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Artificial enzymes, usually created by incorporating one or two abiological catalytic species into protein hosts, are becoming promising tools for biocatalysis. However, they are still far behind natural enzymes due to their limited active sites with low activity and selectivity. Here, we proposed a polymeric approach to generate artificial enzymes by combining proteins with organocatalytic polymers, in which multiple proline organocatalysts are grafted from protein scaffolds via atom-transfer radical polymerization. The resultant artificial polyenzymes, abbreviated as "ArPoly", have similar physicochemical properties as natural enzymes and proved to be effective aldolase mimics for the aqueous asymmetric aldol reaction, in which an optimal ArPoly gave rise to 33% conversion and 94% enantioselectivity. Therefore, we proved the concept of water-soluble proline-based organocatalyst for aqueous asymmetric aldol reactions. This success not only expands the toolbox of asymmetric organocatalysis but also opens other avenues in the field of artificial enzymes where a plethora of proteins/enzymes can be combined with various well-designed organocatalytic polymers for synthetically useful reactions.

Vanadium haloperoxidases play an important catalytic role in the natural production of antibiotics which are difficult to make in the laboratory. Understanding the catalytic mechanism of these enzymes will aid in the production of artificial enzymes useful in bioengineering the synthesis of drugs and useful chemicals. However, the catalytic mechanism remains not fully understood yet. In this paper, we investigate one of the key steps of the catalytic mechanism using QM/MM. Our investigation reveals a new N-halohistidyl intermediate in the catalytic cycle of vanadium chloroperoxidase (VCPO). This new intermediate, in turn, can explain the known inhibition of the enzyme by substrate under acidic conditions (pH[Formula: see text]4). Additionally, we examine the possibility of replacing V in VCPO by Nb or Ta using QM modeling. We report the new result that the Gibbs free energy barriers of several steps of the catalytic cycle are lower in the case of artificial enzymes, incorporating NbO[Formula: see text] or TaO[Formula: see text] instead of VO[Formula: see text]. Our results suggest that these new artificial enzymes may catalyze the oxidation of halide faster than the natural enzyme.

Although great progress has been made in artificial enzyme engineering, their catalytic performance is far from satisfactory as alternatives of natural enzymes. Here, we report a novel and efficient strategy to access high-performance nanozymes via direct atomization of platinum nanoparticles (Pt NPs) into single atoms by reversing the thermal sintering process. Atomization of Pt NPs into single atoms makes metal catalytic sites fully exposed and results in engineerable structural and electronic properties, thereby leading to dramatically enhanced enzymatic performance. As expected, the as-prepared thermally stable Pt single-atom nanozyme (PtTS-SAzyme) exhibited remarkable peroxidase-like catalytic activity and kinetics, far exceeding the Pt nanoparticle nanozyme. The following density functional theory calculations revealed that the engineered P and S atoms not only promote the atomization process from Pt NPs into PtTS-SAzyme but also endow single-atom Pt catalytic sites with a unique electronic structure owing to the electron donation of P atoms, as well as the electron acceptance of N and S atoms, which simultaneously contribute to the substantial enhancement of the enzyme-like catalytic performance of PtTS-SAzyme. This work demonstrates that thermal atomization of the metal nanoparticle-based nanozymes into single-atom nanozymes is an effective strategy for engineering high-performance nanozymes, which opens up a new way to rationally design and optimize artificial enzymes to mimic natural enzymes.

Lithium iron phosphate materials as one of the most representative cathode materials for lithium-ion battery were found to possess intrinsic peroxidase-like activity. LiFePO4 particles were synthesized by a classic hydrothermal process. In this paper, colorless 3,3',5,5'-tetramethylbenzidine is oxidized to a blue product using hydrothermally synthesized LiFePO4 particles (LFP) as a catalyst with a significant absorption peak at 650 nm. Kinetic assay showed that the catalytic performance was consistent with typical Michaelis–Menten kinetics. As a novel peroxidase mimic material, LFP show the advantages of high catalytic efficiency and favorable stability in aqueous solution compared with natural enzymes, thus providing a simple, inexpensive and fast approach to colorimetric detection of H2O2 and glucose.

The in-situ gelation of injectable precursors is desirable in the field of tissue regeneration, especially in the context of irregular defect filling. The current driving forces for fast gelation include the phase-transition of thermally sensitive copolymers, click chemical reactions with tissue components, and metal coordination effect. However, the rapid formation of tough hydrogels remains a challenge. Inspired by aerobic metabolism, we herein propose a tissue-fluid-triggered cascade enzymatic polymerization process catalyzed by glucose oxidase and ferrous glycinate for the ultrafast gelation of acryloylated chondroitin sulfates and acrylamides. The highly efficient production of carbon radicals and macromolecules contribute to auto-accelerated polymerization for soft tissue augmentation in bone defects. The copolymer hydrogel demonstrated the regeneration-promoting capacity of cartilage. As the first example of using artificial enzyme complexes for in-situ polymerization, this work offers a biomimetic approach to the design of strength-adjustable hydrogels for bio-implanting and bio-printing applications.

Enantioselective protonation is conceptually one of the most attractive methods to generate an α-chiral center. However, enantioselective protonation presents major challenges, especially in water as a solvent. Herein, we report an artificial enzyme catalyzed tandem Michael addition and enantioselective protonation reaction of α-substituted acroleins with 2-acyl imidazole derivatives in water. The artificial enzyme uses a synergistic combination of two abiological catalytic sites: a genetically encoded non-canonical p-aminophenylalanine residue and a Lewis acid Cu(II) complex. The exquisite stereochemical control achieved in the protonation of the transient enamine intermediate generated by conjugate addition of the Michael donor is illustrated by the >20:1 dr and up to >99% ee obtained for the products. These results illustrate the potential of exploiting synergistic catalysis in artificial enzymes for challenging reactions.

Based on a biomimetic point of view, a new artificial enzyme model was designed to imitate the biocatalytic behavior of native-chloroperoxidase hemoenzyme. Transition metals catalyzed C–H chlorination and represented the C–H activation process. Cysteine–metal complexes of Mn(II), Fe(III), Co(II), Ni(II), Cu(II) and Zn(II) were prepared and encapsulated in a sodium dodecyl sulfate (SDS)/dodecyl trimethyl ammonium bromide (DTAB) vesicular mixture to imitate a native-chloroperoxidase model at pH 3. Current biocatalysts also behaved as a catalase at pH 3 and as a peroxidase at pH 1. Surprisingly, “Cu(II)–cysteine/SDS/DTAB” nanozyme indicated the maximum chloroperoxidase, catalase and peroxidase activity or efficacy among other mentioned intermediate metal complexes including manganese (2+), cobalt (2+), zinc (2+), iron (3+) and nickel (2+). The performed structural analysis by transmission electron microscopy and dynamic light scattering illustrated different nanozyme shapes and surface charges at distinct pHs. Zeta potential of colloids indicated the presence of Cu(II)–cysteine active site inside the nanozyme. Differential scanning calorimetry shows different stabilities and fluidities of nanozyme at different pHs. FTIR spectroscopy was utilized to investigate the structural complexation of Cu(II)–cysteine active essence. The present study demonstrated that “the sulfur” moiety of l-cysteine is ligated to copper ion at pH 1 and 3 in order to form Cu(II)–cysteine center as a multi-functional active site. Accordingly, the current study presents the preparation of an efficient pseudo-vesicular Cu(II)–cysteine supramolecular system with pH-switchable multi-functional enzymatic ability.

The construction and engineering of artificial enzymes consisting of abiological catalytic moieties incorporated into protein scaffolds is a promising strategy to realize non-natural mechanisms in biocatalysis. Here, we show that incorporation of the noncanonical amino acid para-aminophenylalanine (pAF) into the nonenzymatic protein scaffold LmrR creates a proficient and stereoselective artificial enzyme (LmrR_pAF) for the vinylogous Friedel-Crafts alkylation between α,β-unsaturated aldehydes and indoles. pAF acts as a catalytic residue, activating enal substrates toward conjugate addition via the formation of intermediate iminium ion species, while the protein scaffold provides rate acceleration and stereoinduction. Improved LmrR_pAF variants were identified by low-throughput directed evolution advised by alanine-scanning to obtain a triple mutant that provided higher yields and enantioselectivities for a range of aliphatic enals and substituted indoles. Analysis of Michaelis-Menten kinetics of LmrR_pAF and evolved mutants reveals that different activities emerge via evolutionary pathways that diverge from one another and specialize catalytic reactivity. Translating this iminium-based catalytic mechanism into an enzymatic context will enable many more biocatalytic transformations inspired by organocatalysis.

The interplay between nucleic acids and lipids underpins several key processes in molecular biology, synthetic biotechnology, vaccine technology, and nanomedicine. These interactions are often electrostatic in nature, and much of their rich phenomenology remains unexplored in view of the chemical diversity of lipids, the heterogeneity of their phases, and the broad range of relevant solvent conditions. Here we unravel the electrostatic interactions between zwitterionic lipid membranes and DNA nanostructures in the presence of physiologically relevant cations, with the purpose of identifying new routes to program DNA-lipid complexation and membrane-active nanodevices. We demonstrate that this interplay is influenced by both the phase of the lipid membranes and the valency of the ions and observe divalent cation bridging between nucleic acids and gel-phase bilayers. Furthermore, even in the presence of hydrophobic modifications on the DNA, we find that cations are still required to enable DNA adhesion to liquid-phase membranes. We show that the latter mechanism can be exploited to control the degree of attachment of cholesterol-modified DNA nanostructures by modifying their overall hydrophobicity and charge. Besides their biological relevance, the interaction mechanisms we explored hold great practical potential in the design of biomimetic nanodevices, as we show by constructing an ion-regulated DNA-based synthetic enzyme.

Site-isolation of catalysts can enable incompatible catalysts such as acid and base to be used in one pot for enhanced efficiency and other benefits. Although many synthetic platforms have been reported for this purpose, they generally do not possess the exquisite selectivity of site-isolated enzymes in nature. Here we report water-soluble protein-sized nanoparticles with site-isolated acids in the core and amines on the surface. The catalysts were made through molecular imprinting of cross-linked micelles, followed by facile one-step photoaffinity labeling of the imprinted binding site. With a tunable, substrate-specific active site, the bifunctional artificial enzyme catalyzed highly selective tandem cross aldol reaction between acetone and mixtures of isomeric aryl acetals. It could also transform a less reactive substrate over a more reactive one.

An artificial enzyme with record catalytic activity for organophosphorus degradation has been created from a crown-ether threaded covalent organic framework.

Natural enzymes are efficient and versatile biocatalysts but suffer in their environmental tolerance and catalytic stability. As artificial enzymes, nanozymes can improve the catalytic stability, but it is still a challenge to achieve high catalytic activity. Here, we employed atomic engineering to build the artificial enzyme named Au24Ag1 clusterzyme that hosts an ultrahigh catalytic activity as well as strong physiological stability via atom manipulation. The designed Au24Ag1 clusterzyme activates the Ag-S active site via lattice expansion in the oligomer atom layer, showing an antioxidant property 72 times higher than that of natural antioxidant Trolox. Enzyme-mimicked studies find that Au24Ag1 clusterzyme exhibits high catalase-like (CAT-like) and glutathione peroxidase-like (GPx-like) activity with a maximum reaction rate of 68.9 and 17.8 μM/min, respectively. Meanwhile, the unique catalytic landscape exhibits distinctive reactions against inflammation by inhibiting the cytokines at an early stage in the brain. Atomic engineering of clusterzymes provides a powerful and attractive platform with satisfactory atomic dispersion for tailoring biocatalysts freely at the atomic level.

In recent years, (de)carboxylases that catalyze reversible (de)carboxylation have been targeted for application as carboxylation catalysts. This has led to the development of proof-of-concept (bio)synthetic CO2 fixation routes for chemical production. However, further progress towards industrial application has been hampered by the thermodynamic constraint that accompanies fixing CO2 to organic molecules. In this Review, biocatalytic carboxylation methods are discussed with emphases on the diverse strategies devised to alleviate the inherent thermodynamic constraints and their application in synthetic CO2 -fixation cascades.

Artificial enzyme cascade systems with confinement effect are highly important in synthetic biology and biomedicine. Herein, a framework nucleic acid-based confined enzyme cascade (FNA-CEC) for synergistic cancer therapy in vivo was developed. The FNA-CEC consisted of glucose oxidase and horseradish peroxidase precisely assembled on an addressable DNA tetrahedron scaffold within few nanometers. Glucose oxidase (GOx) can trigger efficient glucose depletion for tumor starvation therapy, and increase the local concentration of H2O2 in situ for enhanced downstream horseradish peroxidase (HRP)-activated prodrug therapy. Due to the spatial-confinement on DNA tetrahedron scaffold, the efficiency of intermediate metabolites transportation between the enzyme cascades was improved. Moreover, FNA-CEC was applied for efficient synergistic cancer therapy in vitro and in vivo . As a simple and efficient approach, the FNA-CEC is expected to expand the toolbox of technologies in synthetic biology and biomedicine.

Synthetic dyes such as malachite green (MG) have a wide range of applications. Meanwhile, they bring great challenges for environmental security and cause potential damages to human health. Compared with traditional approaches, enzymatic catalysis is an emerging technique for wastewater treatment. As alternatives to natural enzymes, artificial enzymes have received much attention for potential applications. In previous studies, we have rationally designed artificial enzymes based on myoglobin (Mb), such as by introducing a distal histidine (F43H mutation) and creating a channel to the heme pocket (H64A mutation). We herein show that the artificial enzyme of F43H/H64A Mb can be successfully applied for efficient biodegradation of MG under weak acid conditions. The degradation efficiency is much higher than those of natural enzymes, such as dye-decolorizing peroxidase and laccase (13–18-fold). The interaction of MG and F43H/H64A Mb was investigated by using both experimental and molecular docking studies, and the biodegradation products of MG were also revealed by UPLC-ESI-MS analysis. Based on these results, we proposed a plausible biodegradation mechanism of MG. With the high-yield of overexpression in E. coli cells, this study suggests that the artificial enzyme has potential applications in the biodegradation of MG in fisheries and textile industries.

Eu3+-Doped nanoceria (Eu:CeO2) with self-integrated catalytic and luminescence sensing functions was synthesized by a simple and gentle one-pot method to build a dual-readout nanozyme platform for organophosphate compound (OPC) sensing in this work. The catalytic degradation of the model substrate of OPC, p-nitrophenyl phosphate (p-NPP), by as-prepared Eu:CeO2 can be completed in 2 min with little influence of temperature and pH values, highlighting the advantages of Eu:CeO2 as an artificial enzyme for dephosphorylation. Most importantly, the characteristic red emission of Eu3+ (592 nm) from Eu:CeO2 can be quenched by p-NPP, accompanied by a color change from colorless to yellow. Based on this, linear ranges of 4-50 μM with a detection limit of 3.3 μM and 1-20 μM with a detection limit of 0.6 μM for p-NPP were obtained by colorimetric and fluorescence methods, respectively. Furthermore, the fluorescence strategy was effectively applied to the determination of ethyl para-nitrophenyl (EPN), one of the most commonly used pesticides, with a detection limit of 5.86 μM. The proposed strategy was also successfully applied to the assay of p-NPP and EPN in real water samples, showing great application prospects in detecting OPC in the environment.

In this work, we describe the application of a synthetic enzyme (synzyme) as the catalyst to promote the multicomponent synthesis of isoxazol-5(4H)-one derivatives. The catalytic system could be used up to 15 times without any notable loss of its activity. Some derivatives showed fluorescence and their photophysical data were evaluated. The mechanism of the reaction was, for the first time, investigated and, among the three reaction pathway possibilities, only one was operating under the developed conditions. ESI-MS(/MS) allowed for both the simultaneous monitoring of the multicomponent reaction (MCR) and the proposition of a kinetic model to explain the transformation. The kinetic model pointed firmly to only one reaction pathway and helped to discard the other two possibilities. The antimicrobial abilities of all synthesized derivatives against Gram-positive and Gram-negative strains were also evaluated. The abilities of functional chromophores (fluorescent compounds) as live cell-imaging probes were verified and one of the multicomponent adducts could stain early endosomes selectively in bioimaging experiments.

A Metal-Organic Framework (MOFs) with large surface area, exposed active site, excellent catalytic performance and high chemical stability has been used as an artificial enzyme and designed for nonenzymatic electrochemical sensors. Here, a strategy of using an enhanced electrochemical sensing platform for the detection of nitic oxide (NO) and hydrogen peroxide (H2O2) was designed via a nano-metalloporphyrinic metal-organic framework (NporMOF(Fe)) as an electrode material. By taking advantage of the small size, high surface area and exposed Fe active site, the obtained NporMOF(Fe) displays excellent electrocatalytic activity toward NO and H2O2. The NporMOF(Fe) modified electrode shows high sensing ability toward the in situ generated NO in NO2- containing phosphate buffer (PB) solution with a wide linear detection range of 5 μM to 200 μM and a very low detection limit of 1.3 μM. Moreover, NporMOF(Fe) exhibits high electrocatalytic activity toward the reduction of H2O2 and the practical detection of H2O2 released from HeLa cells. Furthermore, the NporMOF(Fe) modified electrode shows excellent selectivity toward the detection of NO and H2O2 in the presence of other physiologically important analytes. This method shows excellent biosensing performance, implying the universal applicability of MOFs-based artificial nanozymes for biosensors and the potential application for third generation biosensors.

Peroxidase widely exists in nature and can be applied for the diagnosis and detection of H2O2, glucose, ascorbic acid and other aspects. However, the natural peroxidase has low stability and its catalytic efficiency is easily affected by external conditions. In this work, a copper-based metal–organic framework (Cu-MOF) was prepared by hydrothermal method, and characterized by means of XRD, SEM, FT-IR and EDS. The synthesized Cu-MOF material showed high peroxidase-like activity and could be utilized to catalyze the oxidation of o-phenylenediamine (OPDA) and 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. The steady-state kinetics experiments of the oxidation of OPDA and TMB catalyzed by Cu-MOF were performed, and the kinetic parameters were obtained by linear least-squares fitting to Lineweaver–Burk plot. The results indicated that the affinity of Cu-MOF towards TMB and OPDA was close to that of the natural horseradish peroxidase (HRP). The as-prepared Cu-MOF can be applied for colorimetric detection of H2O2 and glucose with wide linear ranges of 5 to 300 μM and 50 to 500 μM for H2O2 and glucose, respectively. Furthermore, the specificity of detection of glucose was compared with other sugar species interference such as sucrose, lactose and maltose. In addition, the detection of ascorbic acid and sodium thiosulfate was also performed upon the inhibition of TMB oxidation. Based on the high catalytic activity, affinity and wide linear range, the as-prepared Cu-MOF may be used for artificial enzyme mimics in the fields of catalysis, biosensors, medicines and food industry.

Realizing a sustainable future requires intensifying the waste stream conversion, such as converting the greenhouse gas carbon dioxide into value-added products. In this paper, we focus on utilizing formaldehyde as a C1 carbon source for enzymatic C–C bond formation. Formaldehyde can be sustainably derived from other C1 feedstocks, and in this work, we designed a synthetic enzyme cascade for producing the functional C4 sugar erythrulose. This involved tailoring the enzyme formolase, which was optimized for fusing formaldehyde, from a three-carbon producer (dihydroxyacetone) to sets of variants with enhanced two-carbon (glycolaldehyde) or four-carbon (erythrulose) activity. To achieve this, a high-throughput combinatorial screening was developed, and every single variant was evaluated in terms of glycolaldehyde, dihydroxyacetone and erythrulose activity. By applying the two most promising variants in an enzyme cascade, we were able to show for the first time production of ERY starting from a C1 carbon source. In addition, we demonstrated that one of our tailored formolase variants was able to convert 25.0 g L−1 glycolaldehyde to 24.6 g L−1 erythrulose (98% theoretical yield) in a fully atom-economic biocatalytic process. This represents the highest achieved in vitro concentration of erythrulose to date.

Background Earlier studies have attempted to create electronic free insulin delivery systems using different glucose sensing mechanism, no successful clinical translation as hitherto been made. This study aimed to assess the faster responsiveness of the insulin release from this enzyme based nanoparticles which is a self-regulated insulin delivery system constructed by loading with insulin, enzyme glucose oxidase into hyaluronic acid and 2-nitroimidazole forming enzyme-based nanoparticles which works in accordance to the blood glucose level. Materials and method Enzyme-based nanoparticles were prepared by ionic gelation method. Insulin content in the nanoparticles kept for stability study was estimated by human insulin enzyme based immunosorbent assay. In in-vitro studies; different concentrations of glucose were taken and the release study of insulin was recorded. Results This enzyme-based nanoparticles were having average diameter of around 193 nm and stability studies showed that nanoparticles were stable upto 30 days at 4 °C. In-vitro studies showed the release of insulin from nanoparticle conjugates which was effectively correlated with the external glucose concentration created where different concentrations of glucose taken thus facilitating the stabilization of blood glucose levels in the hyperglycemia state which was achieved within 10 min. (400 mg/dL) wherein drug release rate remarkably increased in hyperglycemia state and no specific changes or small amount of release was observed in normoglycemia state (100 mg/dL). Conclusion Overall, this preliminary study of this enzyme-based nanoparticles formulation showed excellent rapid responsiveness towards hyperglycemia which might act as a potential biomimetic system in triggering the release of insulin in sustained manner.

International audience; Performing a heterogeneous catalysis with proteins is still a challenge. Herein, we demonstrate the importance of cross‐linked crystals for sulfoxide oxidation by an artificial enzyme. The biohybrid consists of the insertion of an iron complex into a NikA protein crystal. The heterogeneous catalysts displays a better efficiency‐with higher reaction kinetics, a better stability and expand the substrate scope compared to its solution counterpart. Designing crystalline artificial enzymes represents a good alternative to soluble or supported enzymes for the future of synthetic biology.

Exponentially increasing protein sequence data enables artificial enzyme design using sequence-based protein design methods, including full-consensus protein design (FCD). The success of artificial enzyme design is strongly dependent on the nature of the sequences used. Hence, sequences must be selected from databases and curated libraries prepared to enable a successful design by FCD. In this study, we proposed a selection approach regarding several key residues as sequence motifs. We used l-threonine 3-dehydrogenase (TDH) as a model to test the validity of this approach. In the classification, four residues (143, 174, 188, and 214) were used as key residues. We classified thousands of TDH homologous sequences into five groups containing hundreds of sequences. Utilizing sequences in the libraries, we designed five artificial TDHs by FCD. Among the five, we successfully expressed four in soluble form. Biochemical analysis of artificial TDHs indicated that their enzymatic properties vary; half of the maximum measured enzyme activity (t1/2) and activation energies were distributed from 53 to 65 °C and from 38 to 125 kJ/mol, respectively. The artificial TDHs had unique kinetic parameters, distinct from one another. Structural analysis indicates that consensus mutations are mainly introduced in the secondary or outer shell. The functional diversity of the artificial TDHs is due to the accumulation of mutations that affect their physicochemical properties. Taken together, our findings indicate that our proposed approach can help generate artificial enzymes with unique enzymatic properties.

Nanozymes as one of artificial enzymes show many advantages than natural enzymes. The high Michaelis-Menten constant (Km) to H2O2 is the drawback for nanozymes, which means a high H2O2 concentration to oxidize 3,3',5,5'-tetramethylbenzidine (TMB). For this problem, FeS2/SiO2 double mesoporous hollow spheres (DMHSs) were first synthesized as an artificial peroxidase through a solid reaction. The experimental results demonstrate that Fe3O4 vulcanization and DMHS formation were effective strategies to enhance affinity to H2O2 for the nanozyme. The Km of FeS2/SiO2 DMHSs (H2O2 as the substrate) is 18-fold smaller than that of FeS2 nanoparticles (NPs). The catalytic efficiency (Kcat/Km) of FeS2/SiO2 DMHSs is about 16 times higher than that of FeS2 NPs. FeS2/SiO2 DMHSs can be used as a nanozyme to sensitively and rapidly detect H2O2 and glutathione within 1 min at room temperature.

Developing artificial enzyme mimetics for the detection of small biomolecules are a current research interest because natural enzymes bear some serious disadvantages, such as their catalytic activity can be easily inhibited and they can be digested by proteases. Herein, a heteropoly acids (HPA) encapsulating metal–organic framework (MOF) with metal-carbene structure, [Cu10(H3trz)4(Htrz)4][PMo12VO41] (PMA-MOF) as bifunctional enzyme-mimetic catalyst for colorimetric detection of hydrogen peroxide (H2O2) and ascorbic acid (AA) was designed and synthesized. Thanks to the good stability and the synergistic effect of PMA and MOF, PMA-MOF exhibits the lower limit of detection (0.222 μM towards H2O2 and 0.0046 μM to AA), and the smaller Km value (0.0138 mM for H2O2 and 0.136 mM for o-phenylenediamine) compared to most reported MOF- and HPA-based enzyme-mimetic catalyst, to the best our knowledge.

Artificial enzyme mimics are more stable and cost-effective than bioenzymes, but they usually lack substrate recognition function. Here we report that uranyl (UO22+) features efficient artificial p...

Polymeric carbon nitride (CN) is one of the most promising metal-free photocatalysts to alleviate the energy crisis and environmental pollution. Loading cocatalysts is regarded as an effective way to improve the photocatalytic efficiency of CNs. However, commonly used noble metal cocatalysts limit their applications due to their rarity and high cost. Herein, we present the effective synthesis of single-atom copper-modified CN via supramolecular preorganization with subsequent condensation, which provides effective charge transfer pathways by an "infused" delocalized state with variable-valence catalysis at the same time. The C-Cu-N2 single-atom catalytic site can activate CO2 molecules and reduces the energy barrier toward photocatalytic CO2 reduction. Excellent performance for photocatalytic CO2 reduction was found. This work thereby provides a general protocol of designing a noble-metal-free photocatalyst with infused metal centers toward a wide range of applications.

Acetylcholinesterase inactivating compounds, such as organophosphate (OP) and carbamate (CM) pesticides, are widely used in agriculture to ensure sustainable production of food and feed. As a consequence of their applications, they would result in neurotoxicity, even death. In this essence, the development of enzyme inhibition methods still shows great significance as rapid detection techniques for on-site large-scale screening of OPs and CMs. Initially, mechanisms and applications of various enzyme-inhibition-based methods and devices, including optical colorimetric assay, fluorometric assays, electrochemical biosensors, rapid test card, and microfluidic device, are highlighted in the present overview. Further, to enhance the enzyme sensitivity for detection; alternative enzyme sources or high yield enrichment methods (such as abzyme, artificial enzyme, and recombinant enzyme), as well as enzyme reactivation and identification, are also addressed in this comprehensive overview.

Dinuclear Cu-2(II,II)-cores stabilized by the N3O donorset of HL1 = (2-{[[di(2-pyridyl)methyl](methyl)amino]methyl}phenol), HL2 = 2-({[di(2-pyridyl)methyl] amino}methyl)phenol), and HL3 = 2-({[di(2-pyridyl)methyl]amino}methyl)-4-nitrophenol display a unique superoxide dismutase (SOD) combined with catalase (CAT)-like activity in water, at neutral pH. The Cu2L21 < Cu2L22 < Cu2L23 structure-reactivity trend puts a spotlight on the electron-deficient core of Cu2L23 that exhibits the highest SOD (log k(cat) (O-2(center dot-)) = 7.55) and CAT-like (k(H2O2) = 0.66 M(-1)s(-1)) performance. Time-lapse ESI-MS and EPR experiments indicate that a dimeric core is essential for oxygenic turnover upon H2O2 decomposition.

The authors describe a rapid enzyme-free colorimetric assay for food- and water-borne pathogens by using the multi-functional MnO2-doped magnetic ferrite nanoparticles (MCDs-MnO2 NPs). These nanoparticles not only can recognize, absorb, and separate the analyte from the matrix efficiently but also can directly catalyze the oxidation of TMB into a blue colored product without H2O2. In the presence of target, the nanoparticles preferentially bind to the target surface and the interaction between nanoparticles and TMB can be blocked, providing turn-off sensing of bacteria. By combining the superior capture efficiency of magnetic beads with the high catalytic activity of the enzyme mimic, the turn-off colorimetric assay can detect the gram-positive bacterium and the gram-negative bacterium by the naked eye at a low detection limit (102 cfu mL−1) with a wide line arrange from 10 to 106 cfu mL−1. With the advantage of simplicity, sensitivity, and specificity, this proposed approach showed promise in rapid instrumental and on-site visual detection of bacteria for determination of water contamination.

The protein scaffold around the active site of enzymes is known to influence catalytic activity, but specific scaffold features responsible for favorable influences are often not known. This study ...

A high degree of dependence on fossil fuels is one of the major problems faced by modern societies. d-Glucose and glycerol have emerged in recent years as prospective replacements for fossil fuels ...

Artificial enzymes have provided great antimicrobial activity to combat wound infection. However, the lack of tissue repair capability compromised their treatment effect. Therefore, development of novel artificial enzyme concurrently with the excellent antibacterial activity and the property of promoting wound healing are required. Here, we demonstrated the hydrogel-based artificial enzyme composed of copper and amino acids possessed intrinsic peroxidase-like catalytic activity, which could combat wound pathogen effectively and accelerate wound healing by stimulating angiogenesis and collagen deposition. Furthermore, the system possesses good biocompatibility for practical application. The synergic effect of the hydrogel-based artificial enzyme promises the system as a new paradigm in bacteria-infected wound healing therapy.

Staphylococcus aureus (S. aureus) is an important foodborne pathogen that causes food poisoning and severe infections. Herein, we design a novel and sensitive colorimetric immunoassay for the detection of S. aureus based on the formation of a magnetic bead-based sandwich complex and signal amplification via an enzyme mimic. Magnetic nanoparticles decorated with carbon dots offer rapid enrichment and separation of target bacteria in complex matrices. Sliver nanoclusters are used as an artificial enzyme that can oxidize o-phenylenediamine to form a yellow product with a maximum absorption peak at 421 nm. This method can visually detect S. aureus ranging from 10 to 106 cfu mL−1. Its limit of detection reaches down to 4.9 cfu mL−1. By analyzing food samples, the recovery is from 85.6 to 103.7%.

A systematic structure–activity relationship study was performed to identify the factors that are important to enhancing the transfer hydrogenation efficiency of half-sandwich metal complexes, which is an important class of compounds recently shown to be useful as artificial enzyme cofactors, catalytic drugs, and intracellular catalysts. In general, electron-rich ligands provided catalysts with greater activity and chemical stability than electron-poor ligands under physiologically relevant aqueous conditions. Complexes containing Ir were better catalysts than those containing Ru, Rh, or Os. Kinetic studies by UV-vis absorption spectroscopy revealed that ligand substituent effects could dramatically accelerate the rates of both hydride formation and hydride transfer processes, the latter by up to a remarkable 28-fold. The thermodynamic hydricities of several iridium–hydride species were determined using a hydride exchange method, which showed a strong correlation between the hydride donor strengths of the iridium–hydride species and the transfer hydrogenation yields of their parent complexes. Specifically, this work provides a practical guide for further elaboration of the pentamethylcyclopentadienyl iridium pyridinecarboxamidate catalyst platform. We found that electron-donating groups on the pyridyl ring are conducive to achieving high catalytic rates whereas a wide variety of alkyl and aryl groups on the N-amide moiety are well-tolerated, suggesting that this position might be the best site for conjugation to other functional entities for biological applications.

A new hybrid nanoparticle (NP; fluorenylmethoxycarbonyl-arginine-glycine-aspartate and hemin, Fmoc-RGD/hemin NP) was developed for the simultaneous detection and inhibition of breast cancer cells. Hemin can regulate the reactive oxygen species (ROS), while Fmoc-RGD acts as a scaffold for hemin nanocrystallization. Fmoc groups interact with the porphyrin groups of hemin through hydrophobic and π-π interactions to form a hydrophobic core of the NPs. The hydrophilic RGD chains surround the core to maintain the stability of the nanoparticles in an aqueous medium. The RGD groups of Fmoc-RGD are also selective for tumor cells. This interaction can be exploited to enhance the selectivity of tumor detection. Based on enhanced peroxidase activity, Fmoc-RGD/hemin NPs were developed as signal transducers in a facile and fast point-of-care cancer diagnosis platform. This platform is sensitive to breast cancer cells and hydrogen peroxide (H

L-Histidine (L-His) is an essential amino acid, which is used to synthesize proteins and enzymes. The concentration of L-His in the body is controlled to regulate tissue growth and repair of tissues. In this study, a rapid and sensitive method was developed for colorimetric L-his detection using Cu2+ ions to inhibit the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB)–H2O2 system. H2O2 can oxidize TMB to oxTMB in the presence of copper, and the change in color from colorless (TMB) to blue (oxTMB) is similar to that observed in the presence of peroxidase. However, because the imidazole ring and carboxyl group of L-His can coordinate with Cu2+ ions to form stable L-His–Cu2+ complexes, the color of the TMB–H2O2 solution remains unchanged after the addition of L-His. Therefore, because L-His effectively hinders the colorimetric reaction of TMB with H2O2, this assay can be used to quantitatively determine the concentration of L-His in samples. Under optimized conditions, our colorimetric sensor exhibited two linear ranges of 60 nM to 1 μM and 1 μM to 1 mM for L-His detection and a detection limit of 50 nM (S/N = 3); furthermore, the assay can be performed within 20 min. Moreover, the proposed assay was used to determine the concentration of L-His in urine samples, suggesting that this convenient and label-free colorimetric method presents promising applications in bioanalytical chemistry and clinical diagnosis.

An artificial metalloenzyme acting as a functional biomimic of hydrogenase enzymes was activated by assembly via covalent attachment of the molecular complex, [Ni(PNglycineP)2]2-, within a structured protein scaffold. Electrocatalytic H2 production was observed from pH 3.0 to 10.0 for the artificial enzyme, while no electrocatalytic activity was observed for similar [Ni(PNP)2]2+ systems.

Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy-based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.

Tumor-related exosomes, which are heterogeneous membrane-enclosed nanovesicles shed from cancer cells, have been widely recognized as potential noninvasive biomarkers for early cancer diagnosis. Herein, an artificial enzyme cascade amplification strategy based on a switchable DNA tetrahedral (SDT) scaffold was proposed for quantification of breast cancer-derived exosomes. The SDT scaffold is composed of G-quadruplex mimicking DNAzyme sequences on its two single-stranded edges and glucose oxidase (GOx) on the four termini of the complementary strands. In the initial state, the SDT scaffold is blocked by the switch strand which consists of partial complementary domains with the DNA tetrahedron and a MUC1 aptamer. MCF-7 exosomes could release the quadruplex-forming sequences through the recognition of the MUC1 aptamer. The newly formed DNAzyme brings GOx into spatial proximity and induces high-efficiency enzyme cascade catalytic reactions on the SDT. Consequently, high sensitivity toward MCF-7 exosome analysis was obtained with a wide linear range of 3.8 × 106 to 1.2 × 108 particles per mL and a limit of detection of 1.51 × 105 particles per mL. In addition, such a DNAzyme reconfiguration strategy was able to distinguish MCF-7 exosomes from other breast cancer cell derived exosomes, indicating its excellent method specificity. The proposed enzyme cascade strategy not only provides a novel signal transformation and amplification nanoplatform for quantifying the specific populations of exosomes, but also can be further expanded to the analysis of multiple cancer biomarkers.

A facile one-pot precipitation method was employed to prepare a petal-shaped hybrid under mild conditions. The hybrid is composed of urate oxidase (UOx) encapsulated into a zeolite-like metal–organic framework (MOF) with the doping of a hollow gold nanocage (AuNC). As one of the MOF–enzyme composites, a UOx@MOF(AuNC) hybrid with the features of artificial nanoenzymes was developed as a novel dual-channel biosensing platform for fluorescence (FL) and electrochemical detection of uric acid (UA). As for FL biosensing, enzymatic catalysis of the hybrid in the presence of UA triggered tandem catalysis and oxidation reactions to cause FL quenching. UA was linearly detected in the 0.1–10 μM and 10–300 μM ranges, with the limit of detection (LOD) of 20 nM. As for electrochemical biosensing, the hybrid was dropped on a glassy carbon electrode (GCE) surface to construct a hybrid/GCE platform. Based on the redox reaction of UA on the platform surface, UA was linearly detected in the 0.05–55 μM range, with a LOD of 15 nM. Experimental results confirmed that the hybrid-based dual-channel biosensing platform enabled selective and sensitive responses to UA over potential interferents. The platform has an excellent detection capability in physiological samples. The dual-channel biosensing platform facilitates the exploration of new bioanalysis techniques for early clinical diagnosis of diseases.

Nanozymes own striking merits, including high enzyme-mimicking activity, good stability, and low cost. Due to the powerful and distinguished functions, nanozymes exhibit widespread applications in the field of biosensing and immunoassay, attracting researchers in various fields to design and engineer nanozymes. Recently, nanozymes have been innovatively used to bridge nanotechnology with analytical techniques to achieve the high sensitivity, specificity, and reproducibility. However, the applications of nanozymes in food applications are seldom reviewed. In this review, we summarize several typical nanozymes and provide a comprehensive description of the history, principles, designs, and applications of nanozyme-based analytical techniques in food contaminants detection. Based on engineering and modification of nanozymes, the food contaminants are classified and then discussed in detail via discriminating the roles of nanozymes in various analytical methods, including fluorescence, colorimetric and electrochemical assay, surface-enhanced Raman scattering, magnetic relaxing sensing, and electrochemiluminescence. Further, representative examples of nanozymes-based methods are highlighted for contaminants analysis and inhibition. Finally, the current challenges and prospects of nanozymes are discussed.

Aspartic proteases use a pair of carboxylic acids to activate water molecules for nucleophilic attack. Here we report a nanoparticle catalyst with a similar catalytic motif capable of generating a hydroxide ion in its active site even under acidic reaction conditions. The synthetic enzyme accelerated the hydrolysis of para-nitrophenyl acetate (PNPA) by 91,000 times and could also hydrolyze nonactivated aryl esters at pH 7. The distance between the two acids and, in particular, the flexibility of the catalytic groups in the active site controlled the catalytic efficiency. The synthetic enzyme readily detected the addition of a single methyl on the acyl group of the substrate, as well as the substitution pattern on the phenyl ring.

Cell-free extract and purified enzyme-based systems provide an attractive solution to study biosynthetic strategies towards a range of chemicals. 4-(4-hydroxyphenyl)-butan-2-one, also known as raspberry ketone, is the major fragrance component of raspberry fruit and is used as a natural additive in the food and sports industry. Current industrial processing of the natural form of raspberry ketone involves chemical extraction from a yield of ∼1–4 mg kg−1 of fruit. Due to toxicity, microbial production provides only low yields of up to 5–100 mg L−1. Herein, we report an efficient cell-free strategy to probe into a synthetic enzyme pathway that converts either L-tyrosine or the precursor, 4-(4-hydroxyphenyl)-buten-2-one, into raspberry ketone at up to 100% conversion. As part of this strategy, it is essential to recycle inexpensive cofactors. Specifically, the final enzyme step in the pathway is catalyzed by raspberry ketone/zingerone synthase (RZS1), an NADPH-dependent double bond reductase. To relax cofactor specificity towards NADH, the preferred cofactor for cell-free biosynthesis, we identify a variant (G191D) with strong activity with NADH. We implement the RZS1 G191D variant within a ‘one-pot’ cell-free reaction to produce raspberry ketone at high-yield (61 mg L−1), which provides an alternative route to traditional microbial production. In conclusion, our cell-free strategy complements the growing interest in engineering synthetic enzyme cascades towards industrially relevant value-added chemicals.

Molecular imprinting represents one of the most promising strategies to design artificial enzyme inhibitors. However, the study of molecularly imprinted enzyme inhibitors (MIEIs) remains at a primary stage. Advanced applications of MIEIs for cell regulation have rarely been explored. Using a solid-phase oriented imprinting strategy so as to leave the active site of the enzymes accessible, we synthesized two MIEIs that exhibit high specificity and potent inhibitory effects (inhibition constant at low nM range) towards trypsin and angiogenin. The trypsin MIEI inhibits trypsin activity, tryptic digestion-induced extracellular matrix lysis and cell membrane destruction, indicating its utility in the treatment of active trypsin-dependent cell injury. The angiogenin MIEI blocks cancer cell proliferation by suppressing the ribonuclease activity of angiogenin and decreasing the angiogenin level inside and outside HeLa cells. Our work demonstrates the versatility of MIEIs for both enzyme inhibition and cell fate manipulation, showing their great potential as therapeutic drugs in biomedicine.

In this work, based on the powerful cycle amplification cascades of proximity hybridization-induced hybridization chain reaction and catalyzed hairpin assembly, we engineered a nonenzymatic and ultrasensitive method which combined the Mg2+-DNAzyme recycling signal amplification for the analysis of the human prostate specific antigen. Herein, we adopted PSA-conjugates as triggers in the self-assembly process of two hairpin DNAs (H1, H2) into the products of the CHA which could activate the HCR to induce repeated hybridization. And both ends of each adjacent sequence of the HCR products could form a unit of Mg2+-DNAzyme which in presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus generate observably enhanced fluorescent signal. Benefit from the nucleic acid circuit amplification strategy, PSA of concentration low to 0.73 pg mL−1 was detected in this system. This homogeneous sensing method in solution avoid the use of the sophisticated equipment and complex operation, as well as addition of artificial enzyme, thus greatly reducing the constraints and complexity of experimental conditions. Moreover, considering most protein biomarkers in serum don't have their corresponding aptamers, this sensing method provide a general sensing approach for homogeneous sensitive detection of these important protein biomarkers which transfer rough antigen-antibody interactivity to smart signal amplification sensing strategies, thus exhibiting a remarkable prospect in clinical application.

Utilizing electric fields to catalyze chemical reactions is not a new idea, but in enzymology it undergoes a renaissance, inspired by Warhsel's concept of electrostatic preorganization. According to this concept, the source of the immense catalytic efficiency of enzymes is the intramolecular electric field that permanently favors the reaction transition state over the reactants. Within enzyme design, computational efforts have fallen short in designing enzymes with natural-like efficacy. The outcome could improve if long-range electrostatics (often omitted in current protocols) would be optimized. Here, we highlight the major developments in methods for analyzing and designing electric fields generated by the protein scaffolds, in order to both better understand how natural enzymes function, and aid artificial enzyme design.

Living organisms utilize spatially organized enzyme complexes to carry out signal transduction and metabolic pathways in an efficient and specific way. Herein, inspired by natural enzyme complexes, we report the polymerization-induced coassembly (PICA) of enzyme-polymer conjugates into comicelles with tunable and enhanced cascade activity by using the cascade reaction implemented by glucose oxidase (GOX) and horseradish peroxidase (HRP) as a model system. Notably, the cascade activity of GOX/HRP-polymer comicelles monotonically increases with the GOX/HRP ratio. The cascade activity of GOX/HRP-polymer comicelles is up to 4.9 times higher than that of free GOX and HRP mixtures at the same GOX/HRP ratio. We further demonstrate that our system can quickly detect glucose in contrast with a commercially available glucose assay kit. These findings provide a new and general method of PICA for the controlled construction of artificial enzyme complexes with tunable and enhanced activity in enzyme cascades for advanced biomedical applications.

In order to lower the limit of detection of glucose, a peroxidase-like artificial enzyme with elevated catalysis capacity has been achieved. Fe3O4-doped MnO2 microspheres were fabricated through a two-step hydrothermal method for this purpose. TEM revealed that down-sized Fe3O4 nanoparticles were dispersed throughout the urchin-like MnO2 burrs. An additional XRD peak beyond Fe3O4 and MnO2 nanoparticles was observed, indicating a dislocation structure was formed. The defect in structure as well as the synergistic effect would allow extra enzyme ability. Based on the steady-state kinetic analyses and Michaelis–Menten model, the Michaelis–Menten constants (Km and vmax) were figured out. The results showed that the Fe3O4–MnO2 composite has elevated affinity toward the substrate TMB and H2O2. The limit of detection for glucose was estimated to be 0.75 μmol L−1 based on the Fe3O4–MnO2 composite artificial enzyme. The superparamagnetic properties endowed the material easy separation. The composite of this structure will provide a highly sensitive candidate method for accurate detection of glucose.

Highly-efficient and eco-friendly materials and technologies are urgently needed to meet the requirements of nowadays green development. Photocatalysis with using solar energy and enzymatic catalysis with eco-friendly nature are effective alternatives to address the problem. Notably, beneficial use of the synergistic effect of artificial enzyme and advanced photocatalyst has attracted wide attention. This work presents a biomimetic photocatalytic material, two-dimensional (2D) biomimetic hemin-bismuth tungstate (HBWO). Stable HBWO composites formed by immobilization of monomeric hemin on 2D bismuth tungstate layer, exhibit high photocatalytic performance, better than that of pure 2D bismuth tungstate and unsupported hemin. HBWO shows layered structure with the interlayer spacing at ˜0.35 nm. In the photocatalytic process, hemin can not only act as an electron shuttle, also play an important role in oxygen transfer. Additionally, the synthesized HBWO composites exhibit nice binding affinities and high photocatalytic activity in tetracycline degradation. It is anticipated that beneficial use of synergistic effect of artificial enzyme and photocatalyst via HBWO composites can be a promising eco-friendly and efficient solution for addressing the environmental crisis.