphenyl)ethanone (1a) offers an attractive method to access this important compound. Through screening the available carbonyl reductases, we obtained a carbonyl reductase from Novosphingobium aromaticivorans (CBR), which could completely convert 100 ​g/L of 1a to (S)-1b. Furthermore, a carbonyl reductase from Novosphingobium sp. Leaf2 (NoCR) was identified to completely convert 200 ​g/L of 1a to (S)-1b with excellent enantioselectivity (>99% ee) and 77% isolated yield using FDH/formate system for NADH regeneration. The Km and kcat of recombinant NoCR towards 1a were 0.66 ​mmol/L and 7.5 s-1, and the catalytic efficiency kcat/Km was 11.3 ​mmol/s.L. Meanwhile, NoCR showed high catalytic activity and stereoselectivity towards acetophenone derivatives with halogen or methoxy substitution on the benzene ring, indicating that NoCR is a valuable biocatalyst with potential practical applications.

A Light‐Dark Cascade Procedure for the Regeneration of NADH using Graphitic Carbon Nitride Nanosheets

AbstractPhotocatalytic regeneration of NAD(P)H is a green and promising method which utilizes clean solar energy. However, we found in this study that light exposure of NADH will cause the deactivation of the regenerated NADH. Therefore, we used ultrathin g‐C3N4 nanosheets as photocatalyst and developed a two‐pot light‐dark cascade procedure for NADH regeneration to avoid the light exposure and the deactivation of NADH. This two‐pot procedure included a first light reaction step where triethanolamine was converted to glycolaldehyde through visible light photocatalysis which could reduce NAD+ to NADH through a non‐catalytic process in the followed dark reaction step. Using this procedure, the NADH productivity reached 1.625 mmol ⋅ h−1 ⋅ g−1 and was about 29 times higher that obtained in the one‐pot method. The regenerated NADH using the two‐pot procedure could act as cofactor of alcohol dehydrogenase to catalyze the reduction of acetaldehyde, illustrating the application potential of this procedure to aid enzyme catalyzed redox reactions.

Bioinspired Metalation of the Metal‐Organic Framework MIL‐125‐NH2 for Photocatalytic NADH Regeneration and Gas‐Liquid‐Solid Three‐Phase Enzymatic CO2 Reduction

AbstractCoenzyme NADH regeneration is crucial for sustained photoenzymatic catalysis of CO2 reduction. However, light‐driven NADH regeneration still suffers from the low regeneration efficiency and requires the use of a homogeneous Rh complex. Herein, a Rh complex‐based electron transfer unit was chemically attached onto the linker of the MIL‐125‐NH2. The coupling between the light‐harvesting iminopyridine unit and electron‐transferring Rh‐complex facilitated the photo‐induced electron transfer for the NADH regeneration with the yield of 66.4 % in 60 mins for 5 cycles. The formate dehydrogenase was further deposited onto the hydrophobic layer of the membrane by a reverse filtering technique, which forms the gas‐liquid‐solid reaction interface around the enzyme site. It gave an enhanced formic acid yield of 9.5 mM in 24 hours coupled with the in situ regenerated NADH. The work could shed light on the construction of integrated inorganic‐enzyme hybrid systems for artificial photosynthesis.

Bioinspired Metalation of the Metal-Organic Framework MIL-125-NH2 for Photocatalytic NADH Regeneration and Gas-Liquid-Solid Three-Phase Enzymatic CO2 Reduction.

Coenzyme NADH regeneration is crucial for sustained photoenzymatic catalysis of CO2 reduction. However, light-driven NADH regeneration still suffers from the low regeneration efficiency and requires the use of a homogeneous Rh complex. Herein, a Rh complex-based electron transfer unit was chemically attached onto the linker of the MIL-125-NH2 . The coupling between the light-harvesting iminopyridine unit and electron-transferring Rh-complex facilitated the photo-induced electron transfer for the NADH regeneration with the yield of 66.4 % in 60 mins for 5 cycles. The formate dehydrogenase was further deposited onto the hydrophobic layer of the membrane by a reverse filtering technique, which forms the gas-liquid-solid reaction interface around the enzyme site. It gave an enhanced formic acid yield of 9.5 mM in 24 hours coupled with the in situ regenerated NADH. The work could shed light on the construction of integrated inorganic-enzyme hybrid systems for artificial photosynthesis.

Construction and characterization of BsGDH-CatIB variants and application as robust and highly active redox cofactor regeneration module for biocatalysis

BackgroundCatalytically active inclusion bodies (CatIBs) are known for their easy and cost efficient production, recyclability as well as high stability and provide an alternative purely biological technology for enzyme immobilization. Due to their ability to self-aggregate in a carrier-free, biodegradable form, no further laborious immobilization steps or additional reagents are needed. These advantages put CatIBs in a beneficial position in comparison to traditional immobilization techniques. Recent studies outlined the impact of cooperative effects of the linker and aggregation inducing tag on the activity level of CatIBs, requiring to test many combinations to find the best performing CatIB variant.ResultsHere, we present the formation of 14 glucose dehydrogenase CatIB variants of Bacillus subtilis, a well-known enzyme in biocatalysis due to its capability for substrate coupled regeneration of reduced cofactors with cheap substrate glucose. Nine variants revealed activity, with highest productivity levels for the more rigid PT-Linker combinations. The best performing CatIB, BsGDH-PT-CBDCell, was characterized in more detail including long-term storage at −20 °C as well as NADH cofactor regeneration performance in repetitive batch experiments with CatIB recycling. After freezing, BsGDH-PT-CBDCell CatIB only lost approx. 10% activity after 8 weeks of storage. Moreover, after 11 CatIB recycling cycles in repetitive batch operation 80% of the activity was still present.ConclusionsThis work presents a method for the effective formation of a highly active and long-term stable BsGDH-CatIB as an immobilized enzyme for robust and convenient NADH regeneration.

Photocatalyst-mineralized biofilms as living bio-abiotic interfaces for single enzyme to whole-cell photocatalytic applications.

There is an increasing trend of combining living cells with inorganic semiconductors to construct semi-artificial photosynthesis systems. Creating a robust and benign bio-abiotic interface is key to the success of such solar-to-chemical conversions but often faces a variety of challenges, including biocompatibility and the susceptibility of cell membrane to high-energy damage arising from direct interfacial contact. Here, we report living mineralized biofilms as an ultrastable and biocompatible bio-abiotic interface to implement single enzyme to whole-cell photocatalytic applications. These photocatalyst-mineralized biofilms exhibited efficient photoelectrical responses and were further exploited for diverse photocatalytic reaction systems including a whole-cell photocatalytic CO2 reduction system enabled by the same biofilm-producing strain. Segregated from the extracellularly mineralized semiconductors, the bacteria remained alive even after 5 cycles of photocatalytic NADH regeneration reactions, and the biofilms could be easily regenerated. Our work thus demonstrates the construction of biocompatible interfaces using biofilm matrices and establishes proof of concept for future sustainable photocatalytic applications.

Dehydrogenase-Functionalized Interfaced Materials in Electroenzymatic and Photoelectroenzymatic CO2 Reduction

The simultaneous extenuation of emitted CO2 conversion to chemicals and fuels has purposely motivated researchers toward adapting electrochemical techniques associated with gas diffusion electrodes by using enzyme formate dehydrogenase (FDH). Herein, an in-depth analysis on the background of electroenzymatic CO2 reduction in connection with the nature of binding of FDH on the electrode surface for interfacial direct electron transfer (DET) and mediated electron transfer (MET) are discussed. Types of bioelectrodes with a dehydrogenase enzyme and adapted techniques for their fabrication are emphasized along with new FDH enzymes and their possible effect on CO2 reduction efficacy. Solar-driven photoelectroenzymatic CO2 reduction has emerged as a promising strategy, which is discussed in terms of the rate of CO2 conversion in photoelectrochemical (PEC) cells, enzyme-interfaced materials, NADH regeneration, the role of synthetic photoelectrode materials, and diffusional electron mediators. Advanced applications of electroenzymatic CO2-reduction-driven fuels and their effect are assessed to offer future directions of interfaced materials for electroenzymatic and solar-driven processes.

Mo3+ hydride as the common origin of H2 evolution and selective NADH regeneration in molybdenum sulfide electrocatalysts

Hydride transfers are key to a number of economically and environmentally important reactions, including H2 evolution and NADH regeneration. The electrochemical generation of hydrides can therefore drive the electrification of chemical reactions to improve their sustainability for a green economy. Catalysts containing molybdenum have recently been recognized as among the most promising non-precious catalysts for H2 evolution, but the mechanism by which molybdenum confers this activity remains debated. Here we show the presence of trapped Mo3+ hydride in amorphous molybdenum sulfide (a-MoSx) during the hydrogen evolution reaction and extend its catalytic role to the selective hydrogenation of the biologically important energy carrier NAD to its active 1,4-NADH form. Furthermore, this reactivity applies to other HER-active molybdenum sulfides. Our results demonstrate a direct role for molybdenum in heterogeneous H2 evolution. This mechanistic finding also reveals that molybdenum sulfides have potential as economic electrocatalysts for NADH regeneration in biocatalysis.

Toward cheaper light harvesting systems: Using earth-abundant metal oxide nanoparticles in self-assembled peptide-porphyrin nanofibers.

Cheap artificial light harvesting systems, which competently harvest solar energy and promote efficient energy transfer, are highly sought after in the renewable sector. We report the synthesis of self-assembled peptide-porphyrin fibers (SJ 6) fabricated with iron(III) oxide (Fe3 O4 ) nanoparticles as feasible electron acceptors. Charge-complementarity between the negatively charged peptide (20E) and the protonated Zn-tetraphenyl porphyrin (ZnTPyP) led to an ordered assembly of the ZnTPyP molecules, enabling efficient light harvesting. X-ray diffraction data indicates a more ordered structure in SJ 6 compared to 20E and ZnTPyP. The incorporation of Fe3 O4 nanoparticles into SJ 6 showed significant fluorescence quenching, indicating efficient electron flow from the donor to the acceptor. The SJ 6-nFe3 O4 system performed the light reaction of photosynthesis as confirmed by the reduction of 1 mM NAD+ to 0.180 mM NADH upon exposure to visible light (Xe lamp λ > 420 nm) for 1 h. The photochemical regeneration of NADH using the SJ 6-nFe3 O4 system was coupled to glutamate dehydrogenase-catalyzed conversion of α-ketoglutarate to L-glutamate. These results confirm the successful synthesis of an artificial light harvesting peptide-porphyrin system with Fe3 O4 nanoparticles as promising low-cost electron separators.

Efficient Nicotinamide Adenine Dinucleotide Regeneration with a Rhodium-Carbene Catalyst and Isolation of a Hydride Intermediate.

Regeneration of nicotinamide adenine dinucleotide (NADH) has been the primary interest in the field of enzymatic transformation, especially associating oxidoreductases given the stoichiometric consumption. The synthesized carbene-ligated rhodium complex [(η5-Cp*)Rh(MDI)Cl]+ [Cp* = pentamethylcyclopentadienyl; MDI = 1,1'-methylenebis(3,3'-dimethylimidazolium)] acts as an exceptional catalyst in the reduction of NAD+ to NADH with a turnover frequency of 1730 h-1, which is over twice that of the higher catalytic activity of the commercially available catalyst [Cp*Rh(bpy)Cl]+ (bpy = 2,2'-bipyridine). Offsetting the contentious atmosphere currently taking place over the specific intermediate of the NADH regeneration, this study presents pivotal evidence of a metal hydride intermediate with a bis(carbene) ligand: a stable form of the rhodium hydride intermediate, [(η5-Cp*)Rh(MDI)H]+, was isolated and fully characterized. This enables thorough insight into the possible mechanism and exact intermediate structure in the NAD+ reduction process.

Ultra-efficient synthesis of bamboo-shape porphyrin framework for photocatalytic CO2 reduction and consecutive C-S/C-N bonds formation

The consumption of carbon dioxide (CO2) for direct solar fuel productions and synthesis of fine solar chemicals are gaining leading significance due to the shortage of fossil-fuel and global warming worldwide. Therefore, herein, we report the synthesis and development of solar light harvesting bamboo-shape magnesium tetraphenyl porphyrin based framework (Mg-TPPBF) photocatalyst. The development of Mg-TPPBF photocatalyst-biocatalyst bonded system as a solar light harvesting photocatalyst functions in a extremely efficient manner, increasing to high regeneration of NADH (73.9%), followed by its utilization in exclusive amount of formic acid (HCOOH) production (197.2 μmol) from CO2. The Mg-TPPBF photocatalyst also enables in consecutive C-S/C-N bonds formation with high yield (~96–99%) and selectivity (~97–99%) under solar light with excellent reusability. The current research work highlights the enlargement and application of Mg-TPPBF photocatalyst for direct solar fuel production from CO2 and fine solar chemical synthesis.

Assembly of COFs layer and electron mediator on silica for visible light driven photocatalytic NADH regeneration

Photocatalytic regeneration of the high-cost co-factor NADH is a promising method to couple photo-enzyme catalysis, however, the usage of homogeneous electron mediator ([Cp*Rh(bpy)H2O]2+) which deactivates enzymes deteriorates the whole process efficiency. Herein, we report the immobilization of electron mediator ([Cp*Rh(bpy)H2O]2+) on covalent organic frameworks (COFs) by the sequential assembly method. The resultant bi-functional photocatalyst with controlled cascade electron transfer afforded NADH production rate of 9.8 mmol·gCOF−1·h−1, much higher than previously reported ones with integrated electron mediator. The photo-enzyme coupling system with the photocatalyst and alcohol dehydrogenase could continuously produce butanol for at least 180 min under visible light irradiation, implying the potential application of bi-functional photocatalyst in artificial photosynthesis.

Room-Temperature Persistent Luminescence in Metal Halide Perovskite Nanocrystals for Solar-Driven CO 2 Bioreduction

Room-Temperature Persistent Luminescence in Metal Halide Perovskite Nanocrystals for Solar-Driven CO ₂ Bioreduction

In‐situ prepared S‐doped covalent melem framework as a photocatalyst with improved photocatalytic responses under solar light illumination

AbstractMelem (M) is an emergent metal‐free material due to its nature abundance and easy preparation. Synergetic escalation of band gap generation and charge transfer of M to increase solar chemical synthesis ability remains a hot challenging topic yet. Herein, we designed first time in‐situ prepared sulfur‐doped covalent melem‐based frameworks (S‐CMFs) photocatalyst for NAD(P)H regeneration and 1,3‐oxathiolane‐2‐thiones production under solar light irradiation. S‐CMFs photocatalyst shows excellent photocatalytic stability and activity over the pristine CMFs and M. The presence of sulfur in frameworks drastically improved the photocatalytic efficiency of S‐CMFs due to its unique properties such as enhanced the solar light adsorption ability, molar extinction coefficient, slow charge recombination, fast charge separation and transportation of solar light to generate electrons‐holes. Hence, S‐CMFs photocatalyst has a vital role in the regeneration of NADH and NADPH with 54.84 and 51.05 % catalytic efficiencies and ~ 99 % for yields and selectivity of 1,3‐oxathiolane‐2‐thiones under solar light. This study demonstrates that the Z‐scheme hetero junction and photocatalytic reaction using S‐CMFs would be a suitable route for the production of solar fuel via artificial photosynthetic system.