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.

[Enzymatic synthesis of electroconductive biocomposites based on DNA and optically active polyaniline].

Electroconductive interpolymer polyaniline complexes are synthesized on the DNA matrix, using the method of oxidative polymerization of aniline with two different biocatalyzers: horseradish root peroxidase and micropiroxidase-11 biomimetic. The spectral characteristics and morphology of the acquired biocomposites have been studied. The stereospecificity of the acquired samples of interpolymer complexes is shown, depending on the biocatalyzers used. The results acquired indicate the important role of a biocatalyzer in the formation of the twist direction of an electroconductive polymer spiral on the DNA matrix; i.e., the optical activity of the polymer samples acquired is apparently associated with the biocatalyzer properties.

[Synthesis of electroconductive polyaniline using immobilized laccase].

A new method for synthesis of the conductive complex between polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanosulfonic acid) (PAMPS) was proposed; in this method, the immobilized laccase from the basidiomycete Trametes hirsuta is used as a biocatalyst for aniline oxidative polymerization. The conditions for laccase immobilization on CM cellulose by bifunctional Woodward's reagent were optimized. The catalytic properties of immobilized and native laccases were compared. The immobilized laccase appeared an efficient catalyst for the oxidative radical polymerization of aniline on polysulfonic acid matrix at 4 degrees C. It was demonstrated that the immobilized enzyme could be repeatedly used for enzymatic synthesis of this polymer. Several spectral characteristics of the PANI/PAMPS-complexes synthesized at various pH values were studied. The conductance of PANI specimens produced using immobilized laccase as a catalyst was 13 mS/cm.

[Micellar laccase-catalyzed synthesis of electroconductive polyaniline].

A method of enzymatic synthesis of electroconductive polyaniline on the micelles of dodecylben-zenesulfonic acid sodium salt (DBSNa) is proposed. The high potential laccase from the basidiomycete Trametes hirsuta was used as a biocatalyst. The conditions for polyaniline synthesis were optimized (pH 4.0; reagent concentrations, 10-20 mM; and aniline/DBSNa ratio, 2: 1). The resulting product was electrochemically active in the range of potentials from -200 to 600 mV, electroconductive, and capable of reversible dedoping with a change in pH of solution.

"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.

[Laccase-mediator systems and their applications: a review].

The mechanism of operation of laccase-mediator systems (LMSs) in xenobiotic degradation mediated by "true" redox mediators and laccase enhancing agents is considered. Structural formulae of most common laccase mediators and compounds that can be used as agents enhancing the enzyme operation are presented. Examples of LMS application in biotechnology are described.

[Oxygen can be replaced by artificial electron acceptors in reactions catalyzed by alcohol oxidase].

For the first time, spectrometric and electrochemical studies demonstrated the possibility of using artificial electron acceptors in reactions catalyzed by alcohol oxidase. We report kinetic parameters of homogenous catalytic oxidation of formaldehyde by organic redox compounds belonging to different structural classes (toluidine blue, methylene blue, 2,6-dichlorophenolindo-phenol, and p-benzoquinone) and replacing dioxygen in these reactions. p-Benzoquinone, having the highest redox potential, proved to be the most efficient artificial electron acceptor of all compounds studied.

Purification and characterization of alcohol oxidase from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha.

Alcohol oxidase (AOX) has been purified 8-fold from a genetically constructed over-producing strain of the methylotrophic yeast Hansenula polymorpha C-105 (gcr1 catX) with impaired glucose-induced catabolite repression and completely devoid of catalase. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and HPLC. Some physicochemical and biochemical properties of AOX were studied in detail: molecular weight (approximately 620 kD), isoelectric point (pI 6.1), and UV-VIS, circular dichroism (CD), and fluorescence spectra. The content of different secondary structure motifs of the enzyme has been calculated from the CD spectra using a computer program. It was found that the native protein contains about 50% alpha-helix, 25% beta-sheet, and about 20% random structures. The kinetic parameters for different substrates, such as methanol, ethanol, and formaldehyde, were measured using a Clark oxygen electrode. The rate of enzymatic oxidation of formaldehyde by alcohol oxidase from H. polymorpha is only twice lower compared to the best substrate of the enzyme, methanol.

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.

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.