RESUMEN
Laccases belong to the class of multicopper oxidases catalyzing the oxidation of phenols accompanied by the reduction of molecular oxygen to water without the formation of hydrogen peroxide. The activity of laccases depends on the number of Cu atoms per enzyme molecule. The structure of type 2 copper-depleted laccase from Botrytis aclada has been solved previously. With the aim of obtaining the structure of the native form of the enzyme, crystals of the depleted laccase were soaked in Cu(+)- and Cu(2+)-containing solutions. Copper ions were found to be incorporated into the active site only when Cu(+) was used. A comparative analysis of the native and depleted forms of the enzymes was performed.
Asunto(s)
Botrytis/enzimología , Cobre/metabolismo , Lacasa/metabolismo , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Iones , Lacasa/química , Modelos Moleculares , Datos de Secuencia Molecular , TemperaturaRESUMEN
We have developed and synthesized nanobiocomposite materials based on graphene, poly(3,4-ethylenedioxythiophene), and glucose oxidase immobilized on the surface of various nanomaterials (gold nanoparticles and multi-walled carbon nanotubes) of different sizes (carbon nanotubes of different diameters). Comparative studies of the possible influence of the nanomaterial's nature on the bioelectrocatalytic characteristics of glucose- oxidizing bioanodes in a neutral phosphate buffer solution demonstrated that the bioelectrocatalytic current densities of nanocomposite-based bioanodes are only weakly dependent on the size of the nanomaterial and are primarily defined by its nature. The developed nanobiocomposites are promising materials for new bioelectronic devices due to the ease in adjusting their capacitive and bioelectrocatalytic characteristics, which allows one to use them for the production of dual-function electrodes: i.e., electrodes which are capable of generating and storing electric power simultaneously.
RESUMEN
We unveil experimental evidence that put into question the widely held notion concerning the impact of nanoparticles on the bioelectrocatalytic parameters of enzymatic electrodes. Comparative studies of the bioelectrocatalytic properties of fungal bilirubin oxidase from Myrothecium verrucaria adsorbed on gold electrodes, modified with gold nanoparticles of different diameters, clearly indicate that neither the direct electron transfer rate (standard heterogeneous electron transfer rate constants were calculated to be 31±9 s(-1)) nor the biocatalytic activity of the adsorbed enzyme (bioelectrocatalytic constants were calculated to be 34±11 s(-1)) depends on the size of the nanoparticles, which had diameters close to or larger than those of the enzyme molecules.
RESUMEN
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.
Asunto(s)
Bioquímica/métodos , Cobre/química , Electroquímica/métodos , Lacasa/química , Oxidorreductasas/química , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Sitios de Unión , Biotecnología/métodos , Catálisis , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Conformación Molecular , Datos de Secuencia Molecular , Oxígeno/químicaRESUMEN
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.
Asunto(s)
Activadores de Enzimas/farmacología , Lacasa/metabolismo , Xenobióticos/metabolismo , Biotecnología/métodos , Transporte de Electrón , Lignina/metabolismo , Oxidación-Reducción , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Oxidorreductasas de Alcohol/química , Benzoquinonas/química , Oxidantes/química , Oxígeno/química , Pichia/enzimología , 2,6-Dicloroindofenol/química , Catálisis , Electrodos , Transporte de Electrón , Formaldehído/química , Oro , Azul de Metileno/química , Oxidación-Reducción , Cloruro de Tolonio/químicaRESUMEN
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.
Asunto(s)
Proteínas Fúngicas/análisis , Proteínas Fúngicas/biosíntesis , Lacasa/análisis , Lacasa/biosíntesis , Polyporales/enzimología , Basidiomycota/enzimología , Basidiomycota/crecimiento & desarrollo , Técnicas de Cultivo de Célula , Oxidorreductasas/análisis , Oxidorreductasas/biosíntesis , Polyporales/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Mycobacterium smegmatis/fisiología , Electroquímica/métodos , Electrodos , Mycobacterium smegmatis/citologíaRESUMEN
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.
Asunto(s)
Oxidorreductasas de Alcohol/aislamiento & purificación , Proteínas Fúngicas/aislamiento & purificación , Pichia/enzimología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Etanol/metabolismo , Formaldehído/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metanol/metabolismo , Peso Molecular , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Benzoatos/química , Lacasa/química , Pirazolonas/química , Quinolonas/química , Basidiomycota/enzimología , Benzotiazoles , Electroquímica , Activación Enzimática , Cinética , Estructura Molecular , Oxidación-Reducción , Especificidad por Sustrato , Ácidos Sulfónicos/químicaRESUMEN
A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.
Asunto(s)
Basidiomycota/enzimología , Lacasa/aislamiento & purificación , Lignina/metabolismo , Peroxidasas/aislamiento & purificación , Cromatografía Liquida , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Lacasa/metabolismo , Peroxidasas/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
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.
Asunto(s)
Acrilamidas/química , Alcanosulfonatos/química , Compuestos de Anilina/química , Electrólitos/química , Peroxidasa/química , Arecaceae/enzimologíaRESUMEN
New strains of basidiomycetes producing extracellular laccases (Trametes ochracea 92-78, and Trametes hirsuta 56) have been found by screening of isolates of Trametes fungi. The laccases from T. hirsuta 56 and T. ochracea 92-78 as well as two laccases from previously found and described strains of basidiomycetes, namely Cerrena maxima and Coriolopsis fulvocinerea, were purified to homogeneity. The standard redox potentials of type 1 copper in the enzymes were determined and found to be 780, 790, 750, and 780 mV, respectively. The spectral and biochemical studies showed that the enzymes had no significant differences between the structures of their active sites (T1, T2, and T3). In spite of this fact, the basic biochemical properties as well as the redox potentials of the T1 sites of the enzymes were found to be different. The molecular weights of the laccases range from 64 to 70 kDa, and their pI values range from 3.5 to 4.7. The pH-optima are in the range 3.5-5.2. The temperature optimum for activity is about 50 degrees C. The thermal stabilities of the enzymes were studied. The catalytic and Michaelis constants for catechol, guaiacol, hydroquinone, sinapinic acid, and K(4)Fe(CN)(6) were determined. Based on these results as well as results obtained by comparing with published properties of several laccases, it could be concluded that T. hirsuta and Cerrena maxima laccases have some superior characteristics such as high stability, high activity, and low carbohydrate content, making them attractive objects for further investigations as well as for application in different areas of biotechnology.
Asunto(s)
Basidiomycota/enzimología , Proteínas Fúngicas/química , Lacasa/química , Sitios de Unión , Especificidad por SustratoRESUMEN
Dynamics of transformation of Mycobacterium smegmatis cells by cultivation under nonoptimal conditions (partial starvation) to dormant, nonculturable form has been studied. For this aim, an electrochemical method was developed to detect both viable and 'viable but nonculturable' (VBNC) cells. The current produced by bacteria placed at the electrode surface was measured in the presence of 2,6-dichlorophenol indophenol (DCIP) at the applied potential of 350 mV. It has been established that electrochemical activity changes parallel with the growth of biomass. The transition of M. smegmatis to a dormant, nonculturable state goes along with the decrease of the detection current up to 20% of the maximum level. This means that nonculturable cells have rather high rest metabolic activity. The course of the CFU values has a complicated character during bacterial growth. The placement of the bacterial culture on the solid medium appears to cause a new stress that stops proliferation and stimulates aggregation. Both processes distort CFU measurement results. The quantitative estimation of the viable but nonculturable cells by counting colonies, measuring optical density and current produced by bacteria has been discussed.
Asunto(s)
Mycobacterium smegmatis/crecimiento & desarrollo , 2,6-Dicloroindofenol , Proliferación Celular , Supervivencia Celular , Electroquímica/métodos , Electrodos , Mycobacterium smegmatis/química , Mycobacterium smegmatis/aislamiento & purificaciónRESUMEN
Several red wines were studied to find a correlation between physicochemical parameters characterizing the antioxidant status of wine and total content of phenols in samples. The content of dissolved oxygen (its value varied from 0.75 to 3.28 mg/ml), pH (3.10-3.63), redox potential (-186 to -106 mV), mass concentration of free and total sulfur dioxide (10-30 and 36-200 mg/dm3, respectively), absorption spectra, and total phenol content were determined. The wines fell into two main groups-with a relatively low (1850-2050 mg/dm3) and high (2300-2900 mg/dm3) contents of polyphenols. It was demonstrated that physicochemical parameters (except for the content of sulfur dioxide) correlate with the total phenol content in the wines studied.
Asunto(s)
Flavonoides/análisis , Fenoles/análisis , Vino/análisis , Antioxidantes/análisis , Técnicas Biosensibles , Electroquímica , Concentración de Iones de Hidrógeno , Lacasa , Monofenol Monooxigenasa , Oxidación-Reducción , Oxígeno/análisis , Polifenoles , Dióxido de Azufre/análisis , Vino/normasRESUMEN
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.
Asunto(s)
Basidiomycota/enzimología , Pirazoles/química , Pirazolonas , Ácido Vanílico/análogos & derivados , Xenobióticos/metabolismo , Alcoholes Bencílicos/química , Alcoholes Bencílicos/metabolismo , Peroxidasa de Rábano Silvestre , Lacasa/química , Lacasa/metabolismo , Oxidación-Reducción , Ácido Vanílico/química , Xenobióticos/químicaRESUMEN
The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3'-Sulfophenyl)-3- methylpyrazolone-5 was about 30-40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commercial interest for oxidoreductase-catalyzed biodegradation of organic compounds.
Asunto(s)
Lacasa/metabolismo , Basidiomycota/enzimología , Benzotiazoles , Alcoholes Bencílicos/química , Alcoholes Bencílicos/metabolismo , Cromatografía Líquida de Alta Presión , Electroquímica , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Lacasa/aislamiento & purificación , Peso Molecular , Oxidación-Reducción , Pirazoles/química , Pirazoles/metabolismo , Espectrofotometría , Especificidad por Sustrato , Ácidos Sulfónicos/química , Ácidos Sulfónicos/metabolismoRESUMEN
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 degrees C. The thermal stability of the laccase was studied. The catalytic and Michaelis constants for catechol, hydroquinone, sinapinic acid, and K4Fe(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.
Asunto(s)
Basidiomycota/enzimología , Oxidorreductasas/metabolismo , Basidiomycota/química , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Cinética , Lacasa , Peso Molecular , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , TemperaturaRESUMEN
Method for simultaneous obtaining in homogeneous state of two main forms of metleghemoglobin reductase and main leghemoglobin components from lupine nodules was worked out. The method included steps of saturation with ammonium sulphate (40-80%), gel-filtration on Ultrogel AcA 44, isoelectric focusing and repeated isoelectric focusing. As result the forms of metleghemoglobin reductase with molecular weights 62 and 66 kDa were obtained purified 725 and 402 times respectively and obtained in homogeneous state. The total yield of activity was 37%. The form with 62 kDa molecular weight was more active.
Asunto(s)
Fabaceae/metabolismo , Isoenzimas/aislamiento & purificación , Leghemoglobina/aislamiento & purificación , NADH NADPH Oxidorreductasas/aislamiento & purificación , Raíces de Plantas/metabolismo , Plantas Medicinales , Cromatografía en Gel , Electroforesis en Gel de PoliacrilamidaRESUMEN
An electrochemical cell for simultaneous potentiometric and spectrophotometric measurements of solutions of various substances is described. A schematic diagram of the cell, its advantages, method of use, and possible areas of application are discussed. The cell is suggested to be used for electrochemical potentiometric redox titration of leghemoglobin and electrochemical reduction of organic reductants commonly used in biochemical research.