Native Enzyme Research Articles

Integrating recognition peptide into a nanozyme to mimic uricase binding pocket as a multifunctional nanoplatform for highly selective uric acid recognition, sensing and degrading

Nanozyme based colorimetric sensor for physiological biomarkers detection is becoming an increasingly influential technology in diagnosis, but the selective discrimination of biomarkers using single nanozyme without natural enzyme is greatly challenging. Here, a colorimetric detection platform for biomarker is successfully constructed by integrating recognition peptide (RGPT) into a nanozyme to mimic the binding pocket in natural enzyme. Firstly, RGPT-ultrafine palladium nanoparticles (PdNPs)@reduced graphene oxide (rGO) composite was facilely prepared using an arginine-rich RGPT. RGPT-PdNP@rGO composite displays outstanding laccase-like activity with Km being 0.075 mM at significantly low dosage used, which is one magnitude lower than that of natural enzyme. RGPT-PdNP@rGO nanozyme based one-step sensor can selectively detect uric acid (UA) with a comprehensive linear range from 0.2 to 110 μM and a limit of detection of 120 nM. Moreover, RGPT-PdNP@rGO composite displays an outstanding activity for UA oxidation with Km being 0.024 mM, revealing the nanozyme exhibits a similar affinity towards UA as natural uricase due to the cooperation of the three components. Compared with our previous work, these extremely improved UA recognition, sensing and degrading performances not only support the role of RGPT in enhancing with the recognition ability of the composite nanozyme for targeted UA, but also reveal the recognition role of RGPT is associated closely with inorganic components in the composite. This work provides more definitive and comprehensive information about multifunctional nanozymes for precise biomarker recognition by introducing recognition peptide, which is highly significant for applications beyond mimic-enzyme catalyst, including theranostic, sensing, and energy fields.

Construction of pyrimidine derivatives-copper enzyme mimics as colorimetric sensing elements for efficient detection of phenolic compounds and hydrogen peroxide

As concerns about environmental pollution grow, the rapid identification and quantification of pollutants have become increasingly vital. In this work, a series of pyrimidine derivatives-Cu enzyme mimics (Cytosine-Cu, Cytidine-Cu, and CMP-Cu) with laccase- and peroxidase-like activity were prepared through the coordination of Cu2+ with different pyrimidine derivatives (PDs). The PDs-Cu enzyme mimics contain high levels of Cu+ and N−Cu coordination structures, which provide sufficient catalytic sites for the substrates. Compared with natural enzymes and other nanozymes, PDs-Cu demonstrate superior substrate affinity, catalytic efficiency, stability, and resistance to interference. It was found that PDs-Cu enzyme mimics have different catalytic activities towards different phenolic compounds. Therefore, a three-channel colorimetric sensor array (CSA) was successfully developed utilizing PDs-Cu as the sensing elements. The CSA can accurately identify different phenolic compounds and their mixtures in seawater and simulated wastewater. Additionally, a colorimetric method for detecting H2O2 in eye drops was developed, featuring a detection range of 0.1–10.0μM and a limit of quantification of 0.1μM. This research not only provides a flexible protocol for regulating the catalytic activity of enzyme mimics, but also provides important inspiration for the development of methods for rapid identification and detection of contaminants in the environmental water.

A Nanoparticle-Coated Cellulose Acetate Membrane for Highly Efficient, Low-Cost Circulating Tumor Cell Detection.

Detecting circulating tumor cells has exhibited great significance in treating cancers since its concentration is an index strongly associated with the development and transfer of the tumor. However, the present commercial method for CTC detection is still expensive, because special antibodies and complicated devices must be used for cell separation and imaging. Hence, it is quite necessary to apply alternative materials and methods to decrease the cost of CTC detection. In this article, we coated a cellulose acetate membrane with nanoparticles formed by the polymerization of melamine and furfural, creating a surface with nanoscale roughness for the highly efficient capture of the sparse CTCs in a blood sample. Subsequently, the CTCs on the surface can be quantitatively detected by colorimetry with the aid of a COF-based nanozyme. The detection limit (LOD) can be as low as 3 cells/mL, which is the lowest LOD among the colorimetric methods to our knowledge. Considering the low cost of fabricating the membrane for CTC capture and the robustness of nanozymes compared with natural enzymes, this CTC detection approach displays great potential to decrease the financial burden of commercial CTC detection.

The Biochemistry of Artificial CO2-Fixation Pathways: The Exploitation of Carboxylase Enzymes Alternative to Rubisco

The last decade has registered a rapid development of new artificial CO2-bioconversion processes mirroring natural CO2-fixation by carboxylation and/or reduction reactions. The development of artificial pathways has shown that we have sufficient tools to design and implement, both in vitro and in vivo, complex reaction sequences pointing to construct microbial cell-factories to produce target chemicals at scale. This review is aimed to focus on the most efficient artificial CO2-fixing autotrophic cycles based on the use of carboxylase enzymes that, similarly to Rubisco enzyme, build a C–CO2 bond by reacting an enediolate or an enolate anion with CO2. The development of artificial CO2-fixing autotrophic cycles encompasses the analysis of the complete library of natural carboxylase enzymes taking part in the so called “central” and “assimilation” metabolism to select only those enzymes characterized by high catalytic efficiency, great stability, high substrate affinity, and oxygen tolerability. The review analyzes the biochemistry of the most efficient artificial CO2-fixation pathways implemented up today, evidencing the biosynthetic strategies adopted, the development of replenishing routes, and their integration with cell metabolism.

Chitin membrane for efficient laccase immobilization and synergistic removal of 17α-ethynylestradiol from water-based solutions

A unique chitin–laccase membrane was fabricated as an environmentally friendly biocatalytic platform, utilizing 1-butyl-3-methylimidazolium acetate as the solvent for chitin. Observations using scanning electron microscopy showed that the chitin-laccase membrane possessed a uniform and densely packed structure. Based on the presence of FT-IR signals at 1020 cm−1 and changes in the intensity of signals at 1540 cm−1 and 1645 cm−1, the effectiveness of laccase immobilization was confirmed. FT-IR mapping revealed that the enzyme is evenly distributed on the surface of the membrane. The catalytic activity of the native enzyme and laccase immobilized using the membrane was determined based on a model reaction, and the retention of high activity was confirmed using real solutions. Laccase immobilized using the chitinous membrane retained over 60 % of its initial activity after 30 days of storage at 4 °C. By contrast, the free enzyme retained <40 % of its initial activity. Moreover, the activity of chitin-laccase system remained at 85 % after 5 cycles. This novel chitin–laccase combination was tested in the 17α-ethynylestradiol (EE2) removal from water-based solutions. It was found that EE2 underwent synergistic degradation through concurrent adsorption and biocatalytic transformation, with enzymatic conversion as the dominant mechanism.

Vanadium doped ZIF-L derived V-NC peroxidase-like nanozymes for tanwenic acid sensing

Transition metal-relied nitrogen doping carbon nanozyme has attracted great attention owning to their high potential to replace natural enzymes. In this study, we prepared novel vanadium doped ZIF-L derived N-doped carbon nanozyme (V-NC) via facilely pyrolysis strategy. The cross-shaped morphology endows V-NC with unique V-Nx active sites, hierarchical pore character and large specific surface area, leading to its good peroxidase-mimetic activity. The steady-state kinetics trials revealed the low Km values of V-NC for TMB and H2O2, demonstrating its good affinity to TMB and H2O2. Mechanism research revealed that the excellent peroxidase-like activity of V-NC resulted from co-participation of •OH and O2•−, which were confirmed by reactive oxygen species (ROS) scavenging experiments and EPR trials. Under optimized operational conditions, and by virtue of the antioxidant properties of tannic acid (TA), a colorimetric sensing programme in terms of the peroxidase-imitating function of V-NC was developed for sensitive detection of TA in the 0.1–2.5 µM linear range with low detection limit of 19.8 nM and the relative standard deviation (RSD, n = 3) of below 5 %. This contribution renders a novel idea for designing and developing high activity N-doped carbon nanozyme, and provides an accurate and feasible assay for the analysis of TA in real samples.

Apple polysaccharide stabilized palladium nanoparticles for sensitive detection of organophosphorus pesticide.

The widespread application of organophosphorus pesticides (OPs) has inflicted significant damage on human well-being and food security. Hence, it is imperative to develop a friendly and accessible biosensor for the detection of OPs. Herein, apple polysaccharide (AP) stabilized palladium nanoparticles (AP-PdnNPs) with a particle size of 2.75-5.95nm were prepared using AP as a stabilizer and reducing agent. AP-Pd30NPs exhibited good peroxidase-like activity and effectively decomposed H2O2 to ·OH, which catalyzed the 3,3',5,5'-tetramethylbenzidine system to become blue. The catalytic kinetics of AP-Pd30NPs conformed to the typical Michelis-Menten equation. Furthermore, OPs directly inhibited the peroxidase-like activity of AP-Pd30NPs. Thus, a highly effective colorimetric biosensor was developed for the detection of OPs. The detection range of the biosensor was 0.050μg/L - 200mg/L, and the limit of detection was extremely low to 0.010μg/L. Compared with other nanomaterials, the detection platform based on AP-Pd30NPs can effectively detect organophosphorus pesticides without coupling natural enzymes；this method is more economical and practical. Therefore, this established method explores good perspective for the detection of OPs.

Final E5 to E8 Steps in the Nitrogenase Mechanism for Nitrogen Fixation.

Nitrogenase converts nitrogen in the air to ammonia. It is often regarded as the second most important enzyme in nature after photosystem II. The mechanism for how nitrogenase is able to perform the difficult task of cleaving the strong bond in N2 is debated. It is known that for every electron that is donated to N2, two ATP are hydrolyzed. In the experimentally suggested mechanism, the activation occurs after four reductions of the ground state, but there is no suggestion for how the enzyme uses the hydrolysis energy to perform catalysis. In the theoretical mechanism, it is suggested that hydrolysis is used to reduce the electron donor. In previous papers, the steps leading to the activation of N2 in the so-called E4 state has been investigated, using both the experimental and theoretical mechanism, showing that only the theoretical one leads to agreement with EPR observations for E4. In the present paper, the four steps following E4, leading to the release of two ammonia molecules, are described using the same methodology as used in the previous studies.

PENGARUH METODE FERMENTASI TERHADAP PENINGKATAN KANDUNGAN KIMIA DAN AKTIVITAS FARMAKOLOGI PADA BEBERAPA TANAMAN: Artikel Review

Fermentation is a process that involves the chemical transformation or breakdown of complex organic substances or other food components into simpler compounds through the action of natural enzymes and fermenting microorganisms. The purpose of this literature review is to explore the impact of fermentation methods on the enhancement of phytochemical content and pharmacological activity in various plants. Journal searches were conducted on Google Scholar, PubMed, and ScienceDirect. The results revealed that fermentation methods can increase the chemical content and pharmacological activity of a plant. Microorganisms that are frequently used in the fermentation process, such as Bacillus subtilis and actobacillus brevis, can increase the total phenol content and enhance antioxidant activity. Factors influencing the fermentation process in improving the chemical content and pharmacological activity of a plant include treatment methods, duration of fermentation (incubation), microorganisms used, and the concentration of inoculum used.

Combinatorial optimization of the hybrid cellulase complex structure designed from modular libraries.

Cellulase selectively recognizes cellulose surfaces and cleaves their β-1,4-glycosidic bonds. Combining hydrolysis using cellulase and fermentation can produce alternative fuels and chemical products. However, anaerobic bacteria produce only low levels of highly active cellulase complexes so-called cellulosomes. Therefore, we designed hybrid cellulase complexes from 49 biotinylated catalytic domain (CD) and 30 biotinylated cellulose-binding domain (CBD) libraries on streptavidin-conjugated nanoparticles to enhance cellulose hydrolysis by mimicking the cellulosome structure. The hybrid cellulase complex, incorporating both native CD and CBD, significantly improved reducing sugar production from cellulose compared to free native modular enzymes. The optimal CBD for each hybrid cellulase complex differed from that of the native enzyme. The most effective hybrid cellulase complex was observed with the combination of CD6-4 from Thermobifida fusca YX and CBD46 from the Bacillus halodurans C-125. The hybrid cellulase complex/CD6-4-CBD46 and -CD6-4-CBD2-5 combinations showed increased reducing sugar production. Similar results were also observed in microcrystalline cellulose degradation. Furthermore, clustering on nanoparticles enhanced enzyme thermostability. Our results demonstrate that hybrid cellulase complex structures improve enzyme function through synergistic effects and extend the lifespan of the enzyme.

A novel and facile oxygen-activated time-temperature indicator with wide temperature monitoring range and good stability based on the laccase-like nanozyme

BackgroundTime-Temperature Indicator (TTI) is an indicator device for real-time monitoring of the thermal history of the product. Due to the enzymatic reactions are affected by both time and temperature, enzymatic TTIs have been extensively studied and developed in recent years. However, enzymatic TTIs contain biologically active molecules (enzymes), which require high storage and use conditions. Most of them are designed to mix the system species together and irreversible reaction is undertaken. Nanozymes are the synthetic nanomaterials with similar biocatalytic functions as natural enzymes, which have extensive applications in analytical chemistry, biosensing, and environmental protection due to their facile synthesis, low cost, high stability and durability. ResultsThis work proposed to replace the natural laccase to laccase-like nanozyme, designed a novel and facile O2-activated time-temperature indicator for the first time. Nanozyme had excellent thermal and storage stability, which could maintain fabulous catalytic activity in the wide temperature range of 10–80 °C and after a long-term storage. Based on the O2 was required to participate in the oxidation of laccase-catalyzed substrates, a squeeze-type O2-activated TTI was designed by controlling O2 in the TTI system. The TTI was activated through extruding the O2-coated airbag ruptured and producing an irreversible color reaction. Combined with a smartphone to extract the chromaticity for portable visual real-time monitoring. Five sets of TTIs were prepared based on the concentration of nanozyme, and the activation energies (Ea) ranging from 28.45 to 72.85 kJ mol−1, which were able to be fitted to products with Ea ranging from 3.45 to 97.8 kJ mol−1 and the monitoring-time of less than 7 days. SignificanceCompared to the traditional enzymatic TTI, the TTIs designed based on nanozyme has the advantages of controlled activation, wider temperature monitor range and good stability. Providing a new approach to the development of real-time monitoring of smart devices.

Atomic-scale strain engineering of atomically resolved Pt clusters transcending natural enzymes

Strain engineering plays an important role in tuning electronic structure and improving catalytic capability of biocatalyst, but it is still challenging to modify the atomic-scale strain for specific enzyme-like reactions. Here, we systematically design Pt single atom (Pt1), several Pt atoms (Ptn) and atomically-resolved Pt clusters (Ptc) on PdAu biocatalysts to investigate the correlation between atomic strain and enzyme-like catalytic activity by experimental technology and in-depth Density Functional Theory calculations. It is found that Ptc on PdAu (Ptc-PA) with reasonable atomic strain upshifts the d-band center and exposes high potential surface, indicating the sufficient active sites to achieve superior biocatalytic performances. Besides, the Pd shell and Au core serve as storage layers providing abundant energetic charge carriers. The Ptc-PA exhibits a prominent peroxidase (POD)-like activity with the catalytic efficiency (Kcat/Km) of 1.50 × 109 mM−1 min−1, about four orders of magnitude higher than natural horseradish peroxidase (HRP), while catalase (CAT)-like and superoxide dismutase (SOD)-like activities of Ptc-PA are also comparable to those of natural enzymes. Biological experiments demonstrate that the detection limit of the Ptc-PA-based catalytic detection system exceeds that of visual inspection by 132-fold in clinical cancer diagnosis. Besides, Ptc-PA can reduce multi-organ acute inflammatory damage and mitigate oxidative stress disorder.

Characterisation of seryl tRNA synthetase (srs-2) in Haemonchus contortus and Teladorsagia circumcincta

The aim of the study was to purify and characterise recombinant proteins with the potential as an anti-parasite vaccine. Full-length cDNAs encoding seryl-tRNA synthetase (srs-2) were cloned from Haemonchus contortus (HcSRS-2) and Teladorsagia circumcincta (TcSRS-2). TcSRS-2 and HcSRS-2 cDNA (1458bp) encoded proteins of 486 amino acids, each of which was present as a single band of about 55 kDa on SDS-PAGE. Multiple alignments of the protein sequences showed homology of 94% between TcSRS-2 and HcSRS-2, 76–93% with SRS-2s of eight nematodes and 68% with Mus musculus SRS-2. The predicted three-dimensional structures revealed an overall structural homology of TcSRS-2 and HcSRS-2, highly conserved binding and catalytic sites, and minor differences in the tautomerase binding site residues in other nematode SRS-2 homologues. A phylogenetic tree was constructed using helminth and mammalian SRS-2 sequences. Soluble C-terminal SRS-2 proteins were expressed in Escherichia coli strain AY2.4 and purified. Recombinant HcSRS-2 assay shows that the recombinant enzyme was active and stable. The Km and Vmax for ATP were 3.9 ± 1.0 μM and 2.7 ± 0.1 μmol min−1 mg−1 protein, respectively. Antibodies in serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant HcSRS-2 and TcSRS-2 in enzyme-linked immunosorbent assays. Recognition of the recombinant proteins by antibodies generated by exposure of sheep to the native enzyme indicates similar antigenicity of the two proteins.

Natural enzyme cascade bimetallic sulfide MoCuSx nanozyme for synergistic photothermal/photodynamic enhanced chemodynamic antimicrobial therapy of wound infection

Chemodynamic therapy (CDT) involving highly toxic hydroxyl radical (•OH) with no drug resistance has gained widespread attention for antibacterial applications. However, the CDT effect is seriously confined by overexpressed glutathione (GSH) and limited hydrogen peroxide (H2O2) in the infection sites. Here, we have constructed a cascade enzyme/nanozyme assembly (MCS/GOx) composing of a bimetallic sulfide MoCuSx (MCS) nanozyme loaded with natural glucose oxidase (GOx) for synergistic photothermal/photodynamic enhanced chemodynamic antimicrobial therapy with high performance. The loaded GOx can promote the peroxidase (POD)-like activity of MCS by providing an acidic microenvironment and self-supplying H2O2, achieving cascade generation of •OH for efficient CDT. The CDT effect can be further promoted by a co-catalysis mediated by the Mo4+/Mo6+ redox couple. Upon exposure to an 808 nm near-infrared laser, MCS/GOx provides hyperpyrexia for photothermal antibacterial therapy (PTT), and produce singlet oxygen (1O2) for photodynamic therapy (PDT). Moreover, MCS/GOx can significantly consume antioxidant glutathione via the Cu+/Cu2+ redox couple and PDT-mediated 1O2 production, thereby enhancing CDT antibacterial effect. In vitro experiments exhibit that MCS/GOx has combined antimicrobial effect against Staphylococcus aureus and Escherichia coli. Further in vivo studies demonstrate that MCS/GOx can efficiently eliminate bacterial cells in wound tissues and accelerate wound healing with low inflammatory response and good biosafety. Consequently, the proposed MCS/GOx with multiple bactericidal modalities shows a great potential in fighting wound infections.

Nanozymes in Biomedical Applications: Innovations Originated From Metal-Organic Frameworks.

Nanozymes exhibit significant potential in medical theranostics, environmental protection, energy development, and biopharmaceuticals due to their exceptional catalytic performance. Compared with natural enzymes, nanozymes have the advantages of simple preparation and purification, convenient production and low cost. Therefore, it is very important to prepare nanozymes quickly and efficiently, which not only helps to expand their application scope, but also can further exert their great potential in various fields. Metal-organic frameworks (MOF) materials serve as versatile substrates for constructing nanozymes, offering unique advantages like adjustable structure, high specific surface area, and porous channels. MOF coordination nodes constructed from metal ions or metal clusters have unique properties that can be leveraged to tailor nanozyme characteristics for different applications. This review describes and analyzes recent methods for constructing nanozymes using MOF materials, and explores their application prospects in biomedicine. By expounding the preparation techniques and biomedical applications of nanozymes, this review aims to inspire researchers to develop innovative nanozyme materials and explore new application directions.

Pectin-stabilized nanoceria double coated with lactoferrin/chitosan for management of experimental autoimmune encephalomyelitis

Cerium oxide nanoparticles are a unique antioxidant mimicking the activity of natural antioxidant enzymes. Previous research showed its’ promising effect mitigating free radical damage in neurodegenerative disorders. However, there is still unmet therapeutic needs due to poor BBB penetration, a high accumulation in liver, kidney and spleen. This study aimed to synthesize and optimize nanoceria stabilized by natural bioactive polymers suitable for intranasal administration to manage multiple sclerosis. Among the different employed biopolymers, pectin-stabilized nanoceria exhibited the ideal properties with small particles size 87.20±3.43nm, high zeta potential -56.37±2.39mV and high free radical scavenging activity 85.27±0.07%. Then coating was achieved for the first time by two biopolymers: lactoferrin and chitosan producing a double coated cationic nanoceria. Biological assessment involved using experimental autoimmune encephalomyelitis animal model treated in a dose of 1mg/kg nanoceria for 15 days. Motor function testing in rats revealed 6- and 17-folds increase in latency time in rotating rod and hanging wire tests, respectively. Biochemical analysis revealed significant reduction in lipid peroxidation along with about 1-fold upgrading of the intrinsic antioxidant system. Moreover, histologic examination disclosed decreased degeneration of the brain and spinal cord of treated rats and much decreased liver toxicity.

Reserving Electrons in Cofactor Decorated Coordination Capsules for Biomimetic Electrosynthesis of α-Hydroxy/amino Esters.

Sustainable electricity-to-chemical conversion via the utilization of artificial catalysts inspired by redox biological systems holds great significance for catalyzing synthesis. Herein, we develop a biomimetic electrosynthesis strategy mediated by a nicotinamide adenine dinucleotide (NADH) mimic-containing coordination capsule for efficiently producing α-hydroxy/amino esters. The coordination saturated metal centers worked as an electron relay to consecutively accept single electrons while donating two electrons to the NAD+ mimics simultaneously. The protonation of the intermediate generated active NADH mimics for biomimetic hydrogenation of the substrates via the conventional enzymatic manifold with or without the presence of natural enzymes. The pocket of the capsule encapsulated the substrate and enforced the close proximity between the substrate and the NADH mimics, forming a preorganized intermediate to shift the redox potential by 0.4 V anodically. The cobalt capsule gave methyl mandelate over a range of applied potentials, with an improved yield of 92% when operated at -1.2 V compared to that of Hantzsch ester or natural NADH. Kinetic experiments revealed a Michaelis-Menten mechanism with a Km of 7.5 mM and a Kcat of 1.1 × 10-2 s-1. This extended strategy in tandem with an enzyme exhibited a TON of 650 molE-1 with an initial TOF of 185 molE-1·h-1, outperforming relevant Rh-mediated enzymatic electrosynthesis systems and providing an attractive avenue toward advanced artificial electrosynthesis.

Rational design of Ag-doped MnFe2O4/HNTs for peroxidase-mimicking activity and colorimetric sensing of uric acid in human serum

Mimicking enzyme have significantly advanced sensing assays by replicating native enzyme functions, yet achieving both high catalytic activity and easy recyclability remains a challenge. In this study, Ag-doped MnFe2O4/halloysite nanotubes (HNTs) were rationally designed as a novel nanozyme by depositing in-situ Ag and MnFe2O4 nanoparticles onto HNTs. The resulting nanocomposite exhibited excellent peroxidase-like activity along with magnetic properties. Leveraging these features, a highly efficient and sensitive colorimetric system for detecting uric acid (UA) was developed. The Ag-doped MnFe2O4/HNTs catalyzed the oxidation of 3,3′,5,5′-tetramethylbenzidine in the presence of H2O2, causing a color change from colorless to blue. The system showed a linear absorbance response to UA concentrations ranging from 1 to 20 μM, with a detection limit of 59 nM. Mechanistic studies revealed that reactive oxygen species intermediates (1O2) were generated through the decomposition of H2O2, leading to peroxidase-like activity in the Ag-doped MnFe2O4/HNTs. The assay was successfully applied to detect UA in human serum with recoveries over 99.68 %. This study indicates the successful application of Ag-doped MnFe2O4/HNTs for colorimetric UA detection in human serum. This research introduces a novel approach for designing recyclable, high-performance mimicking enzyme and establishes an effective colorimetric sensing platform for UA detection in human serum.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Hybrid Nanoparticles: Ni and Au Decorated with [FeFe]‐Hydrogenase Mimics

Complexes [(μ‐S₂C₂H₄NHR)Fe₂(CO)₆] (R = p‐C₆H₄‐OCO(CH₂)₉Br (3a); R = p‐C₆H₄‐OCO(CH₂)₈CH₃ (3b)) were used as stabilizing agents in the synthesis of Ni@3 and Au@3 nanoparticles (NPs), which are the first reported stable metallic NPs decorated with [(μ‐S₂C₂H₄NHR)Fe₂(CO)₆] moieties. Electrochemical analysis reveals that incorporating the hydrogenase mimic into the NPs lowers the overpotential and enhances proton reduction electrocatalytic activity in organic media. The NPs act similarly to the [Fe₄S₄] cluster in natural enzymes, functioning as an electron reservoir/relay.