RESUMEN
The pollution of aquatic environments by drugs is a problem for which scarce research has been conducted in regards of their removal. Amycolatopsis sp. Poz 14 presents the ability to biotransformation naphthalene at high efficiency, therefore, in this work this bacterium was proposed as an assimilator of naproxen and carbamazepine. Growth curves at different concentrations of naproxen and carbamazepine showed that Amycolatopsis sp. Poz 14 is able to utilize these drugs at a concentration of 50 mg L-1 as a source of carbon and energy. At higher concentrations, the bacterial growth was inhibited. The transformation kinetics of naproxen showed the total elimination of the compound in 18 days, but carbamazepine was only eliminated in 19.9%. The supplementation with cometabolites such as yeast extract and naphthalene (structure similar to naproxen) at 50 mg L-1, showed that the yeast extract shortened the naproxen elimination to 6 days and reached a higher global consumption rate compared to the naphthalene cometabolite. The biotransformation of carbamazepine was not improved by the addition of cometabolites. The partial sequencing of the genome of Amycolatopsis sp. Poz 14 detected genes encoding putative enzymes for the degradation of cyclic aromatic compounds and the activities of aromatic monooxygenase, catechol 1,2-dioxygenase and gentisate 1,2-dioxygenase exhibited their involving in the naproxen biodegradation. The HPLC-MS analysis detected the 5-methoxysalicylic acid at the end of the biotransformation kinetics. This work demonstrates that Amycolatopsis sp. Poz 14 utilizes naproxen and transforms it to 5-methoxysalicylic acid which is the initial compound for the catechol and gentisic acid metabolic pathway.
Asunto(s)
Actinomycetales/enzimología , Actinomycetales/metabolismo , Redes y Vías Metabólicas , Naproxeno/metabolismo , Actinomycetales/efectos de los fármacos , Actinomycetales/crecimiento & desarrollo , Biodegradación Ambiental , Biotransformación , Carbamazepina/metabolismo , Carbamazepina/farmacología , Carbono/metabolismo , Catecol 1,2-Dioxigenasa , Catecoles , Dioxigenasas , Contaminación Ambiental , Gentisatos , Éteres de Hidroxibenzoatos/metabolismo , Cinética , Oxigenasas de Función Mixta , Naftalenos/metabolismo , Naproxeno/farmacología , Salicilatos/metabolismoRESUMEN
Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments.
Asunto(s)
Pseudomonas putida/enzimología , Pseudomonas putida/genética , Catecol 1,2-Dioxigenasa/genética , Temperatura , Biodegradación Ambiental , Clonación Molecular , Catecol 1,2-Dioxigenasa/análisis , Catecol 1,2-Dioxigenasa/metabolismo , Genes Bacterianos , Concentración de Iones de Hidrógeno , Isoenzimas , MetalesRESUMEN
Fifteen actinomycete strains were evaluated for their potential use in removal of polycyclic aromatic hydrocarbons (PAH). Their capability to degrade of naphthalene, phenanthrene, and pyrene was tested in minimal medium (MM) and MM with glucose as another substrate. Degradation of naphthalene in MM was observed in all isolates at different rates, reaching maximum values near to 76% in some strains of Streptomyces, Rhodococcus sp. 016 and Amycolatopsis tucumanensis DSM 45259. Maximum values of degradation of phenanthrene in MM occurred in cultures of A. tucumanensis DSM 45259 (36.2%) and Streptomyces sp. A12 (20%), while the degradation of pyrene in MM was poor and only significant with Streptomyces sp. A12 (4.3%). Because of the poor performance when growing on phenanthrene and pyrene alone, Rhodococcus sp. 20, Rhodococcus sp. 016, A. tucumanensis DSM 45259, Streptomyces sp. A2, and Streptomyces sp. A12 were challenged to an adaptation schedule of successive cultures on a fresh solid medium supplemented with PAHs, decreasing concentration of glucose in each step. As a result, an enhanced degradation of PAHs by adapted strains was observed in the presence of glucose as co-substrate, without degradation of phenanthrene and pyrene in MM while an increase to up to 50% of degradation was seen with these strains in glucose amended media. An internal fragment of the catA gene, which codes for catechol 1,2-dioxygenase, was amplified from both Rhodococcus strains, showing the potential for degradation of aromatic compounds via salycilate. These results allow us to propose the usefulness of these actinomycete strains for PAH bioremediation in the environment.
Asunto(s)
Actinobacteria/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Actinobacteria/aislamiento & purificación , Biodegradación Ambiental , Catecol 1,2-Dioxigenasa/genética , Catecol 1,2-Dioxigenasa/metabolismo , Medios de Cultivo , Glucosa/metabolismo , Naftalenos/metabolismo , Fenantrenos/metabolismo , Pirenos/metabolismoRESUMEN
Background In biodegradation processes free enzymes often undergo deactivation. Thus, it is very important to obtain highly stable enzymes by different methods. Immobilization allows for successful stabilization of many multimeric enzymes by increasing the rigidity of the enzyme structure. This study aimed to evaluate some environmental factors that affect catechol 1,2-dioxygenase from Stenotrophomonas maltophilia KB2 immobilized in alginate hydrogel. The goal of the present work was to improve the functional stability of the enzyme by increasing its structural rigidity. Results Immobilization yield and expressed activity were 100% and 56%, respectively. Under the same storage conditions, the activity of the immobilized enzyme was still observed on the 28th d of incubation at 4°C, whereas the free enzyme lost its activity after 14 d. The immobilized enzyme required approximately 10°C lower temperature for its optimal activity than the free enzyme. Immobilization shifted the optimal pH from 8 for the soluble enzyme to 7 for the immobilized enzyme. The immobilized catechol 1,2-dioxygenase showed activity against 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol, and 3,5-dichlorocatechol. The immobilization of the enzyme promoted its stabilization against any distorting agents: aliphatic alcohols, phenols, and chelators. Conclusions The entrapment of the catechol 1,2-dioxygenase from S. maltophilia KB2 has been shown to be an effective method for improving the functional properties of the enzyme. Increased resistance to inactivation by higher substrate concentration and other factors affecting enzyme activity as well as broadened substrate specificity compared to the soluble enzyme, makes the immobilized catechol 1,2-dioxygenase suitable for the bioremediation and detoxification of xenobiotic-contaminated environments.
Asunto(s)
Biodegradación Ambiental , Stenotrophomonas maltophilia , Catecol 1,2-Dioxigenasa/metabolismo , Especificidad por Sustrato , Temperatura , Cinética , Técnicas de Cultivo de Célula , Alginatos , Enzimas Inmovilizadas , Geles , Concentración de Iones de HidrógenoRESUMEN
The degradation of phenol (2-30 mM) by free cells and by alginate-immobilized cells of Aureobasidium pullulans FE13 isolated from stainless steel effluents was studied in batch cultures with saline solution not supplemented with nutrients or yeast extract. The rate at which the immobilized cells degrade phenol was similar to the rate at which the suspended cells could degrade phenol, for a concentration of up to 16 mM of phenol. The maximum phenol volumetric degradation rate for 16 mM phenol was found to be 18.35 mg l(-1)h(-1) in the assays with free cells and 20.45 mg l(-1)h(-1) in the assays with alginate-immobilized cells, 18 mM phenol and cellular concentration of 0.176 g/l. At concentrations higher than this, an inhibitory effect was observed, resulting in the lowering of the phenol degradation rates. The immobilization was detrimental to the catechol 1,2-dioxygenase activity. However, the immobilized cells remained viable for a longer period, increasing the efficiency of phenol degradation. The yeast showed catechol 1,2-dioxygenase activity only after growth in the phenol, which was induced at phenol concentrations as low as 0.05 mM and up to 25 mM at 45 h of incubation at 30 degrees C. Phenol concentrations higher than 6mM were inhibitory to the enzyme. Addition of glucose, lactate, succinate, and benzoate reduced the rate at which phenol is consumed by cells. Our results suggest that inoculants based on immobilized cells of A. pullulans FE13 has potential application in the biodegradation of phenol and possibly in the degradation of other related aromatic compounds.
Asunto(s)
Ascomicetos/metabolismo , Fenol/química , Adsorción , Biodegradación Ambiental , Carbono/química , Catecol 1,2-Dioxigenasa/química , Desinfectantes , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Sales (Química)/química , Acero Inoxidable , Temperatura , Factores de TiempoRESUMEN
The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.
Asunto(s)
Técnicas Biosensibles/instrumentación , Catecol 1,2-Dioxigenasa/química , Catecoles/análisis , Cloro/química , Electroquímica/instrumentación , Membranas Artificiales , Nanoestructuras/química , Adsorción , Técnicas Biosensibles/métodos , Catecoles/química , Cristalización/métodos , Electroquímica/métodos , Electrodos , Enzimas Inmovilizadas/química , Diseño de Equipo , Análisis de Falla de Equipo , Nanoestructuras/análisis , Nanotecnología/instrumentación , Nanotecnología/métodos , Unión Proteica , Propiedades de SuperficieRESUMEN
Trifluralin (alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) is a dinitroaniline compound which was first produced in the 1960s and has been used extensively as an agricultural herbicide. There are a few publications on the biodegradation of this xenobiotic compound, but to our knowledge nothing has been documented on the genetic aspects of its catabolism. In this article, we report the analysis of DNA isolated from bacteria previously shown to degrade trifluralin, using as probes the catabolic genes ndoB, todC, xyIX, catA and xyIE which encode the enzymes naphthalene 1,2-dioxygenase, toluene dioxygenase, toluate 1,2-dioxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase respectively. Using PCR and hybridization analysis, the strong hybridization of the ndoB gene with DNA extracted from four trifluralin-degrading isolates was demonstrated, although none of them was able to degrade naphthalene, as indicated by the 'clear zone' test. The results indicated the presence in these bacteria of a dioxygenase gene, whose product could act on trifluralin as its principal substrate, or fortuitously, by cometabolism. This is the first publication on genes in trifluralin-degrading bacteria.
Asunto(s)
Bacterias/genética , Dioxigenasas , Complejos Multienzimáticos/genética , Oxigenasas/genética , Trifluralina/metabolismo , Bacillus megaterium/genética , Bacillus megaterium/metabolismo , Bacterias/metabolismo , Biodegradación Ambiental , Catecol 1,2-Dioxigenasa , Catecol 2,3-Dioxigenasa , Herbaspirillum/genética , Herbaspirillum/metabolismo , Klebsiella oxytoca/genética , Klebsiella oxytoca/metabolismo , Complejos Multienzimáticos/metabolismo , Oxigenasas/metabolismo , Reacción en Cadena de la PolimerasaRESUMEN
In this work, we show that the fungal strain Graphium sp. FIB4 was able to use phenol as the sole carbon source. Higher degradation of phenol was accomplished by alginate-immobilized mycelial mass than by mycelial suspensions of Graphium sp. FIB4. Free mycelium exhibited higher degradation rates when compared with the alginate-immobilized mycelium in the presence of 14 mM of phenol or less. Above this concentration, degradation rates by free mycelium decreased and the immobilized mycelium showed higher values. The maximum degradation rate for 8 mM phenol was found to be 20.13 mg/l x h by free mycelia and 16.24 mg/l x h by immobilized mycelial mass in the presence of 18 mM phenol. When the fungus was grown on medium without phenol, catechol 1,2-dioxygenase activity was not detected. This enzyme activity was induced at phenol concentrations as low as 0.05 mM and up to 6 mM at 24 h incubation at 30 degrees C, suggesting that catechol was oxidized by the ortho type of ring fission. Addition of glucose reduced phenol consumption rate, and both substrates were used simultaneously. Glucose concentrations higher than 0.075% repressed the induction of phenol oxidation by Graphium sp. FIB4 grown on glucose. But glucose did not fully repress utilization of phenol by phenol-pre-induced cells. Immobilization and addition of calcium and barium ions were detrimental to the stability of catechol 1,2-dioxygenase activity and phenol degradation by Graphium sp. FIB4.
Asunto(s)
Ascomicetos/aislamiento & purificación , Ascomicetos/metabolismo , Dioxigenasas , Residuos Industriales , Fenol/metabolismo , Adsorción , Alginatos/metabolismo , Ascomicetos/genética , Ascomicetos/crecimiento & desarrollo , Biodegradación Ambiental , Catecol 1,2-Dioxigenasa , Medios de Cultivo/química , Relación Dosis-Respuesta a Droga , Glucosa/metabolismo , Ácido Glucurónico , Ácidos Hexurónicos , Oxigenasas/metabolismoRESUMEN
Seven strains belonging to genus Pseudomonas were isolated from an enrichment with hydrocarbon mixtures. Tests for enzyme activities showed that five strains used predominantly the catabolic meta-pathway for aromatic hydrocarbon degradation. Furthermore, the xylE gene which encodes a catechol 2,3-dioxygenase was amplified by PCR, and in two strains the nahAc gene, a key enzyme for naphthalene catabolism, was also found. The xylE gene might be a good marker to identify aromatic hydrocarbon degrading bacteria in soils from Patagonia.
Asunto(s)
Dioxigenasas , Genes Bacterianos , Hidrocarburos Aromáticos/metabolismo , Pseudomonas/genética , Microbiología del Suelo , Argentina , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biodegradación Ambiental , Catecol 1,2-Dioxigenasa , Catecol 2,3-Dioxigenasa , ADN Bacteriano/genética , Inducción Enzimática , Naftalenos/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Reacción en Cadena de la Polimerasa , Protocatecuato-3,4-Dioxigenasa/genética , Protocatecuato-3,4-Dioxigenasa/metabolismo , Pseudomonas/clasificación , Pseudomonas/enzimología , Pseudomonas/aislamiento & purificación , Contaminantes del Suelo/metabolismoRESUMEN
Three actinomycete strains isolated from soil treated with 2,4-D were able to degrade the herbicide Diuron in vitro. Strain CCT 4916 was the most efficient, degrading up to 37% of applied Diuron (100 mg Kg-1 soil) in 7 days, as measured by HPLC and UV/VIS spectroscopy. All strains showed protease and urease activity; intracellular activity of metapyrocatechase and pyrocatechase were not found. Actinomycete strain CCT 4916 produced manganese peroxidase, which could be potentially related to degradation of Diuron.