Activity Of Horseradish Peroxidase Research Articles

Impact of an Ionic Liquid Solution on Horseradish Peroxidase Activity.

Horseradish peroxidase (HRP) is an enzyme that oxidizes pollutants from wastewater. A previous report indicated that peroxidases can have an enhancement in initial enzymatic activity in an aqueous solution of 0.26 M 1-ethyl-3-methylimidazolium ethyl sulfate ([EMIm][EtSO4]) at neutral pH. However, the atomistic details remain elusive. In the enzymatic landscape of HRP, compound II (Cpd II) plays a key role and involves a histidine (H42) residue. Cpd II exists as oxoferryl (2a) or hydroxoferryl (2b(FeIV)) forms, where 2a is the predominantly observed form in experimental studies. Intriguingly, the ferric 2b(FeIII) form seen in synthetic complexes has not been observed in HRP. Here, we have investigated the structure and dynamics of HRP in pure water and aqueous [EMIm][EtSO4] (0.26 M), as well as the reaction mechanism of 2a to 2b conversion using polarizable molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations. When HRP is solvated in aq [EMIm][EtSO4], the catalytic water displaces, and H42 directly orients over the ferryl moiety, allowing a direct proton transfer (PT) with a significant energy barrier reduction. Conversely, in neat water, the reaction of 2a to 2b follows the previously reported mechanism. We further investigated the deprotonated form of H42. Analysis of the electric fields at the active site indicates that the aq [EMIm][EtSO4] medium facilitates the reaction by providing a more favorable environment compared with the system solvated in neat water. Overall, the atomic level supports the previous experimental observations and underscores the importance of favorable electric fields in the active site to promote catalysis.

The Energies of Activation and Deactivation of 2,4-Dichlorophenol Degradation by Horseradish Peroxidase Immobilized on the Modified Nanofibrous Membrane

Chlorophenol compounds pose a health risk to many organisms due to their toxicity. The present paper presents the estimation of the activation and deactivation energies and the optimum temperatures of 2,4-dichlorophenol degradation by horseradish peroxidase (HRP). The activities of horseradish peroxidase depending on temperature were analyzed. In a mathematical model, describing 2,4-dichlorophenol degradation by HRP was assumed that both the 2,4-dichlorophenol degradation and the deactivation of HRP were first-order reactions by the enzyme concentration. The parameters of the optimum temperatures Topt, the activation energies Er, and the deactivation energies Ed in the process of 2,4-dichlorophenol degradation by HRP immobilized on a modified nanofibrous membrane were determined kd and t1/2 were determined for HRP immobilized at temperatures in the range of 25 °C to 75 °C. Likewise, thermodynamic parameters such as the change in the enthalpy ∆H#, change in entropy ∆S#, the change in Gibbs free energy ∆G# for native HPR and the change in the enthalpy ∆Hd#, change in entropy ∆Sd#, and the change in Gibbs free energy ∆Gd# for deactivated HRP were determined at 25 °C.

Exploration of the structural mechanism of hydrogen (H2)-promoted horseradish peroxidase (HRP) activity via multiple spectroscopic and molecular dynamics simulation techniques

Horseradish peroxidase (HRP) is an enzyme that is widely used in various fields. In this study, the effects of molecular hydrogen (H2) on the activity and structural characteristics of HRP were investigated by employing multiple spectroscopic techniques, atomic force microscopy (AFM) and molecular dynamics (MD) simulations. The results demonstrated that H2 could enhance HRP activity, especially in 1.5 mg/L hydrogen-rich water (HRW). The structural analysis results showed that H2 might alter HRP activity by affecting the active sites, secondary structure, hydrogen bonding network, CS groups, and morphological characteristics. The MD results also confirmed that H2 could increase the FeN bond distance in the active site, affect the secondary structure, and increase the number of hydrogen bonds. The MD results further suggested that H2 could increase the number of salt bridges, and lengthen the SS bonds in HRP. This study primarily revealed the mechanism by which H2 enhances the HRP activity, providing insight into the interactions between gas and macromolecular proteins. However, some of the results obtained via MD simulations still need to be verified experimentally. In addition, our study also provided a new convenient strategy to enhance enzyme activity.

Catalytic Activity of Horseradish Peroxidase Immobilized on Pristine and Two‐Photon Oxidized Graphene

AbstractBiosensors based on graphene and bio‐graphene interfaces have gained momentum in recent years due to graphene's outstanding electronic and mechanical properties. By introducing the patterning of a single‐layer graphene surface by two‐photon oxidation (2PO), the surface hydrophobicity/hydrophilicity and doping can be varied at the nanoscale while preserving the carbon network, thus opening possibilities to design new devices. In this study, the effect of 2PO on the catalytic activity of the noncovalently immobilized enzyme horseradish peroxidase (HRP) on single‐layer graphene‐coated Si/SiO2 chips is presented. To monitor the activity continuously, a simple well‐plate setup is introduced. Upon controllable 1–2‐layer immobilization, the catalytic activity decreases to a maximum value of 7.5% of the free enzyme. Interestingly, the activity decreases with increasing 2PO area on the samples. Hence, the HRP catalytic activity on the graphene surface is locally controlled. This approach can enable the development of graphene‐bio interfaces with locally varying enzyme activity.

Enzymatic Dimerization-Induced Self-Assembly of Alanine-Tyramine Conjugates into Versatile, Uniform, Enzyme-Loaded Organic Nanoparticles.

Herein, we report a novel enzymatic dimerization-induced self-assembly (e-DISA) procedure that converts alanine-tyramine conjugates into highly uniform enzyme-loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5-12 % standard deviation of as-prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post-synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One-pot multi-enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx-HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π-π, and CH-π interactions. The proposed e-DISA procedure can be widely expanded through the design of diverse enzyme-responsive precursors.

Enzymatic Dimerization‐Induced Self‐Assembly of Alanine‐Tyramine Conjugates into Versatile, Uniform, Enzyme‐Loaded Organic Nanoparticles

AbstractHerein, we report a novel enzymatic dimerization‐induced self‐assembly (e‐DISA) procedure that converts alanine‐tyramine conjugates into highly uniform enzyme‐loaded nanoparticles (NPs) or nanocontainers by the action of horseradish peroxidase (HRP) in an aqueous medium under ambient conditions. The NP formation was possible with both enantiomers of alanine, and the average diameter could be varied from 150 nm to 250 nm (with a 5–12 % standard deviation of as‐prepared samples) depending on the precursor concentration. About 60 % of the added HRP enzyme was entrapped within the NPs and was subsequently utilized for post‐synthetic modification of the NPs with phenolic compounds such as tyramine or tannic acid. One‐pot multi‐enzyme entrapment of glucose oxidase (GOx) and peroxidase (HRP) within the NPs was also achieved. These GOx‐HRP loaded NPs allowed multimodal detection of glucose, including that present in human saliva, with a limit of detection (LoD) of 740 nM through fluorimetry. The NPs exhibited good cytocompatibility and were stable to changes in pH (acidic to basic), temperature, ultrasonication, and even the presence of organic solvent (EtOH) to a certain extent, since they are stabilized by intermolecular hydrogen bonding, π‐π, and CH‐π interactions. The proposed e‐DISA procedure can be widely expanded through the design of diverse enzyme‐responsive precursors.

Annotation of GC-MS Data of Antimicrobial Constituents in the Antarctic Seaweed Phaeurus antarcticus by Molecular Networking.

Phaeurus antarcticus is a member of the Desmarestiaceae family endemic to the Antarctic Peninsula. Reports addressing its chemical composition and biological activities are scarce. Herein, bioactive non-polar compounds of P. antarcticus against pathogenic bacteria, Leishmania amazonensis and Neospora caninum parasites were targeted through GC-MS Molecular Networking and multivariate analysis (OPLS-DA). The effects on horseradish peroxidase (HRP) were also evaluated. P. antarcticus exhibited selective bacteriostatic and bactericidal activities against Staphylococcus aureus with MIC and MBC values from 6.25-100 μg mL-1 . Fractions HX-FC and HX-FD were the most active against L. amazonensis with EC50 ranging from 18.5-62.3 μg mL-1 . Additionally, fractions HX-FC and HX-FD showed potent inhibition of N. caninum at EC50 values of 2.8 and 6.3 μg mL-1 , respectively. All fractions inhibited HRP activity, indicating possible interactions with Heme proteins. It was possible to annotate compounds from tree mains clusters, containing terpenoids, steroids, fatty acids, and alcohols by correlating the spectral data of the GC-MS analysis with Molecular Networking and the OPLS-DA results.

Role of sugars in the inactivation of horseradish peroxidase induced by cold atmospheric plasma

This study investigated the effect of different CAP exposures on the activity of horseradish peroxidase (HRP) in phosphate buffer and in model systems with different concentrations of monosaccharides (glucose, fructose) and disaccharides (sucrose, trehalose) to evaluate the potential role of sugars in enzyme inactivation. Spectroscopic analyses (fluorescence, circular dichroism, UV–Vis absorption) were conducted to highlight structural modifications possibly responsible for HRP inactivation. In phosphate buffer, the inactivation of HRP by CAP resulted dependent on both the treatment time (up to 70% after 30 min) and ozone concentration in the chamber. HRP residual activity was fitted with a very good approximation by the Weibull model. Sugars reduced the CAP efficacy, and this effect was concentration-dependent and much higher for disaccharides than monosaccharides. UV and fluorescence spectra showed that sugars differently preserved HRP tertiary structure, hindering heme degradation and quenching of aromatic amino acids. Likewise, sugars differently affected the loss of secondary structures. These findings only partially explained the diverse protective effects of sugars on HRP. Other factors, such as the different sugars’ abilities to quench plasma reactive species, reduce the system mobility, and stabilize proteins by preserving their hydration shell, were retained to play a role in the reduction of HRP inactivation. This study deepened the knowledge of the effect of CAP on peroxidase activity and demonstrated that the presence of mono and disaccharides, naturally found in fruits and vegetables and widely employed in food and beverage industries, can mitigate the inactivation and structural alterations induced by CAP on peroxidase enzymes.

An ultrahigh-resolution multicolor sensing platform via target-induced etching of gold nanorods for multi-colorimetric analysis of trace silver ions

Heavy metal ion pollution is a pressing global concern with detrimental effects on the environment and human health. To address this issue, we have developed an ultrahigh-resolution multicolor sensing platform for the specific detection of silver ions (Ag(I)). The method employs the nanozyme of platinum nanoparticles (PtNPs) as a link to control the etching of gold nanorods (AuNRs). Through specific binding with Ag(I), the PtNPs hinder the catalase-like activity responsible for hydrogen peroxide (H2O2) decomposition. This inhibition enables the Fenton reaction between H2O2 and ferrous ions, generating highly oxidizing hydroxyl free radicals (·OH). The resulting etching of AuNRs induces a blue-shift in their local surface plasmon resonance (LSPR) absorption spectra. The degree of blue-shift is directly proportional to the Ag(I) concentration, facilitating trace-level detection. Furthermore, the aspect ratio change of the AuNRs yields distinguishable colors. In contrast to common multicolor sensors relying on nanozyme horseradish peroxidase (HRP) activity, which exhibits an inverse relationship between AuNR etching and target concentration, our approach demonstrates a proportional response. This unique characteristic enables ultrahigh-resolution multicolor detection of Ag(I) visible to the naked eye. We anticipate that this innovative sensing platform will pave the way for nanosensor development in environmental monitoring and food safety detection.

The Amphiphilic Property and Structure of β-Amyloid Peptide Contribute to Its Impacts on the Activities of Horseradish Peroxidase and Alkaline Phosphatase.

Recent studies have found that β-amyloid (Aβ) oligomers may play much more important roles than amyloid plaques in the pathogenesis of Alzheimer's disease (AD). However, due to the complexity of Aβ, studying the structural basis of Aβ oligomer toxicity is challenging. Here, we assessed the amphiphilic property and β-hairpin structure of Aβ monomer. The potential impacts of Aβ oligomers and three sequence-modifying peptides on the enzyme activities of horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were further evaluated. We demonstrated that Aβ oligomer possesses the ability to alter the activity of two enzymes. Moreover, modifications on the hydrophobic region and β-turn structure of Aβ monomer significantly alter its impacts on the enzyme activities. In addition, these modifications also change the bonding modes of Aβ monomers or oligomers binding to HRP, as assessed by molecular docking. All of these findings provide direct experimental evidence to reveal the critical roles of the amphiphilic property and β-sheet structure of Aβ monomer in its impacts on protein activity.

Dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase-tethered magnetic microspheres for colorimetric detection of microcystin-LR

A novel dual-amplification system based on CRISPR-Cas12a and horseradish peroxidase (HRP) was developed for colorimetric determination of MC-LR. This dual-amplification was accomplished by combining the nuclease activity of CRISPR-Cas12a with the redox activity of HRP. HRP linked to magnetic beads through an ssDNA (MB-ssDNA-HRP) was used to induce a color change of the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 chromogenic substrate solution. Specific binding of MC-LR with its aptamer initiated the release of a complementary DNA (cDNA), which was designed to activate the trans-cleavage activity of CRISPR-Cas12a. Upon activation, Cas12a cut the ssDNA linker in MB-ssDNA-HRP, causing a reduction of HRP on the magnetic beads. Consequently, the UV–Vis absorbance of the HRP-catalyzed reaction was decreased. The dual-signal amplification facilitated by CRISPR-Cas12a and HRP enabled the colorimetric detection of MC-LR in the range 0.01 to 50 ng·mL−1 with a limit of detection (LOD) of 4.53 pg·mL−1. The practicability of the developed colorimetric method was demonstrated by detecting different levels of MC-LR in spiked real water samples. The recoveries ranged from 86.2 to 118.5% and the relative standard deviation (RSD) was 8.4 to 17.6%. This work provides new inspiration for the construction of effective signal amplification platforms and demonstrates a simple and user-friendly colorimetric method for determination of trace MC-LR.Graphical

Glycerol Flow through a Shielded Coil Induces Aggregation and Activity Enhancement of Horseradish Peroxidase

Glycerol has found its applications as a heat-transfer fluid in heat exchangers, and as a component of functional liquids in biosensor analysis. Flowing non-aqueous fluids are known to be able to induce electromagnetic fields due to the triboelectric effect. These triboelectrically generated electromagnetic fields can affect biological macromolecules. Horseradish peroxidase (HRP) is widely employed as a convenient model object for studying how external electric, magnetic, and electromagnetic fields affect enzymes. Herein, we have studied whether the flow of glycerol in a ground-shielded cylindrical coil affects the HRP enzyme incubated at a 2 cm distance near the coil’s side. Atomic force microscopy (AFM) has been employed in order to study the effect of glycerol flow on HRP at the nanoscale. An increased aggregation of HRP on mica has been observed after the incubation of the enzyme near the coil. Moreover, the enzymatic activity of HRP has also been affected. The results reported that their application can be found in biotechnology, food technology and life sciences applications, considering the development of triboelectric generators, enzyme-based biosensors and bioreactors with surface-immobilized enzymes. Our work can also be of interest for scientists studying triboelectric phenomena, representing one more step toward understanding the mechanism of the indirect action of the flow of a dielectric liquid on biological macromolecules.

An S-scheme CeO2/ foveolate g-C3N4 composite with horseradish peroxidase activity for photo-enzyme synergistic catalytic degradation of phenanthrene

Nanozyme technology is a relatively new technology that has shown promising applications in the removal of water pollutants. This study utilized a CeO2/foveolate g-C3N4 (CeO2/FCN) composite with S-scheme heterojunction to achieve efficient removal of Phenanthrene (PHE) through a combination of photocatalytic and enzymatic reactions. The S-scheme heterojunction was effective in inhibiting the recombination of photogenerated electron hole pairs, which promoted the generation of·OH and·O2-. Additionally, activated H2O2 enables Ce to achieve redox cycle reaction and generate more·OH for catalytic degradation, effectively improving the concentration of active species and promoting the photo-enzyme synergistic catalytic performance. The experimental results showed that PHE degradation efficiency of CeO2/FCN composite could reach 96.6% in 60 min. Furthermore, the photo-enzyme synergistic catalytic mechanism of CeO2/FCN composite and the possible intermediates generated in the degradation process were studied. The highly toxic PHE was converted into non-toxic green degradation products, providing an effective method for the photo-enzyme synergistic catalytic degradation of PAHs.

Kinetics and mechanism of inhibition of horseradish peroxidase activity by N-phosphonomethyl-glycine

The inhibition of horseradish peroxidase activity by N-phosphonomethyl-glycine, an active ingredient in the most extensively used glyphosate-based herbicide (Roundup) in agricultural applications worldwide, was investigated. It has been shown that N-phosphonomethyl-glycine inhibits the activity of peroxidase from horseradish roots. This inhibition is of a competitive nature, the value of Ki = 0.1004 mM. The Michaelis constant (Km = 0.1776 mM) and herbicide concentration (I50 of Roundup = 0.302 mM) were determined. The results demonstrate that N-phosphonomethyl-glycine can suppress the activity of the antioxidant system, and in warm-blooded organisms it may cause the accumulation of free radicals, thereby contributing to expanding knowledge of biological targets Roundup is applied to.

The Kinetics and Mechanism of Inhibition of Horseradish Peroxidase Activity by N-(Phosphonomethyl)glycine

The Kinetics and Mechanism of Inhibition of Horseradish Peroxidase Activity by N-(Phosphonomethyl)glycine

Antibodies from serum and CSF of multiple sclerosis patients bind to oligodendroglial and neuronal cell-lines.

Multiple sclerosis is a highly complex and heterogeneous disease. At the onset it often presents as a clinically isolated syndrome. Thereafter relapses are followed by periods of remissions, but eventually, most patients develop secondary progressive multiple sclerosis. It is widely accepted that autoantibodies are important to the pathogenesis of multiple sclerosis, but hitherto it has been difficult to identify the target of such autoantibodies. As an alternative strategy, cell-based methods of detecting autoantibodies have been developed. The objective of this study was to explore differences in the binding of antibodies from sera and CSF of multiple sclerosis patients and controls to oligodendroglial and neuronal cell-lines, related to antibody type, immunoglobulin (IgG/IgM), matrix (serum/CSF) and disease course. The oligodendroglial and neuronal cell-lines were expanded in tissue culture flasks and transferred to 96-well plates at a concentration of 50 000 cells/well followed by fixation and blocking with bovine serum albumin. Sera and CSF samples, from healthy controls and multiple sclerosis patients, were incubated with the fixed cells. Epitope binding of immunoglobulins (IgG and IgM) in sera and CSF was detected using biotinylated anti-human IgM and IgG followed by avidin conjugated to horseradish peroxidase. Horseradish peroxidase activity was detected with 3,3',5,5'-tetramethylbenzidine substrate. Serum from 76 patients and 30 controls as well as CSF from 62 patients and 32 controls were investigated in the study. The binding was similar between clinically isolated syndrome patients and controls, whereas the largest differences were observed between secondary progressive multiple sclerosis patients and controls. Antibodies from multiple sclerosis patients (all disease course combined) bound more to all investigated cell-lines, irrespectively of matrix type, but binding of immunoglobulin G from CSF to human oligodendroglioma cell-line discriminated best between multiple sclerosis patients and controls with a sensitivity of 93% and a specificity of 96%. The cell-based enzyme linked immunosorbent assay (ELISA) was able to discriminate between multiple sclerosis patients and controls with a high degree of accuracy. The disease course was the major determinant for the antibody binding.

Colorimetric Detection of S1 Nuclease Activity using a Hairpin DNA with Split G-Quadruplex

Hairpin DNA containing split G-quadruplex at its terminus displayed tagging position-dependent horseradish peroxidase activity. In general, the hairpin DNA coupled with one GGG repeat at its 5’-terminus and three GGG repeats at its 3′-terminus demonstrated strong catalytic activity, while slight activity was observed after exchanging the position of split G-quadruplex sequence. Inspired by these observations, a novel colorimetric biosensor for nuclease activity assay was developed by selecting S1 nuclease as the model. Specifically, S1 nuclease cleaves the split G-quadruplex into mono- or oligonucleotide fragments, thereby suppressing the catalytic activity, accompanied by a color change from blue to colorless. Under optimum conditions, S1 nuclease was determined with a linear range from 1.60 to 17.6 U/mL and a detection limit of 1.458 U/mL. The constructed method was simple and did not require complex sequence design, expensive instrumentation, and probe labeling. Most importantly, this work facilitates a better understanding of the catalytic mechanism of the DNAzyme and provides an example of the practical application of G-quadruplex-based DNAzyme.

Sol-Gel Films Doped with Enzymes and Banana Crude Extract as Sensing Materials for Spectrophotometric Determination.

Chromogenic enzymatic reactions are very convenient for the determination of various biochemically active compounds. Sol-gel films are a promising platform for biosensor development. The creation of sol-gel films with immobilized enzymes deserves attention as an effective way to create optical biosensors. In the present work, the conditions are selected to obtain sol-gel films doped with horseradish peroxidase (HRP), mushroom tyrosinase (MT) and crude banana extract (BE), inside the polystyrene spectrophotometric cuvettes. Two procedures are proposed: the use of tetraethoxysilane-phenyltriethoxysilane (TEOS-PhTEOS) mixture as precursor, as well as the use of silicon polyethylene glycol (SPG).In both types of films, the enzymatic activity of HRP, MT, and BE is preserved. Based on the kinetics study of enzymatic reactions catalyzed by sol-gel films doped with HRP, MT, and BE, we found that encapsulation in the TEOS-PhTEOS films affects the enzymatic activity to a lesser extent compared to encapsulation in SPG films. Immobilization affects BE significantly less than MT and HRP. The Michaelis constant for BE encapsulated in TEOS-PhTEOS films almost does not differ from the Michaelis constant for a non-immobilized BE. The proposed sol-gel films allow determining hydrogen peroxide in the range of 0.2-3.5 mM (HRP containing film in the presence of TMB), and caffeic acid in the ranges of 0.5-10.0 mM and 2.0-10.0 mM (MT- and BE-containing films, respectively). BE-containing films have been used to determine the total polyphenol content of coffee in caffeic acid equivalents; the results of the analysis are in good agreement with the results obtained using an independent method of determination. These films are highly stable and can be stored without the loss of activity for 2 months at +4 °C and 2 weeks at +25 °C.

Stepwise Optimization of Inducible Expression System for the Functional Secretion of Horseradish Peroxidase in Saccharomyces cerevisiae.

Horseradish peroxidase (HRP) is a plant-derived glycoprotein that can be developed as a food additive to cross-link proteins or biopolymers. Although Saccharomyces cerevisiae has advantages in the production of food-grade HRP, the low expressional level and inefficient secretion hindered its application values. After comparing the effects of constitutive and inducible expression on cell growth, the strength of HRP expression was roughly tuned by replacing core regions of the promoter in the GAL80-knockout strain and further finely tuned by terminator screening. Additionally, the most suitable signal peptide was selected, and the pre-peptide with pro-peptides was modified to balance the transport of HRP in the endoplasmic reticulum. The extracellular HRP activity of the best strain reached 13 506 U/L at the fermenter level, 330-fold higher than the previous result of 41 U/L in S. cerevisiae. The strategy can be applied to alleviate the inhibition of cell growth caused by the expression of toxic proteins and improve their secretion.

Nanozymes: Definition, Activity, and Mechanisms.

"Nanozyme" is used to describe various catalysts from immobilized inorganic metal complexes, immobilized enzymes to inorganic nanoparticles. Here, the history of nanozymes is dvescribed in detail, and they can be largely separated into two types. Type 1 nanozymes refer to immobilized catalysts or enzymes on nanomaterials, whichweredominant in the first decade since 2004. Type 2 nanozymes, which rely on the surface catalytic properties of inorganic nanomaterials, are the dominating type in the past decade. The definition of nanozymes is evolving, and a definition based on the same substrates and products as enzymes are able to cover most currently claimed nanozymes, although they may have different mechanisms compared to their enzyme counterparts. A broader definition can inspire application-based research to replace enzymes with nanomaterials for analytical, environmental, and biomedical applications. Comparison with enzymes also requires a clear definition of a nanozyme unit. Four ways of defining a nanozyme unit are described, with iron oxide and horseradish peroxidase activity comparison as examples in each definition. Growing work is devoted to understanding thecatalytic mechanism of nanozymes, which provides a basis for further rational engineering of active sites. Finally, future perspective of the nanozyme field is discussed.