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The copolymers of aniline (ANI) and 2-aminophenethyl alcohol (APA) were synthesized on a poly (2-acrylamido-2-methyl-1-propanesulfonic) acid (PAMPS) template with the fungal laccase Trametes hirsuta, which has a high redox potential, as the catalyst. Atmospheric oxygen was the oxidizing agent. The copolymer/PAMPS complexes were pseudo-soluble and were characterized by various physicochemical methods. The electrical conductivity of the copolymers depended on the initial molar ratio of the monomers. The copolymer synthesized at an APA : ANI ratio of 2 : 8 possessed the best characteristics. It is shown that the laccase/mediator system can be to oxidize the primary alcohol groups of the copolymer to reactive aldehyde groups.

The oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) using ammonium peroxydisulfate and the high redox potential laccase from Trametes hirsuta is studied in order to clarify the significant differences in the chemical and enzymatic polymerization reactions of aniline. As opposed to the laccase-catalyzed reaction, the chemical oxidation of TMPD proceeds in two stages. The radical cation formed at the first stage is oxidized by the peroxydisulfate ion to quinoiddiimine that can interact with the initial TMPD. The kinetics of the formation of the radical cation $${\text{TMP}}{{{\text{D}}}^{{\centerdot + }}}$$ is studied by ultraviolet–visible (UV–Vis) and electron paramagnetic resonance spectroscopy. The obtained data allow making an assumption about the difference in the mechanisms of chemical and enzymatic polymerization of aniline.

Template polymerization of aniline was performed using a laccase-mediator system. The high redox-potential laccase from the basidiomycete fungus Trametes hirsuta was a catalyst for polymerization. The aniline dimer served as the redox mediator; atmospheric oxygen served as the oxidant. Sodium dodecylbenzenesulfonate micelles were used as a “soft” template. The biocatalytic polymerization of aniline resulted in conducting polyaniline/sodium dodecylbenzene sulfonate complex that was tested as a protective coating. The efficiency of the inhibition of copper corrosion by the complex was 86–87%, and the dissipation rate of positive and negative charges from cotton fabrics increased by 56 and 27 times, respectively.

The multi-liposomal conjugate with encapsulated enzyme laccase is described, capable of releasing the payload when changing the temperature. For this, thermosensitive cationic microgels were synthesized, differing in the molar content of cross-linker from 2 to 8 mol%. The microgels collapsed when heated beyond the volume phase transition temperature (VPTT) of 38–40 °C, while the degree of the surface area contraction (φ) decreased with increasing the cross-linker content. At lower temperature, each swollen microgel particle, ca. 320 nm in diameter, adsorbed about 200 intact 50 nm anionic liposomes. Over VPTT, the liposomes disrupted and released their payload that was visually detected via a color “development” of a loaded into liposomes laccase substrate in the surrounding solution: the higher φ, the more released enzyme and the higher colored product concentration. So, the correlation between the microgel cross-linking degree and the amount of released liposome payload was demonstrated for the first time. The results we have obtained are of interest for constructing drug carriers, catalysts and diagnostic systems.

The oxidative enzymatic polymerization of 3,4-ethylenedioxythiophene on a biopolymer DNA template is carried out. Laccase from fungus Trametes hirsuta with a high redox potential serves as a catalyst for the monomer polymerization; atmospheric oxygen is used as the oxidizing agent. Phosphate groups of DNA biopolymer, being dopants of the main chain of synthesized poly(3,4-ethylenedioxythiophene) (PEDOT), ensure its electrical conductivity. The physicochemical properties of the obtained complex and its morphology are studied. The conductivity of the PEDOT/DNA is ~0.15 mS/cm. The synthesized electrically conducting biocompatible PEDOT/DNA interpolymer complex can potentially be used for various biomedical purposes.

Polyaniline/carboxylated multiwalled carbon-nanotube composites were synthesized with the use of laccase from the fungus Trametes hirsuta as a catalyst of aniline oxidative polymerization. Atmospheric oxygen was an oxidant. Aniline dimer adsorbed on the carbon material surface served as an enhancer of the enzymatic polymerization of aniline. The composites were synthesized in deionized water without any acidic dopant. The structure, morphology, and electrochemical characteristics of the obtained nanocomposite were examined. It has been shown that the carboxylic groups on the surface of multiwalled carbon nanotubes result in the electrochemical activity of polyaniline in solutions with a neutral pH.

The development of novel materials with improved functional characteristics for supercapacitor electrodes is of current concern and calls for elaboration of innovative approaches. We report on an eco-friendly enzymatic synthesis of a composite based on poly(3,4-ethylenedioxythiophene) (PEDOT) and multi-walled carbon nanotubes (MWCNTs). The redox active compound, sodium 1,2-naphthoquinone-4-sulfonate (NQS), was used as a dopant for the backbone of the polymer. Oxidative polymerization of 3,4-ethylenedioxythiophene (EDOT) was catalyzed by a high redox potential laccase from the fungus Trametes hirsuta. Atmospheric oxygen served as an oxidant. A uniform thin layer of NQS-doped PEDOT formed on the surface of MWCNTs as a result of the enzymatic polymerization. The PEDOT–NQS/MWCNT composite showed a high specific capacitance of ca. 575 F g−1 at a potential scan rate of 5 mV s−1 and an excellent cycling stability within a potential window between −0.5 and 1.0 V, which makes it a promising electrode material for high-performance supercapacitors.

Multicopper oxidases such as bilirubin oxidase (BOD) from Myrothecium verrucaria and laccase (LC) from the basidial fungus Trametes hirsuta have been used as catalysts in dihydroquercetin (DHQ) oxidative polymerization. The conditions selected enabled good yields of DHQ oligomers, which were then analyzed using UV-vis, FTIR, 1Н and 13С NMR spectroscopy. DHQ oligomers synthesized using both enzymes showed higher thermostability as compared with the monomer. Depending on the oxidase, the products of DHQ polymerization differed in physicochemical properties, and as shown by NMR studies, had different structures.

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.

A high-redox potential laccase from fungus Trametes hirsuta was used as a biocatalyst to synthesize poly(3,4-ethylenedioxythiophene) (PEDOT), which is a promising material possessing both electronic and optical properties. This enzyme only requires molecular oxygen as an oxidant, which represents a great advantage for oxidative coupling reactions. The enzymatic oxidative polymerization of 3,4-ethylenedioxythiophene (EDOT) was performed using water-soluble sodium polystyrene sulfonate (PSS) as a template. The pH of the reaction medium and Zn2+ ions influenced the rate of EDOT polymerization. The water-dispersible PEDOT/PSS complex was characterized by UV–Vis and Fourier transformed infrared spectroscopy, as well as by transmission electron microscopy. Enzymatic EDOT polymerization was also carried out in the presence of PSS and multi-walled carbon nanotubes (MWCNT). The PEDOT/PSS/MWCNT composite had a conductivity of ca. 5.7 S/cm and specific electrochemical capacitance of ca. 246 F g−1, calculated from cyclic voltammograms at a potential scan rate of 5 mV s−1.

In this paper, a combination of the pseudocapacitances of both an enzymatically synthesized polyaniline/multi-walled carbon nanotube (PANI/MWCNT) composite with a core shell structure and a gel polymer redox electrolyte was used to improve the specific characteristics of a supercapacitor (SC). The gel of polyvinyl alcohol in sulfuric acid containing sodium 1,2-naphthoquinone-4-sulfonate (PVA/H2SO4/NQS) was used as a gel polymer redox electrolyte. Electrochemical studies have shown that the redox behavior of NQS is a diffusion-controlled and quasi-reversible process. The PANI/MWCNT composite in 13 mM NQS solution had a high specific capacitance of ca. 1100 F g−1 at a scan rate of 5 mV s−1 (three electrode cell configuration). The symmetrical flexible SC device based on the PANI/MWCNT composite and the PVA/NQS gel redox electrolyte had a power density of 1.8 kW kg−1 and an energy density of 23.3 W h kg−1. After 3000 cycles of potential scanning, the specific capacitance of the SC device decreased by less than 7%.

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and characterized seven laccases from different strains of Steccherinum ochraceum obtained from regions of central Russia. Although all seven laccases had the same primary sequences, there was a little variation in their molecular weights and thermostabilities. Moreover, statistically significant differences in laccases’ catalytic parameters of oxidation of phenolic substrates and ABTS were observed. After the deglycosylation of four selected laccases by Endo H and PNGase F, their affinities to pyrocatechol and ABTS became the same, suggesting a substantial role of N-linked glycosylation in moderation of enzymatic properties of laccases.

Deep eutectic solvents (DESs) are an alternative to conventional organic solvents in various biocatalytic reactions. Meanwhile, there have been few studies reporting on synthetic reactions in DESs or DES-containing mixtures involving oxidoreductases. In this work, we have studied the effects of several DESs based on betaine as the acceptor of hydrogen bonds on the catalytic activity and stability of laccase from the basidial fungus Trametes hirsuta and performed enzymatic polymerization of the flavonoid dihydroquercetin (DHQ, taxifolin) in a DES–buffer mixture containing 60 vol.% of betaine-glycerol DES (molar ratio 1:2). The use of the laccase redox mediator TEMPO enabled an increased yield of DHQ oligomers (oligoDHQ), with a number average molecular weight of 1800 g mol−1 and a polydispersity index of 1.09. The structure of the synthesized product was studied using different physicochemical methods. NMR spectroscopy showed that oligoDHQ had a linear structure with an average chain length of 6 monomers. A scheme for enzymatic polymerization of DHQ in a DES–buffer mixture was also proposed.

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.

A laccase (LC) from the basidial fungus Trametes hirsuta has been used for the first time as a catalyst in dihydroquercetin (DHQ) oxidative polymerization. The conditions selected enabled good yields of DHQ oligomers, which were then analyzed using UV–vis, FTIR, 1Н and 13С NMR spectroscopy. It was shown that the first stage of LC-mediated oxidation of DHQ is the removal of hydrogen from 3-OH group and formation of an alkoxy radical, which substitutes hydrogens mainly in positions C2′, C5′ and C6′. The oligomers have the number average molecular weight of 1000 g/mol and the polydispersity index of 1.4.

A new approach is proposed to biocatalytical synthesis of a thin homogeneous layer of conducting polyaniline (PANI) on the surface of MWCNTs using the laccase Trametes hirsuta. Aniline dimer, an enhancer of laccase-catalyzed aniline polymerization, has been adsorbed on MWCNTs. Phytic acid has been used as acidic dopant and gelator. The PANI/MWCNT composite has a core shell structure and shows a high specific capacitance of 554 F g−1 and long-term cycling stability. The approach enables the production of nanostructured composite conducting hydrogel.

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.

We studied bioelectrocatalytic oxidation of ethanol with membrane fractions of Gluconobacter oxydans VKM B-1280 bacterial cells participating as an electrocatalyst. Material of the electrode was graphene-like thermally expanded graphite of a high surface area and low resistivity. Membrane fractions were immobilized on the electrode surface by application in chitosan gel to prevent desorption of the biocatalyst. The bioelectrode characteristics were studied by the voltammetric and chronopotentiometric methods. It was shown that electrooxidation of ethanol on the bioanode could proceed both in the mode of direct mediator-free electrocatalysis and by the mediator mechanism.

In this study, male rats were intragastrically exposed to fullerene C60 for 1-day and 30-day periods at daily doses of 2000 and 250 mg/kg of body weight, respectively. Fullerene was detected in organs and tissues by HPLC after the extraction from biosamples with toluene. No statistically significant differences in hematological and biochemical parameters of control and treated rats were found after single and multiple administrations. Throughout the observation periods no lethality was observed. At necropsy, no pathomorphological changes in internal organs were recorded. Fullerene was found in stomach, small intestine, liver, lungs, spleen, kidneys, and blood. The amounts of the detected fullerene in comparison to the administered doses are far smaller which is the evidence of its efficient excretion. Hence, it can be assumed that fullerene nanoparticles penetrate from the gastrointestinal tract of rats into the bloodstream and translocate into secondary organs with no pronounced toxic effect in experimen...

A new method involving laccase-mediator system has been developed for environmentally friendly synthesis of polyaniline/multi-walled carbon nanotubes (PANI/MWCNT) composite. Fungal laccase, potassium octocyanomolibdate (4 + ) and atmospheric oxygen served as catalyst, redox-mediator and terminal oxidant, respectively. The structure, morphology and electrical conductivity of composites with different PANI content were investigated. The energy storage of enzymatically obtained composite consists of an electrical double layer capacitance as well as pseudocapacitance of conducting polymer. The obtained PANI/MWCNT composite with PANI content ca . 49 wt.% had high specific capacitance and cycle stability during doping/dedoping. The specific capacitance of this composite measured by cyclic voltammetry technique with potential scan rate of 5 mV/s was ca . 440 F/g. The specific capacitance of the composite decreased by less than 7% of its maximum value after 1000 scan cycles between -0.1 and 0.7 V. Supercapacitor (SC) shell was made from flexible adhesive tape (regular Scotch tape) and current collector was formed after its separation from the surface of graphite foil. The ethanol dispersion of PANI/MWCNT composite was deposited on the current collector surface. The gel polymer electrolyte (polyvinyl alcohol in 1 M phosphoric acid) was employed as both electrolyte medium and separator. The energy and power densities under an operating window of 0.7 V were ca . 7.03 Wh/kg and 5.2 kW/kg, respectively.

A new method for the enzymatic synthesis of oligoaniline soluble in organic solutions is developed. The high-redox potential laccase from the fungus Trametes hirsuta is used as a biocatalyst and atmospheric oxygen serves as oxidant. Aniline oligomers showed a high inhibition of copper corrosion in aqueous HCl and NaCl solutions.

14 Single- and multi-walled carbon nanotubes from different sources were characterized in detail, and the characteristics obtained were carefully analyzed. The carbon material with the highest capacitance, and also other superior properties (“Taunit-M” from “NanoTechCenter”, Russia), was chosen for further modification and fabrication of buckypaper based electrodes. These electrodes were biomodified with plant and fungal laccases, as well as fungal bilirubin oxidase. The designed biocathodes were investigated in simple buffers and also in a complex physiological fluid (human serum). Biocathodes based on immobilized fungal laccase were bioelectrocatalytically inactive in chloride containing media at neutral pH. In spite of the quite high current densities realized using biodevices based on plant laccase and fungal bilirubin oxidase, the limited thermal stability of the enzymes renders the biocathodes inadequate for practical applications in implanted situations.

Laccase-mediated system based on potassium octocyanomolybdate (4+) was first used for acceleration of the enzymatic aniline polymerization. The enzymatic reaction yielded oxidized octocyanomolybdate (5+) which can oxidize the aniline monomer to the aniline radical cation. This resulted in the formation of conducting polyaniline with the concomitant regeneration of the redox mediator. The proposed method is environmentally benign, permits a higher degree of control over the kinetics of the reaction and, hence, the synthesis of a conducting polymer with improved physicochemical properties. The optimal conditions for the laccase-mediated synthesis have been found. The redox mediator does not modify the backbone of conducting polyaniline (PANI). The characterization of the polymer obtained by either a laccase-catalyzed method or a laccase-mediator method was carried out using UV–vis, FTIR spectroscopy, TEM investigation and the MALDI-TOF mass spectrometry. The advantage of a laccase-mediated synthesis of PANI compared with the synthesis catalyzed by laccase alone has been shown.

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.

We have studied oxidative polymerization of aniline dimer (N-phenyl-1,4-phenylenediamine) catalyzed by high-redox potential laccase isolated from the fungi Trametes hirsuta ( Wulfen ) Pilat CF-28. Enzymatic aniline dimer polymerization was performed in aqueous micellar solution of sodium dodecylbenzenesulfonate, the atmospheric oxygen serving as an oxidizer. The resultant dispersion was stable for at least 6 months. The products synthesized were characterized using Fourier transform infrared and UV–vis spectroscopies. MALDI TOF analysis has shown that aniline dimers polymerize to mainly form aniline oligomers with the m/z ratio up to 2180, which corresponds to a polymerization degree of 24 (in terms of aniline subunits). Enzymatically formed aniline oligomers consist for the most part of para-directed units in the form of emeraldine salt. The end product structure depends on the reaction medium pH. Transmission electron microscopy has revealed granular nanoparticles of the reaction product.

The optical properties and morphology of complexes based on polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), depending on their synthesis conditions, have been characterized by UV-visible spectroscopy and atomic force microscopy. The dependence of the electron absorption spectra of PANI/PAMPS complexes and the surface topography of their films on the initiation way of PANI formation (chemical and enzymatic) and the use of promoters of aniline polymerization has been investigated. The aniline polymerization kinetics with and without polymerization promoters has been studied. All PANI/PAMPS complexes are found to have a nanocomposite time-stable structure.

Enzymatic polymerization of aniline was performed in micellar solutions of the anionic surfactant sodium dodecylbenzenesulfonate (SDBS). High-redox–potential oxidase–laccase isolated from the fungi, Trametes hirsuta, was used as a catalyst in oxidative polymerization of aniline, the atmospheric oxygen serving as an oxidizing agent. It is shown that the laccase-catalyzed reaction of monomer polymerization is kinetically controllable and its mechanism is very distinctly different from that of chemical polymerization. The synthesized PANI/SDBS complexes were characterized using Fourier transform infrared and UV–vis spectroscopy, cyclic voltammetry, termogravimetry, transmission electron microscopy, and electron diffraction. The antistatic properties of the prepared PANI/SDBS composite were also studied. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

In the present work, physico-chemical properties of conducting polyaniline (PANI) prepared by laccase-catalyzed oxidative polymerization in water dispersions of sodium dodecylbenzenesulfonate micellar solutions were studied. The polymer was characterized using FTIR spectroscopy, cyclic voltammetry, termogravimetry, transmission electron microscopy, and electron diffraction. The antistatic properties of the obtained polyaniline were also studied.

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal 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°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.

A method of enzymatic synthesis of electroconductive polyaniline on the micelles of dodecylbenzenesulfonic 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.

One step, simple and environmentally friendly enzymatic synthesis of optically active polyaniline in the bulk solution and on the surface of glass slides (in situ deposition) was developed. Fungal laccase from Trametes hirsuta was used as the catalyst of the oxidative polymerisation of aniline. The reaction was carried out under aerobic condition in the weak acidic aqueous solution in the presence of the chiral dopant, such as (S)- or (R)-camphorsulfonic acid. The doping/de-doping procedure of optically active polyaniline was reversible. It was shown that a chiral conformation of the polyaniline was quite variable, and the optical activity of the polymer could be changed by the replacement of camphorsulfonic acid enantiomer in the polyaniline structure.

Amorphous cellulose was used as a specific carrier for the deposition of self-assembled multienzyme complexes capable of catalyzing coupled reactions. Naturally glycosylated fungal cellobiohydrolases (CBHs) of glycosyl hydrolase families 6 and 7 were specifically deposited onto the cellulose surface through their family I cellulose-binding modules (CBM). Naturally glycosylated fungal laccase was then deposited onto the preformed glycoprotein layer pretreated by ConA, through the interaction of mannosyl moieties of fungal glycoproteins with the multivalent lectin. The formation of a cellulase-ConA-laccase composite was proven by direct and indirect determination of activity of immobilized laccase. In the absence of cellulases and ConA, no laccase deposition onto the cellulose surface was observed. Finally, basidiomycetous cellobiose dehydrogenase (CDH) was deposited onto the cellulose surface through the specific interaction of its FAD domain with cellulose. The obtained paste was applied onto the surface of a Clark-type oxygen electrode and covered with a dialysis membrane. In the presence of traces of catechol or dopamine as mediators, the obtained immobilized multienzyme composite was capable of the coupled oxidation of cellulose by dissolved oxygen, thus providing the basis for a sensitive assay of the mediator. Swollen amorphous cellulose plays three different roles in the obtained biosensor as: (i) a gelforming matrix that captures the analyte and its oxidized intermediate, (ii) a specific carrier for protein self-assembly, and (iii) a source of excess substrate for a pseudo-reagent-less assay with signal amplification. The detection limit of such a tri-enzyme biosensor is 50-100 nM dopamine.

The electroenzymatic reactions of Trametes hirsuta laccase in the pure organic solvent dimethyl sulfoxide (DMSO) have been investigated within the framework for potential use as a catalytic reaction scheme for oxygen reduction. The bioelectrochemical characteristics of laccase were investigated in two different ways: (i) by studying the electroreduction of oxygen in anhydrous DMSO via a direct electron transfer mechanism without proton donors and (ii) by doing the same experiments in the presence of laccase substrates, which display in pure organic solvents both the properties of electron donors as well as the properties of weak acids. The results obtained with laccase in anhydrous DMSO were compared with those obtained previously in aqueous buffer. It was shown that in the absence of proton donors under oxygenated conditions, formation of superoxide anion radicals is prevented at bare glassy carbon and graphite electrodes with adsorbed laccase. The influence of the time for drying the laccase solution at the electrode surface on the electroreduction of oxygen was studied. Investigating the electroenzymatic oxidation reaction of catechol and hydroquinone in DMSO reveals the formation of various intermediates of the substrates with different electrochemical activity under oxygenated conditions. The influence of the content of aqueous buffer in the organic solvent on the electrochemical behaviour of hydroquinone/1,4-benzoquinone couple was also studied.

Electrochemical properties of two multiforms of laccase from Trametes pubescens basidiomycete (LAC1 and LAC2) have been studied. The standard redox potentials of the T1 sites of the enzymes were found to be 746 and 738 mV vs. NHE for LAC1 and LAC2, respectively. Bioelectroreduction of oxygen based on direct electron transfer between each of the two forms of Trametes pubescens laccase and spectrographic graphite electrodes has been demonstrated and studied. It is concluded that the T1 site of laccase is the first electron acceptor, both in solution (homogeneous case) and when the enzymes are adsorbed on the surface of the graphite electrode (heterogeneous case). Thus, the previously proposed mechanism of oxygen bioelectroreduction by adsorbed fungal laccase was additionally confirmed using two forms of the enzyme. Moreover, the assumed need for extracellular laccase to communicate directly and electronically with a solid matrix (lignin) in the course of lignin degradation is discussed. In summary, the possible roles of multiforms of the enzyme based on their electrochemical, biochemical, spectral, and kinetic properties have been suggested to consist in broadening of the substrate specificity of the enzyme, in turn yielding the possibility to dynamically regulate the process of lignin degradation according to the real-time survival needs of the organism.

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.

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.

An express electrochemical method for determining the metabolic activity of live cells based on the possibility of an electron exchange between an electrode and elements of the biological electron transfer chain in the presence of a mediator is proposed. This method is useful for studying any live cells (animal, plant, and microbial), including anaerobic, dormant, and spore cells. The sample preparation and measurement itself does not take more than 30 min. The detection limit in a volume of 15 ml amounts to 105 cells/ml. The applicability of the assessment method of the metabolic activity level during the transition of the bacteria Mycobacterium smegmatis into an uncultivable dormant state was demonstrated. This method is of special value for medicine and environmental control, detecting latent forms of pathogens. An optimal combination of the methods for the express analysis of latent pathogens is proposed.

This paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. The issue that is reconsidered here is the previously proposed existence of ''active" and "resting" forms of laccases. The presence of fungal laccases with partly reduced active sites is demonstrated. Of further interest is that an aggregated state in solution, not to our knowledge previously noted for laccase, has been found by using small-angle X-ray scattering as well as thorough analysis of the results of several biochemical experiments. Under some conditions, this aggregated state may correlate with the resting form of the laccases, although this resting form could have a broader significance. It was shown that Trametes ochracea laccase had some anomalous characteristics, which could be correlated with the high concentration of the "resting" enzyme. The mechanism of formation of resting laccase is suggested. Knowledge of the resting state is of importance for in vitro studies. Additionally, a suggestion about the possible regulatory role of this form in vivo is mentioned. (c) 2006 Elsevier SAS. All rights reserved.

The interaction between fungal laccases from Trametes basidiomycetes and different lignins has been studied using electrochemical and chromatographic methods. The kinetic parameters for the oxidation reaction of hydroquinone and for Kraft lignin by molecular oxygen catalysed by Trametes hirsuta laccase have been investigated. Polymerisation of lignin by laccase was shown. The process was independent on the source of lignin as well as on the origin and purity of the enzymes. A complex pattern of interaction between the laccase-mediator system (LMS) and lignin was shown. Along with the polymerisation of lignin, the ability to degrade lignin using the LMS was observed. Data generated by combining size exclusion chromatographic separation of lignin with the use of a laccase modified electrode as post column detector, strongly suggest that laccase can oxidise natural lignin by a long-range electron transfer mechanism. The influence of the origin of lignin as well as source of the enzyme on this process was also shown. (c) 2006 Elsevier Inc. All rights reserved. (Less)

Four different ways to detect Kraft and soluble pine lignin using either soluble or immobilised Trametes hirsuta laccase have been investigated. In the first, the concentration of lignin was measured using laccase in homogeneous medium with a Clark electrode. In the second, laccase immobilised onto aminated porous glass beads was used in a reactor submerged into the lignin containing solution and the reaction was followed with a Clark electrode. The third and fourth techniques involved lignin detection using laccase modified spectrographic graphite electrodes, either used in flow injection mode or in batch mode, where lignin solutions were injected into an electrochemical cell. It was shown that the use of laccase modified graphite electrodes in conjunction with flow injection for amperometric lignin detection is an attractive perspective and our data form the basis for designing laccase-based biosensors to be applied for measuring lignin and its model compounds in waste water, e.g. from the pulp and paper industry.

Mediatorless (direct) electron transfer between bare and thiol-modified gold electrodes and fungal laccases from different sources has been demonstrated. The electrochemical activity of the enzymes from basidiomycetes Trametes hirsuta, Trametes ochracea, and Cerrena maxima under aerobic and anaerobic conditions can clearly be observed using cyclic voltammetry and spectroelectrochemistry. Bioelectroreduction of oxygen by T. hirsuta laccase immobilized on amino-thiophenol-modified gold electrodes, starting at +625 mV vs. NHE, is demonstrated and differences in bioelectrocatalysis of the enzyme immobilized on bare and thiol-modified electrodes are shown. It was found that hydrogen peroxide was one of the products of oxygen electroreduction on gold electrodes modified with fungal laccases, whereas no significant peroxide formation was observed for T. hirsuta laccase immobilised on thiol-modified gold electrodes. Thus, a hypothesis about two different mechanisms of oxygen electroreduction by fungal laccases adsorbed on bare and thiol-modified electrodes is proposed., The work has been financially supported by the Swedish Research Council, by the Swedish Institute (SI), by the Consejería de Educación de la Comunidad de Madrid, and the European Social Funding (F.S.E.). The SI is acknowledged for the support of a postdoctoral fellowship for S.S.

Electrochemical studies of laccases from basidiomycetes, i.e., Trametes hirsuta, Trametes ochracea, Coriolopsis fulvocinerea, Cerrena maxima, and Cerrena unicolor, have been performed. Direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. For all mentioned high potential laccases direct electron transfer (DET) has been registered at spectrographic graphite and highly ordered pyrolytic graphite electrodes. The characteristics of DET reactions of the enzymes were analysed under aerobic and anaerobic conditions. It is shown that the T I site of the laccase is the primary electron acceptor, both in solution (homogenous case) and at surface of the graphite electrode (heterogeneous case). A mechanism of ET for the process of the electroreduction of oxygen at the laccase-modified graphite electrodes is proposed and the similarity of this heterogeneous process to the laccase catalysed oxygen reduction homogeneous reaction is concluded. (c) 2005 Elsevier B.V. All rights reserved. (Less)

An amperometric enzyme electrode based on laccase for determining phenolic compounds is proposed. The following three types of polymer materials were used for enzyme immobilization on the surface of a glassy-carbon electrode: positively charged cetyl ethyl poly (ethyleneimine) (CEPEI) and negatively charged commercial Nafion and Eastman AQ 29D polymers. The advantages and disadvantages of each of the above polymers for enzyme immobilization are discussed. The detection limits of the model phenolic compounds hydroquinone and pyrocatechol in a buffer solution on laccase immobilization in a Nation membrane were 3.5 x 10(-8) and 5.0 x 10(-8) M, respectively, at a signal-to-noise ratio of 3. Electrodes with laccase immobilized in Nation and Eastman AQ 29D membranes exhibited the shortest response time. The operating stability and the stability in storage can be significantly improved by the additional incorporation of gelatin in the polymer matrices. Gelatin prevents enzyme inactivation as a result of enzyme modification by the free-radical oxidation products of phenolic compounds. (Less)

The electrochemistry of some copper-containing proteins and enzymes, viz. azurin, galactose oxidase, tyrosinase (catechol oxidase), and the "blue" multicopper oxidases (ascorbate oxidase, bilirubin oxidase, ceruloplasmin, laccase) is reviewed and discussed in conjunction with their basic biochemical and structural characteristics. It is shown that long-range electron transfer between these enzymes and electrodes can be established, and the mechanistic schemes of the DET processes are proposed. (c) 2004 Elsevier B. V. All rights reserved.

Mediatorless, electrochemically driven, redox transformations of T1 (type 1) and T2 copper sites in Trametes hirsuta laccase were studied by cyclic voltammetry and spectroelectrochemical redox titrations using bare gold electrode. DET (direct electron transfer) between the electrode and the enzyme was observed under anaerobic conditions. From analysis of experimental data it is concluded that the T2 copper site is in DET contact with gold. It was found that electron transfer between the gold surface and the T1 copper site progresses through the T2 copper site. From EPR measurements and electrochemical data it is proposed that the redox potential of the T2 site for high-potential ‘blue’ laccase is equal to about 400 mV versus NHE (normal hydrogen electrode) at pH 6.5. The hypothesis that the redox potentials of the T2 copper sites in low- and high-potential laccases/oxidases from totally different sources might be very similar, i.e. approx. 400 mV, is discussed.

Catalytic properties of monoamino oxidase (MAO) in a homogeneous reaction of oxidation and a heterogeneous reaction of bioelectrocatalytic oxidation of benzylamine by derivatives of phenothiazine and phenoxazine (artificial electron acceptors) is studied. The efficiency of electroenzymic catalysis involving Methylene Blue and MAO immobilized on the electrode is comparable to that of homogeneous enzymic catalysis. When immobilized in a film of polymer electrolyte Eastman AQ-29, Methylene Blue and Toluidine Blue efficiently carry electrons from the reduced redox center of immobilized MAO onto a glassy-carbon electrode. Dependence of the oxidation current of these compounds on the concentration of the benzylamine substrate is studied and it is shown that the enzymic reaction is the limiting stage in a bioelectrocatalytic process. This conclusion gives grounds for using the dependence of the anodic current on the monoamine concentration as a calibration plot when assaying biogenic amines.