Laccase-Catalyzed Heterocoupling of Dihydroquercetin and p-Aminobenzoic Acid: Effect of the Reaction Product on Cultured Cells.

Derivatization of the natural flavonoid dihydroquercetin with p-aminobenzoic acid was carried out in an ethyl acetate/citric buffer biphasic system using laccase from the fungus Trametes hirsuta. The main reaction product yield was ~68 mol %. The product was characterized by 1H NMR, 13C NMR, and liquid chromatography-mass spectroscopy, and its structure was elucidated. The reaction product affected viability of cultured human rhabdomyosarcoma cells (RD cell line) in a dose-dependent manner and, therefore, can be of interest to pharmaceutical industry.

[Dihydroquercetin polymerization using laccase immobilized into an ionic liquid].

It was shown that the laccase (LC) included into hydrophobic ionic liquid (IL) can be reused for the biotransformation of dihydroquercetin (DHQ). The physicochemical characteristics of DHQ oligomers synthesized using LC/IL did not differ from the characteristics of the oligomers obtained with native laccase. The synthesized oligomers have a number average molecular weight of 1050 g/mol and a polydispersity index of 1.41. Oligomers possess higher antioxidant activity than the monomer.

[Aniline Polymerization on Multiwall Carbon Nanotubes with Immobilized Laccase].

A composite material consisting of electrically conductive polyaniline deposited on the surface of multiwall carbon nanotubes has been synthesized. Enzymatic synthesis was carried out in the presence of Trametes hirsuta laccase immobilized on the nanotube surface. The obtained composite was morphologically uniform, and its electrochemical capacity and stability were much higher than those of a composite synthesized according to the conventional chemical procedure.

Enzymatic polymerization of dihydroquercetin using bilirubin oxidase.

Dihydroquercetin (or taxifolin) is one of the most famous flavonoids and is abundant in Siberian larch (Larix sibirica). The oxidative polymerization of dihydroquercetin (DHQ) using bilirubin oxidase as a biocatalyst was investigated and some physicochemical properties of the products were studied. DHQ oligomers (oligoDHQ) with molecular mass of 2800 and polydispersity of 8.6 were obtained by enzymatic reaction under optimal conditions. The oligomers appeared to be soluble in dimethylsulfoxide, dimethylformamide, and methanol. UV-visible spectra of oligoDHQ in dimethylsulfoxide indicated the presence of highly conjugated bonds. The synthesized oligoDHQ was also characterized by FTIR and (1)H and (13)C NMR spectroscopy. Comparison of NMR spectra of oligoDHQ with DHQ monomer and the parent flavonoids revealed irregular structure of a polymer formed via the enzymatic oxidation of DHQ followed by nonselective radical polymerization. As compared with the monomer, oligoDHQ demonstrated higher thermal stability and high antioxidant activity.

Biocatalytic synthesis of conducting polymers and prospects for its application.

Enzymatic methods of synthesis of conducting polymers, physicochemical properties of the resulting products, and mechanisms of the reactions are considered. The enzymes involved in oxidative polymerization of monomers are briefly characterized. Examples of practical application of enzymatically synthesized conducting polymers are given.

"Blue" laccases.

This review concerns copper-containing oxidases--laccases. Principal biochemical and electrochemical properties of laccases isolated from different sources are described, as well as their structure and mechanism of catalysis. Possible applications of laccases in different fields of biotechnology are discussed.

Electrochemistry and kinetics of fungal laccase mediators.

The screening of potential redox mediators for laccase was performed using homogeneous Trametes hirsuta laccase. Heterogeneous (electrochemical) and homogeneous (oxidation by laccase) reactions of the different types of the enhancers (mediators) of the enzyme were investigated. It was discovered that derivatives of phenyl-methyl-pyrazolones and benzoic acid, as well as N-hydroxynaphthalimide were efficient substrates for the laccase. The characterization of several representatives from each class was carried out using electrochemical and enzyme kinetics methods. The kinetic parameters for the oxidation of phenyl-methyl-pyrazolones and 3-(6-hylroxy)-aminobenzoic acid were comparable to those for 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by the laccase, whereas the rate of enzymatic oxidation of N-hydroxynaphthalimide was sufficiently lower. Electrochemical experiments demonstrated that only oxidation of phenyl-methyl-pyrazolones and N-hydroxynaphthalimide yielded several high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, whereas derivatives of benzoic acid showed low-potential intermediate, which was not able to oxidized lignin model compound. Phenyl-methyl-pyrazolones was about 50% as effective in degrading veratryl alcohol compared to ABTS as judged from HPLC kinetic studies, whereas N-hydroxynaphthalimide showed the same efficiency as ABTS. Phenyl-methyl-pyrazolones and hydroxynaphthalimides may be of commercial interest for oxidoreductase-catalyzed biodegradation of different xenobiotics.

Isolation and Purification of enzymes from ligninolytic complex of the basidial fungus Trametes pubescens (Schumach.) Pilát and study of their properties.

A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.

Comparison of physico-chemical characteristics of four laccases from different basidiomycetes.

New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50 degrees C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K(4)Fe(CN)(6) were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.

Electrochemical investigation of the dynamics of Mycobacterium smegmatis cells' transformation to dormant, nonculturable form.

Dynamics of transformation of Mycobacterium smegmatis cells by cultivation under nonoptimal conditions (partial starvation) to dormant, nonculturable form has been studied. For this aim, an electrochemical method was developed to detect both viable and 'viable but nonculturable' (VBNC) cells. The current produced by bacteria placed at the electrode surface was measured in the presence of 2,6-dichlorophenol indophenol (DCIP) at the applied potential of 350 mV. It has been established that electrochemical activity changes parallel with the growth of biomass. The transition of M. smegmatis to a dormant, nonculturable state goes along with the decrease of the detection current up to 20% of the maximum level. This means that nonculturable cells have rather high rest metabolic activity. The course of the CFU values has a complicated character during bacterial growth. The placement of the bacterial culture on the solid medium appears to cause a new stress that stops proliferation and stimulates aggregation. Both processes distort CFU measurement results. The quantitative estimation of the viable but nonculturable cells by counting colonies, measuring optical density and current produced by bacteria has been discussed.

Novel laccase redox mediators: spectral, electrochemical, and kinetic properties.

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3'-Sulfophenyl)-3- methylpyrazolone-5 was about 30-40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commercial interest for oxidoreductase-catalyzed biodegradation of organic compounds.

On applicability of laccase as label in the mediated and mediatorless electroimmunoassay: effect of distance on the direct electron transfer between laccase and electrode.

Applicability of laccase as enzyme-label has been investigated. It was shown that the property of laccase to catalyze the oxygen electroreduction at an electrode allows to develop a mediatorless and pseudoreagentless electro-enzyme-immunoassay (EEIA). In this case the electrode acts as an electron-donor substrate. When the bioelectrocatalytic reaction takes place, some electric charge is collected on the electrode. A method of determination of the electrode charge as well as the concentration of oxidized form of the mediator at the electrode surface has been elaborated. For this aim a technique of the measurement of current-surge was employed. Human immunoglobulin G and insulin were taken as model in this investigation. A back titration schemes without any mediator and in the presence of o-carboxybenzoylferrocene as a mediator was applied. The antibody carbon-black and the antigen glassy-carbon electrodes were used. The limits of detection were found to be 0.3 and 1.6 nM, respectively. The advantage of the mediatorless assay is that the charge leakage is imperceptible by open circuit for a long time and the accumulation of the charge occurs linearly with time. The charge accumulation for a long time allows to diminish the limit of detection. However, there is a limitation of the method. The direct electron transfer slows down with increasing the distance between the enzyme molecule and the electrode surface. This effect reduces the sensitivity of the method. The decrease of the electron transfer rate with distance has been estimated. Monolayer of hemoglobin dividing the laccase molecule from the electrode surface decreases the rate by four times. The electron transfer rate for the antibody electrode with associated antigen-laccase conjugate is less than that for the analogous electrode, covered with monolayer of covalently attached laccase, by 210 times. The current-surge peak was expected to decrease with distance by an equation of the form I = I0 exp[-r/r0]. The parameter r0 is equal to 2.2 +/- 0.8 nm. The possibility of the sensitivity increase in the mediatorless mode by 'wiring' through the multilayer film of immunoproteins immobilized on the electrode is discussed.

Laccase from Coriolus hirsutus as alternate label for enzyme immunoassay. Determination of pesticide 2,4-dichlorophenoxyacetic acid.

A new label--laccase from the fungus Coriolus hirsutus--was applied for solid-phase enzyme-linked immunosorbent assays of the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). Two proposed assays are based on (1) competitive binding of antibody-laccase conjugate with immobilized 2,4-D-protein conjugate and 2,4-D in tested sample, and (2) competition of 2,4-D and 2,4-D-laccase conjugate for binding with immobilized antibodies. Kinetic and concentration dependencies for these reactions were studied, and the ELISAs were optimized in accordance with the data obtained. The elaborated systems permit the detection of 2,4-D in concentrations down to 10-20 ng/mL; time of the assays is 1.5-2 h. The main advantage of the laccase label, in comparison with the widely used peroxidase one, lies in the lack of hydrogen peroxide from substrate mixture, because dissolved oxygen plays the role of oxidizer.

Modulatory effects of ceruloplasmin on lymphocytes, neutrophils and monocytes of patients with altered immune status.

We investigated the effect of plasma ceruloplasmin (Cp) on the different types of lymphocyte rosetting, and phagocytosis of polystyrene particles and culture Candida albicans by peripheral blood neutrophils and monocytes. Lymphocytes, neutrophils, and monocytes were isolated from the blood of patients with elevated immuno-status (n = 9), healthy donors (n = 21), and patients with reduced immuno-status (n = 21). The ability of Cp to decrease the number of lymphocytes forming E- and EAC-rosettes and rosettes with auto-erythrocytes was shown for both patients and healthy donors. The maximal decrease of the number of E-rosettes (by 35%) and EAC-rosettes (by 57%) was shown for lymphocytes of the patients with elevated immuno-status. Cp had an effect on rosetting only when lymphocytes were preincubated with it, suggesting that Cp binding to lymphocytes was responsible for these effects. The decrease in all types of rosetting caused by Cp was dose-related, with a maximum effect at physiological concentration of Cp (300 micrograms/ml). We demonstrated an enhancing effect of Cp on phagocytosis of Candida albicans and polystyrene particles by neutrophils (with a maximum enhancement by 180% for neutrophils of the patients with decreased immuno-status) and monocytes (with a maximum of 89% for monocytes of healthy donors). Cp enhances phagocytosis of neutrophils and monocytes by binding these cells, not by opsonizing ingested particles as a conventional opsonin (ie. lipopolysaccharide from E.coli). The stimulating effect of Cp on phagocytosis was three times higher than that of LPS from E.coli.

The mechanism of interaction of ceruloplasmin with superoxide radicals.

1. The mechanism of interaction of CP with O2- radicals in chemical and enzymatic systems of superoxide radical generation as well as in the pulse radiolysis technique was studied. 2. It is found that CP does not exert any kinetic influence on the decomposition of superoxide radical and, unlike SOD, cannot catalyze the reaction of disproportionation of these radicals in systems with chemical and enzymatic generation of O2-. 3. The data obtained confirm the suggestion that CP interacts with precursors of O2- radicals. 4. The irradiation of CP does not change its inhibiting activity in the reaction of the formation of superoxide radicals in systems with enzymatic O2- generation, but decreases its oxidase activity. 5. The results obtained demonstrated that the increase in the radiation dose resulted in the decrease of the inhibiting activity of SOD, whereas the activity of CP did not change.

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.

Bioelectrocatalysis: the electrochemical kinetics of hydrogenase action.

A hydrogen electrode based on a hydrogenase immobilized on a carbon material has been developed. The equilibrium hydrogen potential was reached in an atmosphere of H2 on the enzyme electrode according to the mechanism of direct exchange of electrons between the electrode and the enzyme active center. The electrochemical kinetics of the hydrogen enzyme electrode action is presented.

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.

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode. The formation of the complex between laccase-labelled antibody and antigen on the electrode surface resulted in a considerable (more than 300 mV) shift of the electrode potential. The rate of the increase of the electrode potential was inversely proportional to the concentration of the free antigen in the sample. The non-specific adsorption of conjugate and other proteins on the electrode could be eliminated by using a polyethylenimine-based polymer on the electrode surface. Insulin was used as a model analyte. The sensitivity limit for this antigen was approximately 3 micrograms ml-1.

Immunoenzyme determination of total serum ceruloplasmin. Application to Wilson disease.

Enzyme immunoassay for ceruloplasmin (CP)*, employing monospecific CP antibodies labeled with horse radish peroxidase was developed. This method permits to determine total content of CP, which is present in Wilson disease patients' blood in enzymatically active and enzymatically inactive forms. The evidence is presented that the method can be used for a direct determination of CP in blood serum. The minimal CP concentration which may be determined by enzyme immunoassay (IEA) is 5.10(-9) g/ml. The method was used for determination of CP concentrations in Wilson disease patients' blood with different disease severity. Analysis of blood samples taken from 6 Wilson disease patients with the use of IEA method revealed similar total CP concentrations. At the same time, the oxidase activities of CP in the blood of different patients varied more than sevenfold.