O. V. Koroleva

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.

Mitigating Activity of Humic Substances: Direct Influence on Biota

Mitigating activity of HS can be defined as a phenomenon of lowering the adverse effects of contaminants toxicity and of those of abiotic stress factors such as unfavourable temperature, pH, salinity, etc. As a rule, it is related to the detoxifying properties of HS or to their beneficial effects on biota. This review focuses on the latter effects and considers the possible mechanisms of mitigating activity of humic materials in terms of biological activity of HS. The beneficial effects of HS on biota are segregated into the four categories according to the underlying mechanism of their action: an influence on the organism development; an enhancement in nutrient supply; catalysis of the biochemical reactions; and an antioxidant activity. The published data on the mentioned essential biological functions of HS are reviewed.

Characterization of plant phenolic compounds by cyclic voltammetry

Phenolic acids and flavonoids were characterized by cyclic voltammetry and total antioxidant activity in the reaction with the ABTS cation radical. Anode peak voltages (Eap) and their pH dependences were determined for the studied phenolic acids and flavonoids. The Eap and Trolox equivalent antioxidant capacity (TEAC) values were found to correlate for polyphenols, which react with the ABTS cation radical in two steps. Correlation between the half-wave potential (E1/2) and TEAC was determined for electrochemically irreversible compounds. Mechanisms of the reaction of phenolics on the electrode involving one-and two-electron oxidation are proposed.

Structure of native laccase from Trametes hirsuta at 1.8 Å resolution

This paper describes the structural analysis of the native form of laccase from Trametes hirsuta at 1.8 A resolution. This structure provides a basis for the elucidation of the mechanism of catalytic action of these ubiquitous proteins. The 1.8 A resolution native structure provided a good level of structural detail compared with many previously reported laccase structures. A brief comparison with the active sites of other laccases is given.

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.

Laccase and Mn-peroxidase production by Coriolus hirsutus strain 075 in a jar fermentor.

The white-rot fungus Coriolus hirsutus strain 075 excretes considerable amounts of laccase and Mn-peroxidase into culture broth over a brief production time. The effects of agitation speed, temperature, aeration and inoculum amount on laccase production using a 10-l fermentor were studied. The optimum fermentation conditions were a 15% inoculum, an aeration rate of 0.88 vvm, an agitation speed of 160 rpm, and a temperature of 28 degrees C. By optimizing the fermentation conditions, the laccase activity reached 80+/-3 U/ml in 3 d and the purified enzyme output was 30 mg/l. The laccase and Mn-peroxidase were purified by means of isoelectrofocusing and ion-exchange chromatography. The pIs of the laccase isoenzymes were 4.2 and 4.5. Mn-peroxidase had only one isoenzyme with a pI of 3.2. The optimum pH was 4.5 for laccase with syringaldazine as the substrate and 5.0-5.3 for Mn-peroxidase with Mn(+2) and H2O2 as the substrates. The laccase and Mn-peroxidase retained 50% of their activities at 50 degrees C after 55 h and 12 h of incubation time, respectively.

Isolation and Study of Some Properties of Laccase from the Basidiomycetes Cerrena maxima

A new strain producing extracellular laccase (Cerrena maxima 0275) was found by screening of isolates of Basidiomycetes, and the dynamics of laccase biosynthesis by this strain was studied. The enzyme was purified to homogeneity. The molecular weight of the enzyme is 57 kD, and its pI is 3.5. The activity is constant at pH values in the range 3.0-5.0. The temperature optimum for activity is 50°C. The thermal stability of the laccase was studied. The catalytic and Michaelis constants for catechol, hydroquinone, sinapinic acid, and K4 Fe(CN)6 were determined. The standard redox potential of type 1 copper in the enzyme is 750 ± 5 mV. Thus, the investigated laccase is a high redox potential laccase.

Extremophilic microorganisms: Biochemical adaptation and biotechnological application (review)

In this review, we analyzed modern data on the biochemical adaptation of microorganisms to living under extreme conditions. Special attention is given to analysis of adaptation responses of microorganisms under exposure to increased radiation at molecular and cellular levels. Data on the practical use of extremophiles as well as extremoenzymes, biologically active compounds, biopolymers, etc., synthesized by them are systematized.

Laccases from Basidiomycetes: Physicochemical Characteristics and Substrate Specificity towards Methoxyphenolic Compounds

Laccases from the Basidiomycetes Coriolus hirsutus, Coriolus zonatus, Cerrena maxima, and Coriolisimus fulvocinerea have been isolated and purified to homogeneity and partially characterized. The kinetics of oxidation of different methoxyphenolic compounds by the fungal laccases has been studied. As laccase substrates, such methoxyphenolic compounds as 4-hydroxy-3,5-dimethoxycinnamic acid (sinapinic acid), 4-hydroxy-3-methoxycinnamic acid (ferulic acid), and 2-methoxyphenol (guaiacol) were used. The stoichiometries of the enzymatic reactions were determined: guaiacol and sinapinic acid are one-electron donors and their oxidation apparently results in the formation of dimers. It was established that kcat/Km, which indicates the effectiveness of catalysis, increases in the series guaiacol, ferulic acid, and sinapinic acid. This fact might be connected with the influence of substituents of the phenolic ring of the substrates. This phenomenon was established for fungal laccases with different physicochemical properties, amino acid composition, and carbohydrate content. This suggests that all fungal laccases possess the same mechanism of interaction between organic substrate electron donors and the copper-containing active site of the enzyme and that this interaction determines the observed values of the kinetic parameters.

Intramolecular electron transfer in laccases

Rate constants and activation parameters have been determined for the internal electron transfer from type 1 (T1) to type 3 (T3) copper ions in laccase from both the fungus Trametes hirsuta and the lacquer tree Rhus vernicifera, using the pulse radiolysis method. The rate constant at 298 K and the enthalpy and entropy of activation were 25 ± 1 s−1, 39.7 ± 5.0 kJ·mol−1 and −87 ± 9 J·mol−1·K−1 for the fungal enzyme and 1.1 ± 0.1 s−1, 9.8 ± 0.2 kJ·mol−1 and −211 ± 3 J·mol−1·K−1 for the tree enzyme. The initial reduction of the T1 site by pulse radiolytically produced radicals was direct in the case of T. hirsuta laccase, but occured indirectly via a disulfide radical in R. vernicifera. The equilibrium constant that characterizes the electron transfer from T1 to T3 copper ions was 0.4 for T. hirsuta laccase and 1.5 for R. vernicifera laccase, leading to full reduction of the T1 site occurring at 2.9 ± 0.2 electron equivalents for T. hirsuta and 4 electron equivalents for R. vernicifera laccase. These results were compared with each other and with those for the same process in other multicopper oxidases, ascorbate oxidase and Streptomyces coelicolor laccase, using available structural information and electron transfer theory.