A. I. Yaropolov

Flow-injection analysis of phenols at a graphite electrode modified with co-immobilised laccase and tyrosinase

Abstract The kinetic parameters of the oxidation reaction of phenolic compounds by molecular oxygen catalysed by fungal laccase have been studied. An amperometric biosensor for detection of phenols in environmental analysis is proposed. The enzymes laccase and tyrosinase were co-immobilised by adsorption onto a spectrographic graphite electrode. The bienzyme electrode was used as a sensor in a single-line flow-injection system. The analysis is based on the amperometric detection of the enzymatic reaction products at a potential of -0.05 V vs. Ag AgCl . The experimental parameters were optimised. The joint use of laccase and tyrosinase in the analytical procedure allows the detection of a large group of phenolic compounds.

Laccase-catalyzed synthesis of conducting polyaniline

Laccase isolated from Coriolus hirsutus was first used in the synthesis of water-soluble conducting polyaniline. The laccase-catalyzed polymerization of aniline was performed in the presence of sulfonated polystyrene (SPS) as a template. Laccase shows remarkable advantages in the synthesis of conducting polyaniline compared to the commonly used horseradish peroxidase due to its high activity and stability under acidic conditions. The characterization of the polyelectrolyte complex of polyaniline and SPS has been carried out using UV-Vis and FTIR spectroscopy. Cyclic voltammetry and dc conductivity measurements confirmed that electroactive polyaniline was synthesized by the laccase-catalyzed polymerization of aniline.

Phosphorescent polymer films for optical oxygen sensors

Abstract Hydrophobic phosphorescent Pt-porphyrins have been used for the development of luminescent polymer films designed for fibre-optic oxygen sensors. Luminescent and quenching characteristics of several Pt-porphyrins incorporated into polymer matrices have been studied to optimize the preparation of sensitive coatings for fibre-optical sensors. The films thus obtained have been used for fibre-optical oxygen monitoring in solutions. They proved to be effective and have some practical advantages in comparison with the oxygen-sensitive probes and coatings used at present.

Electrochemical properties of some copper-containing oxidases

Abstract The electrochemical behaviour of copper-containing oxidases (two laccases, tyrosinase, ceruloplasmin and ascorbate oxidase) has been investigated at graphite and carbon electrodes. These enzymes catalyse the oxidation of organic and inorganic substrates by molecular oxygen in homogeneous solutions. Molecular oxygen is reduced directly to water in a four-electron mechanism. Direct (mediatorless) bioelectrocatalysis of oxygen reduction was observed with electrodes modified with fungal and lacquer tree laccase. It was found that when the electrode modified with fungal laccase was used oxygen electroreduction started at a potential approximately 0.35 V more positive than that observed with the electrode modified with lacquer tree laccase. The effect of different pretreatments of the electrode surface before adsorption of laccase was investigated. It was established that the optimum pH for the electrocatalytic reaction of oxygen reduction in the presence of lacquer tree laccase is shifted towards the alkaline range compared with that with fungal laccase. No redox transformations of the prosthetic groups of the two laccases were revealed in anaerobic conditions and in the presence of various promoters. No bioelectrocatalytic reaction of oxygen was revealed with the other oxidases. An electrochemical response was obtained at electrodes coated with only tyrosinase or ceruloplasmin in the absence of oxygen. The conditions which can influence the bioelectrocatalysis have been discussed.

Laccase-based biosensor for determination of polyphenols: determination of catechols in tea

A new method of amperometric determination of phenolic compounds using an enzyme electrode is proposed. The latter represents the combination of the oxygen electrode and immobilized laccase. Analytical systems of flow injection and batch types were considered. A method of immobilization was developed that provided an increase in the stability of the enzyme. Optimal conditions for biosensor operation were found. The time needed for analysis in the flow injection mode was below 100 s. A column with immobilized enzyme could be used for up to 500 determinations of phenolic compounds without decrease of the enzyme activity. The practical validity of the method was demonstrated by tannin analysis in tea of different brands.

293 - Electrocatalysis of a cathodic oxygen reduction by laccase

Summary The effect of laccase adsorbed on carbon-black electrodes on the electrochemical reduction of molecular O2 has been studied. It is shown that laccase introduced into O2-saturated solution (acetate-phosphate buffer, pH=5,5) shifts the potential towards the positive side and accelerates the O2 electroreduction in the 1.2-0.6 V range. With the use of carbon-black adsorbed laccase, the potential reaches 1.2 V, which is close to the oxygen equilibrium potential. The dependence of the stationary potential on the pH and on the oxygen pressure indicates that the parameters ϖU/ϖpH and dU/d log po2 correspond to the coefficients in the Nernst equation for the O2−H2O couple. A scheme for the oxygen reduction with immobilized laccase isproposed on the basis of experimentally measured kinetic parameters.

The mechanism of interaction of carnosine with superoxide radicals in water solutions

The antiradical activity and the radiation stability of carnosine in water solutions was studied by the pulse radiolysis technique with spectrophotometric registration of absorbance. The transient spectra were recorded in the range 245-670 nm during 2 x 10(-6)-20 s after the pulse using a flow system for continuous change and saturation of the samples by different gases. Also, the spectra of the stable products of radiolysis were studied. The results obtained give evidence that carnosine in water solutions in the presence of oxygen behaves like a multifunctional antioxidant. Even at low concentrations, dipeptide forms a charge-transfer complex (Car ... O2-., lambda max = 265 nm) with the superoxide radical which changes the reactivity of O2-.. The absorbance band of the complex was shifted towards lower energy as compared to superoxide radical lambda max = 255 nm). The interaction of carnosine with OH-radicals proceeding at very high rate and resulting in the formation of a stable product suggested another type of dipeptide activity. The kinetic mechanism of the interaction of carnosine with products of radiolysis of water in aerobic conditions is discussed.

Production of lignin modifying enzymes by co-cultivated White-rot fungi Cerrena maxima and Coriolus hirsutus and characterization of laccase from Cerrena maxima

The lignin modifying enzymes production by co-cultivated C. hirsutus and Cerrena maxima, pure fungal cultures Coriolus hirsutus and Cerrena maxima in defined medium under static and shaken conditions, in media with birch sawdust and sulfonate lignin and under SSF of birch sawdust was comparatively studied. Analysis of the structure of residual lignin before and after fungal treatment indicated that the oxidation of phenolic component of lignin occurred and the residual lignin is enriched in carboxylic acid groups. Three laccase isoenzymes are produced by Cerrena maxima growing in defined media. The isoenzyme with pI 3.5 was largely present and its molecular mass, isoelectric point, metal content and carbohydrate composition were determined. The laccase was characterized to have a broad substrate specificity. It belongs to high redox potential laccases (750 mV vs. normal hydrogen electrode) and might be useful in industrial applications.

Direct electron transfer effect biosensors

A review of biosensors based on the direct electron transfer effect is carried out in this work. Different redox enzymes and proteins using the direct electron transport mechanism for electrocatalytical processes are described. The tunneling of electrons as a possible explanation of the direct electron transfer effect is also discussed.

MECHANISM OF H2-ELECTROOXIDATION WITH IMMOBILIZED HYDROGENASE

Abstract The reaction of hydrogen electrooxidation on carbon-black electrodes with hydrogenase has been studied. It is shown that in the presence of hydrogenase a hydrogen equilibrium potential equal to 0.0 V is established on a carbon-black electrode in phosphate buffer, pH 7.5, saturated with hydrogen. The kinetic parameters have been studied and the mechanism of the hydrogen electroenzymic oxidation reaction has been determined.