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1.
Protein Expr Purif ; 188: 105964, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34454050

RESUMO

The gene of catechol 1, 2-dioxygenase was identified and cloned from the genome of Oceanimonas marisflavi 102-Na3. The protein was expressed in Escherichia coli BL21 (DE3) and purified to homogeneity of a dimer with molecular mass of 69.2 kDa. The enzyme was highly stable in pH 6.0-9.5 and below 45 °C and exhibited the maximum activity at pH 8.0 and 30 °C. Being the first characterized intradiol dioxygenase from marine bacteria Oceanimonas sp., the enzyme showed catalytic activity for catechol, 3-methylcatechol, 4-methylcatechol, 3-chlorocatechol, 4-chlorocatechol and pyrogallol. For catechol, Km and Vmax were 11.2 µM and 13.4 U/mg of protein, respectively. The enzyme also showed resistance to most of the metal ions, surfactants and organic solvents, being a promising biocatalyst for biodegradation of aromatic compounds in complex environments.


Assuntos
Aeromonadaceae/enzimologia , Proteínas de Bactérias/genética , Catecol 1,2-Dioxigenase/genética , Catecóis/metabolismo , Aeromonadaceae/química , Aeromonadaceae/classificação , Aeromonadaceae/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Catecol 1,2-Dioxigenase/química , Catecol 1,2-Dioxigenase/isolamento & purificação , Catecol 1,2-Dioxigenase/metabolismo , Catecóis/química , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Filogenia , Multimerização Proteica , Pirogalol/química , Pirogalol/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
2.
Microb Cell Fact ; 20(1): 114, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34098954

RESUMO

BACKGROUND: The current shift from a fossil-resource based economy to a more sustainable, bio-based economy requires development of alternative production routes based on utilization of biomass for the many chemicals that are currently produced from petroleum. Muconic acid is an attractive platform chemical for the bio-based economy because it can be converted in chemicals with wide industrial applicability, such as adipic and terephthalic acid, and because its two double bonds offer great versatility for chemical modification. RESULTS: We have constructed a yeast cell factory converting glucose and xylose into muconic acid without formation of ethanol. We consecutively eliminated feedback inhibition in the shikimate pathway, inserted the heterologous pathway for muconic acid biosynthesis from 3-dehydroshikimate (DHS) by co-expression of DHS dehydratase from P. anserina, protocatechuic acid (PCA) decarboxylase (PCAD) from K. pneumoniae and oxygen-consuming catechol 1,2-dioxygenase (CDO) from C. albicans, eliminated ethanol production by deletion of the three PDC genes and minimized PCA production by enhancing PCAD overexpression and production of its co-factor. The yeast pitching rate was increased to lower high biomass formation caused by the compulsory aerobic conditions. Maximal titers of 4 g/L, 4.5 g/L and 3.8 g/L muconic acid were reached with glucose, xylose, and a mixture, respectively. The use of an elevated initial sugar level, resulting in muconic acid titers above 2.5 g/L, caused stuck fermentations with incomplete utilization of the sugar. Application of polypropylene glycol 4000 (PPG) as solvent for in situ product removal during the fermentation shows that this is not due to toxicity by the muconic acid produced. CONCLUSIONS: This work has developed an industrial yeast strain able to produce muconic acid from glucose and also with great efficiency from xylose, without any ethanol production, minimal production of PCA and reaching the highest titers in batch fermentation reported up to now. Utilization of higher sugar levels remained conspicuously incomplete. Since this was not due to product inhibition by muconic acid or to loss of viability, an unknown, possibly metabolic bottleneck apparently arises during muconic acid fermentation with high sugar levels and blocks further sugar utilization.


Assuntos
Carboxiliases/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Hidroliases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Sórbico/análogos & derivados , Xilose/metabolismo , Carboxiliases/genética , Catecol 1,2-Dioxigenase/genética , Clonagem Molecular , DNA Fúngico , Fermentação , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Hidroliases/genética , Hidroxibenzoatos/metabolismo , Microbiologia Industrial , Engenharia Metabólica/métodos , Redes e Vias Metabólicas , Piruvato Descarboxilase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ácido Chiquímico/análogos & derivados , Ácido Chiquímico/metabolismo , Ácido Sórbico/isolamento & purificação , Ácido Sórbico/metabolismo
3.
Appl Environ Microbiol ; 87(6)2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33397706

RESUMO

Salicylic acid plays an important role in the plant immune response, and its degradation is therefore important for plant-pathogenic fungi. However, many nonpathogenic microorganisms can also degrade salicylic acid. In the filamentous fungus Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid, followed by decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). A. niger cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form cis,cis-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of A. niger grown on salicylic acid to identify genes (shyA and crcA) involved in salicylic acid metabolism. Heterologous production in Escherichia coli followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that cis,cis-muconic acid can be produced from salicylic acid. In addition, the in vivo roles of shyA, dhbA, and crcA were studied by creating A. niger deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid.IMPORTANCE Nonrenewable petroleum sources are being depleted, and therefore, alternative sources are needed. Plant biomass is one of the most abundant renewable sources on Earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as cis,cis-muconic acid. cis,cis-Muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins, and lubricants. Currently, cis,cis-muconic acid is mainly produced through chemical synthesis from petroleum-based chemicals. Here, we show that two enzymes from fungi can be used to produce cis,cis-muconic acid from salicylic acid and contributes in creating alternative methods for the production of platform chemicals.


Assuntos
Aspergillus niger/enzimologia , Proteínas Fúngicas/metabolismo , Oxigenases de Função Mista/metabolismo , Ácido Salicílico/metabolismo , Aspergillus niger/genética , Carboxiliases/genética , Catecol 1,2-Dioxigenase/genética , Proteínas Fúngicas/genética , Oxigenases de Função Mista/genética , Filogenia
4.
Int J Mol Sci ; 21(24)2020 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-33316871

RESUMO

Study of the potential of Antarctic microorganisms for use in bioremediation is of increasing interest due to their adaptations to harsh environmental conditions and their metabolic potential in removing a wide variety of organic pollutants at low temperature. In this study, the psychrotolerant bacterium Rhodococcus sp. strain AQ5-07, originally isolated from soil from King George Island (South Shetland Islands, maritime Antarctic), was found to be capable of utilizing phenol as sole carbon and energy source. The bacterium achieved 92.91% degradation of 0.5 g/L phenol under conditions predicted by response surface methodology (RSM) within 84 h at 14.8 °C, pH 7.05, and 0.41 g/L ammonium sulphate. The assembled draft genome sequence (6.75 Mbp) of strain AQ5-07 was obtained through whole genome sequencing (WGS) using the Illumina Hiseq platform. The genome analysis identified a complete gene cluster containing catA, catB, catC, catR, pheR, pheA2, and pheA1. The genome harbours the complete enzyme systems required for phenol and catechol degradation while suggesting phenol degradation occurs via the ß-ketoadipate pathway. Enzymatic assay using cell-free crude extract revealed catechol 1,2-dioxygenase activity while no catechol 2,3-dioxygenase activity was detected, supporting this suggestion. The genomic sequence data provide information on gene candidates responsible for phenol and catechol degradation by indigenous Antarctic bacteria and contribute to knowledge of microbial aromatic metabolism and genetic biodiversity in Antarctica.


Assuntos
Catecóis/metabolismo , Genoma Bacteriano , Rhodococcus/genética , Aclimatação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Catecol 1,2-Dioxigenase/genética , Catecol 1,2-Dioxigenase/metabolismo , Temperatura Baixa , Rhodococcus/metabolismo
5.
Enzyme Microb Technol ; 141: 109663, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33051016

RESUMO

In this study, three psychrotolerant phenol-degrading yeast strains Candida subhashii (strain A011), Candida oregonenis (strain B021) and Schizoblastosporion starkeyi-henricii (strain L012) isolated from Rucianka peatland were examined to determine which alternative metabolic pathway for phenol biodegradation is used by these microorganisms. All yeast strains were cultivated in minimal salt medium supplemented with phenol at 500, 750 and 1000 mg l-1 concentration with two ways of conducting phenol biodegradation experiments: with and without the starving step of yeast cells. For studied yeast strains, no catechol 2,3-dioxygenase activities were detected by enzymatic assay and no products of catechol meta-cleavage in yeast cultures supernatants (GC-MS analysis), were detected. The detection of catechol 1,2-dioxygenase activity and the presence of cis,cis-muconic acid in the analyzed samples revealed that all studied psychrotolerant yeast strains were able to metabolize phenol via the ortho-cleavage pathway. Therefore, they may be tested in terms of their use to develop biotechnology for the production of cis,cis-muconic acid, a substrate used in the production of plastics (PET) and other valuable goods.


Assuntos
Redes e Vias Metabólicas , Fenol/metabolismo , Saccharomycetales/metabolismo , Microbiologia do Solo , Biodegradação Ambiental , Catecol 1,2-Dioxigenase/metabolismo , Catecóis/análise , Catecóis/metabolismo , Polônia , Saccharomycetales/classificação , Saccharomycetales/enzimologia , Saccharomycetales/isolamento & purificação , Ácido Sórbico/análogos & derivados , Ácido Sórbico/análise , Ácido Sórbico/metabolismo
6.
Int J Mol Sci ; 21(9)2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32392868

RESUMO

2,4-Dichlorophenol (2,4-DCP) is a ubiquitous environmental pollutant categorized as a priority pollutant by the United States (US) Environmental Protection Agency, posing adverse health effects on humans and wildlife. Bioremediation is proposed as an eco-friendly, cost-effective alternative to traditional physicochemical remediation techniques. In the present study, fungal strains were isolated from marine invertebrates and tested for their ability to biotransform 2,4-DCP at a concentration of 1 mM. The most competent strains were studied further for the expression of catechol dioxygenase activities and the produced metabolites. One strain, identified as Tritirachium sp., expressed high levels of extracellular catechol 1,2-dioxygenase activity. The same strain also produced a dechlorinated cleavage product of the starting compound, indicating the assimilation of the xenobiotic by the fungus. This work also enriches the knowledge about the mechanisms employed by marine-derived fungi in order to defend themselves against chlorinated xenobiotics.


Assuntos
Basidiomycota/fisiologia , Clorofenóis/metabolismo , Invertebrados/microbiologia , Animais , Organismos Aquáticos/microbiologia , Basidiomycota/enzimologia , Basidiomycota/isolamento & purificação , Biodegradação Ambiental , Catecol 1,2-Dioxigenase/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Simbiose , Poluentes Químicos da Água/metabolismo
7.
Sci Rep ; 10(1): 5279, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210346

RESUMO

Melia azedarach-rhizosphere mediated degradation of benzo(a)pyrene (BaP), in the presence of cadmium (Cd) was studied, using efficient rhizobacterial isolate. Serratia marcescens S2I7, isolated from the petroleum-contaminated site, was able to tolerate up to 3.25 mM Cd. In the presence of Cd, the isolate S2I7 exhibited an increase in the activity of stress-responsive enzyme, glutathione-S-transferase. Gas Chromatography-Mass spectroscopy analysis revealed up to 59% in -vitro degradation of BaP after 21 days, while in the presence of Cd, the degradation was decreased by 14%. The bacterial isolate showed excellent plant growth-promoting attributes and could enhance the growth of host plant in Cd contaminated soil. The 52,41,555 bp genome of isolate S. marcescens S2I7 was sequenced, assembled and annotated into 4694 genes. Among these, 89 genes were identified for the metabolism of aromatic compounds and 172 genes for metal resistance, including the efflux pump system. A 2 MB segment of the genome was identified to contain operons for protocatechuate degradation, catechol degradation, benzoate degradation, and an IclR type regulatory protein pcaR, reported to be involved in the regulation of protocatechuate degradation. A pot trial was performed to validate the ability of S2I7 for rhizodegradation of BaP when applied through Melia azedarach rhizosphere. The rhizodegradation of BaP was significantly higher when augmented with S2I7 (85%) than degradation in bulk soil (68%), but decreased in the presence of Cd (71%).


Assuntos
Benzo(a)pireno/metabolismo , Biodegradação Ambiental/efeitos dos fármacos , Cádmio/toxicidade , Melia azedarach/efeitos dos fármacos , Rizosfera , Serratia marcescens/metabolismo , Microbiologia do Solo , Poluentes do Solo/toxicidade , Proteínas de Bactérias/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Catecol 2,3-Dioxigenase/metabolismo , Catecóis/metabolismo , DNA Bacteriano/genética , Cromatografia Gasosa-Espectrometria de Massas , Genoma Bacteriano , Glutationa Transferase/metabolismo , Hidroxibenzoatos/metabolismo , Melia azedarach/crescimento & desenvolvimento , Óperon , Filogenia , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/genética , Serratia marcescens/isolamento & purificação , Ácido Succínico/farmacologia
8.
Prep Biochem Biotechnol ; 50(5): 486-493, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31900038

RESUMO

Catechol 1,2-dioxygenases catalyze catechol ring-opening, a critical step in the degradation of aromatic compounds. Cupriavidus campinensis BJ71, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterial strain, was previously isolated from an environment contaminated with 2,4-D. In this study, catA encoding a catechol 1,2-dioxygenase was cloned from the BJ71 strain. The gene was 939 bp long and encoded a polypeptide of 312 amino acids with a molecular weight of 34 kDa. To investigate its enzymatic characteristics, CatA was heterologously expressed in Escherichia coli. Optimal reaction conditions for the pure enzyme were 35 °C and pH 8.0. The enzyme remained stable within a range of 25 °C-45 °C and pH 6.0-9.0, thus indicating that CatA has wide temperature and pH adaptability. After incubation at 45 °C, the enzyme activity of CatA decreased to 37.12%, but its activity was not affected by incubation at pH 9.0. The pure enzyme was able to use catechol, 4-methyl-catechol and 4-chlorocatechol as substrates. Enzyme kinetic parameters Km and Vmax were 39.97 µM and 10.68 U/mg, respectively. This is the first report of the cloning of a gene encoding a catechol 1,2-dioxygenase from a 2,4-D-degrading bacterial strain.


Assuntos
Proteínas de Bactérias/química , Catecol 1,2-Dioxigenase/química , Cupriavidus/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Catecol 1,2-Dioxigenase/genética , Catecol 1,2-Dioxigenase/isolamento & purificação , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Filogenia , Alinhamento de Sequência , Temperatura
9.
J Gen Appl Microbiol ; 66(3): 188-194, 2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-31723074

RESUMO

The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn2+ and Mn2+, and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K+ slightly increased the activity.


Assuntos
Burkholderia cepacia/enzimologia , Catecol 1,2-Dioxigenase/genética , Catecol 1,2-Dioxigenase/metabolismo , Burkholderia cepacia/genética , Catecol 1,2-Dioxigenase/antagonistas & inibidores , Catecol 1,2-Dioxigenase/química , Catecóis/metabolismo , Clonagem Molecular , Dioxinas/análise , Genes Bacterianos , Concentração de Íons de Hidrogênio , Metais/farmacologia , Microbiologia do Solo , Poluentes do Solo/análise , Tensoativos/farmacologia , Temperatura
10.
World J Microbiol Biotechnol ; 35(12): 186, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31728655

RESUMO

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.


Assuntos
Actinomycetales/enzimologia , Actinomycetales/metabolismo , Redes e Vias Metabólicas , Naproxeno/metabolismo , Actinomycetales/efeitos dos fármacos , Actinomycetales/crescimento & desenvolvimento , Biodegradação Ambiental , Biotransformação , Carbamazepina/metabolismo , Carbamazepina/farmacologia , Carbono/metabolismo , Catecol 1,2-Dioxigenase , Catecóis , Dioxigenases , Poluição Ambiental , Gentisatos , Éteres de Hidroxibenzoatos/metabolismo , Cinética , Oxigenases de Função Mista , Naftalenos/metabolismo , Naproxeno/farmacologia , Salicilatos/metabolismo
11.
Environ Toxicol Pharmacol ; 67: 108-116, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30807915

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant hazardous environmental contaminants. Various strategies, including chemical and physical like oxidation, fixation, leaching, and electrokinetic or biological-based techniques are used for remediation of polluted sites. Bioremediation of PAHs, via PAH-degrading endophytic and rhizospheric microbes, represent a time-/cost-effective way for ecorestoration. Four bacterial strains were isolated from contaminated soil on MSM supplemented with anthracene, alpha-naphthalene or catechol as sole carbon sources. These isolates were identified with 16S rRNA as Bacillus anthracis, B. cereus, B. mojavensis and B. subtilis. The degradation efficiency on the selected aromatic compounds was tested by HPLC analysis. B. subtilis showed the highest degradation efficiency of anthracene (99%) after five days of incubation. B. subtilis showed the highest catechol 1, 2 dioxygenase activity in MSM supplemented with anthracene. The enzyme was purified by gel filtration chromatography and characterized (70 kD, Km 2.7 µg and Vmax 178U/mg protein). The catechol 1,2 dioxygenase gene from the identified four bacterial strains were isolated and submitted to GenBank (accession numbers MG255165-MG255168). The gene expression level of catechol 1,2 dioxygenase was upregulated 23.2-fold during the 72 h of incubation period. Furthermore, B. subtilis is a promising strain to be used in bioremediation of aromatic compounds-contaminated environments.


Assuntos
Bacillus/metabolismo , Proteínas de Bactérias/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Poluentes do Solo/metabolismo , Bacillus/genética , Bacillus/isolamento & purificação , Proteínas de Bactérias/genética , Biodegradação Ambiental , Catecol 1,2-Dioxigenase/genética , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Microbiologia do Solo
12.
Int J Mol Sci ; 20(1)2018 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-30586858

RESUMO

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver⁻Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.


Assuntos
Proteínas de Bactérias/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Pseudomonas chlororaphis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Catecol 1,2-Dioxigenase/química , Catecol 1,2-Dioxigenase/classificação , Catecóis/metabolismo , Clorofenóis/química , Clorofenóis/metabolismo , Cromatografia em Gel , Cromatografia por Troca Iônica , Homogentisato 1,2-Dioxigenase/química , Homogentisato 1,2-Dioxigenase/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metais/química , Metais/metabolismo , Filogenia , Estabilidade Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Temperatura
13.
Antonie Van Leeuwenhoek ; 111(12): 2293-2301, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29959655

RESUMO

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413-416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.


Assuntos
Proteínas de Bactérias/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Catecol 2,3-Dioxigenase/metabolismo , Dioxigenases/metabolismo , Sphingomonadaceae/enzimologia , Proteínas de Bactérias/genética , Benzoatos/metabolismo , Compostos de Bifenilo/metabolismo , Catecol 1,2-Dioxigenase/genética , Catecol 2,3-Dioxigenase/genética , Catecóis/metabolismo , Clonagem Molecular , Dioxigenases/genética , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Naftalenos/metabolismo , Pentaclorofenol/metabolismo , Fenantrenos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microbiologia do Solo , Sphingomonadaceae/genética , Sphingomonadaceae/isolamento & purificação , Especificidade por Substrato , Tolueno/metabolismo , Xilose/metabolismo
14.
Electron. j. biotechnol ; 34: 83-90, july. 2018. tab, ilus, graf
Artigo em Inglês | LILACS | ID: biblio-1047375

RESUMO

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.


Assuntos
Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Catecol 1,2-Dioxigenase/genética , Temperatura , Biodegradação Ambiental , Clonagem Molecular , Catecol 1,2-Dioxigenase/análise , Catecol 1,2-Dioxigenase/metabolismo , Genes Bacterianos , Concentração de Íons de Hidrogênio , Isoenzimas , Metais
15.
J Hazard Mater ; 351: 71-79, 2018 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-29518654

RESUMO

A moving bed biofilm reactor (MBBR) effectively removes pollutants and even runs under extreme conditions. However, the pH shock resistance of a biofilm in MBBRs has been rarely reported. In this study, simulated phenol wastewater with acidic shock (pH 7.5-3.0) was used. In the pH shock phase, the phenol and COD removal efficiencies initially decreased and gradually increased to more than 90%. Microscopic studies showed that the superficial biofilm was mainly composed of fungi (yeasts) in the acidic pH shock phase. The microbial community composition in the acidic pH shock phase was significantly different from those in other phases. Firmicutes and Ascomycota were the dominant bacterial and fungal phyla in this stage, respectively. 16S rRNA gene-based functional annotation indicated that functional profiles related to aromatic compound degradation existed in all of the stages. Therefore, MBBRs show potential for the treatment of phenolic wastewater exposed to pH shock.


Assuntos
Reatores Biológicos , Fenol/metabolismo , Poluentes Químicos da Água/metabolismo , Bactérias/genética , Bactérias/metabolismo , Análise da Demanda Biológica de Oxigênio , Catecol 1,2-Dioxigenase/metabolismo , Fungos/genética , Fungos/metabolismo , Concentração de Íons de Hidrogênio , Microbiota , RNA Ribossômico 16S
16.
Metab Eng ; 46: 13-19, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29474840

RESUMO

Muconic acid (MA) is a dicarboxylic acid used for the production of industrially relevant chemicals such as adipic acid, terephthalic acid, and caprolactam. Because the synthesis of these polymer precursors generates toxic intermediates by utilizing petroleum-derived chemicals and corrosive catalysts, the development of alternative strategies for the bio-based production of MA has garnered significant interest. Plants produce organic carbon skeletons by harvesting carbon dioxide and energy from the sun, and therefore represent advantageous hosts for engineered metabolic pathways towards the manufacturing of chemicals. In this work, we engineered Arabidopsis to demonstrate that plants can serve as green factories for the bio-manufacturing of MA. In particular, dual expression of plastid-targeted bacterial salicylate hydroxylase (NahG) and catechol 1,2-dioxygenase (CatA) resulted in the conversion of the endogenous salicylic acid (SA) pool into MA via catechol. Sequential increase of SA derived from the shikimate pathway was achieved by expressing plastid-targeted versions of bacterial salicylate synthase (Irp9) and feedback-resistant 3-deoxy-D-arabino-heptulosonate synthase (AroG). Introducing this SA over-producing strategy into engineered plants that co-express NahG and CatA resulted in a 50-fold increase in MA titers. Considering that MA was easily recovered from senesced plant biomass after harvest, we envision the phytoproduction of MA as a beneficial option to add value to bioenergy crops.


Assuntos
Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ácido Sórbico/análogos & derivados , Arabidopsis/genética , Catecol 1,2-Dioxigenase/genética , Catecol 1,2-Dioxigenase/metabolismo , Liases/biossíntese , Liases/genética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Plantas Geneticamente Modificadas/genética , Ácido Salicílico/metabolismo , Ácido Sórbico/metabolismo
17.
Microbiol Res ; 205: 118-124, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28942837

RESUMO

Phenolic acids can enhance the mycotoxin production and activities of hydrolytic enzymes related to pathogenicity of soilborne fungus Fusarium oxysporum. However, characteristics of phenolic acid-degrading bacteria have not been investigated. The objectives of this study were to isolate and characterize bacteria capable of growth on benzoic and vanillic acids as the sole carbon source in the peanut rhizosphere. Twenty-four bacteria were isolated, and the identification based on 16S rRNA gene sequencing revealed that pre-exposure to phenolic acids before sowing shifted the dominant culturable bacterial degraders from Arthrobacter to Burkholderia stabilis-like isolates. Both Arthrobacter and B. stabilis-like isolates catalysed the aromatic ring cleavage via the ortho pathway, and Arthrobacter isolates did not exhibit higher C12O enzyme activity than B. stabilis-like isolates. The culture filtrate of Fusarium sp. ACCC36194 caused a strong inhibition of Arthrobacter growth but not B. stabilis-like isolates. Additionally, Arthrobacter isolates responded differently to the culture filtrates of B. stabilis-like isolates. The Arthrobacter isolates produced higher indole acetic acid (IAA) levels than B. stabilis-like isolates, but B. stabilis-like isolates were also able to produce siderophores, solubilize mineral phosphate, and exert an antagonistic activity against peanut root rot pathogen Fusarium sp. ACCC36194. Results indicate that phenolic acids can shift their dominant culturable bacterial degraders from Arthrobacter to Burkholderia species in the peanut rhizosphere, and microbial interactions might lead to the reduction of culturable Arthrobacter. Furthermore, increasing bacterial populations metabolizing phenolic acids in monoculture fields might be a control strategy for soilborne diseases caused by Fusarium spp.


Assuntos
Arachis/microbiologia , Bactérias/metabolismo , Agentes de Controle Biológico , Hidroxibenzoatos/metabolismo , Doenças das Plantas/prevenção & controle , Rizosfera , Antibiose , Arachis/crescimento & desenvolvimento , Arthrobacter/enzimologia , Arthrobacter/crescimento & desenvolvimento , Arthrobacter/isolamento & purificação , Arthrobacter/metabolismo , Bactérias/genética , Bactérias/isolamento & purificação , Ácido Benzoico/metabolismo , Burkholderia/enzimologia , Burkholderia/crescimento & desenvolvimento , Burkholderia/isolamento & purificação , Burkholderia/metabolismo , Catecol 1,2-Dioxigenase/metabolismo , Catecol 2,3-Dioxigenase/metabolismo , Fusarium/patogenicidade , Hidroxibenzoatos/farmacologia , Ácidos Indolacéticos/metabolismo , Fosfatos/metabolismo , Doenças das Plantas/microbiologia , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Sideróforos/metabolismo , Solo , Microbiologia do Solo , Ácido Vanílico/metabolismo
18.
J Basic Microbiol ; 57(10): 883-895, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28745827

RESUMO

Catechol 1,2-dioxygenase is the key enzyme that catalyzes the cleavage of the aromatic ring of catechol. We explored the genetic diversity of catechol 1,2-dioxygenase in the fecal microbial metagenome by PCR with degenerate primers. A total of 35 gene fragments of C12O were retrieved from microbial DNA in the feces of pygmy loris. Based on phylogenetic analysis, most sequences were closely related to C12O sequences from Acinetobacter. A full-length C12O gene was directly cloned, heterologously expressed in Escherichia coli, and biochemically characterized. Purified catPL12 had optimum pH and temperature pH 8.0 and 25 °C and retained 31 and 50% of its maximum activity when assayed at 0 and 35 °C, respectively. The enzyme was stable at 25 and 37 °C, retaining 100% activity after pre-incubation for 1 h. The kinetic parameters of catPL12 were determined. The enzyme had apparent Km of 67 µM, Vmax of 7.3 U/mg, and kcat of 4.2 s-1 for catechol, and the cleavage activities for 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol were much less than for catechol, and no activity with hydroquinone or protocatechuate was detected. This study is the first to report the molecular and biochemical characterizations of a cold-adapted catechol 1,2-dioxygenase from a fecal microbial metagenome.


Assuntos
Catecol 1,2-Dioxigenase/genética , Catecol 1,2-Dioxigenase/metabolismo , Fezes/microbiologia , Variação Genética , Metagenoma , Acinetobacter/enzimologia , Acinetobacter/genética , Animais , Catecol 1,2-Dioxigenase/classificação , Catecóis/metabolismo , Clonagem Molecular , Primers do DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Lorisidae/microbiologia , Filogenia , Reação em Cadeia da Polimerase/métodos
19.
Plant Physiol ; 171(2): 914-31, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27208235

RESUMO

Norway spruce (Picea abies) is periodically attacked by the bark beetle Ips typographus and its fungal associate, Endoconidiophora polonica, whose infection is thought to be required for successful beetle attack. Norway spruce produces terpenoid resins and phenolics in response to fungal and bark beetle invasion. However, how the fungal associate copes with these chemical defenses is still unclear. In this study, we investigated changes in the phenolic content of Norway spruce bark upon E. polonica infection and the biochemical factors mediating these changes. Although genes encoding the rate-limiting enzymes in Norway spruce stilbene and flavonoid biosynthesis were actively transcribed during fungal infection, there was a significant time-dependent decline of the corresponding metabolites in fungal lesions. In vitro feeding experiments with pure phenolics revealed that E. polonica transforms both stilbenes and flavonoids to muconoid-type ring-cleavage products, which are likely the first steps in the degradation of spruce defenses to substrates that can enter the tricarboxylic acid cycle. Four genes were identified in E. polonica that encode catechol dioxygenases carrying out these reactions. These enzymes catalyze the cleavage of phenolic rings with a vicinal dihydroxyl group to muconoid products accepting a wide range of Norway spruce-produced phenolics as substrates. The expression of these genes and E. polonica utilization of the most abundant spruce phenolics as carbon sources both correlated positively with fungal virulence in several strains. Thus, the pathways for the degradation of phenolic compounds in E. polonica, initiated by catechol dioxygenase action, are important to the infection, growth, and survival of this bark beetle-vectored fungus and may play a major role in the ability of I. typographus to colonize spruce trees.


Assuntos
Ascomicetos/fisiologia , Carbono/metabolismo , Fenóis/metabolismo , Picea/microbiologia , Doenças das Plantas/microbiologia , Gorgulhos/microbiologia , Animais , Ascomicetos/patogenicidade , Catecol 1,2-Dioxigenase/genética , Catecol 1,2-Dioxigenase/metabolismo , Catecóis/química , Catecóis/metabolismo , Flavonoides/química , Flavonoides/metabolismo , Fenóis/química , Picea/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Resinas Vegetais/química , Resinas Vegetais/metabolismo , Estilbenos/química , Estilbenos/metabolismo , Terpenos/química , Terpenos/metabolismo , Fatores de Virulência
20.
Prep Biochem Biotechnol ; 46(7): 673-8, 2016 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26760080

RESUMO

The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858 bp encoding a polypeptide of 285 amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the Km and Vmax of recombinant catechol 1,2-dioxygenase were 9.2 µM and 0.987 µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.


Assuntos
Candida tropicalis/enzimologia , Catecol 1,2-Dioxigenase/genética , Fenóis/metabolismo , Sequência de Aminoácidos , Candida tropicalis/metabolismo , Catecol 1,2-Dioxigenase/química , Catecol 1,2-Dioxigenase/metabolismo , Clonagem Molecular , Concentração de Íons de Hidrogênio , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Oxigenases de Função Mista/metabolismo , Homologia de Sequência de Aminoácidos , Temperatura
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