Artificial Enzymes Research Articles

The Detection of Inactivated p53 Protein by a Biosensor

P53 is a gene that closely correlates with human tumors. In this paper, we explore how various biology techniquescan be applied to detect the malfunction of p53. Firstly, we insert the GFP gene, as correctly translated p53 proteinwill undergo fluorescence. Secondly, we apply artificial enzymes such as an antibody, and the structure of functionalp53 protein differs from those that are incorrectly translated. The enzyme can detect this as its active site matches thestructure of the functional p53 protein. Lastly, we aim to detect not the p53 protein but its side product. Caspase 3 is theone we will target to find. In this paper, we carefully compare and contrast these three methods, and the choice shouldalways be considere with different experimental conditions. The detection using GFP should be the best method amongthe rest, as it gives a visually accessible result, is also easy, and can produce the result very quickly.

Visual Sensor Arrays for Distinction of Phenolic Acids Based on Two Single-Atom Nanozymes.

Although great achievements have been made in the study of artificial enzymes, the design of nanozymes with high catalytic activities of natural enzymes and the further establishment of sensitive biosensors still remain challenging. Here, two nanozymes, i.e., ZnCoFe three-atom nanozyme (TAzyme) and Sn single-atom nanozyme (SAzyme)/Ti3C2Tx, are developed, which show peroxidase-like catalytic activities by catalyzing the reaction of hydrogen peroxide (H2O2), 4-aminoantipyrine (4-AAP), and phenolic acids to generate colorimetric reactions. The involvement of different phenolic acids leads to the generation of different color products. These subtle color-variation profiles between these phenolic acids prompt us to exploit an electronic tongue based on the two nanozymes to distinguish phenolic acids. Data interpretation by the pattern recognition method, such as linear discriminant analysis (LDA), displays good clustering separation of six different phenolic acids at concentrations of 0.1 μM to 1 mM, validating the effectiveness of the colorimetric nanozyme sensor array.

Defect-Rich CoFe-Layered Double Hydroxides as Superior Peroxidase-like Nanozymes for the Detection of Ascorbic Acid.

The development of artificial enzymes with superior catalytic properties to natural enzymes has been a long-standing goal of chemists. Herein, defect-rich CoFe-layered double hydroxides (d-CoFe-LDHs) nanosheets are developed and used as superior peroxidase-like nanozymes for the detection of ascorbic acid (AA). The d-CoFe-LDHs with an average thickness of ∼3 nm and a lateral size of ∼20 nm are synthesized through rapid nucleation in a colloid mill, which exhibited abundant unsaturated sites (oxygen vacancies and cobalt vacancies). Impressively, d-CoFe-LDHs exhibited excellent peroxidase-mimicking performance with strong substrate affinity and robustness in a wide pH range. Density functional theory calculations show that d-CoFe-LDHs have lower H2O2 adsorption energy, which promotes the decomposition of H2O2, thereby improving the catalytic activity. The chromogenic system of d-CoFe-LDHs and 3,3',5,5'-tetramethylbenzidine can be used to accurately detect the content of AA, and the detection limit is about 3.6 μM. This study opens up a new approach for the construction of highly active defective LDH peroxidases for the detection of biomolecules.

Aggregation‐induced emission artificial enzyme (AIEzyme) with DNase‐like activity: Imaging and matrix cleavage for combating bacterial biofilm

AbstractDNase‐catalyzed hydrolysis of extracellular DNA (eDNA) has been widely employed to eradicate intractable biofilms. Although aggregation‐induced emission (AIE) has become the ideal tool for killing planktonic bacteria, AIE luminogens (AIEgens) often lack DNase‐mimetic activity, so as to suffer from poor anti‐biofilm capacity. Here, an “AIEzyme”, a kind of AIE nanomaterial with enzyme‐like activity, is designed and synthesized, where the AIEgens are used as the ligands of Zr‐based coordination polymer nanoparticles. Not only does AIEzyme have enduring DNase‐mimetic activity with low activation energy, but also structural rigidity‐stabilized fluorescence. Due to the long‐acting hydrolysis for eDNA in biofilm, AIEzyme can efficiently disorganize the established biofilms with good penetrability and realize the healing of superbug‐infected wounds under only one dose of AIEzyme. This work provides a strategy to endow ordinary AIEgens with DNase‐like and anti‐biofilm activities. Moreover, AIEzymes can be observed by virtue of their own AIE character, facilitating the study on self‐positioning and residual amount of AIEzymes in wounds. The concept “AIEzyme” would hopefully stimulate the tremendous expansion for the tool kits and the application of AIEgens and artificial enzymes.

Continuous levodopa production by an artificial enzyme.

Continuous levodopa production by an artificial enzyme.

Current progress, challenges, and future perspectives of language models for protein representation and protein design

The sequence-structure-function paradigm of protein is the basis of molecular biology. What is the underlying mechanism of such sequence and structure/function corresponding relationship? We reviewed the methods for protein representation and protein design. With these protein representation models, we can accurately predict many properties of proteins, such as stability and binding affinity. Progen, Chroma, RF Diffusion, SCUBA, and other protein design models have demonstrated how human-designed artificial proteins can have desired biological functions. The protein design will revolutionize drug development. And more efficient artificial enzymes that break down industrial waste or plastics will contribute to carbon neutrality. We also discussed the three greatest challenges of protein design in future and possible solutions.

Vanadium‐Incorporated Mesoporous Silica as Oxidase Mimic for Colorimetric Dopamine Detection and Anticancer Activity

AbstractThe intriguing features of artificial enzymes made nanozymes attractive in the field of biosensing and biomedical. Here, vanadium‐incorporated dendritic mesoporous silica (VMSN) as an oxidase mimicking nanozyme was utilized as a sensitive and selective colorimetric biosensor to detect dopamine (DA) and treat cancer cells. The nanozyme was successfully fabricated through the in‐situ incorporation of vanadium ions in the silica framework via a simple sol‐gel method using CTAB and mesitylene. The excellent oxidase‐like activity of VMSN was monitored by the oxidation of TMB. The kinetics, mechanism of the catalytic reaction and the effect of temperature, pH, and concentration on oxidase‐like activity were investigated thoroughly and further, based on the oxidase‐like activity, a fast and sensitive colorimetric sensing of dopamine developed with a LOD of 0.0021 μM and a linear range of 0–50 μM. The proposed colorimetric method was successfully used to determine DA concentration in commercially available dopamine hydrochloride injection.VMSN was further modified with folic acid and utilized against cancer cells in vitro. Neuroblastoma cells (SHSY‐5Y) show ROS generated by VMSN in the presence of oxygen, leading to enhanced cytotoxicity detrimental for SHSY‐5Y cells.

Au/CeO2 NR Restricted Inside Cu-MOFs: A Three-in-One Artificial Enzyme with Synergistically Enhanced Peroxidase-Like Activity for Dual-Mode Sensing of Multiple Biomarkers

Nanomaterials with inherent enzyme-mimetic properties have been extensively studied in biosensing field, but it is still challenging to overcome their natively weak enzyme-mimetic activity and further promote their stabilities. In this work, a three-in-one artificial enzyme with synergistically enhanced peroxidase-like activity and outstanding stability is constructed by restricting Au-loaded CeO2 nanorods (NR) (1% Au:CeO2 mass ratio) inside Cu-based metal–organic frameworks (Cu(PABA)), termed Au1/CeO2 NR@Cu(PABA). It has been demonstrated that the artificial enzyme possesses significantly improved performance in catalytic decomposition of H2O2, with ∼2.41 and ∼1.49 times higher activity than its individual constituents (Au1/CeO2 NR and Cu(PABA)), benefiting from the synergistic effect and the unique restricted structure; owns strong affinities to substrates, with separately ∼8.21- and ∼3.13-fold lower Michaelis constants toward H2O2 and o-phenylenediamine, respectively, than horseradish peroxidase; and exhibits desirable pH (4–12), thermal (30–80 °C), organic solvent (N,N-dimethylformamide, acetone, methanol, etc.), and long-term storage (30 days) stabilities, advantaging practical applications. Taking aforementioned superior properties of Au1/CeO2 NR@Cu(PABA), a universal colorimetric/fluorometric dual-mode sensing platform is built and utilized for detection of biomarkers (e.g., glucose, galactose, and cholesterol) in biological fluids with satisfactory recoveries (90.2–108%). This work offers new horizons in designing high-efficiency, stable, and credible biomimetic catalysts to accelerate future advanced engineering of nanozymes.

In situ continuous Dopa supply by responsive artificial enzyme for the treatment of Parkinson’s disease

Oral dihydroxyphenylalanine (Dopa) administration to replenish neuronal dopamine remains the most effective treatment for Parkinson’s disease (PD). However, unlike the continuous and steady dopamine signaling in normal neurons, oral Dopa induces dramatic fluctuations in plasma Dopa levels, leading to Dopa-induced dyskinesia. Herein, we report a functional nucleic acid-based responsive artificial enzyme (FNA-Fe3O4) for in situ continuous Dopa production. FNA-Fe3O4 can cross the blood-brain barrier and target diseased neurons relying on transferrin receptor aptamer. Then, FNA-Fe3O4 responds to overexpressed α-synuclein mRNA in diseased neurons for antisense oligonucleotide treatment and fluorescence imaging, while converting to tyrosine aptamer-based artificial enzyme (Apt-Fe3O4) that mimics tyrosine hydroxylase for in situ continuous Dopa production. In vivo FNA-Fe3O4 treatment results in recovery of Dopa and dopamine levels and decrease of pathological overexpressed α-synuclein in PD mice model, thus ameliorating motor symptoms and memory deficits. The presented functional nucleic acid-based responsive artificial enzyme strategy provides a more neuron friendly approach for the diagnosis and treatment of PD.

Selective Oxidation of Halophenols Catalyzed by an Artificial Miniaturized Peroxidase.

The development of artificial enzymes for application in sustainable technologies, such as the transformation of environmental pollutants or biomass, is one of the most challenging goals in metalloenzyme design. In this work, we describe the oxidation of mono-, di-, tri- and penta-halogenated phenols catalyzed by the artificial metalloenzyme Fe-MC6*a. It promoted the dehalogenation of 4-fluorophenol into the corresponding 1,4-benzoquinone, while under the same experimental conditions, 4-chloro, 4-bromo and 4-iodophenol were selectively converted into higher molecular weight compounds. Analysis of the 4-chlorophenol oxidation products clarified that oligomers based on C-O bonds were exclusively formed in this case. All results show that Fe-MC6*a holds intriguing enzymatic properties, as it catalyzes halophenol oxidation with substrate-dependent chemoselectivity.

NiCo2O4 with changed catalytic process and enhanced catalytic property as mimic enzyme by introducing Mo6 +

The modification of transition metal oxide by introducing with heteroatoms is a valid strategy to enhance artificial enzyme activities benefited from the change of the catalytic process and electronic properties. Herein, a simple hydrothermal method has been used to fabricate Mo-NiCo2O4 (MNCO) via introducing Mo6+ into NiCo2O4 (NCO). Compared with pure NiCo2O4, the acquired NiCo2O4 (MNCO) with the introduction of Mo exhibits better catalytic activity of peroxidase mimic enzyme. More interesting, only·O2- and h+ can be captured during catalytic process when MNCO as artificial enzyme, while·OH,·O2- and h+ can be found in the presence of NCO. Meanwhile, after the introduction of Mo, electron transfer has been drastically accelerated which contributed to the enhanced peroxidase-like activity. The Km value of H2O2 of NCO is 49-fold higher than that of MNCO, indicating that better affinity between MNCO and H2O2. And the LOD (detection limit) of MNCO is 22 times lower than that of NCO in the detection of H2O2. Based on excellent peroxidase-like active, a sensing platform for detecting hydroquinone in seawater is designed, confirming the great practicability in sensing fields.

The development of a novel copper-loaded mesoporous silica nanoparticle as a peroxidase mimetic for colorimetric biosensing and its application in H2O2 and GSH assay.

In recent years, the development of nanomaterials-based peroxidase mimics as enzyme sensors has been attracting considerable interest due to their outstanding features, including potent stability, and cost-effectiveness toward natural enzymes. In this work, mesoporous silica nanoparticles functionalized by copper (Cu-MSN) were prepared as a new artificial enzyme for the first time through the sol-gel procedure. A comprehensive investigation of the catalytic activity of Cu-MSN was done through the oxidation of chromogenic peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), and (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), in the presence of H2O2. The results indicate that the peroxidase-like activity of the as-prepared sample is significantly higher than other nanoparticles. Additionally, for the study, a facile and rapid sensing method based on the enzyme-like activity of Cu-MSN to detect H2O2 and glutathione (GSH) was developed to examine the potency of the proposed biosensor. Preliminary analysis revealed that the limit of detection (LOD) of H2O2 and GSH is 0.2 and 0.0126μM, in the range of 0.9-100 and 0.042-1μM, respectively. These findings support the claims for the efficiency of the sensor in detection fields. Also, human serum was utilized as the real sample to obtain additional evidence.

New bis[MoO2] and [MoO(O2)] compounds: An artificial enzyme with peroxidase activity against o-phenylenediamine and dopamine

In this study, two binuclear dioxido- and oxidoperoxido molybdenum (VI) complexes, [{MoVIO2}2(L)(H2O)2] 1 and [{MoVIO(O2)}2(L)(H2O)2] 2, were synthesized. Complex 1 was obtained through a 1:2 reaction of ligand I with MoO2(acac)2, while complex 2 was synthesized in situ by reacting MoO3 with H2O2 in a 1:2 ratio. The structures and characteristics of the complexes were examined employing several techniques such as elemental (CHN) analysis, spectroscopy (FT-IR, UV–Vis, 1H, and 13CNMR), and thermal study (TGA). SC-XRD analysis of complex 1a revealed that the molybdenum central atom adopts an octahedral geometry and is bonded to phenolic oxygen, enolate oxygen, and azomethine nitrogen atoms. Powder X-ray diffraction was used to determine the purity of the bulk material, and the results were compared to single crystal data. Computational calculations were performed using density functional theory (DFT) at the B3LYP/6-31G(d, p) level of theory for the ligand and the LANL2DZ level of theory for the complexes, yielding geometry optimized structures that were then employed in frequency and NMR-calculations. These theoretical findings were compared to the experimental results and showed a good correlation. Furthermore, the complexes exhibited peroxidase-like activity in the presence of hydrogen peroxide as evidenced by the oxidation of o-phenylenediamine and dopamine.

Engineering Single-Atom Nanozymes for Catalytic Biomedical Applications.

Nanomaterials with enzyme-mimicking properties, coined as nanozymes, are a promising alternative to natural enzymes owing to their remarkable advantages, such as high stability, easy preparation, and favorable catalytic performance. Recently, with the rapid development of nanotechnology and characterization techniques, single atom nanozymes (SAzymes) with atomically dispersed active sites, well-defined electronic and geometric structures, tunable coordination environment, and maximum metal atom utilization are developed and exploited. With superior catalytic performance and selectivity, SAzymes have made impressive progress in biomedical applications and are expected to bridge the gap between artificial nanozymes and natural enzymes. Herein, the recent advances in SAzyme preparation methods, catalytic mechanisms, and biomedical applications are systematically summarized. Their biomedical applications in cancer therapy, oxidative stress cytoprotection, antibacterial therapy, and biosensing are discussed in depth. Furthermore, to appreciate these advances, the main challenges, and prospects for the future development of SAzymes are also outlined and highlighted in this review.

Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies.

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27-42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.

Engineering the Hydrophobic Microenvironment in Polystyrene-Supported Artificial Catalytic Triad Nanocatalysts: An Effective Strategy for Improving Catalytic Performance

Hydrophobic environments have been identified as one of the main parameters affecting the catalytic performance of artificial catalytic triads but are often ignored as an approach to engineering these catalysts. Here, we have developed a simple yet powerful strategy to engineer the hydrophobic environment in polystyrene-supported artificial catalytic triad (PSACT) nanocatalysts. Hydrophobic copolymers containing either oligo(ethylene glycol) side chains or hydrocarbon side chains were synthesized and used for the preparation of nanocatalysts through nanoprecipitation in aqueous media. By using the hydrolysis of 4-nitrophenyl acetate (4NA) as a model reaction, we studied the influence of chemical structures and effective constituent ratios of hydrophobic copolymers on the catalytic performance of PSACT nanocatalysts. Additionally, PSACT nanocatalysts could catalyze the hydrolysis of a few carboxylic esters, even polymers, and be reused for five consecutive runs without significant loss of catalytic activity. This strategy may open an avenue for engineering other artificial enzymes, and these PSACT nanocatalysts have potential applications for the hydrolysis of carboxylic esters.

Self-adaptive Metal-Organic Framework Assembles Di-iron Active Sites to Mimic Monooxygenases.

Despite significant efforts, it remains a challenge to design artificial enzymes that can mimic both structures and functions of natural enzymes. Here, we report the post-synthetic construction of binuclear iron catalysts in MOF-253 to mimic natural di-iron monooxygenases. The adjacent bipyridyl (bpy) linkers in MOF-253 can freely rotate to form the [(bpy)FeIII(μ2-OH)]2 active site in a self-adaptive fashion. The composition and structure of the [(bpy)FeIII(μ2-OH)]2 active sites in MOF-253 were characterized by a combination of inductively coupled plasma-mass spectrometry, thermogravimetric analysis, X-ray absorption spectrometry, and Fourier-transform infrared spectroscopy. The MOF-based artificial monooxygenase effectively catalyzed oxidative transformations of organic compounds, including C-H oxidation and alkene epoxidation reactions, using O2 as the only oxidant, which indicates the successful recapitulation of the structure and functions of natural monooxygenases using readily accessible MOFs. The di-iron system exhibited at least 27 times higher catalytic activity than the corresponding mononuclear control. DFT calculations showed that the binuclear system had a 14.2 kcal/mol lower energy barrier than the mononuclear system in the rate-determining C-H activation process, suggesting the importance of cooperativity of the iron centers in the [(bpy)FeIII(μ2-OH)]2 active site in the rate-determining step. The stability and recyclability of the MOF-based artificial monooxygenase were also demonstrated.

DNA-Programmed Tuning of the Growth and Enzyme-Like Activity of a Bimetallic Nanozyme and Its Biosensing Applications.

Nanozymes, which combine the merits of both nanomaterials and natural enzymes, have aroused tremendous attention as new representatives of artificial enzyme mimics. However, it still remains to be a great challenge to rationally engineer the morphologies and surface properties of nanostructures that lead to the desired enzyme-like activities. Here, we report a DNA-programming seed-growth strategy to mediate the growth of platinum nanoparticles (PtNPs) on gold bipyramids (AuBPs) for the synthesis of a bimetallic nanozyme. We find that the preparation of a bimetallic nanozyme is in a sequence-dependent manner, and the encoding of a polyT sequence allows the successful formation of bimetallic nanohybrids with greatly enhanced peroxidase-like activity. We further observe that the morphologies and optical properties of T15-mediated Au/Pt nanostructures (Au/T15/Pt) change over the reaction time, and the nanozymatic activity can be tuned by controlling the experimental conditions. As a concept application, Au/T15/Pt nanozymes are used to establish a simple, sensitive, and selective colorimetric assay for the determination of ascorbic acid (AA), alkaline phosphatase (ALP), and the inhibitor sodium vanadate (Na3VO4), demonstrating excellent analytical performance. This work provides a new avenue for the rational design of bimetallic nanozymes for biosensing applications.

Stereodivergent Biocatalytic Formal Reduction of α-Angelica Lactone to (R)- and (S)-γ-Valerolactone in a One-Pot Cascade.

The formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing two Old Yellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41 % conversion and up to 91 % ee. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84 % ee and 41 % overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical.

Gum tragacanth-mediated synthesis of metal nanoparticles, characterization, and their applications as a bactericide, catalyst, antioxidant, and peroxidase mimic

Abstract Among biogenic methods employed for synthesizing various nanoparticles (NPs), gum tragacanth (TGC)-mediated NP production is important. The gum TGC not only qualifies the principles of green chemistry but also embraces unique qualities. In this perspective, the current review concentrates on the composition, uses, and exploitation of gum towards synthesizing metal NP of silver (Ag), gold (Au), palladium (Pd), platinum (Pt), and their characterization (UV-visible absorption spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and transmission electron microscopy). In addition, applications of synthesized NP as a bactericide, catalyst, antioxidant, and peroxidase mimic are emphasized. Ag NP (13 nm) showed antibacterial action against Gram-negative and Gram-positive bacteria at 2–12 μg‧mL−1. The exploitation of Ag NP as a bactericide makes it a candidate of choice for medicinal and pharmacological applications. The catalytic activity of Pd NP (14 nm) demonstrated borohydride reduction of methylene blue. The gum reduced/capped metal and metal oxide NP serve as redox and photocatalysts for the remediation of toxic pigments and dyes in industrial effluents. At 15 μg‧mL−1, Pd NP exhibited 1,1-diphenyl-2-picrylhydrazyle radical scavenging activity (95.8%) and served as an artificial enzyme mimic for colorimetric sensing of hydrogen peroxide. The industrial applications of other TGC-based nanocomposites, such as heavy metal sorption, wound dressing, drug carrier, tissue engineering, etc., are mentioned.