Kinetics of phenol oxidation by peroxidase

Studies of the kinetic behavior of horseradish peroxidase (HRP) at pH 8 and at room temperature indicate that the reaction of phenol with H₂O₂ catalyzed by HRP exhibits normal Michaelis-Menten saturation kinetics. An irreversible reaction mechanism for the steady-state kinetics of HRP, which is consistent with the experimental data, is considered. The second-order rate constants for the reactions of HRP with H₂O₂ and compound II with phenol are 4.14 × 10⁵ M^-1s^-1 and 5.54 × 10⁴M^-1s^-1, respectively.     

Biocatalytic System Made of 3D Chitin,Silica Nanopowder and Horseradish Peroxidase for the Removal of 17α-Ethinylestradiol: Determination of Process Efficiency and Degradation Mechanism

The occurrence of 17α-ethinylestradiol (EE2) in the environment and its removal have drawn special attention from the scientific community in recent years, due to its hazardous effects on human and wildlife around the world. Therefore, the aim of this study was to produce an efficient enzymatic system for the removal of EE2 from aqueous solutions. For the first time, commercial silica nanopowder and 3D fibrous chitinous scaffolds from Aplysina fistularis marine sponge were used as supports for horseradish peroxidase (HRP) immobilization. The effect of several process parameters onto the removal mechanism of EE2 by enzymatic conversion and adsorption of EE2 were investigated here, including system type, pH, temperature and concentrations of H₂O₂ and EE2. It was possible to fully remove EE2 from aqueous solutions using system SiO₂(HRP)–chitin(HRP) over a wide investigated pH range (5–9) and temperature ranges (4–45 °C). Moreover, the most suitable process conditions have been determined at pH 7, temperature 25 °C and H₂O₂ and EE2 concentrations equaling 2 mM and 1 mg/L, respectively. As determined, it was possible to reuse the nanoSiO₂(HRP)–chitin(HRP) system to obtain even 55% EE2 degradation efficiency after five consecutive catalytic cycles.     

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.     

Peroxidase-dependent fluorescence decrease of carboxyfluorescein and its enhancement by liposome encapsulation

The fluorescence intensity of 5(6)-carboxyfluorescein (CF) was decreased by addition of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The reaction inside a liposome containing CF and HRP on addition of H₂O₂ was measured fluorometrically after destruction of the liposome with Triton X-100. The reaction efficiency was higher than that without liposome because CF and HRP were concentrated inside the latter. The determination of H₂O₂ can be performed with a smaller amount of HRP by liposome encapsulation.     

Dual Enzyme‐Triggered In Situ Crosslinkable Gelatin Hydrogels for Artificial Cellular Microenvironments

Horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂)‐mediated crosslinking reaction has become an attractive method to create in situ forming hydrogels. While the crosslinking system has been widely utilized, there are certain issues require improvement to extend their biomedical applications, including creation of stiff hydrogels without compromising cytocompatibility due to initially high concentrations of H₂O₂. A gelatin‐based hydrogels formed through a dual enzyme‐mediated crosslinking reaction using HRP and glucose oxidase (GOx) as an H₂O₂‐generating enzyme to gradually supply a radical source in HRP‐mediated crosslinking reaction is reported. The physicochemical properties can be controlled by varying enzyme concentrations. Furthermore the hydrogel matrices provide 3D microenvironments for supporting the growth and spreading of human dermal fibroblasts with minimized cytotoxicity, despite the cells being encapsulated within stiff hydrogels. These hydrogels formed with HRP/GOx have great potential as artificial microenvironments for a wide range of biomedical applications.

     

Composite Multienzyme Amperometric Biosensors for an Improved Detection of Phenolic Compounds

A biosensor design, in which glucose oxidase and peroxidase are coimmobilized by simple physical inclusion into the bulk of graphite‐Teflon pellets, is reported for the detection of phenolic compounds. This design allows the “in situ” generation of the H₂O₂ needed for the enzyme reaction with the phenolic compounds, which avoids several problems detected in the performance of single peroxidase biosensors as a consequence of the presence of a high H₂O₂ concentration. So, a much lower surface fouling was found at the GOD‐HRP biosensor in comparison with a graphite‐Teflon‐HRP electrode, suggesting that the controlled generation of H₂O₂ makes more difficult the formation of polymers from the enzyme reaction products. The construction of trienzyme biosensors, in which GOD, HRP and tyrosinase were coimmobilized into the graphite‐Teflon matrix is also reported, and their performance was compared with that of GOD‐HRP bienzyme electrodes. The practical applicability of the composite multienzyme amperometric biosensors was evaluated by the estimation of the phenolic compounds content in waste waters from a refinery, and the results were compared with those obtained by using a colorimetric official method based on the reaction with 4‐aminoantipyrine.     

磁性固定化酶处理含酚废水的研究

研究了磁性壳聚糖微球(磁性CS-M)及壳聚糖微球(CS-M)固定化辣根过氧化物酶(HRP)对模拟含酚废水的催化效果,探讨了反应时间、酶活力、H2O2浓度及酚浓度对反应的影响。对均相与非均相酶处理酚效果进行比较,显示固定化酶处理含酚废水具有很大的优越性,且磁性酶的效果最佳。     

Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

Immunoassay is one of the biochemical analytical techniques using the specific antigen antibody com-plexation for analytical purposes. It has extensive ap-plication in clinical diagnostics, prevention and cure of diseases, and virus diagnostics. The presentation and progress of immunoassay methodology are one of the greatest achievements of bioanalytical chemistry. It is estimated that several-hundred millions of immuno-analytical determinations are carried out every year all over the world. E…     

Hierarchical Hybrid Peroxidase Catalysts for Remediation of Phenol Wastewater

We report a new family of hierarchical hybrid catalysts comprised of horseradish peroxidase (HRP)–magnetic nanoparticles for advanced oxidation processes and demonstrate their utility in the removal of phenol from water. The immobilized HRP catalyzes the oxidation of phenols in the presence of H₂O₂, producing free radicals. The phenoxy radicals react with each other in a non‐enzymatic process to form polymers, which can be removed by precipitation with salts or condensation. The hybrid peroxidase catalysts exhibit three times higher activity than free HRP and are able to remove three times more phenol from water compared to free HRP under similar conditions. In addition, the hybrid catalysts reduce substrate inhibition and limit inactivation from reaction products, which are common problems with free or conventionally immobilized enzymes. Reusability is improved when the HRP–magnetic nanoparticle hybrids are supported on micron‐scale magnetic particles, and can be retained with a specially designed magnetically driven reactor. The performance of the hybrid catalysts makes them attractive for several industrial and environmental applications and their development might pave the way for practical applications by eliminating most of the limitations that have prevented the use of free or conventionally immobilized enzymes.     

Determination of glucose using a coupled-enzymatic reaction with new fluoride selective optical sensing polymeric film coated in microtiter plate wells

The determination of glucose in beverages is demonstrated using newly developed fluoride selective optical sensing polymeric film that contains aluminum (III) octaethylporphyrin (Al[OEP]) ionophore and the chromoionophore ETH7075 cast at the bottom of wells of a 96-well polypropylene microtiter plate. The method uses a dual enzymatic reaction involving glucose oxidase enzyme (GOD) and horseradish peroxidase (HRP), along with an organofluoro-substrate (4-fluorophenol) as the source of fluoride ions. The concentration of fluoride ions after enzymatic reaction is directly proportional to the glucose level in the sample. The method has a detection limit of 0.8 mmol L⁻¹, a linear range of 0.9-40 mmol L⁻¹ and a sensitivity of 0.125 absorbance/decade of glucose concentration. Glucose levels in several beverage samples determined using the proposed method correlate well with a reference spectrophotometric enzyme method based on detection of hydrogen peroxide using bromopyrogallol red dye (BPR). The new method can also be used to determine H₂O₂ concentrations in the 0.1-50 mmol L⁻¹ range using a single enzymatic reaction involving H₂O₂ oxidation of 4-fluorophenol catalyzed by HRP. The methodology could potentially be used to detect a wide range of substrates for which selective oxidase enzymes exist (to generate H₂O₂), with the high throughput of simple microtiter plate detection scheme.     

3‐Indoxyl Phosphate as an Electrochemical Substrate for Horseradish Peroxidase

In this work 3‐indoxyl phosphate (3‐IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3‐IP in presence of hydrogen peroxide (H₂O₂) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of ?0.15 V (vs. Ag pseudo‐reference electrode) when a screen‐printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V_max, K_M) for both substrates (3‐IP and H₂O₂) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86×10^?12 and 5.68×10^?12 M, respectively. Thus, 3‐IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.     

Catalysis‐Electrochemical Detection of Horseradish Peroxidase at Zeptomole Level in Capillary Zone Electrophoresis

Capillary zone electrophoresis with catalysis‐electrochemical detection has been developed and applied to determining horseradish peroxidase (HRP) at zeptomole levels. In this method, an on‐line enzyme catalysis reactor with a reaction capillary was designed. Isoenzymes of HRP were separated by capillary zone electrophoresis, and then they catalyzed the enzyme substrate 3,3′,5,5′‐tetramethylbenzide (TMB(Red)) and H₂O₂ in the reaction capillary. The reaction product, TMB(Ox), could be determined using amperometric detection on a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Because of enzyme amplification, a significant amount of TMB(Ox) could be produced for detection. Therefore, the limit of detection (LOD) of HRP is very low. The optimum conditions of the method are 1.5×10^?2 mol/L borate (pH 7.4) for the run buffer, 2×10^?3 mol/L for the concentration of H₂O₂, 2×10^?4 mol/L TMB(Red)+2.0×10^?2 mol/L citrate‐phosphate (pH 5.0) for the substrate solution, 40 cm for the liquid pressure height, 20 kV for the separation voltage, 100 mV for the detection potential. HRP could be measured with a detection limit of 4.8×10^?12 mol/L or 47.5 zmol (S/N=3). The linear range is from 2.40×10^?11 to 2.40×10^?8 mol/L. Using this method, commercial HRP was measured at zeptomole within ten minutes.     

Enhancement of a conducting polymer-based biosensor using carbon nanotube-doped polyaniline

A biosensor with improved performance was developed through the immobilization of horseradish peroxidase (HRP) onto electropolymerized polyaniline (PANI) films doped with carbon nanotubes (CNTs). The effects of electropolymerization cycle and CNT concentration on the response of the biosensor toward H₂O₂ were investigated. It was found that the application of CNTs in the biosensor system could increase the amount and stability of the immobilized enzyme, and greatly enhanced the biosensor response. Compared with the biosensor without CNTs, the proposed biosensor exhibited enhanced stability and approximately eight-fold sensitivity. A linear range from 0.2 to 19 μM for the detection of H₂O₂ was observed for the proposed biosensor, with a detection limit of 68 nM at a signal-to-noise ratio of 3 and a response time of less than 5 s.     

Liposomal Enzyme Nanoreactors Based on Nanoconfinement for Efficient Antitumor Therapy

Enzymatic reactions can consume endogenous nutrients of tumors and produce cytotoxic species and are therefore promising tools for treating malignant tumors. Inspired by nature where enzymes are compartmentalized in membranes to achieve high reaction efficiency and separate biological processes with the environment, we develop liposomal nanoreactors that can perform enzymatic cascade reactions in the aqueous nanoconfinement of liposomes. The nanoreactors effectively inhibited tumor growth in vivo by consuming tumor nutrients (glucose and oxygen) and producing highly cytotoxic hydroxyl radicals (⋅OH). Co-compartmentalization of glucose oxidase (GOx) and horseradish peroxidase (HRP) in liposomes could increase local concentration of the intermediate product hydrogen peroxide (H₂O₂) as well as the acidity due to the generation of gluconic acid by GOx. Both H₂O₂ and acidity accelerate the second-step reaction by HRP, hence improving the overall efficiency of the cascade reaction. The biomimetic compartmentalization of enzymatic tandem reactions in biocompatible liposomes provides a promising direction for developing catalytic nanomedicines in antitumor therapy.     

Kinetics and reaction mechanism of phenol hydroxylation catalyzed by La-Cu<Subscript>4</Subscript>FeAlCO<Subscript>3</Subscript>

The present work synthesizes La-Cu₄FeAICO₃ catalyst under microwave irradiation and characterizes its structure using XRD and IR techniques. The results show that the obtained La-Cu₄FeAICO₃ has a hydrotalcite structure. In the phenol hydroxylation with H₂O₂ catalyzed by La-Cu₄FeAICO₃, the effects of reaction time and phenol/H₂O₂ molar ratio on the phenol hydroxylation, and relationships between the initial hydroxylation rate with concentration of the catalyst, phenol, H₂O₂ and reaction temperature are also investigated in details. It is shown the phenol conversion can reach 50.09% (mol percent) in the phenol hydroxylation catalyzed by La-Cu₄FeAICO₃, under the reaction conditions of the molar ratio of phenol/H₂O₂1/2, the amount ratio of phenol/catalyst 20, reaction temperature 343 K, reaction time 120 min, 10 ml_ distilled water as solvent. Moreover, a kinetic equation of v = k[La-Cu₄FeAlCO₃][C₆H₅OH][H₂O₂]. and the activation energy of E _a=58.37 kJ/mol are obtained according to the kinetic studies. Due to the fact that the HO-Cu⁺-OH species are detected in La-Cu₄FeAICO₃/H₂O₂ system by XPS, the new mechanism about the generation of hydroxyl free radicals in the phenol hydroxylation is proposed, which is supposed that HO-Cu⁺-OH species are transition state in this reaction.     

Monitoring H2O2 on the Surface of Single Cells with Liquid Crystal Elastomer Microspheres

Live‐imaging of signaling molecules released from living cells is a fundamental challenge in life sciences. Herein, we synthesized liquid crystal elastomer microspheres functionalized with horse‐radish peroxidase (LCEM‐HRP), which can be immobilized directly on the cell membrane to monitor real‐time release of H₂O₂ at the single‐cell level. LCEM‐HRP could report H₂O₂ through a concentric‐to‐radial (C‐R) transfiguration, which is due to the deprotonation of LCEM‐HRP and the break of inter or intra‐chain hydrogen bonding in LCEM‐HRP caused by HRP‐catalyzed reduction of H₂O₂. The level of transfiguration of LCEM‐HRP revealed the different amounts of H₂O₂ released from cells. The estimated detection sensitivity was ≈2.2×10^?7 μm for 10 min of detection time. The cell lines and cell–cell heterogeneity was explored from different configurations. LCEM‐HRP presents a new approach for in situ real‐time imaging of H₂O₂ release from living cells and can be the basis for seeking more advanced chemical probes for imaging of various signaling molecules in the cellular microenvironment.     

Kinetic study of acrylamide radical polymerization initiated by the horseradish peroxidase–mediated system

The polymerization kinetics of acrylamide (AAM) in water initiated by a ternary enzymatic system of horseradish peroxidase (HRP)/H₂O₂/acetylacetone (ACAC) was investigated. Conversion–time plots were obtained by dilatometry under different conditions of reaction temperatures and initial concentrations of HRP, ACAC, H₂O₂, and AAM. The results showed that the effect of the initial concentration of ACAC on the inhibition period was significant. The inhibition period decreases with increasing the initial concentration of ACAC. The inhibition period can be even eliminated by the use of a comparatively large amount of ACAC. From the conversion–time plots, the polymerization rate equation was obtained. Some kinetic features were explained on the basis of analysis of the reaction mechanism. © 2012 Wiley Periodicals, Inc. Int J Chem Kinet 44: 475–481, 2012     

Hydroxylation of phenol over ts-2, a titanium silicate molecular sieve

The influence of different process parameters on the hydroxylation of phenol with H₂O₂ over the titanium silicate molecular sieve TS-2 has been investigated. Apart from the primary products vis., p- and o-dihydroxybenzenes, the corresponding quinones are also formed. Higher Ti contents in the catalyst (TS-2) and higher catalyst concentrations lower the formation of the secondary oxidation products. Solvents have an influence on the relative amounts of the two dihydroxybenzenes in the product. H₂O₂ efficiency of up to 74% is obtainable at ∼ 28% phenol conversion to dihydroxybenzenes.     

Preparation of the Cationic Dendrimer-Based Hydrogels for Controlled Heparin Release

We introduce a cationic polyamidoamine (PAMAM) dendrimers and tetronic (Te) based hydrogels in which precursor copolymers were prepared with simple methods. In the synthetic process, tyramine-conjugated tetronic (TTe) was prepared via activation of its four terminal hydroxyl groups by nitrophenyl chloroformate (NPC) and then substitution of tyramine (TA) into the activated product to obtain TTe. Cationic PAMAM dendrimers G3.0 functionalized with p-hydroxyphenyl acetic acid (HPA) by use of carbodiimide coupling agent (EDC) to obtain Den-HPA. ¹H-NMR confirmed the amount of HPA and TA conjugations. The aqueous TTe and Den-HPA copolymer solution rapidly formed the cationic hydrogels in the presence of horseradish peroxidase enzyme (HRP) and hydrogen peroxide (H₂O₂) at physiological conditions. The gelation time of the hydrogels could be modulated ranging from 7 to 73 secs, when the concentrations of HRP and H₂O₂ varied. The hydrogels exhibited minimal swelling degree and low degradation under physical condition. In vitro cytotoxicity study indicated that the hydrogels were highly cytocompatible as prepared at 0.15 mg/mL HRP and 0.063 wt% of H₂O₂ concentration. Heparin release profiles show that the cationic hydrogels can sustainably release the anionic anticoagulant drug. The obtained results demonstrated a great potential of the cationic hydrogels for coating medical devices or delivering anionic drugs.     

Bioelectrochemical Characterization of Horseradish and Soybean Peroxidases

Heme peroxidase are ubiquitous enzymes catalyzing the oxidation of a broad range of substrates by hydrogen peroxide. In this paper the bioelectrochemical characterization of horseradish peroxidase (HRP) and soybean peroxidase (SBP), belonging to class III of the plant peroxidase superfamily, was studied. The homogeneous reactions between peroxidases and some common redox mediators in the presence of hydrogen peroxide have been carried out by cyclic voltammetry. The electrochemical characterization of the reactions involving enzyme, substrate and mediators concentrations allowed us to calculate the kinetic parameters for the substrate–enzyme reaction (K_MS) and for the redox mediator–enzyme reaction (K_MM). A full characterization of the direct electron transfer kinetic parameters between the electrode and enzyme active site was also performed by opportunely modeling data obtained from cyclic voltammetry and square wave voltammetry experiments. The experimental data obtained with immobilized peroxidases show enhanced direct electron transfer and excellent electrocatalytical performance for H₂O₂. Despite the structural similarities and common catalytic cycle, HRP and SBP exhibit differences in their substrate affinity and catalytic efficiency. Basing on our results, it can be concluded that peroxidase from soybean represents an interesting alternative to the classical and largely employed one obtained from horseradish as biorecognition element of electrochemical mediated biosensors.