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Thermophilic Methanothermobacter spp. are used as model microbes to study the physiology and biochemistry of the conversion of molecular hydrogen and carbon dioxide into methane (i.e., hydrogenotrophic methanogenesis). Yet, a genetic system for these model microbes was missing despite intensive work for four decades. Here, we report the successful implementation of genetic tools for Methanothermobacter thermautotrophicus ΔH. We developed shuttle vectors that replicated in Escherichia coli and M. thermautotrophicus ΔH. For M. thermautotrophicus ΔH, a thermostable neomycin resistance cassette served as the selectable marker for positive selection with neomycin, and the cryptic plasmid pME2001 from Methanothermobacter marburgensis served as the replicon. The shuttle-vector DNA was transferred from E. coli into M. thermautotrophicus ΔH via interdomain conjugation. After the successful validation of DNA transfer and positive selection in M. thermautotrophicus ΔH, we demonstrated heterologous gene expression of a thermostable β-galactosidase-encoding gene (bgaB) from Geobacillus stearothermophilus under the expression control of four distinct synthetic and native promoters. In quantitative in-vitro enzyme activity assay, we found significantly different β-galactosidase activity with these distinct promoters. With a formate dehydrogenase operon-encoding shuttle vector, we allowed growth of M. thermautotrophicus ΔH on formate as the sole growth substrate, while this was not possible for the empty-vector control. IMPORTANCE The world economies are facing permanently increasing energy demands. At the same time, carbon emissions from fossil sources need to be circumvented to minimize harmful effects from climate change. The power-to-gas platform is utilized to store renewable electric power and decarbonize the natural gas grid. The microbe Methanothermobacter thermautotrophicus is already applied as the industrial biocatalyst for the biological methanation step in large-scale power-to-gas processes. To improve the biocatalyst in a targeted fashion, genetic engineering is required. With our shuttle-vector system for heterologous gene expression in M. thermautotrophicus, we set the cornerstone to engineer the microbe for optimized methane production but also for production of high-value platform chemicals in power-to-x processes.

An auto-inducing expression system was developed that could express target genes in S. marcescens MG1. Using this system, MG1 was constructed as a whole-cell biocatalyst to produce 2,3-butanediol/acetoin. Formate dehydrogenase (FDH) and 2,3-butanediol dehydrogenase were expressed together to build an NADH regeneration system to transform diacetyl to 2,3-butanediol. After fermentation, the extract of recombinant S. marcescens MG1ABC (pETDuet-bdhA-fdh) showed 2,3-BDH activity of 57.8 U/mg and FDH activity of 0.5 U/mg. And 27.95 g/L of 2,3-BD was achieved with a productivity of 4.66 g/Lh using engineered S. marcescens MG1(Pswnb+pETDuet-bdhA-fdh) after 6 h incubation. Next, to produce 2,3-butanediol from acetoin, NADH oxidase and 2,3-butanediol dehydrogenase from Bacillus subtilis were co-expressed to obtain a NAD+ regeneration system. After fermentation, the recombinant strain S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC) showed AR activity of 212.4 U/mg and NOX activity of 150.1 U/mg. We obtained 44.9 g/L of acetoin with a productivity of 3.74 g/Lh using S. marcescens MG1ABC (pSWNB+pETDuet-bdhA-yodC). This work confirmed that S. marcescens could be designed as a whole-cell biocatalyst for 2,3-butanediol and acetoin production.

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.

Sertkaya, Simge (Balikesir Author), Syngas fermentation via the Wood-Ljungdahl (WL) pathway is a promising approach for converting gaseous pollutants (CO and CO2) into high-value commodities. Because the WL involves several enzymes with trace metal components, it requires an adequate supply of micronutrients in the fermentation medium for targeted bioprocessing such as bioethanol production. Plackett-Burman statistical analysis was performed to examine the most efficient trace elements (Ni, Mg, Ca, Mn, Co, Cu, B, W, Zn, Fe, and Mo) and their concentrations for Clostridium ljungdahlii on ethanol production. Overall, 1.5 to 2.5 fold improvement in ethanol production could be achieved with designed trace element concentrations. The effects of tungsten and copper on ethanol and biomass production were determined to be the most significant, respectively. The model developed was statistically significant and has the potential to significantly decrease the cost of trace element solutions by 18-22%. This research demonstrates the critical importance of optimizing the medium for syngas fermentation in terms of product distribution and economic feasibility., Ege University FDK-2020-22039 ED481D 2019/033

The immobilization of the non-metallic enzyme formate dehydrogenase from Candida boidinii (CbFDH) into a nanoporous carbon with appropriate pore structure was explored for the bioelectrochemical conversion of CO2 to formic acid (FA). Higher FA production rates were obtained upon immobilization of CbFDH compared to the performance of the enzyme in solution, despite the lower nominal CbFDH to NADH (β-nicotinamide adenine dinucleotide reduced) cofactor ratio and the lower amount of enzyme immobilized. The co-immobilization of the enzyme and a rhodium complex as mediator in the nanoporous carbon allowed the electrochemical regeneration of the cofactor. Preparative electrosynthesis of FA carried out on biocathodes of relatively large dimensions (ca. 3 cm × 2 cm) confirmed the higher production rate of FA for the immobilized enzyme. Furthermore, the incorporation of a Nafion binder in the biocathodes did not modify the immobilization extent of the CbFDH in the carbon support. Coulombic efficiencies close to 46% were obtained for the electrosynthesis carried out at −0.8 V for the biocathodes prepared using the lowest Nafion binder content and the co-immobilized enzyme and rhodium redox mediator. Although these values may yet be improved, they confirm the feasibility of these biocathodes in larger scales (6 cm2) beyond most common electrode dimensions reported in the literature (ca. a few mm2). NH, VM and JI thank Spanish MINICINN (projects CTQ2016-76231-C2-2-R and PID2019-108136RB-C32) for financial support. COA thanks the financial support of the European Research Council through a Consolidator Grant (PHOROSOL 684161).

Many organisms reductively assimilate selenite to synthesize selenoprotein. Although the thioredoxin system, consisting of thioredoxin 1 (TrxA) and thioredoxin reductase with NADPH, can reduce selenite and is considered to facilitate selenite assimilation, the detailed mechanism remains obscure. Here, we show that selenite was reduced by the thioredoxin system from Pseudomonas stutzeri only in the presence of the TrxA (PsTrxA), and this system was specific to selenite among the oxyanions examined. Mutational analysis revealed that Cys33 and Cys36 residues in PsTrxA are important for selenite reduction. Free thiol-labeling assays suggested that Cys33 is more reactive than Cys36. Mass spectrometry analysis suggested that PsTrxA reduces selenite via PsTrxA-SeO intermediate formation. Furthermore, an in vivo formate dehydrogenase activity assay in Escherichia coli with a gene disruption suggested that TrxA is important for selenoprotein biosynthesis. The introduction of PsTrxA complemented the effects of TrxA disruption in E. coli cells, only when PsTrxA contained Cys33 and Cys36. Based on these results, we proposed the early steps of the link between selenite and selenoprotein biosynthesis via the formation of TrxA–selenium complexes.

The design of bioinspired synthetic inorganic molecular complexes is challenging, due to a lack of understanding of enzyme action and the degree to which that action can be translated into mimics. Exemplary of this challenge is the reversible conversion of formate into CO2 by formate dehydrogenase (FDH) enzymes with Mo/W centers in large molybdopterin cofactors. Despite numerous efforts to synthesize Mo/W-containing molecular complexes, none have been demonstrated to reproduce the full reactivity of FDH. Here, we carry out a large-scale, high-throughput screening study on all mononuclear Mo/W complexes currently deposited in Cambridge Structural Database (CSD). Using density functional theory, we systematically investigate the individual effects of metal identity, ligand identity, oxidation state, and coordination number on structural, electronic and catalytic properties. We compare our results on molecular complexes with quantum mechanics/molecular mechanics simulations on a representative FDH enzyme to further elucidate the influence of the enzyme environment. These comparisons reveal that the enzyme environment primarily influences the metal-local geometry, and these metal-local structural variations can improve catalysis. Through a series of computational mutations on molecular complexes, we extend beyond the CSD structures to further identify the limits of varied chalcogen and metal identity. This broad set and comparison reveal relatively little variation of electronic properties of the metal center due to the presence of the enzyme environment or changes in metal-distant ligand chemistry. Instead, these properties are found to be much more sensitive to the identity of the metal and the nature of the bound terminal chalcogen.

Biocatalytic conversion of greenhouse gases such as carbon dioxide into commercial products is one of the promising key approaches to solve the problem of climate change. Microbial enzymes, including carbonic anhydrase, NAD-dependent formate dehydrogenase, ribulose bisphosphate carboxylase, and methane monooxygenase, have been exploited to convert atmospheric gases into industrial products. Carbonic anhydrases are Zn2+-dependent metalloenzymes that catalyze the reversible conversion of CO2 into bicarbonate. They are widespread in bacteria, algae, plants, and higher organisms. In higher organisms, they regulate the physiological pH and contribute to CO2 transport in the blood. In plants, algae, and photosynthetic bacteria carbonic anhydrases are involved in photosynthesis. Converting CO2 into bicarbonate by carbonic anhydrases can solidify gaseous CO2, thereby reducing global warming due to the burning of fossil fuels. This review discusses the three-dimensional structures of carbonic anhydrases, their physiological role in marine life, their catalytic mechanism, the types of inhibitors, and their medicine and industry applications.

Microbial synthesis of plant-based (-)-menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (-)-limonene and (+)-cis-isopulegone. Herein, the first artificial and effective system was developed for (+)-cis-isopulegone biosynthesis from (-)-limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene-3-hydroxylase, and isopiperitenol dehydrogenase. A cofactor self-regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)-cis-isopulegone (up to 281.2 mg L-1 ) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (-)-menthol.

International audience; Only two enzymes are capable of directly reducing CO 2 : the CO dehydrogenase, which produces CO at a [NiFe 4 S 4 ] active site, and the formate dehydrogenase, which produces formate at a mononuclear W or Mo active site. Both metalloenzymes are very rapid, energy-efficient and specific in terms of product. They have been connected to electrodes with two different objectives. A series of studies used protein film electrochemistry to learn about different aspects of the mechanism of these enzymes (reactivity with substrates, inhibitors…). Another series focused on taking advantage of the catalytic performance of these enzymes to build biotechnological devices, from CO 2-reducing electrodes to full photochemical devices performing artificial photosynthesis. Here, we review all these works. Marta Meneghello obtained her Ph.D. in Chemistry from the University of Southampton (UK) in 2018, under the supervision of Prof P.N. Bartlett, working on the immobilization of redox enzymes at electrode surfaces. Since 2018, she is a postdoctoral researcher in the group of Christophe Leger and Vincent Fourmond at the CNRS of Marseille (France). Her current research focuses on kinetics studies of metalloenzymes, such as CO dehydrogenase and formate dehydrogenase, using electrochemical methods. Dr Christophe Léger obtained his Ph.D. from the University of Bordeaux and was a postdoc in the group of Fraser Armstrong from 1999 to 2002. He is "Directeur de Recherche" at CNRS. His interests lie in kinetic and mechanistic studies of complex metalloenzymes. Vincent Fourmond obtained his Ph.D. at the interface of physics and biology from Université Paris Diderot in 2007. He held postdoctoral positions first in the group of Christophe Léger in Marseille and then Vincent Artero in Grenoble, before coming back to Marseille as a permanent CNRS researcher in 2011. His research interests revolve around the use of kinetic techniques, principally protein film electrochemistry, to understand the mechanisms of metalloenzymes (hydrogenases, CO dehydrogenases, molybdenum enzymes), the development of methodological aspects of PFE, and the development and maintenance of the open source data analysis software QSoas [1] .

A non-natural cofactor and formate driven system for reductive carboxylation of pyruvate is presented. A formate dehydrogenase (FDH) mutant, FDH*, that favors a non-natural redox cofactor, nicotinamide cytosine dinucleotide (NCD), for generation of a dedicated reducing equivalent at the expense of formate were acquired. By coupling FDH* and NCD-dependent malic enzyme (ME*), the successful utilization of formate is demonstrated as both CO2 source and electron donor for reductive carboxylation of pyruvate with a perfect stoichiometry between formate and malate. When 13 C-isotope-labeled formate was used in in vitro trials, up to 53 % of malate had labeled carbon atom. Upon expression of FDH* and ME* in the model host E. coli, the engineered strain produced more malate in the presence of formate and NCD. This work provides an alternative and atom-economic strategy for CO2 fixation where formate is used in lieu of CO2 and offers dedicated reducing power.

Background In times of global climate change, the conversion and capturing of inorganic CO2 have gained increased attention because of its great potential as sustainable feedstock in the production of biofuels and biochemicals. CO2 is not only the substrate for the production of value-added chemicals in CO2-based bioprocesses, it can also be directly hydrated to formic acid, a so-called liquid organic hydrogen carrier (LOHC), by chemical and biological catalysts. Recently, a new group of enzymes were discovered in the two acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which catalyze the direct hydrogenation of CO2 to formic acid with exceptional high rates, the hydrogen-dependent CO2 reductases (HDCRs). Since these enzymes are promising biocatalysts for the capturing of CO2 and the storage of molecular hydrogen in form of formic acid, we designed a whole-cell approach for T. kivui to take advantage of using whole cells from a thermophilic organism as H2/CO2 storage platform. Additionally, T. kivui cells were used as microbial cell factories for the production of formic acid from syngas. Results This study demonstrates the efficient whole-cell biocatalysis for the conversion of H2 + CO2 to formic acid in the presence of bicarbonate by T. kivui. Interestingly, the addition of KHCO3 not only stimulated formate formation dramatically but it also completely abolished unwanted side product formation (acetate) under these conditions and bicarbonate was shown to inhibit the membrane-bound ATP synthase. Cell suspensions reached specific formate production rates of 234 mmol g protein −1 h−1 (152 mmol g CDW −1 h−1), the highest rates ever reported in closed-batch conditions. The volumetric formate production rate was 270 mmol L−1 h−1 at 4 mg mL−1. Additionally, this study is the first demonstration that syngas can be converted exclusively to formate using an acetogenic bacterium and high titers up to 130 mM of formate were reached. Conclusions The thermophilic acetogenic bacterium T. kivui is an efficient biocatalyst which makes this organism a promising candidate for future biotechnological applications in hydrogen storage, CO2 capturing and syngas conversion to formate.

Formate dehydrogenase from Candida boidinii (EC.1.2.1.2; CbFDH) is a commercially available enzyme and can be easily handled as a catalyst for the CO2 reduction to formate in the presence of NADH, single-electron reduced methylviologen (MV+˙) and so on. It was found that the formate oxidation to CO2 with CbFDH was suppressed using the oxidized MV as a co-enzyme and the single-electron reduced MV (MV+˙) was effective for the catalytic activity of CbFDH for the CO2 reduction to formate compared with that using the natural co-enzyme of NADH [Y. Amao, Chem. Lett., 2017, 46, 780-788]. The CO2 reduction to formate catalyzed by CbFDH requires two molecules of the MV+˙. In order to clarify the two-electron reduction process using MV+˙ in the CO2 reduction to formate catalyzed with CbFDH, we attempted enzyme reaction kinetics, electrochemical and quantum chemical analyses. Kinetic parameters obtained from the enzymatic kinetic analysis metric revealed an index of affinity of MV+˙ for CbFDH in the CO2 reduction to formate. From the results of the electrochemical analysis, it was predicted that only one molecule of MV+˙ was bound to CbFDH, and the MV bound to CbFDH was to be necessarily re-reduced by the electron source outside of CbFDH to supply the second electron in the CO2 reduction to formate. From the results of docking simulation and density functional theory (DFT) calculations, it was indicated that one molecule of MV bound to the position close to CO2 in the inner part of the substrate binding pocket of CbFDH contributed to the two-electron CO2 reduction to formate.

Formate is produced, in plants, by various biochemical pathways and it is degraded by Formate Dehydrogenase FDH, in presence of NAD+, into CO2 and NADH. FDH has been proposed as one of the enzymes regulating molybdenum (Mo) and iron (Fe) homeostasis. Here we explored the impact of FDH perturbation on Mo and Fe plant nutritional status and FDH relevance on the plant responses against abiotic stresses, by using in silico and experimental approaches. The characterization of different Arabidopsis thaliana and Nicotiana tabacum FDH transgenic lines suggests that FDH promoter activity is dependent on both Mo and Fe nutritional supply and that FDH overexpression alters Mo concentrations in seeds and Fe concentration in seeds, leaves and stems. Also, FDH overexpression delays the dark-induced senescence whereas the lack of FDH accelerates its progression. FDH is therefore a multifaceted enzyme with impact on Mo and Fe homeostasis and regulation of dark-induced senescence.

Biocatalytic transamination is widely used in industrial production of chiral chemicals. Here, we constructed a novel multi-enzyme system to promote the conversion of the amination reaction. Firstly, we constructed the ArR-ωTA/TdcE/FDH/LDH multi-enzyme system, by combination of (R)-selective ω-transaminase derived from Arthrobacter sp. (ArR-ωTA), formate dehydrogenase (FDH) derived from Candida boidinii, formate acetyltransferase (TdcE) and lactate dehydrogenase (LDH) derived from E. coli MG1655. This multi-enzyme system was used to efficiently remove the by-product pyruvate by TdcE and LDH to facilitate the transamination reaction. The TdcE/FDH pathway was found to dominate the by-product pyruvate removal in the transamination reaction. Secondly, we optimized the reaction conditions, including D-alanine, DMSO, and pyridoxal phosphate (PLP) with different concentration of 2-pentanone (as a model substrate). Thirdly, by using the ArR-ωTA/TdcE/FDH/LDH system, the conversions of 2-pentanone, 4-phenyl-2-butanone and cyclohexanone were 84.5%, 98.2% and 79.3%, respectively.

Formate dehydrogenase from Candida boidinii (CbFDH) reversibly catalyzes formate to carbon dioxide with the redox couple NAD+/NADH. There have been many studies on CbFDH-catalyzed oxidation of formate to carbon dioxide in the presence of NAD+. On the other hand, there are few studies on the detailed mechanism of the carbon dioxide reduction to formate catalyzed by CbFDH in the presence of NADH. In addition, it is not clear whether CbFDH can reduce carbon dioxide, bicarbonate or carbonate to formate. In this study, formate production with CbFDH in the presence of NADH was investigated in solutions with different ratios of carbon dioxide, bicarbonate and carbonate. The reaction rate of formate production with CbFDH increased in proportion to the concentration of carbon dioxide in the reaction solution. On the other hand, the formate production with CbFDH was suppressed in proportion to the concentration of bicarbonate or carbonate in the reaction solution. Thus, CbFDH was found to catalytically reduce only carbon dioxide to formate among the three types of carbonate species.

Enzymatic production of xylitol is a promising alternative to the chemical hydrogenation process. However, it encounters problems that are largely due to protein susceptibility to environmental factors. In this study, to develop a robust, practical enzymatic process for xylitol production, a coupled enzyme system consisting of formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and xylose reductase (XR) was constructed, wherein the alkaline product produced by FDH and the acidic product produced by GDH could neutralize each other during cofactor regeneration. After optimization of conditions, a pH-neutralization, redox-balanced process was developed that could be carried out in pure water requiring no pH regulation. As a result, a xylitol production of 273.6 g/L that is much higher than those yet reported was obtained from 2 M xylose in 24 h, with a relatively high productivity of 11.4 g/(L h). The strategy demonstrated here can be adapted for the production of other NADH-consuming products.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O-2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo=S) and oxo (Mo=O) bonds, were observed in the presence of azide (N-3(-)) or cyanate (OCN-). Azide provided best protection against O-2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

Graphene oxide‐based nanomaterials are promising for enzyme immobilization due to the possibilities of functionalizing surface. Polyethylenimine‐grafted graphene oxide was constructed as a novel scaffold for immobilization of formate dehydrogenase. Compared with free formate dehydrogenase and graphene oxide adsorbed formate dehydrogenase, thermostability, storage stability, and reusability of polyethylenimine‐grafted graphene oxide‐formate dehydrogenase were enhanced. Typically, polyethylenimine‐grafted graphene oxide‐formate dehydrogenase remained 47.4% activity after eight times’ repeat reaction. The immobilized capacity of the polyethylenimine‐grafted graphene oxide was 2.4‐folds of that of graphene oxide. Morphological and functional analysis of polyethylenimine‐grafted graphene oxide‐formate dehydrogenase was performed and the assembling mechanism based on multi‐level interactions was studied. Consequently, this practical and facile strategy will likely find applications in biosynthesis, biosensing, and biomedical engineering.

With the widespread concerns on CO2 capture and utilization, various approaches have been developed to convert CO2 into valuable products. To achieve fast and efficient conversion, a hybrid CO2 electroreduction system was constructed, in which a cofactor-dependent formate dehydrogenase (FDH) was responsible for CO2 enzymatic reduction to formate and Cu nanoparticles (CuNPs) was used for enzyme immobilization and cofactor regeneration. A polyethylene glycol swing arm was further adopted to construct an enzyme-cofactor conjugate and promote the overall reaction. This electron-mediator-free hybrid system containing immobilized FDH-cofactor conjugates and CuNPs achieved a formate production of 8.5 mM and a productivity as high as 11.8 μM mU−1 h−1 with a faraday efficiency of 22.8%, several folds better than systems with unconjugated FDHs alone or with unconjugated FDHs and CuNPs. These results suggest that such hybrid systems may represent a potentially viable alternative for many cofactor-dependent bioelectrochemical applications.

A microbial battery (MB) breaks down waste organics and stores the liberated electrons in a solid-state cathode. Here, we demonstrate that these electrons can be utilized, along with the products (e.g., carbon dioxide, CO2) of the MB, to electrochemically generate higher-value products from waste. We utilize a series-stacked MB with mediated enzymatic electrosynthesis (EES) to produce either hydrogen (H2) from protons (H+) or formate (HCOO–) from CO2 with the electrons and carbon directly derived from waste organics. We also demonstrate that a reduced solid-state cathode can be used as a solid-state anode as an electron source for EES. This work shows that higher-value products can be electrochemically generated at high faradaic efficiencies (up to 91% for hydrogenase and 72% for formate dehydrogenase) using the energy, electrons, and carbon contained in the waste organic input without the need for continuous oxygen, potentiostatic control, or supplemental power.

Isotope substitution of enzymes has become a means of addressing the participation of protein motions in enzyme-catalyzed reactions. The idea is that only the enzyme mass will be altered and not the electrostatics, so that the protein dynamics are essentially the same but at lower frequencies because of the mass change. In this study, we variably label all carbon atoms in formate dehydrogenase (FDH) with

A facile approach for the construction of reagent-free electrochemical dehydrogenase-based biosensors is presented. Enzymes and cofactors (NAD+ and Fe(CN)63-) were immobilized by modification of screen-printed carbon electrodes with graphene oxide (GO) and an additional layer of cellulose acetate. The sensor system was exemplarily optimized for an l-lactate electrode in terms of GO concentration, working potential, and pH value. The biosensor exhibited best characteristics at pH 7.5 in 100 mM potassium phosphate buffer at an applied potential of +0.250 V versus an internal pseudo Ag reference electrode. Thereby, sensor performance was characterized by a linear working range from 0.25 to 4 mM and a sensitivity of 0.14 μA mM-1. The detection principle was additionally evaluated with three other dehydrogenases (d-lactate dehydrogenase, alcohol dehydrogenase, and formate dehydrogenase, respectively). The developed reagentless biosensor array enabled simultaneous and cross-talk free determination of l-lactate, d-lactate, ethanol, and formate.

(R)-Amination mediated by (R)-specific ω-transaminases generally requires costly d-alanine in excess to obtain the desired chiral amines in high yield. Herein, a one-pot, trienzymatic cascade comprising an (R)-specific ω-transaminase, an amine dehydrogenase, and a formate dehydrogenase was developed for the economical and eco-friendly synthesis of (R)-chiral amines. Using inexpensive ammonium formate as the sole sacrificial agent, the established cascade system enabled efficient ω-transaminase-mediated (R)-amination of various ketones, with high conversions and excellent ee (>99%); water and CO2 were the only waste products.

The present research work has been carried out for investigation of genetic diversity and phenetic relationship among nine Bangladeshi cultivars of cucumber (Cucumis sativus L.) using some isozymes and protein profiling by SDS-PAGE. Seventeen isozymes including acid phosphatase (ACP), alcohol dehydrogenase (ADH), alkaline phosphatase (ALP), amino peptidase (AMP), aspartate amino transferase (AAT), β-galactosidase (GAL), β-glucosidase (GLU), catalase (CAT), esterases (EST), formate dehydrogenase (FDH), fructose bisphosphatase (FBP), fumarate hydratase (FUM), hexokinase (HEX), malate dehydrogenase (MDH-NAD+), peroxidase (PRX), superoxide dismutase (SOD) and tyrosinase (TYR) were assayed. A total of 48 bands were detected resolved in 17 isozymes, out of which 21 were polymorphic. No polymorphism has been found in acid phosphatase, β-galactosidase, formate dehydrogenase, fumarate hydratase and peroxidase. A total of 23 protein bands were resolved, out of which 22 were polymorphic. Seven isozymes banding patterns were observed in Superoxide dismutase which was the highest among the 17 enzyme systems. Dendrograms and three-dimensional plots based on isozymes, SDS-PAGE and combined dataset clearly separated almost all the cultivars. From the phonetic analysis, it was revealed that the cultivars ‘Alavy-1’, ‘Baromasse’, ‘Raj-1’ and ‘Dinajpuri’ are somewhat more closely related than the other cultivars. The cultivar ‘Green king’ produced six cultivar specific bands in SDS-PAGE. This study will help successful breeding programmes in cucumber in Bangladesh.

Three differently immobilized preparations of an enzyme extract from Candida boidinii containing a NAD+-dependent formate dehydrogenase (CbFDH) were prepared by encapsulation using calcium alginate (CbFDH-Alg) or polyvinyl alcohol (CbFDH-PVA) or by adsorption on montmorillonite K 10 (CbFDH-Mont). All FDH preparations were characterized in terms of both oxidation and reduction activities. For the formic oxidation activity, all immobilized FDHs had 100% activity at pH 8.0 and 50 °C, as the soluble enzyme. Among the immobilized biocatalyst, only CbFDH-PVA showed activity in the reduction of hydrogen carbonate and had 100% activity at pH 6.0 and 40 °C, as the soluble enzyme. The half-life values of immobilized enzymes increased by at least 3.1-folds compared to the soluble enzyme. Formic acid was produced from HCO3 - as a source of carbon dioxide using soluble CbFDH and CbFDH-PVA and the formic acid yields were determined as 80 and 92%, respectively for soluble CbFDH and CbFDH-PVA after 240 min reaction. CbFDH-Alg, CbFDH-PVA and CbFDH-Mont retained 40.1, 49.8 and 26.1% of their initial activities after 10 reuses in formate oxidation reaction. The remaining reduction activity of CbFDH-PVA was 30% after 10 reuses. These results show the CbFDH immobilization following different protocols strongly alter their oxidation/reduction activities. © 2019 Elsevier Ltd CTQ2017-86170-R Ministerio de Economía y Competitividad The authors thank to Dr. Baris Binay (Gebze Technical University) for the kind gift of the enzyme. We also gratefully recognize the support from the MINECO from Spanish Government, (project number CTQ2017-86170-R). The help and suggestions from Dr. Angel Berenguer (Departamento de Química Inorgánica, Universidad de Alicante) are gratefully recognized.

Oil sludge is one kind of toxic and persistent contamination to ecology system from petroleum industry. In order to recycle contaminated sands and reduce environmental impacts at a lower operating cost, enzyme cocktail 21/CbFDH including NADH regeneration system for oily sludge bioremediation was constructed for the first time. The intracellular enzymes of oil-degrading strain Acinetobacter calcoaceticus 21 were prepared and the formate dehydrogenase gene Cbfdh from Candida boidinii was cloned and functionally expressed in E.coli BL21 induced by lactose. The activity and stability of CbFDH was enhanced through self-induction medium optimization using Box-Behnken design. The CbFDH activity was 12.2 times increased and was only decreased 3.9% upon storage at 30 °C for 5 d. The CbFDH increased the degradation rate of oil in high concentration. For the sludge with 10% oil (w/w), the degradation rate achieved 35.6% after 12 h using enzyme 21/CbFDH with the protein ratio of 1:4. The results will provide novel perspectives for creation and operation of petroleum-degrading enzymes involving formate dehydrogenase with higher efficiency and lower cost comparing to current microbial strains or consortium.

Summary Pectobacterium atrosepticum SCRI1043 is a phytopathogenic Gram‐negative enterobacterium. Genomic analysis has identified that genes required for both respiration and fermentation are expressed under anaerobic conditions. One set of anaerobically expressed genes is predicted to encode an important but poorly understood membrane‐bound enzyme termed formate hydrogenlyase‐2 (FHL‐2), which has fascinating evolutionary links to the mitochondrial NADH dehydrogenase (Complex I). In this work, molecular genetic and biochemical approaches were taken to establish that FHL‐2 is fully functional in P. atrosepticum and is the major source of molecular hydrogen gas generated by this bacterium. The FHL‐2 complex was shown to comprise a rare example of an active [NiFe]‐hydrogenase‐4 (Hyd‐4) isoenzyme, itself linked to an unusual selenium‐free formate dehydrogenase in the final complex. In addition, further genetic dissection of the genes encoding the predicted membrane arm of FHL‐2 established surprisingly that the majority of genes encoding this domain are not required for physiological hydrogen production activity. Overall, this study presents P. atrosepticum as a new model bacterial system for understanding anaerobic formate and hydrogen metabolism in general, and FHL‐2 function and structure in particular., Pectobacterium atrospecticum contains the genes for formate hydrogenlyase‐2, considered the ancient progenitor of Complex I. Here, P. atrosepticum was harnessed as a new model system for advancing new knowledge in FHL‐2. The complex was found to contain an unusual selenium‐free formate dehydrogenase and a [NiFe]‐hydrogenase‐4 with a large membrane arm. FHL‐2 was established as the major source of hydrogen gas; however, some components of the membrane arm were surprisingly not essential for this activity.

Nitrogen-fixing bacterial strain, designated B2T, was isolated from methane-oxidation enrichment originating from a Sphagnum-dominated raised peatland in Tver region, Russia, and its phenotypic, chemotaxonomic and genomic characteristics were investigated. Cells of isolate were Gram-negative, aerobic, rod or spiral-shaped, with motility provided by a single polar flagellum in liquid media and peritrichous flagella on solid media. Strain was able to grow at 15–40 °C, pH 5.5–8.5 and tolerated NaCl to 2.0 % (w/v). Strain B2T gave positive amplification for dinitrogen reductase (nifH gene) and acetylene reduction activity was recorded up to 1250 nmol ethylene h–1 (mg protein)–1. Analysis of 16S rRNA showed that B2T represents a member of the genus Azospirillum and had the highest sequence similarity with A. humicireducens SgZ-5T (97.92 %). The predominant quinone system was ubiquinone Q-10 and the major fatty acids were C18 : 1ω7, C16 : 1ω7 and C16 : 0. The strain was facultative methylotrophic and used methanol and formate for the growth. Genome sequencing revealed a genome size of 8.0 Mbp and a G+C content of 67.8 mol%. The mxaFI genes encoding methanol dehydrogenase were absent, but a homologous xoxF gene was detected. The genes encoding enzymes involved in the biosynthesis of tetrahydromethanopterin (H4MPT) (formaldehyde oxidation) and NAD-linked formate dehydrogenase (fdsABG) were identified. Pairwise determined whole genome average nucleotide identity (gANI) values confirmed that strain B2T represents a novel species, for which we propose the name Azospirillum palustre sp. nov. with the type strain B2T (VKM B-3233T, КСТС 62613Т).

Enzymatic reduction of CO2 is of great significant, which involves an efficient multienzyme cascade system (MECS). In this work, formate dehydrogenase (FDH), glutamate dehydrogenase (GDH), and reduced pyridine nucleotide (NADH) (FDH&GDH&NADH), formaldehyde dehydrogenase (FalDH), GDH, and NADH (FalDH&GDH&NADH), and alcohol dehydrogenase (ADH), GDH, and NADH (ADH&GDH&NADH) were embedded in ZIF-8 (one kind of metal organic framework) to prepare three kinds of enzymes and coenzymes/ZIF-8 nanocomposites. Then by dead-end filtration these nanocomposites were sequentially located in a microporous membrane, which was combined with a pervaporation membrane to timely achieve the separation of product methanol. Incorporation of the pervaporation membrane was helpful to control reaction direction, and the methanol amount increased from 5.8 ± 0.5 to 6.7 ± 0.8 μmol. The reaction efficiency of an immobilized enzymes-ordered distribution in a membrane was higher than that disordered distribution in the membrane, and the methanol amount increased from 6.7 ± 0.8 to 12.6 ± 0.6 μmol. Moreover, it appeared that introduction of NADH into ZIF-8 enhanced the transformation of CO2 to methanol from 12.6 ± 0.6 to 13.4 ± 0.9 μmol. Over 50% of their original productivity was retained after 12 h of use. This method has wide applicability and can be used in other kinds of multienzyme systems.

Presently, the world is facing energy crisis so there is a strong need of in silico mining and analysis of potential bacteria leading to renewable energy generation i.e. bio hydrogen production using bio-wastes as substrate. Hydrogenase (Hyd), a key enzyme mediates hydrogen evolution, is supported with multiple maturation proteins like HypA, HypB, HypC, HypD, HypE, HypF, HycE, and Formate dehydrogenase in hydrogen-producing bacteria. Simple sequence repeats (SSRs) are the large source of genetic markers and it contains tandemly repeated short units of 1–6 bp. It is the first time to find out the SSRs (Simple Sequence Repeats) in the hydrogenase maturation protein supported genes (hypABCDEF), hycE and formate dehydrogenase (fdhF), as well as the organization of these gene(s) supported operon, depending on its presence in different hydrogen-producing bacteria i.e. in E. coli, Clostridium and Rhodobacter. Four operons i.e. eco-b2726 (hypABCDE), eco-b2713 (hypF), eco-b2724 (hycE) and eco-b4079 (fdhF) in E. coli; 4 operons i.e. CBO0432 (hypA), cbo-CBO0431 (hypB), cbo-CBO1837 (hypED), and cbo-CBO1837 (hydA) in Clostridium; and 2 operons i.e. rsp- RSP_0503 (hypABCDE) and rsp- RSP_0491 (hypF) in Rhodobacter were used in the current study. In this paper, we have performed an intensive investigation to find out the SSRs in all the above subunits, and it was concluded that dinucleotides were the most frequent repeat class followed by trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides. A total of 467, 332, and 387 small repeats were detected in entire maturation protein-assisted genes of E. coli, Clostridium, and Rhodobacter respectively. The length of the screened repeats was short owing to the small length of the maturation protein supported genes and none of the long length SSRs was identified in this whole study. It has been concluded that the heptamer (GCCGATC)2 in fdhF of E. coli, as well as the hexamer (GGGAGT)2 in hypA of Clostridium, may serve as an ideal marker. We have employed SSRIT (Simple Sequence Repeat Identification Tool) software for isolation of the SSRs. The resulted SSR markers may facilitate in the genetic identification of different potential unknown hydrogen-producing bacteria in a short time.