Horseradish peroxidase‐based hybrid nanoflowers with enhanced catalytical activities for polymerization reactions of phenol derivatives

Catalytic activity and stability of HRP‐Cu²⁺ hybrid nanoflowers (hCu‐NFs) in the polymerization reactions of phenol derivatives was investigated. It was observed that the catalytic activity and stability of hybrid nanoflowers on the polymerization of the phenol derivatives was considerably higher compared to free Horseradish peroxidase (HRP) enzyme. The hCu‐NFs effectively polymerized phenolic compounds as a novel nanobiocatalyst and led to polymers having quite high yields, molecular weights, and thermal stabilities compared to free HRP enzyme. The hCu‐NFs provide substantial repeated use and showed some degree of catalytic activity even after fourth cycle experiment in the polymerization reactions.     

Stabilizing and suicide-peroxide protecting effect of Ni2+ on horseradish peroxidase

The present research discusses the structure stabilizing and protecting effects of Ni²⁺ against suicide-peroxide inactivation of horseradish peroxidase (HRP). Suicide inactivation of HRP by hydrogen peroxide (3 mM) was monitored by measuring change in the absorbance of the colored product (tetraguaiacol) of the catalytic reaction cycle at 470 nm. Progress curves of the catalytic reaction cycle were obtained at 27 °C, phosphate buffer (5 mM), pH 7.0. The corresponding kinetic parameters (e.g., initial enzyme activity (α_o) and the apparent rate constant (k_i) of suicide inactivation of HRP by peroxide) were evaluated using a kinetic equation derived in this study. Comparative activatory and inhibitory effects of Ni²⁺ on the kinetics of suicide-peroxide inactivation of HRP are discussed.     

Determination of Lead Employing Simple Flow Injection AAS with Monolithic Alginate-Polyurethane Composite Packed In-Valve Column

A simple flow injection FlameAAS for lead determination with an alginate-polyurethane composite (ALG-PUC) monolithic in-valve column has been developed. The ALG-PUC monolithic rod was prepared by mixing methylene diphenyl diisocyanate with polyol and sodium alginate with the ratio of 2:1:1 by weight for a 5 min polymerization reaction. It was then put into a column (0.8 cm i.d × 11 cm length) situated in a switching valve for the FI set up. A single standard calibration could be obtained by plotting the loaded µg Pb²⁺ vs. FI response (absorbances). The loaded µg Pb²⁺ is calculated: μg Pb²⁺ = FR_load × LT × C_Pb²⁺, where the FR load is the flow rate of the loading analyte solution (mL min⁻¹), LT is the loading time (min), and C_Pb²⁺ is the Pb²⁺ concentration (µg mL⁻¹). A linear calibration equation was obtained: FI response (absorbances) = 0.0018 [µg Pb²⁺] + 0.0032, R² = 0.9927 for 1–150 µg Pb²⁺, and RSD of less than 20% was also obtained. Application of the developed procedure has been demonstrated in real samples.     

Discovery of New Coumarin-Based Lead with Potential Anticancer,CDK4 Inhibition and Selective Radiotheranostic Effect: Synthesis, 2D & 3D QSAR,Molecular Dynamics,In Vitro Cytotoxicity,Radioiodination, and Biodistribution Studies

Novel 6-bromo-coumarin-ethylidene-hydrazonyl-thiazolyl and 6-bromo-coumarin-thiazolyl-based derivatives were synthesized. A quantitative structure activity relationship (QSAR) model with high predictive power r² = 0.92, and RMSE = 0.44 predicted five compounds; 2b, 3b, 5a, 9a and 9i to have potential anticancer activities. Compound 2b achieved the best ΔG of –15.34 kcal/mol with an affinity of 40.05 pki. In a molecular dynamic study 2b showed an equilibrium at 0.8 Å after 3.5 ns, while flavopiridol did so at 0.5 Å after the same time (3.5 ns). 2b showed an IC₅₀ of 0.0136 µM, 0.015 µM, and 0.054 µM against MCF-7, A-549, and CHO-K1 cell lines, respectively. The CDK4 enzyme assay revealed the significant CDK4 inhibitory activity of compound 2b with IC₅₀ of 0.036 µM. The selectivity of the newly discovered lead compound 2b toward localization in tumor cells was confirmed by a radioiodination biological assay that was done via electrophilic substitution reaction utilizing the oxidative effect of chloramine-t. ¹³¹I-2b showed good in vitro stability up to 4 h. In solid tumor bearing mice, the values of tumor uptake reached a height of 5.97 ± 0.82%ID/g at 60 min p.i. ¹³¹I-2b can be considered as a selective radiotheranostic agent for solid tumors with promising anticancer activity.     

Effect of Physiological Concentrations of Vitamin C on the Inhibitation of Hydroxyl Radical Induced Light Emission from Fe2+-EGTA-H2O2 and Fe3+-EGTA-H2O2 Systems In Vitro

Ascorbic acid (AA) has antioxidant properties. However, in the presence of Fe²⁺/Fe³⁺ ions and H₂O₂, it may behave as a pro-oxidant by accelerating and enhancing the formation of hydroxyl radicals (^•OH). Therefore, in this study we evaluated the effect of AA at concentrations of 1 to 200 µmol/L on ^•OH-induced light emission (at a pH of 7.4 and temperature of 37 °C) from 92.6 µmol/L Fe²⁺—185.2 µmol/L EGTA (ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid)—2.6 mmol/L H₂O₂, and 92.6 µmol/L Fe³⁺—185.2 µmol/L EGTA—2.6 mmol/L H₂O₂ systems. Dehydroascorbic acid (DHAA) at the same range of concentrations served as the reference compound. Light emission was measured with multitube luminometer (AutoLumat Plus LB 953) for 120 s after automatic injection of H₂O₂. AA at concentrations of 1 to 50 µmol/L and of 1 to 75 µmol/L completely inhibited light emission from Fe²⁺-EGTA-H₂O₂ and Fe³⁺-EGTA-H₂O₂, respectively. Concentrations of 100 and 200 µmol/L did not affect chemiluminescence of Fe³⁺-EGTA-H₂O₂ but tended to increase light emission from Fe²⁺-EGTA-H₂O₂. DHAA at concentrations of 1 to 100 µmol/L had no effect on chemiluminescence of both systems. These results indicate that AA at physiological concentrations exhibits strong antioxidant activity in the presence of chelated iron and H₂O₂.     

Copper doped ceria porous nanostructures towards a highly efficient bifunctional catalyst for carbon monoxide and nitric oxide elimination

Copper doped ceria porous nanostructures with a tunable BET surface area were prepared using an efficient and general metal–organic-framework-driven, self-template route. The XRD, SEM and TEM results indicate that Cu²⁺ was successfully substituted into the CeO₂ lattice and well dispersed in the CeO₂:Cu²⁺ nanocrystals. The CeO₂:Cu²⁺ nanocrystals exhibit a superior bifunctional catalytic performance for CO oxidation and selective catalytic reduction of NO. Interestingly, CO oxidation reactivity over the CeO₂:Cu²⁺ nanocrystals was found to be dependent on the Cu²⁺ dopants and BET surface area. By tuning the content of Cu²⁺ and BET surface area through choosing different organic ligands, the 100% conversion temperature of CO over CeO₂:Cu²⁺ nanocrystals obtained from thermolysis of CeCu–BPDC nanocrystals can be decreased to 110 °C. The porous nanomaterials show a high CO conversion rate without any loss in activity even after five cycles. Furthermore, the activity of the catalysts for NO reduction increased with the increase of BET surface, which is in accordance with the results of CO oxidation.     

Enzyme repurposing of a hydrolase as an emergent peroxidase upon metal binding

As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase 6-PGLac bearing a putative metal binding site leads to the emergence of peroxidase activity (k_cat 7.8 × 10^–2 s^–1, K_M 1.1 × 10^–5 M). Both X-ray crystallographic and EPR data of the copper-loaded enzyme Cu·6-PGLac reveal a bis-histidine coordination site, located within a shallow binding pocket capable of accommodating the o-dianisidine substrate.     

Facile Synthesis of Platinum–Cerium(IV) Oxide Hybrids Arched on Reduced Graphene Oxide Catalyst in Reverse Micelles with High Activity and Durability for Hydrolysis of Ammonia Borane

Highly dispersed Pt‐CeO₂ hybrids arched on reduced graphene oxide (Pt‐CeO₂/rGO) were facilely synthesized by a combination of the reverse micelle technique and a redox reaction without any additional reductant or surfactant. Under a N₂ atmosphere, the redox reaction between Ce³⁺ and Pt²⁺ occurs automatically in alkaline solution, which results in the formation of Pt‐CeO₂/rGO nanocomposites (NCs). The as‐synthesized Pt‐CeO₂/rGO NCs exhibit superior catalytic performance relative to that shown by the free Pt nanoparticles, Pt/rGO, Pt‐CeO₂ hybrid, and the physical mixture of Pt‐CeO₂ and rGO; furthermore, the nanocomposites show significantly better activity than the commercial Pt/C catalyst toward the hydrolysis of ammonia borane (NH₃BH₃) at room temperature. Moreover, the Pt‐CeO₂/rGO NCs have remarkable stability, and 92 % of their initial catalytic activity is preserved even after 10 runs. The excellent activity of the Pt‐CeO₂/rGO NCs can be attributed not only to the synergistic structure but also to the electronic effects of the Pt‐CeO₂/rGO NCs among Pt, CeO₂, and rGO.     

Synthesis and characterization of biodegradable palm palmitic acid based bioplastic

This study involves the quantitative analysis of high free fatty acid crude palm oil, the separation of palmitic acid and synthesis of palm palmitic acid-based bioplastic. Synthesis of dimethyl 2-tetradecylmalonate (DMTDM) using methyl palmitate (MP) with sodium hydride (NaH) in the presence of reactive solvent of dimethyl carbonate (DMC) was carried out. The reaction conditions comprise at a mole ratio of MP: DMC: NaH: dimethylformamide (DMF) (0.1:2:0.25:1) at 60 °C for 14 h with 88.3 ± 1.4% yield. FTIR spectra of DMTDM showed the ester carbonyl group at 1740 cm^–1. The polymerization of DMTDM with 1,6-hexandiol or 1,12-dodecandiol was carried out using titanium (IV) isopropoxide Ti(OiPr)₄ as the catalyst and reaction time of 24 h. The results showed that the poly(dodecyl 2-tetradecylmalonte) (PDTDM) exhibited good thermal properties compared to poly(hexyl 2-tetradecylmalonte) (PHTDM). The increase of the chain length of diol in PDTDM improved the thermal properties of polyester with glass transition, T_g of 13 ºC and melting point of 51 ºC with a molecular weight of 12508 Da and polydispersity index (PDI) of 1.4. In general, the synthetic polyesters can be used as internalplasticizer in bio-based industry.     

Biomimetic versus enzymatic high-potential electrocatalytic reduction of hydrogen peroxide on a functionalized carbon nanotube electrode

We report the non-covalent functionalization of a multi-walled carbon nanotube (MWCNT) electrode with a biomimetic model of the horseradish peroxidase (HRP) active site. By modifying the MWCNT electrode surface with imidazole-modified polypyrrole, a new biomimetic complex of HRP was synthesized on the MWCNT sidewalls via the coordination of imidazole (Im) to the metal centre of iron protoporphyrin IX, affording (Im)(PP)Fe^III. Compared to the pi-stacking of non-coordinated (PP)Fe^III on a MWCNT electrode, the (Im)(PP)Fe^III-modified MWCNT electrode exhibits higher electrocatalytic activity with an I _max = 0.52 mA cm^–2 for the reduction of H₂O₂, accompanied by a high onset potential of 0.43 V vs. Ag/AgCl. The performances of these novel surface-confined HRP mimics were compared to those of a MWCNT electrode modified by HRP. Although the enzyme electrode displays a higher electrocatalytic activity towards H₂O₂ reduction, the (Im)(PP)Fe^III-modified MWCNT electrode exhibits a markedly higher operational stability, retaining 63% of its initial activity after one month.     

Effect of proton donors on direct electron transfer in the system gold electrode–horseradish peroxidase

The effect of proton donors (PD) on the direct electron transfer (ET) reaction between polycrystalline Au electrodes and horseradish peroxidase (HRP) was investigated. HRP was immobilised directly on the bare Au surface. The pH of the contacting solution was varied at a constant ionic strength and the following different PDs were used as additives: H₃O⁺, NH₄⁺, [La(H₂O)]³⁺, [Y(H₂O)]³⁺, [Lu(H₂O)]³⁺. The kinetics of the bioelectrocatalytic reduction of H₂O₂ catalysed by HRP was studied with linear sweep voltammetry (LSV) in the potential range from 700 to −100 mV vs. SCE as well as amperometrically at −50 mV vs. Ag|AgCl with the HRP-modified Au electrodes placed in a wall-jet flow through electrochemical cell. An increase of [H₃O⁺] results in an enhancement of the current of the bioelectroreduction of H₂O₂ due to a more facilitated direct ET between Au and the enzyme over the potential range involved. It is shown that at high overvoltages (E<0.4 V) the PDs do not affect the rate of the enzymatic reduction of H₂O₂ but rather increase significantly the rate of direct ET between Au and HRP and the efficiency of acting as a PD is strongly correlated with their PD properties. The dependence of the apparent heterogeneous rate constant of direct ET, k_s, on [H₃O⁺] makes it possible to suggest that the reaction mechanism involves the participation of a proton in the elementary step of the charge transfer.     

Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (K_m = 26.06 mg·mL⁻¹ and V_max = 5.647 U·mg⁻¹). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.     

A novel mimetic peroxidase catalyst by using magnetite-containing silica nanoparticles as carriers

A method for coating magnetite and mimetic enzyme (hemin) with amorphous silica to form a novel mimetic peroxidase (magnetite–hemin/SiO₂) has been developed by combining reverse microemulsion and the modified Stöber method. The magnetic silica nanoparticle supported hemin has a long-term stability toward temperature and good reusability. They can be easily separated from the reaction solution by using an external magnetic field and reused directly for next round of reaction. The peroxidase activity of the magnetite–hemin/SiO₂ was studied based on its catalytic effect on the reaction of p-hydroxyphenylacetic acid and H₂O₂. The results indicated that the catalytic activity of the new mimetic enzyme catalyst is higher than that of the free hemin. The possibility of its application was proven by the determination of H₂O₂, with the detection limits of 7.3 nmol L^?1 H₂O₂.     

Microperoxidase-11/NH2-FSM16 as a H2O2-resistant heterogeneous nanobiocatalyst: a suicide-inactivation study

The catalytic activity of heme peptides is an area of intense investigation. They are utilized for exploring the fine details of structural and functional properties of an active site, and to create minimized and industrial catalysts. The peroxidase activity and kinetics of suicide-inactivation of microperoxidase-11/FSM16 as a heterogeneous nanobiocatalyst in oxidation reaction of guaiacol were studied in the presence of high concentration of hydrogen peroxide (2?mM), as its natural suicide-substrate. The substrate concentration was first-order in relation to aromatic substrate (AH), and the ratio of suicide-substrate (H₂O₂) was kept much higher than the benign substrate (guaiacol). The results of kinetic analysis confirmed a similar mechanism for suicide-peroxide inactivation of horseradish peroxidase (HRP), microperoxidase (MP-11) and MP-11/NH₂-FSM16. Inactivation kinetic parameters, including intact activity of MP-11/NH₂-FSM16, ??_i, and the apparent inactivation rate constant (k _i) were obtained as 0.229?±?0.009?min^?1 and 0.651?±?0.041?min^?1 at [H₂O₂]?=?2.0?mM, respectively, in 5.0?mM phosphate buffer solution (PBS; pH 7.0) at 27?°C. Our results indicated that covalent immobilization of microperoxidase onto NH₂-FSM16 protected the heme group against peroxide inactivation resulting in generation of an efficient peroxide-resistant heterogeneous nanobiocatalyst.     

Investigation on lipase-catalyzed solution polymerization of cyclic carbonate

In this study, 26-membered macrocyclic carbonate, cyclobis(decamethylene carbonate) [(DMC)₂] was attempted to undergo ring-opening polymerization by lipase catalysis in toluene. Novozym-435 exhibited even higher catalytic activity towards (DMC)₂ polymerization compared with SnOCt₂ while high molecular weight (M_n) of 5.4 × 10⁴ and yield of 99% was still achieved at ultra-low enzyme/substrate (E/S) weight ratio of 1/200. ¹H NMR spectra demonstrated the existence of terminal hydroxyl group. Solid phase polymerization in the absence of toluene unexpectedly took place at the temperature lower than (DMC)₂’s melting point of 110 °C. Compared with solvent-free case, the addition of toluene solvent resulted in marked increase in reaction rate. As to the polymerization during 48 h with the E/S weight ratio of 1/100, a region existed at around toluene/carbonate (vol/wt, ml/g) ratio of 1∼2 where the polymerizations gave optimal results in terms of both higher molecular weight and monomer conversion. It was found that much higher molecular weight polymers may be obtained by decreasing enzyme concentrations. Plots of ln{[M]₀:[M]_t} versus reaction time were in linear agreement, indicating no chain termination, and monomer consumption follows a first-order rate law. The Novozym-435 catalyzed polymerization of (DMC)₂ in toluene presented pseudo-living characteristic. Compared with 6-membered trimethylene carbonate, much lower reaction activity of large-sized (DMC)₂ is observed, which is opposite to the result concerning the enzymatic polymerization of lactones with different ring-size.     

Characterization of the CYP3A4 Enzyme Inhibition Potential of Selected Flavonoids

Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor: IC₅₀ = 2.5 ± 0.6 µM, K_i = 2.4 ± 1.0 µM, k_inact = 0.07 ± 0.01 min⁻¹, k_inact/K_i = 0.03 min⁻¹ µM⁻¹. Chrysin caused the highest reduction of heme (94.5 ± 0.5% residual concentration). None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.     

An H2O2 Biosensor Based on Immobilization of Horseradish Peroxidase Labeled Nano‐Au in Silica Sol‐Gel/Alginate Composite Film

Abstract

A novel approach to assemble an H₂O₂ amperometric biosensor was introduced. The biosensor was constructed by entrapping horseradish peroxidase (HRP) labeled nano‐scaled particulate gold (nano‐Au) (HRP‐nano‐Au electrostatic composite) in a new silica sol‐gel/alginate hybrid film using glassy carbon electrode as based electrode. This suggested strategy fully merged the merits of sol‐gel derived inorganic‐organic composite film and the nano‐Au intermediator. The silica sol‐gel/alginate hybrid material can improve the properties of conventional sol‐gel material and effectively prevent cracking of film. The entrapment of HRP in the form of HRP‐nano‐Au can not only factually prevent the leaking of enzyme out of the film but also provide a favorable microenvironment for HRP. With hydroquinone as an electron mediator, the proposed HRP electrode exhibited good catalytic activity for the reduction of H₂O₂. The parameters affecting both the qualities of sol‐gel/alginate hybrid film and the biosensor response were optimized. The biosensor exhibited high sensitivity of 0.40 Al mol^?1 cm^?2 for H₂O₂ over a wide linear range of concentration from 1.22×10^?5 to 1.46×10^?3 mol L^?1, rapid response of <5 s and a detection limit of 0.61×10^?6 mol L^?1. The enzyme electrode has remarkable stability and retained 86% of its initial activity after 45 days of storage in 0.1 mol L^?1 Tris‐HCl buffer solutions at pH 7.     

Non-Toxic Dimeric Peptides Derived from the Bothropstoxin-I Are Potent SARS-CoV-2 and Papain-like Protease Inhibitors

The COVID-19 outbreak has rapidly spread on a global scale, affecting the economy and public health systems throughout the world. In recent years, peptide-based therapeutics have been widely studied and developed to treat infectious diseases, including viral infections. Herein, the antiviral effects of the lysine linked dimer des-Cys¹¹, Lys¹²,Lys¹³-(pBthTX-I)₂K ((pBthTX-I)₂K)) and derivatives against SARS-CoV-2 are reported. The lead peptide (pBthTX-I)₂K and derivatives showed attractive inhibitory activities against SARS-CoV-2 (EC₅₀ = 28–65 µM) and mostly low cytotoxic effect (CC₅₀ > 100 µM). To shed light on the mechanism of action underlying the peptides’ antiviral activity, the Main Protease (M^pro) and Papain-Like protease (PL^pro) inhibitory activities of the peptides were assessed. The synthetic peptides showed PL^pro inhibition potencies (IC₅₀s = 1.0–3.5 µM) and binding affinities (K_d = 0.9–7 µM) at the low micromolar range but poor inhibitory activity against M^pro (IC₅₀ > 10 µM). The modeled binding mode of a representative peptide of the series indicated that the compound blocked the entry of the PL^pro substrate toward the protease catalytic cleft. Our findings indicated that non-toxic dimeric peptides derived from the Bothropstoxin-I have attractive cellular and enzymatic inhibitory activities, thereby suggesting that they are promising prototypes for the discovery and development of new drugs against SARS-CoV-2 infection.     

Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in sol–gel-derived tin oxide/gelatin composite films

Horseradish peroxidase (HRP) was immobilized into a new type of sol–gel-derived nano-sized tin oxide/gelatin composite film (SnO₂ composite film) using a sol–gel film/enzyme/sol–gel film “sandwich” configuration. Direct electrochemistry and electrocatalysis of HRP incorporated into the composite films were investigated. HRP/SnO₂ composite film exhibited a pair of stable and quasi-reversible cyclic voltammetric peaks for the HRP Fe(III)/HRP Fe(II) redox couple with a formal potential of about −0.25 V (vs. SCE) in a pH 6.0 phosphate buffer solution. The electron transfer between the enzyme and the underlying electrode was greatly enhanced in the microenvironment with nano-SnO₂ particles and nanoporous structures. Morphologies and microstructures of the composite films and HRP/composite films were characterized with TEM, AFM. Electrochemical impedance spectroscopy (EIS) was also used to feature the HRP incorporated into composite films. FTIR and UV–Vis spectroscopy demonstrated that HRP in the composite film could retain its native secondary structure. With the advantages of organic–inorganic hybrid materials, the HRP/SnO₂ composite film modified electrode displayed good stability and electrocatalytic activity to the reduction of H₂O₂, The apparent Michaelis-Menten constant was estimated to be 0.345 mM, indicating a high affinity of HRP entrapped into the composite film toward H₂O₂.