Search

Abstract: Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes. This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly. [Copyright &y& Elsevier]

Abstract: Up to date, enzymatic polymerization of phenolic compounds bearing different moieties such as ferrocene, sugar, furamide, and male-imide have been reported. In this study, horseradish peroxidase (HRP)-catalyzed polymerization of another hydroxy substituted aromatic compound, 2-hydroxycarbazole (HC), was firstly presented. The structure of the polymer (PHC) was verified by using nuclear magnetic resonance and infrared techniques. Further characterization was carried out by means of TG, DSC, CV (cyclic voltammetry), SEM (scanning electron microscopy), fluorescence analyses, and solid state conductivity measurements. Average molecular weight of the polymer was determined by gel permeation chromatography and was around two thousands. The optical and electrochemical band gaps of PHC were dramatically lower than those of HC. The photochemical behaviors of HC and PHC were investigated by recording the fluorescence spectra in two different solvents. Solid state conductivity measurement showed that the electrical conductivity value of hydroxyl-functionalized carbazole polymer is relatively higher than those of the previously reported phenol polymers in the literature. Moreover, the conductivity of PHC increased by doping with iodine vapour. [Copyright &y& Elsevier]

Abstract: A convenient and effective strategy for fabrication of hydrogen peroxide biosensor based on sodium alginate (SA) and polyvinyl butyral (PVB) as matrices was reported in this paper. The horseradish peroxidase (HRP) and SA were electro-co-deposited onto the surface of gold electrode, and the HRP–SA/Au electrode was further coated with PVB. The interaction between HRP and SA was characterized by UV–vis absorption spectroscopy, and the fabricating process of biosensor was characterized by electrochemical impedance spectroscopy (EIS). The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Experimental conditions were investigated which influence the performance of the biosensor, such as pH, and applied potential. The biosensor showed a linear response to H2O2 over a concentration range from 7.0×10−6 to 4.1×10−3 M with a detection limit of 1.8×10−6 M based on a signal-to-noise ratio of 3 under optimum conditions. The value of HRP in the composite was evaluated to be 1.38mM. The biosensor obtained from this study possesses high sensitivity, good reproducibility, and long-term stability. [Copyright &y& Elsevier]

Abstract: Effects of immobilization pH and pore characters of mesoporous silicas (MPSs), MCM-41, SBA-15, and MCF, were simultaneously investigated for the immobilization of horseradish peroxidase (HRP; EC 1.11.1.7). MCM-41 and SBA-15 were rod-like with respective average pore diameters of 32, and 54Å, while that of MCF with spherical cell and frame structure was 148Å. Moreover, the MPSs synthesized were of identical surface functional groups and similar contents of free silanol groups. At immobilization pH 6 and 8 almost 100% HRP loadings were obtained and insignificant leaching were observed for all types of supports at pH 6. However, MCF was found to give both the highest enzyme loading and leaching at pH 10. Maximum and minimum HRP activities were obtained at respective immobilization pH 8, and 6. Activities of immobilized HRP increased with support pore diameters in the order: MCM-41

Abstract: A novel amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase (HRP) on DNA–silver nanohybrids (DNA–Ag) and poly(diallyldimethylammonium chloride) (PDDA)-protected gold nanoparticles (PDDA–Au) was successfully fabricated by combining the self-assembly technique with an in situ electrochemical reduction of the DNA–Ag+ complex. The preparation process of modified electrode was characterized with UV–vis absorption spectroscopy, transmission electron microscopy (TEM) and atomic force microscope (AFM). The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Experimental conditions influencing the biosensor performance such as pH, potential were optimized. The resulting biosensor (HRP/DNA–Ag/PDDA–Au/DNA–Ag/Au electrode) showed a linear response to H2O2 over a concentration range from 7.0μM to 7.8mM with a detection limit of 2.0μM (S/N=3) under optimized conditions. The apparent Michaelis–Menten constant () was evaluated to be 1.3mM. The sensor exhibited high sensitivity, good accuracy and an acceptable stability. [Copyright &y& Elsevier]

Abstract: A detailed comparative stability study of the purified bitter gourd peroxidase and commercially available pure horseradish peroxidase has been undertaken against various denaturants. Stability of the enzymes was monitored spectrophotometrically as well as by ellipticity changes in far-circular dichroism region. Bitter gourd peroxidase was more thermo-stable. The disruption of secondary structure and enzymatic activity at various temperatures was greater for horseradish peroxidase. Bitter gourd peroxidase retained remarkably greater fraction of catalytic activity as compared to horseradish peroxidase in the alkaline pH range. The difference in catalytic activity of bitter gourd peroxidase by varying the pH was related to the change in secondary structure as manifested by the change in CD value at 222nm. It was further complemented by the far UV-CD spectra, which showed greater retention of secondary structure at pH 6.0 and 10.0. BGP had remarkable stability in the presence of urea, sodium dodecyl sulphate and dimethyl formamide. In view of its higher stability, bitter gourd peroxidase can serve as a better alternative to horseradish peroxidase in clinical and environmental analyses as well as in various biotechnological applications. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase catalyzed an oxidative coupling reaction of 4′-hydroxyphenyl β-glucoside (arbutin) and 2,5-dihydroxybenzoic acid sodium salt (gentisate) using H2O2 as an electron acceptor to yield a precipitating yellow compound. An approximate arbutin/gentisate ratio of 1:2 was effective for the synthesis. The addition of 100–300mM H2O2 to the mixtures of 100mM arbutin and 200mM gentisate attained optimized yields of 50–60% in the initial arbutin. Mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance data revealed that the precipitated compound was a novel glycoside bound between the C3′-position of the hydroxyphenyl moiety of arbutin and the C6-position of gentisate, followed by the intramolecular esterification between the phenolic hydroxyl group of arbutin moiety and the carboxyl group of gentisate moiety. The product exerted anti-oxidation and competitive inhibition against mushroom tyrosinase higher than arbutin, while it inhibited mouse melanoma tyrosinase lower than arbutin. [Copyright &y& Elsevier]

Abstract: The reactivity of horseradish peroxidase (HRP) with water insoluble phenolic compounds has been studied in 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4])/water mixtures. The enzyme retained some catalytic activity up to 90% ionic liquid in water at 25°C only at pH values higher than 9.0. Activity steadily decreased towards neutral and acidic conditions, as judged by 4-aminoantypirin/phenol activity tests. Inhibition of horseradish peroxidase under neutral acidic condition was due to the binding of fluoride anions released from tetrafluoroborate anion to the heme iron as demonstrated by the sharp UV–visible absorption transition diagnostic of the conversion from a five coordinated to a six coordinated high spin ferric heme iron. Thus, reactions with water insoluble phenols were carried out under alkaline reaction conditions and 75% [BMIM][BF4]/water mixture. Under these conditions, the distribution of the reaction products was much narrower with respect to that observed in aqueous buffers or water/dimethylformamide or water/dimethylsulfoxide mixtures, and polymeric species other than dimers were not observed. Technical scale preparations of a novel 4-phenylphenol ortho dimer [2,2′-bi-(4-phenylphenol)] with a high yield of the desired product were obtained. [Copyright &y& Elsevier]

Abstract: In this work a new enzyme–zeolite interaction was examined. Horseradish peroxidase (HRP-C), a heme-containing enzyme, shows an activity increase for HRP-C catalysed phenol and 4-aminoantipyrine co-oxidation in aqueous medium, in the presence of NaY zeolite, although the attempts to use this enzyme immobilized on the zeolite resulted in an almost complete loss of activity. Pseudo-homogeneous mechanical mixtures of enzyme and zeolite in solution were compared at five different temperatures (10, 20, 25, 30 and 35°C), and have shown possible interactions which influence the enzyme activity. In order to better analyse the interactions effects, co-immobilization of the enzyme and zeolite was performed in gelatin particles; in this case the enhancement in enzymatic activity was much larger than when pseudo-homogeneous mixtures were used. HRP-C immobilized in gelatin showed activity increases as large as 100% when zeolite NaY is co-immobilized in the same particles, depending on the experimental conditions. Although the mechanism by which the zeolite induces this effect still requires clarification, one possible explanation is that some ionization of OH groups of the enzyme occurs, due to ion exchange of Na-ions of the zeolite and protons of the enzyme, an effect which might also occur in the gelatin matrix; the zeolite may also influence the conformation of the enzyme by means of the electrical fields that are generated at the surface of these materials, which are known to influence the catalytic action of zeolites. [Copyright &y& Elsevier]

Abstract: Bromophenol blue and methyl orange removal capabilities of citraconic anhydride-modified horseradish peroxidase were compared with those of native horseradish peroxidase. Citraconic anhydride-modified horseradish peroxidase showed higher decolorization efficiencies for both dyes than native horseradish peroxidase. Upon the chemical modification, the decolorization efficiencies were increased by 1.8% and 12.4% for bromophenol blue and methyl orange, respectively. The quantitative relationships between decolorization efficiencies of dyes and reaction conditions were also investigated. Experimental data revealed that aqueous phase pH, reaction time, temperature, enzyme concentration and ratio of dye and H2O2 play a significant role on the dye degradation. Lower dose of citraconic anhydride-modified horseradish peroxidase was required than that of native enzyme for the decolorizations of both dyes to obtain the same decolorization efficiencies. Citraconic anhydride-modified HRP exhibited a good decolorization of dye over a wide range of dye concentration from 8 to 24 or 32μmoll−1 at 300μmoll−1 H2O2, which would match industrial expectations. Kinetic constants for two different dyes were also determined. Citraconic anhydride-modified horseradish peroxidase shows greater affinity and catalytic efficiency than native horseradish peroxidase for both dyes. [Copyright &y& Elsevier]

Abstract: Different peroxidases were used to catalyze the synthesis of neolignan. Horseradish peroxidase (type I, Sigma) was employed successfully to catalyze the formation of magnolol by the phenolic oxidative coupling of allyphenol. The effects of organic co-solvents, pH, addition rate of H2O2, and the ratio of enzyme/substrate on the yields of dimer were studied. After the peroxidase-catalyzed reaction was performed, different neolignans were isolated and identified as magnolol (3), the trimeric ortho–ortho-coupled product dunnianol (4), the ortho-O-coupled product isomagnolol (5), and the Pummerer''s ketone derivative (6). The inhibitory activities of magnolol against cyclo-oxygenase and lipoxygenase were also reported. [Copyright &y& Elsevier]

The influence of the regeneration of horseradish peroxidase activity at 4 °C on both the thermal inactivation and the reactivation kinetics of the enzyme was studied in phosphate buffer and a mixture of this buffer with 10% (v/v) N,N-dimethylformamide, at temperatures ranging from 70 to 85 °C. A series-type model was fitted to the experimental data that exhibited typical biphasic patterns, which became less pronounced as enzyme activity was regenerated. The kinetic parameters were found to be, in general, significantly different in both inactivation media. The magnitude of the activation energies for both the reactions of formation and inactivation of the intermediate enzyme indicated that conformational changes might play a major role in both processes when the solvent is added. A logarithmic model, although lacking a theoretical background, was able to predict the enzyme activity regeneration upon storage of the inactivated samples at 4 °C. There was evidence that the rate of reactivation may be dependent on the amount of intermediate formed in the first reaction or remaining after inactivation, as suggested by both the inactivation temperatures and extent of heating time relationships verified in this work. [Copyright &y& Elsevier]

Horseradish peroxidase was modified by phthalic anhydride and glucosamine hydrochloride. The thermostabilities and removal efficiencies of phenolics by native and modified HRP were assayed. The chemical modification of horseradish peroxidase increased their thermostability (about 10- and 9-fold, respectively) and in turn also increased the removal efficiency of phenolics. The quantitative relationships between removal efficiency of phenol and reaction conditions were also investigated using modified enzyme. The optimum pH for phenol removal is 9.0 for both native and modified forms of the enzyme. Both modified enzyme could suffer from higher temperature than native enzyme in phenol removal reaction. The optimum molar ratio of hydrogen peroxide to phenol was 2.0. The phthalic anhydride modified enzyme required lower dose of enzyme than native horseradish peroxidase to obtain the same removal efficiency. Both modified horseradish peroxidase show greater affinity and specificity of phenol. [Copyright &y& Elsevier]

A fused protein consisting of a cellulose-binding domain (CBD) and horseradish peroxidase (HRP) was bound to cellulose beads and evaluated in aqueous-organic solvent systems. The CBD–HRP fusion protein containing two different functionalities, a catalytic domain and a binding domain, preserved both capabilities in this non-conventional environment. A six-fold increase in the half-life of the enzyme in buffer resulted from immobilization onto cellulose via CBD. The immobilized enzyme was also more stable than the native enzyme in increasing concentrations of acetone (0–92%). There was a general decrease in activity as the solvent concentration in the mixture increased (in all solvent types: THF, acetone, acetonitrile and ethanol). However, the immobilized enzyme was at all times more active than the soluble enzyme forms. The thermostability of the enzyme in buffer, at 40–60 °C, was also improved by immobilization. The soluble CBD–HRP fusion protein exhibited greater stability (both to organic solvents and temperature), but lower activity, in comparison with the native HRP. This work demonstrates for the first time the use of a cellulose-bound CBD-enzyme as a catalyst in aqueous-organic solvent media. [Copyright &y& Elsevier]

Encapsulation of horseradish peroxidase (HRP) by the sol–gel method into silica (SiO2) or alkylated silica (RSiO2, in which R is Me or Pr) yields biocatalytic glasses designated HRP@SiO2 and HRP@RSiO2. These new enzyme composite materials catalyze one-electron oxidation by H2O2 of the dye 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) into the radical cation ABTS⋅+. In the presence of excess H2O2, ABTS⋅+ is converted into a mixture of at least five compounds. This undesirable side-reaction can be suppressed by using stoichiometric amount of H2O2. The effects of ABTS concentration and ionic strength of the buffer on the ABTS⋅+ yield and the apparent rate constant were investigated. The catalyst HRP@MeSiO2 achieves higher ABTS⋅+ yields (70–85%) than HRP@SiO2 and HRP@PrSiO2 (30–40%) do. The apparent rate constants for HRP@MeSiO2 are 7–10 times higher than those for HRP@SiO2 and HRP@PrSiO2. When the ionic strength of the buffer is raised, the rate constants increase for HRP@SiO2 and HRP@PrSiO2, and do not change for HRP@MeSiO2. The diminished electrostatic interaction between the negatively-charged substrate, ABTS, and the surface of methylated silica is the main cause for the increased catalytic efficiency. These results are important for understanding of encapsulated and otherwise immobilized enzymes. The possible electrostatic effects should be taken into consideration in the choice of the matrix materials used in the design of biosensors, supported catalysts, and other composite materials. [Copyright &y& Elsevier]

Abstract: Poly(4-fluoro-2-methoxyphenol) has been synthesized using horseradish peroxidase biocatalyst in a buffer/1-butyl-3-methylimidazoliumtetrafluoroborate solvent mixture. Experimental observations indicate that the enzymatically synthesized polyfluorophenol is a suitable photoconductive material. The photocurrency obtained under 532nm light intensity is about 0.82W/cm2. The work is relevant to the use of enzymes to produce polymeric materials with potential uses as components in photoelectronic devices owing to polymer characteristics in its mild and eco-friendly production process. [Copyright &y& Elsevier]