G. P. Shumakovich

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.

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 (∼620 kD), isoelectric point (pI6.1), 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% α-helix, 25% β-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.

Micellar laccase-catalyzed synthesis of electroconductive polyaniline

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.

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 oxidation of manganese ions catalysed by laccase.

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.

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°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.

Laccase-catalyzed synthesis of aniline oligomers and their application for the protection of copper against corrosion

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.

Electrochemical investigation of binding sites of plantacyanin: blue, copper-containing protein of plants

Abstract The binding site structure of plantacyanin (plant blue Cu-protein) was studied using voltammetry at the electrodes modified with sulphur-containing compounds (thioglycolic acid, dipyridyl disulfide, cysteamine, β-mercaptoethanol). The scanning of the functional groups of the modifying layer was used to reveal the most efficient modifications of the electrode and to determine the character of the binding groups of the protein. A comparison of the electron exchange characteristics of plantacyanin at the different electrodes with that of the other proteins of the known binding site structure (plastocyanin, cytochrome c, cytochrome c553, high potential ion-sulfur protein) was carried out. By means of this comparison and also using literary data about plantacyanin tertiary structure, the localization of the protein binding sites and their hydrophobicity were evaluated. Two poles was suggested to exist with a high permeability to electrons. One of these binding sites is of a high hydrophobicity (4.1–6.7 kj mol−1). The other is hydrophilic and contains several charged amino groups. The problem of how to search the partners of plantacyanin in the electron transport chain was discussed.

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 1H and 13C 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.

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.