RESUMO
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.
Assuntos
Compostos de Anilina/síntese química , Biomimética/métodos , DNA/química , Peroxidase do Rábano Silvestre/química , Nanocompostos/química , Biocatálise , Dicroísmo Circular , Condutividade Elétrica , Eletroforese em Gel de Ágar , Peroxidase do Rábano Silvestre/metabolismo , Microscopia de Força Atômica , Oxirredução , Espectroscopia de Infravermelho com Transformada de Fourier , EstereoisomerismoRESUMO
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.
Assuntos
Compostos de Anilina/síntese química , Lacase/química , Polímeros/síntese química , Ácidos Sulfônicos/síntese química , Trametes/enzimologia , Compostos de Anilina/química , Celulose/química , Reagentes de Ligações Cruzadas/química , Condutividade Elétrica , Enzimas Imobilizadas/química , Isoxazóis/química , Oxirredução , Polímeros/química , Ácidos Sulfônicos/química , Propriedades de SuperfícieRESUMO
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.
Assuntos
Compostos de Anilina/síntese química , Basidiomycota/enzimologia , Benzenossulfonatos/química , Proteínas Fúngicas/química , Lacase/química , Compostos de Anilina/química , Condutividade Elétrica , Concentração de Íons de Hidrogênio , MicelasRESUMO
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.
Assuntos
Ativadores de Enzimas/farmacologia , Lacase/metabolismo , Xenobióticos/metabolismo , Biotecnologia/métodos , Transporte de Elétrons , Lignina/metabolismo , Oxirredução , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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.
Assuntos
Oxirredutases do Álcool/química , Benzoquinonas/química , Oxidantes/química , Oxigênio/química , Pichia/enzimologia , 2,6-Dicloroindofenol/química , Catálise , Eletrodos , Transporte de Elétrons , Formaldeído/química , Ouro , Azul de Metileno/química , Oxirredução , Cloreto de Tolônio/químicaRESUMO
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 10-5 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.
Assuntos
Mycobacterium smegmatis/fisiologia , Eletroquímica/métodos , Eletrodos , Mycobacterium smegmatis/citologiaRESUMO
Twenty strains of the wood-degrading fungi from the genus Trametes Fr., capable of synthesizing laccases, were screened according to the changes in the oxidase activity in a submerged culture. The range of maximal efficiency of various species with respect to extracellular oxidase activity was determined. The absence of correlation between the oxidase activity in a submerged culture and the size of colored zone on agar media (Bavendamm reaction) was demonstrated. The most efficient strains, T. hirsita 56 and T. ochracea 92-78, were used to produce laccases, homogeneous according to SDS electrophoresis data. A number of biochemical parameters characteristic of these enzymes were determined.
Assuntos
Proteínas Fúngicas/análise , Proteínas Fúngicas/biossíntese , Lacase/análise , Lacase/biossíntese , Polyporales/enzimologia , Basidiomycota/enzimologia , Basidiomycota/crescimento & desenvolvimento , Técnicas de Cultura de Células , Oxirredutases/análise , Oxirredutases/biossíntese , Polyporales/crescimento & desenvolvimentoRESUMO
An enzymatic method of producing a conducting polyelectrolyte complex of polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) was developed. Acidic stable peroxidase isolated from royal palm tree (Roystonea regia L.) leaves was used as a catalyst in the oxidative polymerization of aniline at pH 2.8. The synthesis procedure was optimized. Spectroscopic and electrochemical characteristics of nanoparticles of obtained PANI/PAMPS complexes at different pH were studied. It was shown that the acidity of the medium affects their properties.
Assuntos
Acrilamidas/química , Alcanossulfonatos/química , Compostos de Anilina/química , Eletrólitos/química , Peroxidase/química , Arecaceae/enzimologiaRESUMO
Optimal conditions for preparing laccase conjugates by the periodate method have been selected. The effect of the initial molar ratio of IgG to laccase and pH of the medium on the composition of laccase conjugates was studied by the HPLC method. The maximum yield of the conjugates was observed, when laccase was oxidized with 0.12 M sodium periodate the pH of the medium was 8.5, and the initial molar ratio of IgG to laccase was 2:1. The conjugates can be stored for one year without any loss in immunological activity.
Assuntos
Imunoglobulina G/química , Oxirredutases/química , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Técnicas Imunoenzimáticas , Lacase , Oxirredutases/imunologia , Ácido Periódico/químicaRESUMO
Light-addressable potentiometric sensors were prepared by immobilizing glucose oxidase or alpha-chymotrypsin on the surface of a pH-sensitive electrode. Two methods of immobilization were used: incorporation into a hydrophilic matrix of bovine serum albumin and incorporation into a hydrophobic matrix of modified polyethylenimine. The glucose oxidase and alpha-chymotrypsin preparations immobilized in the polyethylenimine matrix were characterized by the Michaelis constant values of 6.0 and 3.1. mM, respectively, and by pH optimums at 6.0-7.2 and 7.0-8.0, respectively. These values are consistent with results obtained by other methods. Therefore, light-addressable potentiometric sensors can be used in combination with both hydrophilic and hydrophobic matrices. The ability to combine light-addressable potentiometric sensors with hydrophobic matrices provides an opportunity for the development of potentiometric biosensors for detecting substances poorly soluble in water.
Assuntos
Técnicas Biossensoriais , Quimotripsina/metabolismo , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/metabolismo , Eletrodos , Concentração de Íons de Hidrogênio , Cinética , Luz , Penicillium/enzimologia , PolímerosRESUMO
A study to determine optimal molar ratios of Protein A to laccase for the synthesis of their conjugate by periodate method is presented. No loss of enzymatic or immunological activity of the conjugate developed was observed during 6 month. The conjugate could be effectively used in various techniques of enzyme immunoassay (competitive or sandwich techniques, dot immunoblotting) for immunoglobulin G. The detection limits of the assays for immunoglobulins were better than 1 ng/ml.
Assuntos
Oxirredutases/química , Proteínas Recombinantes de Fusão/síntese química , Proteína Estafilocócica A/química , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G , Lacase , Ácido Periódico/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The possibility of using homovanillic acid as a substrate of laccase (produced by the basidiomycete Coryolus hirsutus) has been demonstrated for the first time. The reaction was shown to result in the formation of a fluorescent product. Several kinetic parameters and optimal conditions were determined for this enzymatic reaction. The use of homovanillic acid as the substrate was found to increase the enzyme immunoassay sensitivity by an order of magnitude, compared to conventional substrates.
Assuntos
Técnicas Imunoenzimáticas , Oxirredutases/metabolismo , Basidiomycota/enzimologia , Ácido Homovanílico/metabolismo , Cinética , Lacase , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
The kinetics and action mechanism of the galactose oxidase from Fusarium graminearum were studied. pH-optimum of the enzyme activity and stability was 7.0, the activity and stability of the galactose oxidase being decreased at any other values of pH. The enzyme is destabilized at acidic pH that is connected with protonization of its ionogenic group with pK 4.7. The temperature optimum of the galactose oxidase is 35 degrees C. When studying the enzyme thermoinactivation, it was found that at temperatures below 30 degrees C the energy of activation of denaturation was about 40 kcal/mole and at temperatures ranging from 30 to 70 degrees C - 13 kcal/mole. On the basis of the data obtained it was concluded that a low-temperature form of the galactose oxidase, possessing a higher energy of activation of denaturation, is more active than a high-temperature form. The value of Km for the enzyme in respect to galactose was 0.19 M, and the value of Vmax = 360 mumole/min per g of the preparation.
Assuntos
Fusarium/enzimologia , Galactose Oxidase/metabolismo , Estabilidade de Medicamentos , Galactose/metabolismo , Galactose Oxidase/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , TemperaturaRESUMO
A new immunochemical reagent is proposed which contains laccase, isolated from the culture liquid of the basidial fungus Coriolus hirsutus, as a marker enzyme. The feasibility of immunolaccase conjugates for different variants of immunoassay, i.e. "sandwich", competitive and indirect, is demonstrated. The comparison of immunolaccase and immunoperoxidase conjugates showed that the absolute sensitivity of laccase-antibody conjugates was 3 times higher than that of antibody-peroxidase conjugates (7.7 x 10(-11) M and 2.3 x 10(-10) M, respectively). The assay based on antibody-laccase conjugates is simpler than that employing antibody-peroxidase conjugates, since in the former case air oxygen in used as the second substrate of the enzymatic reaction.
Assuntos
Basidiomycota/enzimologia , Técnicas Imunoenzimáticas , Oxirredutases , Animais , Calibragem , Estudos de Avaliação como Assunto , Imunoglobulinas/análise , Indicadores e Reagentes , Insulina/análise , Anticorpos Anti-Insulina/análise , Lacase , Ligantes , Camundongos , Oxirredutases/imunologia , Oxirredutases/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
An approach was developed to screening organic compounds for putative activity of redox mediators of oxidoreductases, including laccases and peroxidases, applicable for xenobiotic degradation. The study was carried out with a homogenous laccase preparation from the basidiomycete Trametes hirsuta and horse-radish root peroxidase. Compounds belonging to 1-phenyl-3-methylpyrazolones were selected. Spectroscopic and electrochemical investigation of two of the compounds, sodium 1-phenyl-2,3-dimethyl-4-aminopyrazolon 5n(4)-methanesulfonate (PPNa) and 1-(3'-sulfophenyl)-3-methylpyrazolone (SPP), was performed. Electrochemical oxidation of both PPNa and SPP gave rise to high-potential intermediates capable of oxidizing veratryl alcohol; a lignin-modeling compound. Kinetic indices of these compounds were determined in enzymatic reactions with the presence of laccase. It was shown that enzymatic oxidation of SPP by laccase produced high-potential intermediates capable of oxidizing veratryl alcohol to veratric acid. Veratryl alcohol did not oxidize during enzymatic oxidation of SPP by peroxidase. This points to a difference between the mechanisms of enzymatic oxidation of PPNa and SPP by laccase and peroxidase.
Assuntos
Basidiomycota/enzimologia , Pirazóis/química , Pirazolonas , Ácido Vanílico/análogos & derivados , Xenobióticos/metabolismo , Álcoois Benzílicos/química , Álcoois Benzílicos/metabolismo , Peroxidase do Rábano Silvestre , Lacase/química , Lacase/metabolismo , Oxirredução , Ácido Vanílico/química , Xenobióticos/químicaRESUMO
The effects of various factors on the biosynthesis of extracellular laccase (EC 1.14.18.1) by the basidiomycete Coriolus hirsutus (Wulf.: Fr.) Quel. no. 072 during submerged cultivation were examined. Optimal parameters for cultivation in a fermenter of 10 l were determined: temperature, 28 degrees C; stirrer rotation speed, 160 rpm; and the inoculum volume, 15% of the working volume of the fermenter. The filtrate contained peroxidase, laccase, and phenol oxidase activities and displayed a high thermal stability.
Assuntos
Basidiomycota/metabolismo , Oxirredutases/biossíntese , Basidiomycota/citologia , Meios de Cultura , LacaseAssuntos
Técnicas Biossensoriais , Óxidos/química , Potenciometria , Tantálio/química , Acetobacteraceae/metabolismo , Quimotripsina/metabolismo , Éter/química , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Luz , Oxirredução , Tirosina/análogos & derivados , Tirosina/química , Tirosina/metabolismoRESUMO
It is known that human ceruloplasmin (CP) is made up of several isoforms which differ by the structure of their carbohydrate fragment. One of these isoforms, CP1, which makes up to approximately 40% of the native CP molecule and which contains a carbohydrate fragment, [formula: see text] is specifically bound to human erythrocyte (ER) receptors. This isoform was isolated by using lectin affinity chromatography. It was found that CP1 produces a much stronger protective effect on ER during Cu(2+)-induced lysis as compared with CP. A kinetic analysis of Cu2+ accumulation and reduced glutathione (GSH) decline in ER revealed that the lack of correlation between these two processes. It was found that in the presence of CP and CP1 the GSH concentration is not critical for the hemolytic resistance of ER. In the presence of CP1 ER hemolysis occurs at a slower rate whereas the GSH decline at a much faster rate than in the presence of CP.
Assuntos
Ceruloplasmina/fisiologia , Cobre/toxicidade , Eritrócitos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Ceruloplasmina/metabolismo , Cromatografia de Afinidade , Radicais Livres , Glutationa/metabolismo , Humanos , Cinética , Superóxidos/metabolismoRESUMO
The effects of ligands of active and inhibitory centers of homogeneous aldose reductase from cattle eye lens on glucose reduction were studied. Using spectrophotometric titration and equilibrium gel filtration, the interaction of the enzyme active center with substrates was investigated. It was shown that the reaction kinetics obeys a mechanism with a quasi-equilibrium non-ordered attachment of substrates and isomerization of enzyme complexes with nicotinamide dinucleotide phosphates in the course of the catalytic act. It was found that the NADPH in equilibrium NADP equilibrium in the enzyme active center is shifted to the right; however, NADP dissociation may occur only as a result of the aldehyde reduction. The mechanisms of regulation of the enzyme activity by NADP, ADP and alpha-glycerol phosphate were proposed. It was shown that the binding of catalin and morine to the enzyme results in the inhibition of the enzymatic reaction and in the isomerization blocking. It was found that the inhibitory site of the isomeric form of aldose reductase displays a lower affinity for morine.
Assuntos
Aldeído Redutase/metabolismo , Difosfato de Adenosina/farmacologia , Aldeído Redutase/antagonistas & inibidores , Animais , Sítios de Ligação , Bovinos , Cromatografia em Gel , Inibidores Enzimáticos , Flavonoides/farmacologia , Indicadores e Reagentes , Isomerismo , Cinética , Cristalino/enzimologia , NADP/metabolismo , Oxazinas/farmacologia , OxirreduçãoRESUMO
A homogeneous aldose reductase was isolated from bovine eye lens tissue by using affinity chromatography on blue agarose. A kinetic analysis of the initial rates of NADPH oxidation at 0.5-100 mM glucose and at 1.2-10 microM NADPH was carried out. The Line-weaver-Burk plots for glucose concentration were nonlinear at fixed concentrations of NADPH and linear at fixed concentrations of glucose. It was shown that the experimental plots reflect the mechanisms, in which substrate regulation of enzyme activity is effectuated by glucose binding to the regulatory site or is due to the shift of the equilibrium between the isomeric forms of aldose reductase.