RESUMEN
Chloroperoxidase (CPO) is a haeme-thiolate enzyme able to catalyse the halogenation and oxidation of a wide range of organic substrates. In this work, the CPO-catalysed chlorination and bromination reaction of natural estrogens was characterised. Estradiol, estrone and equiline were efficiently converted to halogenated compounds in the presence of chloride or bromide and hydrogen peroxide. The catalytic efficiency of CPO in this reaction is similar to that measured for other aromatic substrates; as expected the bromination reaction proceeds more efficiently than the chlorination reaction. Three major products were detected for chlorination of estradiol; two of them were monohalogenated compounds while a third product was a dihalogenated compound at positions 2 and 4 of the aromatic ring A. Chlorinated compounds are not substrates for tyrosinase, suggesting that the halogenated form of estrogens is less susceptible to form o-quinones.
Asunto(s)
Cloruro Peroxidasa , Bromuros , Catálisis , Cloruro Peroxidasa/química , Cloruro Peroxidasa/metabolismo , Cloruros , Estradiol , Estrógenos , Estrona , Halogenación , Peróxido de Hidrógeno , Monofenol Monooxigenasa , QuinonasRESUMEN
BACKGROUND: Tetrabromobisphenol (TBBPA), a flame retardant compound, is considered a ubiquitous pollutant, with potential impact on the environment and human health. Several technologies have been applied to accelerate its degradation and minimize environmental impacts. Due to its aromaticity character, peroxidase enzymes may be employed to carry out its transformation in mild conditions. Therefore, the purpose of this work was to determine the capacity of the enzyme chloroperoxidase (CPO) to oxidize TBBPA in several water samples. METHODS: The oxidation capacity of CPO was evaluated in catalytic conditions using water samples from surface and groundwater, as well as effluents from wastewater treatment plants. The biocatalytic performance of CPO was improved due to its immobilization on nanofibers composed of polyvinyl alcohol and chitosan (PVA/chitosan). RESULTS: Free and immobilized CPO were able to transform more than 80% in short reaction times (60 min); producing more biodegradable and less toxic products. Particularly, the immobilized enzyme was catalytically active in a wider range of pH than the free enzyme with the possibility of reusing it up to five times. CONCLUSIONS: The biocatalytic oxidation of TBBPA under environmental conditions is highly efficient, even in complex media such as treated effluents of wastewater treatment plants.
Asunto(s)
Cloruro Peroxidasa/química , Enzimas Inmovilizadas/química , Retardadores de Llama , Nanofibras/química , Bifenilos Polibrominados/química , Contaminantes Ambientales/química , Oxidación-Reducción , Aguas ResidualesRESUMEN
The synthesis of halogenated nucleosides and nucleobases is of interest due to their chemical and pharmacological applications. Herein, the enzymatic halogenation of nucleobases and analogues catalysed by microorganisms and by chloroperoxidase from Caldariomyces fumago has been studied. This latter enzyme catalysed the chlorination and bromination of indoline and uracil. Pseudomonas, Citrobacter, Aeromonas, Streptomyces, Xanthomonas, and Bacillus genera catalysed the chlorination and/or bromination of indole and indoline. Different products were obtained depending on the substrate, the biocatalyst and the halide used. In particular, 85% conversion from indole to 5-bromoindole was achieved using Streptomyces cetonii.
Asunto(s)
Ascomicetos/metabolismo , Cloruro Peroxidasa/metabolismo , Hidrocarburos Halogenados/metabolismo , Indoles/metabolismo , Uracilo/análogos & derivados , Ascomicetos/enzimología , Bacterias/metabolismo , Biocatálisis , Ciclohexanonas/metabolismo , Halogenación , Uracilo/metabolismoRESUMEN
Heme peroxidases are subject to a mechanism-based oxidative inactivation. During the catalytic cycle, the heme group is activated to form highly oxidizing species, which may extract electrons from the protein itself. In this work, we analyze changes in residues prone to oxidation owing to their low redox potential during the peroxide-mediated inactivation of chloroperoxidase from Caldariomyces fumago under peroxidasic catalytic conditions. Surprisingly, we found only minor changes in the amino acid content of the fully inactivated enzyme. Our results show that tyrosine residues are not oxidized, whereas all tryptophan residues are partially oxidized in the inactive protein. The data suggest that the main process leading to enzyme inactivation is heme destruction. The molecular characterization of the peroxide-mediated inactivation process could provide specific targets for the protein engineering of this versatile peroxidase.
Asunto(s)
Ascomicetos/enzimología , Cloruro Peroxidasa/antagonistas & inhibidores , Hemo/química , Peróxido de Hidrógeno/farmacología , Cloruro Peroxidasa/química , Cloruro Peroxidasa/metabolismo , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Activación Enzimática/efectos de los fármacos , Peróxido de Hidrógeno/química , Modelos Moleculares , Oxidación-Reducción , Ingeniería de ProteínasRESUMEN
Among intrinsically conducting polymers, polyaniline is traditionally synthesized by chemical or electrochemical methods. Recently enzymatic synthesis of conducting polymers has been explored. In this work, polymers were synthesized using chloroperoxidase from Caldariomyces fumago and substituted anilines such as 2,6-dimethylaniline, 2,6-dichloroaniline, and 2,3,5,6-tetrachloroaniline (TCA), in order to promote a linear polymerization. These polymers were doped with (1S)-(+)-10-camphorsulfonic acid, dodecylbenzenesulfonic acid, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) with molar ratios of 1:0.25 and 1:0.5. Doped polymers showed conductivity corresponding to the semiconductors. TCA polymer doped with AMPSA showed the higher conductivity values. Different AMPSA concentrations have been tested, and the highest conductivity value of 1.6 x 10(-2) S*m(-1) was obtained for the complex with molar ratio of 1:0.5. This value is similar to those found with other substituted anilines with sulfonic groups. In addition, the enzymatically synthesized polymeric film showed combined transparency and semiconducting properties.
Asunto(s)
Compuestos de Anilina/química , Ascomicetos/enzimología , Cloruro Peroxidasa/química , Proteínas Fúngicas/química , Polímeros/química , SemiconductoresRESUMEN
We have identified an atypical kinetic behavior for the oxidative halogenation of several polycyclic aromatic hydrocarbons (PAHs) by chloroperoxidase (CPO) from Caldariomyces fumago. This behavior resembles the capacity of some members of the P450 family to simultaneously recognize several substrate molecules at their active sites. Indeed, fluorometric studies showed that PAHs exist in solution as monomers and pi-pi dimers that interact to different extents with CPO. The dissociation constants of dimerization were evaluated for every single PAH by spectrofluorometry. Furthermore, docking studies also suggest that CPO might recognize either one or two substrate molecules in its active site. The atypical sigmoidal kinetic behavior of CPO in the oxidative halogenation of PAHs is explained in terms of different kinetic models for non-heteroatomic PAHs (naphthalene, anthracene and pyrene). The results suggest that the actual substrate for CPO in this study was the pi-pi dimer for all evaluated PAHs.
Asunto(s)
Cloruro Peroxidasa/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Ascomicetos/enzimología , Fenómenos Biofísicos , Dimerización , Cinética , Modelos Moleculares , Oxidación-Reducción , Hidrocarburos Policíclicos Aromáticos/química , Espectrometría de Fluorescencia , Especificidad por SustratoRESUMEN
Peroxidase transformations of widely distributed pollutants, tetra- and penta-chlorinated phenols and anilines, were studied using different peroxidases. Chloroperoxidase from Caldariomyces fumago was able to transform tetra- and penta-chlorinated phenols and anilines, while horseradish peroxidase, lignin peroxidase from Phanerochaete chrysosporium and versatile peroxidase from Bjerkandera adusta were able only to transform the halogenated phenols. Chloroperoxidase showed a specific activity on pentachlorophenol two orders of magnitude higher than lignin peroxidase and horseradish peroxidase, and one order of magnitude higher than versatile peroxidase. The main product from peroxidase oxidation in all cases was a polymeric and insoluble material. The insolubilization of halogenated phenols and anilines permits their removal, reduces their bioavailability, and thus reduces their environmental impact. The other minor products from the enzymatic transformation of highly chlorinated compounds were determined by mass spectrometry. Tetrachloroquinone, dimers and trimers of halogenated compounds were also identified. Chloroperoxidase was able to halogenate tetrachloroaniline to form pentachloroaniline.
Asunto(s)
Cloruro Peroxidasa/metabolismo , Hidrocarburos Aromáticos/metabolismo , Hidrocarburos Halogenados/metabolismo , Compuestos de Anilina/química , Compuestos de Anilina/metabolismo , Ascomicetos/enzimología , Clorofenoles/química , Clorofenoles/metabolismo , Hidrocarburos Aromáticos/química , Hidrocarburos Halogenados/química , Espectrometría de Masas , Estructura Molecular , Pentaclorofenol/química , Pentaclorofenol/metabolismoRESUMEN
The catalytic potential of chloroperoxidase (CPO) immobilized on mesoporous materials was evaluated for the oxidation of 4,6-dimethyldibenzothiophene in water/acetonitrile mixtures. Two different types of materials were used for the immobilization: a metal containing Al-MCM-41 material with a pore size of 26 A and SBA-16 materials with three different pore sizes: 40, 90 and 117 A. The SBA-16 40 A did not retain any CPO. The nature and the pore size of the material affected the catalytic activity of the enzyme as well as its stability. Compared to the free enzyme, the thermal stability of CPO at 45 degrees C was two and three times higher than when immobilized on Al-MCM-41 and SBA-16 90 A, respectively.
Asunto(s)
Ascomicetos/enzimología , Cloruro Peroxidasa/química , Tiofenos/química , Activación Enzimática , Enzimas Inmovilizadas/química , Compuestos Orgánicos/química , Oxidación-Reducción , Porosidad , Compuestos de Azufre/químicaRESUMEN
A sigmoidal kinetic behavior of chloroperoxidase for the oxidation of 4,6-dimethyldibenzothiophene (4,6-DMDBT) in water-miscible organic solvent is for the first time reported. Kinetics of 4,6-DMDBT oxidation showed a cooperative profile probably due to the capacity of chloroperoxidase to recognize a substrate dimer (pi-pi dimer) in its active site. Experimental evidence is given for dimer formation and its presence in the active site of chloroperoxidase. The kinetic data were adjusted for a binding site able to interact with either monomer or dimer substrates, producing a cooperative model describing a one-site binding of two related species. Determination of kinetics constants by iterative calculations of possible oxidation paths of 4,6-DMDBT suggests that kinetics oxidation of dimer substrate is preferred when compared to monomer oxidation. Steady-state fluorometry of substrate in the absence and presence of chloroperoxidase, described by the spectral center of mass, supports this last conclusion.
Asunto(s)
Cloruro Peroxidasa/metabolismo , Tiofenos/química , Tiofenos/metabolismo , Catálisis , Dimerización , Cinética , Oxidación-Reducción , Análisis EspectralRESUMEN
Chloroperoxidase from Caldariomyces fumago was crystallized. The crystals were modified with several cross-linkers, but only glurataldehyde was able to produce catalytically active and insoluble crystals. Unlike other immobilized chloroperoxidase preparations, these catalytic crystals are more thermostable than the unmodified soluble enzyme. The enhanced stability is probably due to the structure conservation in the crystalline matrix. In addition, non-cross-linked chloroperoxidase crystals retained more activity than the soluble enzyme after incubation in an organic solvent with low water content. Although the cross-linked crystals were catalytically active, they showed lower specific activity than the soluble enzyme. This low activity may be due to non-specific reactions between the cross-linker and essential residues for catalysis. Alternative cross-linking strategies are discussed.
Asunto(s)
Cloruro Peroxidasa/química , Catálisis , Cloruro Peroxidasa/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Cristalización , Hongos/enzimología , Glutaral/química , Peróxido de Hidrógeno/química , Microscopía Electrónica de Rastreo , Unión Proteica , Temperatura , Agua/químicaRESUMEN
This study investigated the degradation of 4-chlorophenol (4-CP) by Caldariomyces fumago chloroperoxidase (CPO). Enzymatic oxidations were studied in reaction mixtures at pH 3.0, 4.0, and 6.0 in the presence and absence of Cl- containing 3.5 IU of CPO and 4-CP and hydrogen peroxide concentrations within the range of 0.5-50 and 0.005-50 mM, respectively. Distinct patterns of products regarding color, concentration, and solubility were observed. Reaction mixtures at pH 6.0 containing 3.5 IU of CPO and 5.0 mM 4-CP and H2O2 (1:1 stoichiometry) showed the highest 4-CP removal of 95% and the highest formation of a dark precipitate.
Asunto(s)
Archaea/metabolismo , Clorofenoles/farmacocinética , Biodegradación Ambiental , Cloruro Peroxidasa/metabolismo , Fermentación , Peróxido de Hidrógeno/metabolismo , CinéticaRESUMEN
Chloroperoxidase from Caldariomyces fumago was able to chlorinate 17 of 20 aromatic hydrocarbons assayed in the presence of hydrogen peroxide and chloride ions. Reaction rates varied from 0.6 min(-1) for naphthalene to 758 min(-1) for 9-methylanthracene. Mono-, di- and tri-chlorinated compounds were obtained from the chloroperoxidase-mediated reaction on aromatic compounds. Dichloroacenaphthene, trichloroacenaphthene, 9,10-dichloroanthracene, chloropyrene, dichloropyrene, dichlorobiphenylene and trichlorobiphenylene were identified by mass spectral analyses as products from acenaphthene, anthracene, pyrene and biophenylene respectively. Polycyclic aromatic hydrocarbons with 5 and 6 aromatic rings were also substrates for the chloroperoxidase reaction. The importance of the microbial chlorination of aromatic pollutants and its potential environmental impact are discussed.
Asunto(s)
Cloruro Peroxidasa/metabolismo , Hongos/enzimología , Hidrocarburos Clorados/metabolismo , Catálisis , Hidrocarburos Clorados/química , CinéticaRESUMEN
Organophosphorus pesticides are the most common classes involved in poisonings related to pesticides. We used enzymatic activity of chloroperoxidase on the metabolism of some phosphorothioate pesticides published previously and molecular mechanics methods to perform a theoretical approach of the mechanism of biological oxidation of this class of pesticides. The molecular structure of eight pesticides were optimized by molecular mechanics methods using the CAChe program package for biomolecules, ver. 3.11 (Oxford Molecular Ltd., Campbell, CA). Total energy resulted from the structure optimization process and the partial charges of both phosphorus and sulfur were computed for every pesticide. Phosphorus partial charge and enzymatic activity were significantly related by linear regression analysis (r=0.82, P<0.05). Analyzing our results and using previously reported enzymatic activity of chloroperoxidase on these pesticides, we deduced chemical events involved in activation of the active site of chloroperoxidase and proposed a novel mechanism of oxidation for this class of pesticides. This mechanism will also help to understand the oxidation process of pesticides by cytochrome P450, and production of toxic metabolites.