RESUMO
Dye contamination in printing and dyeing wastewater has long been a major concern due to its serious impact on both the environment and human health. In the quest for bioremediation of these hazardous dyes, biological resources such as biodegradation bacteria and enzymes have been investigated in severely polluted environments. In this context, the triphenylmethane transporter gene (tmt) was identified in six distinct clones from a metagenomic library of the printing and dyeing wastewater treatment system. Escherichia coli expressing tmt revealed 98.1% decolorization efficiency of triphenylmethane dye malachite green within 24 h under shaking culture condition. The tolerance to malachite green was improved over eightfold in the Tmt strain compared of the none-Tmt expressed strain. Similarly, the tolerance of Tmt strain to other triphenylmethane dyes like crystal violet and brilliant green, was improved by at least fourfold. Site-directed mutations, including A75G, A75S and V100G, were found to reinforce the tolerance of malachite green, and double mutations of these even further improve the tolerance. Therefore, the tmt has been demonstrated to be a specific efflux pump for triphenylmethane dyes, particularly the malachite green. By actively pumping out toxic triphenylmethane dyes, it significantly extends the cells tolerance in a triphenylmethane dye-rich environment, which may provide a promising strategy for bioremediation of triphenylmethane dye pollutants in the environments.
Assuntos
Biodegradação Ambiental , Corantes , Escherichia coli , Corantes de Rosanilina , Compostos de Tritil , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes/metabolismo , Compostos de Tritil/metabolismo , Corantes de Rosanilina/metabolismo , Poluentes Químicos da Água/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMO
Malachite green dye belongs to the triphenylmethane group and is a common environmental pollutant that threatens non-target organisms. We report the potential of the early colonizing marine bacterium Pseudomonas sp. ESPS40 isolated from the Arabian Sea, India, to decolorize malachite green (MG). The bacterium ESPS40 exhibited a higher ability for MG degradation (86-88%) at varying NaCl concentrations (1-3%). The highest MG degradation (~ 88%) was observed at 1% NaCl. The bacterial strain ESPS40 showed degradation up to 800 mg L-1 MG. Further, enzyme activities such as tyrosinase (63.48-526.52 U L-1) and laccase (3.62-28.20 U L-1) were also analyzed with varying concentrations (100 mg L-1-1000 mg L-1) of MG during the degradation process. The dye degradation was confirmed by Fourier transform infrared spectroscopy (FTIR) and high-performance liquid chromatography (HPLC). The outcome of the present study demonstrated Pseudomonas sp. ESPS40 as a potential strain for the efficient degradation of MG at higher concentrations. Thus, Pseudomonas sp. ESPS40 can be utilized as a potential candidate for the biodegradation of MG in wastewater treatment.
Assuntos
Corantes de Rosanilina , Cloreto de Sódio , Cloreto de Sódio/metabolismo , Corantes de Rosanilina/química , Corantes de Rosanilina/metabolismo , Biodegradação Ambiental , Pseudomonas/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , ÍndiaRESUMO
Laccases highlight for xenobiotic bioremediation, as well as application in the fine chemical, textile, biofuel and food industries. In a previous work, we described the preliminary characterization of laccase LacMeta, a promising enzyme for the bioremediation of dyes, able to decolorization malachite green (MG), trypan blue, methylene blue. Here we demonstrate that LacMeta is indeed suitable for the complete degradation and detoxification of MG dye, not just for its discoloration, since some works show false positives due to the formation of colorless intermediates such as leucomalachite. The optimal pH and temperature parameters of LacMeta were 5.0 and 50 °C, respectively (MG as substrate). LacMeta was tolerant of up to 10 mmol L- 1 EDTA (82%) and up to 5% (V/V) acetone (91%) and methanol (71%), while SDS promoted severe inhibition. For ions, a high tolerance to cobalt, zinc, manganese, and calcium (10 mmol L- 1) was also observed (> 90%). Even under high-salinity conditions (1 mol L- 1 NaCl), the residual bleaching activity of the dye remained at 61%. Furthermore, the bleaching product of MG did not inhibit the germination of sorghum and tomato seeds and was inert to the vegetative structures of the germinated seedlings. Additionally, this treatment effectively reduced the cytotoxic effect of the dye on microorganisms (Escherichia coli and Azospirillum brasilense), which can be explained by H-NMR spectral analysis results since LacMeta completely degraded the peak signals corresponding to the aromatic rings in the dye, demonstrating extreme efficiency in the bioremediation of the xenobiotic at high concentrations (50 mg L- 1).
Assuntos
Lacase , Xenobióticos , Lacase/metabolismo , Corantes de Rosanilina/metabolismo , Corantes/metabolismo , Biodegradação AmbientalRESUMO
While mechanical stimulation is known to regulate a wide range of biological processes at the cellular and tissue levels, its medical use for tissue regeneration and rehabilitation has been limited by the availability of suitable devices. Here we present a mechanically active gel-elastomer-nitinol tissue adhesive (MAGENTA) that generates and delivers muscle-contraction-mimicking stimulation to a target tissue with programmed strength and frequency. MAGENTA consists of a shape memory alloy spring that enables actuation up to 40% strain, and an adhesive that efficiently transmits the actuation to the underlying tissue. MAGENTA activates mechanosensing pathways involving yes-associated protein and myocardin-related transcription factor A, and increases the rate of muscle protein synthesis. Disuse muscles treated with MAGENTA exhibit greater size and weight, and generate higher forces compared to untreated muscles, demonstrating the prevention of atrophy. MAGENTA thus has promising applications in the treatment of muscle atrophy and regenerative medicine.
Assuntos
Músculo Esquelético , Adesivos Teciduais , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Adesivos Teciduais/metabolismo , Corantes de Rosanilina/metabolismo , Atrofia Muscular/prevenção & controle , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Contração MuscularRESUMO
Understanding the spatial distribution of bioactive small molecules is indispensable for elucidating their biological or pharmaceutical roles. Here, a rapid and effective analysis strategy was introduced to study the distribution of veterinary drugs in aquatic products. Malachite green (MG), one of the most widely used veterinary drugs in aquaculture, was selected as the targeted compound. Zebrafish (Danio rerio) was used as a model organism. After an exposure test, the matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technique was applied to directly analyze the content changes of malachite green in zebrafish tissues. The reliable relationship of exposure time and content change of MG was described precisely by the extended Freundlich equation. The process of modeling was discussed in detail, and some important parameters or trend information was obtained, including the maximum content of MG in different fish tissues, time to maximum content, elimination time, equilibrium content, and so on. With a simplification of sample pretreatment, this research strategy can be used for monitoring the spatial distribution of veterinary drugs and related metabolites of laboratory-exposed fish. The obtained model can provide a perspective for rational drug use in aquaculture and precise drug residue detection in production activities.
Assuntos
Corantes de Rosanilina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Modelos Químicos , Padrões de Referência , Corantes de Rosanilina/normas , Peixe-ZebraRESUMO
The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113th arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.
Assuntos
Aminoácidos/química , Aminoácidos/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/genética , Corantes de Rosanilina/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Estrutura Secundária de Proteína , Corantes de Rosanilina/metabolismoRESUMO
Chronic ulceration of the colon is associated with the activation of TLR4/NF-κB and P2X7R/NLRP3 signaling pathways. We investigated the effect of individual or combined administration of BBG, a P2X7R blocker, and OLT1177, a selective NLRP3 inhibitor, in the dextran sodium sulfate-induced ulcerative colitis (UC) rat model. The ulcerative rats were treated orally with brilliant blue G (BBG) (50 mg/kg/day) or OLT1177 (200 mg/kg/day) or a combination of both. Myd88 and NF-κB levels were measured by ELISA, qRT-PCR, and immunohistochemical staining. Cytokines known to be associated with TLR4/NF-κB or P2X7R/NLRP3 signaling were measured by ELISA. P2X7R and NLRP3 expression were measured by ELISA and qRT-PCR. The administration of BBG or OLT1177 ameliorated the toxic effects of DSS on the colon as they restored normal colonic macroscopic and microscopic morphology. BBG administration, but not OLT1177, reduced the expression of Myd88, NF-κB, IL-6, and TNF-α in addition to lowering P2X7R and oxidative stress levels. Individual BBG or OLT1177 administration decreased NLRP3 inflammasome recruitment and subsequent activation of caspase-1, IL-1ß, and IL-18. However, the combined administration of OLT1177 with BBG potentiated its inhibitory effect on the NLRP3, which was reflected by the additional suppressive effect on caspase-1, IL-1ß, IL-18 levels. In conclusion, BBG/OLT1177 exhibited complementary effects and effectively ameliorated UC. This novel approach provides a basis for the clinical application of this combination for the treatment of IBDs and might also be promising for the pharmacological intervention of other NLRP3 inflammasome-dependent inflammatory conditions.
Assuntos
Colite Ulcerativa/tratamento farmacológico , Nitrilas/farmacologia , Corantes de Rosanilina/farmacologia , Animais , Caspase 1/metabolismo , Colite/induzido quimicamente , Colite Ulcerativa/metabolismo , Citocinas/metabolismo , Sulfato de Dextrana/farmacologia , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nitrilas/metabolismo , Ratos , Ratos Wistar , Receptores Purinérgicos P2X7/metabolismo , Corantes de Rosanilina/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The wide use of malachite green (MG) as a dye has caused substantial concern owing to its toxicity. Bacillus cereus can against the toxic effect of MG and efficiently decolourise it. However, detailed information regarding its underlying adaptation and degradation mechanisms based on proteomic data is scarce. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ)-facilitated quantitative method was applied to analyse the molecular mechanisms by which B. cereus degrades MG. Based on this analysis, 209 upregulated proteins and 198 downregulated proteins were identified with a false discovery rate of 1% or less during MG biodegradation. Gene ontology and KEGG analysis determined that the differentially expressed proteins were enriched in metabolic processes, catalytic activity, antioxidant activity, and responses to stimuli. Furthermore, real-time qPCR was utilised to further confirm the regulated proteins involved in benzoate degradation. The proteins BCE_4076 (Acetyl-CoA acetyltransferase), BCE_5143 (Acetyl-CoA acetyltransferase), BCE_5144 (3-hydroxyacyl-CoA dehydrogenase), BCE_4651 (Enoyl-CoA hydratase), and BCE_5474 (3-hydroxyacyl-CoA dehydrogenase) involved in the benzoate degradation pathway may play an important role in the biodegradation of MG by B. cereus. The results of this study not only provide a comprehensive view of proteomic changes in B. cereus upon MG loading but also shed light on the mechanism underlying MG biodegradation by B. cereus.
Assuntos
Bacillus cereus/genética , Bacillus cereus/metabolismo , Proteínas de Bactérias/genética , Corantes de Rosanilina/metabolismo , 3-Hidroxiacil-CoA Desidrogenase/genética , 3-Hidroxiacil-CoA Desidrogenase/metabolismo , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Proteínas de Bactérias/metabolismo , Enoil-CoA Hidratase/genética , Enoil-CoA Hidratase/metabolismo , ProteômicaRESUMO
Dye-decolorization is one of the most important steps in dye-polluted wastewater treatment. The dye-decolorization bacteria were isolated from active sludge collected from wastewater treating pond of a dyeing and printing plant using serial dilution method. Among the 44 bacteria isolates from the active sludge, the strain Bacillus amyloliquefaciens W36 was found to have strong ability in dye-decolorization. The effects of carbon source, nitrogen sources, C/N, metal ions, temperature, pH, and rotation speed for dye-decolorization were investigated. The optimum decolorization conditions were that the strain was grown in enriched mineral salt medium (EMSM) using maltose 1 g/L, (NH4)2SO4 1 g/L as carbon and nitrogen source respectively, supplemented with 100 mg/L different dyes (pH 6.0), at 30 °C, 200 rpm from 48 to 96 h. The bacteria could aerobically decolorize dyes, such as Coomassie brilliant blue (95.42%), Bromcresol purple (93.34%), Congo red (72.37%) and Sarranine (61.7%), within 96 h. The dyes decolorization products were analyzed by ultra-violet and visible (UV-vis) spectroscopy before and after decolorization, which indicated that the four dyes were significantly degraded by the strain. The results indicated that the bacteria Bacillus amyloliquefaciens W36 could be used in dye-polluted wastewater treatment.
Assuntos
Bacillus amyloliquefaciens/isolamento & purificação , Bacillus amyloliquefaciens/metabolismo , Corantes/metabolismo , Esgotos/microbiologia , Descoloração da Água/métodos , Bactérias/genética , Bactérias/isolamento & purificação , Bactérias/metabolismo , Biodegradação Ambiental , Púrpura de Bromocresol/metabolismo , Carbono/metabolismo , Vermelho Congo/metabolismo , Nitrogênio/metabolismo , Corantes de Rosanilina/metabolismo , Águas Residuárias/microbiologia , Purificação da ÁguaRESUMO
The development and function of tissues, blood, and the immune system is dependent upon proximity for cellular recognition and communication. However, the detection of cell-to-cell contacts is limited due to a lack of reversible, quantitative probes that can function at these dynamic sites of irregular geometry. Described here is a novel chemo-genetic tool developed for fluorescent detection of protein-protein proximity and cell apposition that utilizes the Fluorogen Activating Protein (FAP) in combination with a Dye Activated by Proximal Anchoring (DAPA). The FAP-DAPA system has two protein components, the HaloTag and FAP, expressed on separate protein targets or in separate cells. The proteins function to bind and activate a compound that has the hexyl chloride (HexCl) ligand connected to malachite green (MG), the FAP fluorogen, via a poly(ethylene glycol) spacer spanning up to 28 nm. The dehalogenase protein, HaloTag, covalently binds the HexCl ligand, locally concentrating the attached MG. If the FAP is within range of the anchored fluorogen, it will bind and activate MG specifically when the bath concentration is too low to saturate the FAP receptor. A new FAP variant was isolated with a 1000-fold reduced KD of â¼10-100 nM so that the fluorogen activation reports proximity without artificially enhancing it. The system was characterized using purified FRB and FKBP fusion proteins and showed a doubling of fluorescence upon rapamycin induced complex formation. In cocultured HEK293 cells (HaloTag and FAP-expressing) fluorescence increased at contact sites across a broad range of labeling conditions, more reliably providing contact-specific fluorescence activation with the lower-affinity FAP variant. When combined with suitable targeting and expression constructs, this labeling system may offer significant improvements in on-demand detection of intercellular contacts, potentially applicable in neurological and immunological synapse measurements and other transient, dynamic biological appositions that can be perturbed using other labeling methods that stabilize these interactions.
Assuntos
Corantes Fluorescentes/metabolismo , Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Corantes de Rosanilina/metabolismo , Cumarínicos/química , Cumarínicos/metabolismo , Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Hexanos/química , Hexanos/metabolismo , Humanos , Hidrocarbonetos Clorados/química , Hidrocarbonetos Clorados/metabolismo , Hidrolases/química , Ligantes , Microscopia de Fluorescência , Polietilenoglicóis/química , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Corantes de Rosanilina/química , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Mannanases are ubiquitous enzymes and are being explored for diverse industrial applications. In this study, a novel bacterial strain Microbacterium sp. CIAB417 was identified and characterized for extracellular production of mannanase. Microbacterium sp. CIAB417 was found to produce maximum mannanase after 36 h of incubation at 37 °C. Mannanase produced by the isolate was observed for maximum activity at optimum pH of 6 and optimum temperature of 50 °C. Crude mannanase was found to be capable of producing mannooligosachharides (MOS) by hydrolyzing hemicellulose from locust bean gum and Aloe vera. The produced MOS was characterized and found to be mixture of mannobiose to mannohexose units. Mannanase was also explored for decolorization of dyes. Bromophenol blue and coomassie blue R-250 were observed to be decolorized to the extent of 45.40 and 42.75%, respectively. Hence, the identified bacterial strain producing mannanase could be of great significance for applications in food and textile industry.
Assuntos
Microbacterium/metabolismo , Oligossacarídeos/metabolismo , beta-Manosidase/metabolismo , Azul de Bromofenol/metabolismo , Corantes , Galactanos/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Mananas/metabolismo , Gomas Vegetais/metabolismo , Corantes de Rosanilina/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
Malachite green (MG) and crystal violet (CV) are the typical triphenylmethane dyes, which are recalcitrant molecules exerting mutagenic and carcinogenic effects on living organisms. Characterization of the residues of MG, CV, and their metabolites in biological organisms is of importance, especially for in vivo and in situ characterization. In this study, a method for determination of trace MG, CV, and their leuco metabolites in zebrafish by surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) was developed. A microscale solid-phase microextraction (SPME) probe was developed and used for extraction and enrichment of trace MG, CV, and their leuco metabolites in zebrafish after exposure. After that, the loaded SPME probe was directly employed for nanoESI-MS analysis under ambient and open-air conditions. Under the optimum conditions, the method demonstrated good linearity, with correlation coefficient values (r2) no less than 0.9925. The limits of detection and quantification were 0.014-0.023 ng mL-1 and 0.046-0.077 ng mL-1, respectively. By using the proposed method, the bioaccumulation of MG and CV in zebrafish was investigated, and the distribution of MG, CV, and their leuco metabolites in different organs of zebrafish was studied. MG, CV, and their leuco metabolites were all found in zebrafish tissues including brain, muscle, heart, and kidney after exposure, with highest concentration in intestine followed in ovary.
Assuntos
Violeta Genciana/análise , Nanotecnologia , Corantes de Rosanilina/análise , Microextração em Fase Sólida , Animais , Violeta Genciana/metabolismo , Estrutura Molecular , Tamanho da Partícula , Corantes de Rosanilina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Propriedades de Superfície , Peixe-ZebraRESUMO
Malachite green (MG), a triphenylmethane dye is extensively used for coloring silk, aquaculture and textile industries, it has also has been reported toxic to life forms. This study aimed to investigate the biodegradation potential of MG by actinobacteria. The potent actinobacterial strain S20 used in this study was isolated from forest soil (Sabarimala, Kerala, India) and identified as Streptomyces chrestomyceticus based on phenotype and molecular features. Strain S20 degraded MG up to 59.65 ± 0.68% was studied in MSM medium and MG (300 mg l-1) and degradation was increased (90-99%) by additions of 1% glucose and yeast extract into the medium at pH 7. The treated metabolites from MG by S20 characterized by FT-IR and GC-MS. The results showed MG has been degraded into nontoxic compounds evaluated by (1) phytotoxic assay on Vigna radiata, (2) microbial toxicity on Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Streptococcus sp. and Escherichia coli, (3) cytotoxicity assay in a human cell line (MCF 7). The toxicity studies demonstrated that the byproducts from MG degradation by S. chrestomyceticus S20 were no toxic to plants and microbes and less toxic to human cells as compared to the parent MG. Perhaps this is the first work reported on biodegradation of MG by S. chrestomyceticus which could be a potential candidate for the removal of MG from various environments.
Assuntos
Corantes de Rosanilina , Streptomyces/metabolismo , Humanos , Células MCF-7 , Corantes de Rosanilina/metabolismo , Corantes de Rosanilina/toxicidade , Vigna/crescimento & desenvolvimentoRESUMO
BACKGROUND: Biodegradation of toxic organic dye using nanomaterial-based microbial biocatalyst is an ecofriendly and promising technique. MATERIALS AND METHODS: Here, we have investigated the novel properties of functionalized Au-Ag bimetallic nanoparticles using extremophilic Deinococcus radiodurans proteins (Drp-Au-AgNPs) and their degradation efficiency on the toxic triphenylmethane dye malachite green (MG). RESULTS AND DISCUSSION: The prepared Drp-Au-AgNPs with an average particle size of 149.8 nm were capped by proteins through groups including hydroxyl and amide. Drp-Au-AgNPs demonstrated greater degradation ability (83.68%) of MG than D. radiodurans cells and monometallic AuNPs. The major degradation product was identified as 4-(dimethylamino) benzophenone, which is less toxic than MG. The degradation of MG was mainly attributed to the capping proteins on Drp-Au-AgNPs. The bimetallic NPs could be reused and maintained MG degradation ability (>64%) after 2 cycles. CONCLUSION: These results suggest that the easily prepared Drp-Au-AgNPs have potential applications as novel nanomedicine for MG detoxification, and nanomaterial for biotreatment of a toxic polyphenyl dye-containing wastewater.
Assuntos
Proteínas de Bactérias/metabolismo , Deinococcus/química , Nanopartículas Metálicas/química , Corantes de Rosanilina/metabolismo , Proteínas de Bactérias/química , Corantes/química , Corantes/metabolismo , Difusão Dinâmica da Luz , Cromatografia Gasosa-Espectrometria de Massas , Ouro/química , Ouro/metabolismo , Tamanho da Partícula , Corantes de Rosanilina/química , Prata/química , Prata/metabolismo , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Malachite green is a common environmental pollutant that poses a great threat to non-target organisms, including humans. This study reports the characterization of a bacterial strain, Pseudomonas veronii JW3-6, which was isolated from a malachite green enrichment culture. This strain degraded malachite green efficiently in a wide range of temperature and pH levels. Under optimal degradation conditions (32.4 °C, pH 7.1, and inoculum amount of 2.5 × 107 cfu/mL), P. veronii JW3-6 could degrade 93.5% of 50 mg/L malachite green within seven days. Five intermediate products from the degradation of malachite green were identified: leucomalachite green, 4-(dimethylamino) benzophenone, 4-dimethylaminophenol, benzaldehyde, and hydroquinone. We propose a possible degradation pathway based on these findings. The present study is the first to report the degradation of malachite green by P. veronii and the identification of hydroquinone as a metabolite in the degradation pathway.
Assuntos
Biodegradação Ambiental , Redes e Vias Metabólicas , Pseudomonas/metabolismo , Corantes de Rosanilina/metabolismo , Biodiversidade , Microbiologia Ambiental , Cinética , Estrutura Molecular , Filogenia , Pseudomonas/classificação , Pseudomonas/genética , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S , Corantes de Rosanilina/químicaRESUMO
In this study, mutant CotA-laccase SF was successfully expressed in Escherichia coli by co-expression with phospholipase C. The optimized extracellular expression of CotA-laccase SF was 1257.22 U/L. Extracellularly expressed CotA-laccase SF exhibits enzymatic properties similar to intracellular CotA-laccase SF. CotA-laccase SF could decolorize malachite green (MG) under neutral and alkaline conditions. The Km and kcat values of CotA-laccase SF to MG were 39.6 mM and 18.36 s-1. LC-MS analysis of degradation products showed that MG was finally transformed into 4-aminobenzophenone and 4-aminophenol by CotA-laccase. The toxicity experiment of garlic root tip cell showed that the toxicity of MG metabolites decreased. In summary, CotA-laccase SF had a good application prospect for degrading malachite green.
Assuntos
Corantes/metabolismo , Lacase/metabolismo , Corantes de Rosanilina/metabolismo , Corantes/toxicidade , Escherichia coli/genética , Escherichia coli/metabolismo , Lacase/genética , Mutação , Corantes de Rosanilina/toxicidadeRESUMO
Malachite green is a carcinogenic dye that has been detected in fish tissues and freshwater. Here we evaluated the malachite green decoloring ability of a photoautotrophic cyanobacterium, Synechococcus elongatus PCC 7942 (Synechococcus), that lives in freshwater. Results show that 99.5% of the dye was removed by Synechococcus through bioabsorption and bioaccumulation; however, the dye was not degraded or chemically modified. Next, we established an engineered Synechococcus strain to degrade the dye after uptake. The triphenylmethane reductase gene katmr was heterologously expressed, resulting in high production of a soluble recombinant protein. The engineered strain showed advanced decoloring abilities at a low cell density and in stressful environments. It degraded malachite green into the smaller molecules 4-methylaminobenzoic acid and 4-hydroxyl-aniline. After treatment with the engineered cyanobacterium, the growth of wheat seeds was fully recovered in the presence of malachite green. These results demonstrate the potential application of the engineered Synechococcus as a photosynthetic cell factory for the removal of malachite green from wastewater.
Assuntos
Proteínas de Bactérias/genética , Corantes/metabolismo , Oxirredutases/genética , Corantes de Rosanilina/metabolismo , Synechococcus/metabolismo , Poluentes Químicos da Água/metabolismo , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Enterobacter aerogenes/enzimologia , Enterobacter aerogenes/genética , Engenharia Metabólica , Oxirredutases/metabolismo , Fotobiorreatores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Synechococcus/genética , Compostos de Tritil/metabolismoRESUMO
Laccases are multi copper oxidases that can oxidize both phenolic and nonphenolic lignin related compounds. Consequently, there has been continuous demand for laccases for the oxidative degradation of phenolic dyes in effluents. In view of this, the present work was focused on laccase production by solid substrate fermentation using a newly isolated fungus Perenniporia tephropora-L168. To intensify the laccase production, the process parameters pH, nitrogen, inducer, and substrate: water ratio were optimized by using statistical model. A set of optimal conditions noted were pH 3, nitrogen 0.001 g/L; inducer 0.5% and substrate: water ratio (1:10), which yielded laccase 1,160 U/g. The crude laccase exhibited noteworthy potential to degrade a triaryl-methane dye especially Malachite green. Also, during bioremediation studies, the statistical process optimization could achieve 81% decolourization within 180 min. The laccase treatment brought chemical transformation in malachite green as evident from UV-Visible spectra, FTIR, HPLC while toxicity against bacteria and fungi was also reduced. During phytotoxicity study, effect of treated and untreated dye on germination of seed was analyzed. Interestingly, the germination index for Vigna aconitifolia and Vigna radiata was increased by two and fourfold, respectively. Overall, this work demonstrates optimized production of laccase using Perenniporia tephropora-L168 and its efficient bioremediation potential for triaryl-methane dye.
Assuntos
Corantes/metabolismo , Fermentação , Lacase/biossíntese , Polyporaceae/metabolismo , Corantes de Rosanilina/metabolismo , Corantes/química , Concentração de Íons de Hidrogênio , Lacase/química , Polyporaceae/isolamento & purificação , Corantes de Rosanilina/químicaRESUMO
The active laccases of ascomycetous fungus Setosphaeria turcica were identified by Native-PAGE and ESI-MS/MS, and one of these isozymes Stlac2 was heterologous expressed to investigate the decolorization of malachite green. Setosphaeria turcica produced three active laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and the recombinant Stlac2 exhibited activity of 28.23â¯U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60⯰C. The activity of recombinant Stlac2 was inhibited by 10â¯mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10â¯mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20â¯mg/L malachite green in 15â¯min without any mediators. CONCLUSIONS: Generally, recombinant protein of fungal laccase Stlac2 was active without glycosylation and decolorize malachite green efficiently, which has potential industrial applications.
Assuntos
Ascomicetos/enzimologia , Lacase/genética , Lacase/metabolismo , Corantes de Rosanilina/metabolismo , Sequência de Aminoácidos , Cor , Expressão Gênica , Glicosilação , Concentração de Íons de Hidrogênio , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Lacase/química , Modelos Moleculares , Pichia/genética , Conformação Proteica , TemperaturaRESUMO
In this study, a CotA laccase from Bacillus subtilis cjp3 was successfully immobilized onto magnetic graphene oxide (MGO) nanomaterials via covalent bonding with hydrochloride/N-hydroxysuccinimide (EDC/NHS). The morphology, structure, and properties of the MGO-laccase were then characterized by scanning-electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray-photoelectron spectroscopy (XPS), and a magnetic-property-measurement system (MPMS). The magnetic composite exhibited an extremely high binding capacity of ~145.04mg/g and maintained maximal relative enzyme activities at 25°C, pH7, and a reaction time of 2h. The pH, thermal, operational, and storage stabilities of MGO-laccase were significantly improved over those of free laccase. Moreover, MGO-laccase exhibited a higher tolerance than that of free laccase in the presence of organic solvents, inhibitors, metal ions, and salts. Furthermore, MGO-laccase showed good decolorization performance of malachite green (MG), with decolorization rates reaching 99% after 5h of reaction at 30°C and pH6. In addition, the maximum saturation magnetization of MGO-laccase was 27.7emu/g, allowing for rapid magnetic separation. Accordingly, magnetic separation allowed MGO-laccase to maintain 75% of its activity after ten consecutive decolorization cycles.