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1.
Ecotoxicol Environ Saf ; 196: 110561, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32276163

RESUMO

A ternary catalysis system was investigated to evaluate the comparative degradation of toxic fungicide metabolite 3,5-dichloroaniline (3,5-DCA) by laccase and MnO2 with mediators. In this study, copper based fungal enzyme laccase (Trametes versicolor origin) and metal catalyst MnO2 with various combinations of phenolic mediators (catechol, syringaldehyde, syringic acid, caffeic acid and gallic acid) were monitored to optimize and screen the better one for 3,5-DCA degradation assay. Catechol showed better potentiality in reduction of 3,5-DCA among the studied mediators. Catechol (2mM) showed the highest reduction rate (99-100%) followed by syringaldehyde (40.51%) with 2U/mL of laccase at 25 °C within 24 h reaction time. Similarly, complete degradation of 3,5-DCA was obtained by catechol (2mM) with 2 mg/mL of MnO2 in MnO2-mediator assay. The notable finding of current study indicated the triggering of catechol for better 3,5-DCA degradation at higher pH condition but inertness in laccase-mediator assay due to laccase destabilization. The reaction pathways of optimized mediator-based catalysis for laccase and MnO2 were proposed. Finally, the optimized laccase-catechol based degradation was considered as a pioneer green catalysis approach to reduce the toxic metabolite 3,5-DCA concentrations in aqueous medium as compared to MnO2-catechol catalysis.


Assuntos
Compostos de Anilina/análise , Fungicidas Industriais/análise , Lacase/metabolismo , Compostos de Manganês/química , Óxidos/química , Trametes/enzimologia , Compostos de Anilina/metabolismo , Benzaldeídos/química , Catálise , Catecóis/química , Fungicidas Industriais/metabolismo , Fenóis/química
2.
Ecotoxicol Environ Saf ; 195: 110419, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32182526

RESUMO

Ecotoxicological evaluations require the use of assays with several bioindicators from different trophic levels. Only a few ecotoxicological tests using fungi have been developed, reason why, detection of adverse effects from compounds that exert fungicide action may be overlooked. This work developed a toxicity test based on the inhibition of laccase enzymatic activity in the fungus Trametes versicolor. The test was applied to several fungicides and succeeded to determine inhibition values (half maximum effective concentration, EC50) for most of them (flusilazole, imazalil, pyrimethanil, tetraconazole), though a clear dose-response was not evident for others (thiabendazole, metalaxyl). The application on atrazine (herbicide), imidacloprid (insecticide) and oxytetracycline (antibiotic), proved the proposed test is suitable towards other agrochemicals. The test was also used to estimate the detoxification resulting from two different approaches employed in the removal of agrochemicals. (a) First, in the liquid-phase elimination by fungal biomass simultaneously removing atrazine, imazalil, tebuconazole and triadimenol, the test showed a significant decrease in toxicity by biodegradation (adsorption contribution to detoxification was negligible). (b) Second, a solid-phase biomixture (used for pesticide degradation from agricultural wastewater) partially removed atrazine, imazalil, metalaxyl and pyrimethanil after 33 d; nonetheless, this system could not reduce the toxicity of the matrix, and higher laccase inhibition was detected after the treatment. The design test increases the battery of available bioassays to determine the toxicity of agrochemicals, and provides an interesting tool to monitor biodegradation processes.


Assuntos
Ecotoxicologia/métodos , Monitoramento Ambiental/métodos , Fungicidas Industriais/toxicidade , Lacase/antagonistas & inibidores , Praguicidas/análise , Poluentes do Solo/análise , Trametes/efeitos dos fármacos , Agricultura , Biodegradação Ambiental , Bioensaio , Fungicidas Industriais/análise , Trametes/enzimologia
3.
Biochim Biophys Acta Proteins Proteom ; 1868(2): 140335, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31785381

RESUMO

Pyranose oxidase (POx) catalyzes the oxidation of d-glucose to 2-ketoglucose with concurrent reduction of oxygen to H2O2. POx from Trametes ochracea (ToPOx) is known to react with alternative electron acceptors including 1,4-benzoquinone (1,4-BQ), 2,6-dichlorophenol indophenol (DCPIP), and the ferrocenium ion. In this study, enzyme variants with improved electron acceptor turnover and reduced oxygen turnover were characterized as potential anode biocatalysts. Pre-steady-state kinetics of the oxidative half-reaction of ToPOx variants T166R, Q448H, L545C, and L547R with these alternative electron acceptors were evaluated using stopped-flow spectrophotometry. Higher kinetic constants were observed as compared to the wild-type ToPOx for some of the variants. Subsequently, the variants were immobilized on glassy carbon electrodes. Cyclic voltammetry measurements were performed to measure the electrochemical responses of these variants with glucose as substrate in the presence of 1,4-BQ, DCPIP, or ferrocene methanol as redox mediators. High catalytic efficiencies (Imaxapp/KMapp) compared to the wild-type POx proved the potential of these variants for future bioelectrocatalytic applications, in biosensors or biofuel cells. Among the variants, L545C showed the most desirable properties as determined kinetically and electrochemically.


Assuntos
Desidrogenases de Carboidrato/metabolismo , Técnicas Eletroquímicas/métodos , 2,6-Dicloroindofenol/química , Benzoquinonas/química , Biocatálise , Desidrogenases de Carboidrato/química , Desidrogenases de Carboidrato/genética , Domínio Catalítico , Eletrodos , Compostos Ferrosos/química , Glucose/química , Glucose/metabolismo , Cinética , Metalocenos/química , Mutagênese Sítio-Dirigida , Oxirredução , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Trametes/enzimologia
4.
Chemosphere ; 239: 124779, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31521934

RESUMO

Laccase mediator system (LMS), a very attractive candidate for refractory organics biodegradation, harbors tremendous potential on industry application. However, the performance of LMS usually varies with the discrepancy of mediators and substrates in their chemical structures. Here, we adopt electrochemical analysis that is able to assess the degradation performance of various LMS on three different dyes by quantitative analysis of reaction outcome. Two mechanisms were suggested to explain the grafting of three mediators (1-Hydroxybenzotriazole, Violuric Acid and Acetosyringone), involving the transformation of proton or electron to produce active moieties, which subsequently react with target substrates. A thorough electrochemical insight into the redox features of mediators and its change in the presence of laccase and substrates were carried out using electrochemical analysis. The effectiveness of each kind of LMS on substrates was preliminarily evaluated by analyzing the change of the peak current and potential of mediators. The actual conversion rate of dyes was used to verify the analysis results, which confirms the important role of the stability of the oxidized form as well as their redox potential of the mediators in determining the mechanism of substrate oxidation. The application of electrochemical analysis in efficiency evaluation of LMS shed new light on effective selection of suitable mediators for degradation of refractory organics. It was therefore possible to prejudge the efficacy of LMS by analyzing the electrochemical parameters of target substances and mediators, which undoubtedly has broad further application prospects of LMS.


Assuntos
Corantes/química , Lacase/química , Poluentes Químicos da Água/química , Acetofenonas/química , Antraquinonas/química , Barbitúricos/química , Biodegradação Ambiental , Cor , Vermelho Congo/química , Técnicas Eletroquímicas , Lacase/metabolismo , Oxirredução , Corantes de Rosanilina/química , Trametes/enzimologia , Triazóis/química
5.
Artigo em Inglês | MEDLINE | ID: mdl-31757086

RESUMO

Laccases have attracted a great deal of interest because of their remarkable ability for the degradation of synthetic dyes present in wastewaters. New laccase producing sources with robust operational and functional properties are being continuously explored. In this work, the potential for the decolorization and detoxification of synthetic dyes was evaluated in two Mexican strains of the genus Trametes. The decolorization capacity of Trametes maxima LE130 and Trametes sp. LA1 was tested in solid and liquid media. The phytotoxicity of the degradation products was determined using Raphanus sativus and Pisum sativum seeds. In solid media, both strains showed a higher decolorization capacity (p ≤ 0.05) than Phanerochaete chrysosporium ATCC 24725, which is known to be very efficient in lignin and dye-degradation. They produced laccase as the main ligninolytic enzyme; T. maxima LE130 secreted a single isoform of 43.9 kDa, while Trametes sp. LA1 produced three isoforms of 67.3, 58.6 and 52.7 kDa, respectively. Trametes sp. LA1 culture fluids were capable of decolorizing and detoxifying chemically diverse dyes (anthraquinonic dye Remazol Brilliant Blue R, azoic Reactive Black 5 and triphenylmethane Crystal Violet) without the addition of redox mediators. Therefore, this could be considered as a new laccase source which could be potentially competitive in the bioremediation of dye-containing wastewaters.


Assuntos
Biodegradação Ambiental , Corantes/metabolismo , Inativação Metabólica , Lacase/metabolismo , Trametes/enzimologia , Águas Residuárias/química , Descoloração da Água/métodos , Corantes/química , Corantes/toxicidade , Raphanus/efeitos dos fármacos
6.
J Agric Food Chem ; 67(43): 12054-12060, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31560529

RESUMO

The purpose of current research is to design and acquire novel biological macromolecule materials with enhanced functional properties. Chitosan-ferulic acid binary conjugate (CFC) was synthesized based on the carbodiimide-mediated coupling reaction, and then ß-lactoglobulin-ferulic acid-chitosan ternary conjugate (BFCC) was fabricated by laccase induction. Furthermore, the impact of laccase concentration on the formation mechanism of BFCC was investigated by the analyses of reaction group content, ultraviolet-visible (UV-vis) absorption, circular dichroism (CD), and fluorescence spectroscopy. Results showed that hetero- and homo-conjugates between CFC and ß-lactoglobulin (ß-LG) were achievable at the low concentration (≤4 U/mL) and high concentration (≥6 U/mL) of laccase, respectively. The CD spectrum indicated that the interaction with CFC made ß-LG more disorderly. Functional evaluation results revealed that the antioxidant activity and thermal stability of BFCC were improved compared with ß-LG. The knowledge obtained in the present study provided an effective method to acquire innovative biological macromolecule materials with desirable functional characteristics.


Assuntos
Quitosana/química , Ácidos Cumáricos/química , Lacase/química , Lactoglobulinas/química , Animais , Biocatálise , Bovinos , Dicroísmo Circular , Proteínas Fúngicas/química , Conformação Proteica , Trametes/enzimologia
7.
Ecotoxicol Environ Saf ; 183: 109555, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31419699

RESUMO

In this study, we investigated the transformation of atenolol (ATL) by the naturally occurring laccase from Trametes versicolor in aqueous solution. Removal efficiency of ATL via laccase-catalyzed reaction in the presence of various laccase mediators was examined, and found that only the mediator 2, 2, 6, 6-tetramethyl-1-piperidinyloxy (TEMPO) was able to greatly promote ATL transformation. The influences of TEMPO concentration, laccase dosage, as well as solution pH and temperature on ATL transformation efficiency were tested. As TEMPO concentrations was increased from 0 to 2000 µM, ATL transformation efficiency first increased and then decreased, and the optimal TEMPO concentration was determined as 500 µM. ATL transformation efficiency was gradually increased with increasing laccase dosage. ATL transformation was highly pH-dependent with an optimum pH of 7.0, and it was almost constant over a temperature range of 25-50 °C. Humic acid inhibited ATL transformation through competition reaction with laccase. The presence of anions HCO3- and CO32- reduced ATL transformation due to both anions enhanced solution pHs, while Cl-, SO42-, and NO3- at 10 mM showed no obvious influence. The main transformation products were identified, and the potential transformation pathways were proposed. After enzymatic treatment, the toxicity of ATL and TEMPO mixtures was greatly reduced. The results of this study might present an alternative clean strategy for the remediation of ATL contaminated water matrix.


Assuntos
Atenolol/análise , Óxidos N-Cíclicos/química , Lacase/metabolismo , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Atenolol/metabolismo , Catálise , Substâncias Húmicas/análise , Oxirredução , Temperatura , Trametes/enzimologia , Poluentes Químicos da Água/metabolismo
8.
Adv Mater ; 31(32): e1901677, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215110

RESUMO

Untethered actuation is important for robotic devices to achieve autonomous motion, which is typically enabled by using batteries. Using enzymes to provide the required electrical charge is particularly interesting as it will enable direct harvesting of fuel components from a surrounding fluid. Here, a soft artificial muscle is presented, which uses the biofuel glucose in the presence of oxygen. Glucose oxidase and laccase enzymes integrated in the actuator catalytically convert glucose and oxygen into electrical power that in turn is converted into movement by the electroactive polymer polypyrrole causing the actuator to bend. The integrated bioelectrode pair shows a maximum open-circuit voltage of 0.70 ± 0.04 V at room temperature and a maximum power density of 0.27 µW cm-2 at 0.50 V, sufficient to drive an external polypyrrole-based trilayer artificial muscle. Next, the enzymes are fully integrated into the artificial muscle, resulting in an autonomously powered actuator that can bend reversibly in both directions driven by glucose and O2 only. This autonomously powered artificial muscle can be of great interest for soft (micro-)robotics and implantable or ingestible medical devices manoeuvring throughout the body, for devices in regenerative medicine, wearables, and environmental monitoring devices operating autonomously in aqueous environments.


Assuntos
Glucose/química , Músculos/química , Oxigênio/química , Polímeros/química , Polivinil/química , Pirróis/química , Aspergillus niger/enzimologia , Fontes de Energia Bioelétrica , Biocombustíveis , Técnicas Biossensoriais , Condutividade Elétrica , Eletricidade , Técnicas Eletroquímicas , Glucose Oxidase/química , Ouro/química , Humanos , Lacase/química , Oxirredução , Estresse Mecânico , Trametes/enzimologia
9.
Enzyme Microb Technol ; 128: 34-39, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186108

RESUMO

Utilization of polyphenol oxidases (laccase, tyrosinase) in biosensor technology is an efficient approach towards phenol detection, which is significant in numerous fields such as environmental monitoring, food industry etc. The use of crude extract instead of pure enzyme eliminates the need for costly and laborious processes of enzyme separation and purification. This study employs polyphenol oxidase extract, biosynthesized by white-rot fungi Trametes pubescens (TP) for the development of amperometric biosensors for catechol detection. The catalytic activity of the crude extract was firstly used to induce the bio-synthesis of conducting polymer - polypyrrole (Ppy), resulting in the self-encapsulation of the enzyme extract within the conducting material. The viability and biological integrity of the enzyme extract was preserved after the synthesis and was able to efficiently detect phenolic compounds such as catechol. Comparative evaluations between the biosynthesized Ppy based biosensor (bio-Ppy) and the biosensor based on bio-PPy with additional enzyme extract (bio-Ppy-TP) were performed. Lastly, the performance of these two biosensors was compared with that of a third one, based on chemically synthesized Ppy with enzyme extract (chem-Ppy-TP). All three types of biosensors proved high efficiency for catechol detection at low concentration (1-60 µM) and were employed for real sample detection in fruit wines showing linear correlation with the spectrophotometric results obtained with the Folin-Ciocalteau standard test.


Assuntos
Técnicas Biossensoriais/métodos , Catecol Oxidase/metabolismo , Catecóis/análise , Misturas Complexas/metabolismo , Enzimas Imobilizadas/metabolismo , Trametes/enzimologia , Misturas Complexas/isolamento & purificação , Polímeros , Pirróis
10.
Colloids Surf B Biointerfaces ; 181: 470-479, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31176119

RESUMO

HYPOTHESIS: The development of enzymatic conjugates with industrial applications require approaches with good scalability and batch-to-batch reproducibility. Hereof, nearly monodisperse iron oxide nanoparticles can be synthesized by thermal decomposition with high yields. A mixture of gallic and polyacrylic acid is used for the direct water transfer and later immobilization of laccase (Trametes versicolor). EXPERIMENTS: Nanoparticles were synthesized by thermal decomposition (13.1 nm by TEM, 50 nm by DLS) and later transferred to water by a ligand exchange method with polyacrylic acid and a polyacrylic acid/gallic acid mixture. Laccase was immobilized on water dispersions of both nanoparticles via a carbodiimide coupling. FINDINGS: The nanoparticles exhibited superparamagnetic behavior with insignificant values of iHc. The presence of gallic acid hindered the formation of multiple polyacrylic acid layers, therefore improving the colloidal stability of the nanoparticles (100 nm by DLS) after weeks of storage. Nanoparticles containing only polyacrylic acid showed poor activity (60% loading, 4.5% activity), while nanoparticles with both polyacrylic and gallic acids showed enzymatic activity values 4.4 times higher than the free enzyme (13% loading, 57% activity). The nanoparticles improved the storage stability (8 times) of the enzyme, its thermoresistance (4 times), and its reactivity against azo dyes Camalgite and Congo Red (21 and 27% increase, respectively). In addition to some improved catalytic properties in comparison to similar works, this is the first report of the use of gallic acid for both the direct transfer to water and enzyme immobilization on highly monodisperse, batch-to-batch reproducible superparamagnetic nanoparticles.


Assuntos
Enzimas Imobilizadas/metabolismo , Compostos Férricos/metabolismo , Lacase/metabolismo , Nanopartículas de Magnetita/química , Nanopartículas/metabolismo , Trametes/enzimologia , Compostos Férricos/química , Ácido Gálico/química , Ácido Gálico/metabolismo , Estrutura Molecular , Nanopartículas/química , Tamanho da Partícula , Propriedades de Superfície
11.
Protein Sci ; 28(6): 1143-1150, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30972861

RESUMO

Trametes versicolor glutathione transferase Omega 3S (TvGSTO3S) catalyzes the conjugation of isothiocyanates (ITC) with glutathione (GSH). Previously, this isoform was investigated in depth both biochemically and structurally. Structural analysis of complexes revealed the presence of a GSH binding site (G site) and a deep hydrophobic binding site (H site) able to bind plant polyphenols. In the present study, crystals of apo TvGSTO3S were soaked with glutathionyl-phenethylthiocarbamate, the product of the reaction between GSH and phenethyl isothiocyanate (PEITC). On the basis of this crystal structure, we show that the phenethyl moiety binds in a new site at loop ß2 -α2 while the glutathionyl part exhibits a particular conformation that occupies both the G site and the entrance to the H site. This binding mode is allowed by a conformational change of the loop ß2 -α2 at the enzyme active site. It forms a hydrophobic slit that stabilizes the phenethyl group at a distinct site from the previously described H site. Structural comparison of TvGSTO3S with drosophila DmGSTD2 suggests that this flexible loop could be the region that binds PEITC for both isoforms. These structural features are discussed in a catalytic context.


Assuntos
Glutationa Transferase/química , Glutationa/biossíntese , Isotiocianatos/metabolismo , Trametes/enzimologia , Sítios de Ligação , Biocatálise , Glutationa/química , Glutationa Transferase/metabolismo , Isotiocianatos/química , Modelos Moleculares , Estrutura Molecular
12.
N Biotechnol ; 52: 1-8, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-30922999

RESUMO

Lignin and lignin components of woody biomass have been identified as an attractive alternative to fossil fuels. However, the complex composition of this plant polymer is one of the drawbacks that limits its exploitation. Biocatalysis of lignin to produce platform chemicals has been receiving great attention as it presents a sustainable approach for lignin valorisation. Aligned with this area of research, in the present study we have applied ultra-high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) to identify the preferred lignin substrates of a ligninolytic enzyme, a laccase produced by the terrestrial fungus Trametes versicolor. A commercial lignin was incubated with the laccase and acetosyringone (a laccase mediator) for up to 168 h and direct infusion electrospray FT-ICR MS enabled the identification of thousands of molecular species present in the complex lignin sample at different incubation time points. Significant changes in the chemical composition of lignin were detected upon laccase treatment, which resulted in a decrease in the molecular mass distribution of assigned species, consistent with laccase lytic activity. This reduction was predominantly in species classified as lignin-like (based on elemental ratios) and polymeric in nature (>400 Da). Of particular note was a fall in the number of species assigned containing sulfur. Changes in the chemical composition/structure of the lignin polymer were supported by FT-IR spectroscopy. We propose the use of FT-ICR MS as a rapid and efficient technique to support the biotechnological valorisation of lignin as well as the development and optimization of laccase-mediator systems for treating complex mixtures.


Assuntos
Ciclotrons , Análise de Fourier , Lacase/metabolismo , Lignina/metabolismo , Acetofenonas/metabolismo , Íons , Análise de Componente Principal , Espectroscopia de Infravermelho com Transformada de Fourier , Enxofre , Espectrometria de Massas em Tandem , Trametes/enzimologia
13.
Chemosphere ; 224: 743-750, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30851526

RESUMO

As a typical class of environmental endocrine disruptors, bisphenol A poses a potential threat to the sustainable survival and reproduction of living beings and human beings. In this study, the interaction between Trametes versicolor laccase and bisphenol A (BPA) was studied by molecular docking simulation, and the catalytic degradation of BPA was verified by experiments. The conditions for the laccase production of T. versicolor were optimized by orthogonal design, and the degradation of BPA was studied using its crude enzyme solution. The optimum degradation conditions were obtained by response surface methodology (RSM). Ultimately, the transformation products after 3 and 6 h of reaction were detected by gas chromatography-mass spectrometry. Docking results demonstrated that the reaction between laccase and BPA was spontaneous, and the degradation rate in 24 h reached 88.76%. RSM results showed that the highest BPA degradation rate of 97.68% was reached after 1 h reaction at 44.6 °C, 5 mg/L initial BPA concentration, and pH 5.20. The intermediate products of BPA catalyzed by laccase included ethylbenzene, p-xylene, and cyclohexanone 1-methyl-4-isopropenyl-2-cyclohexenol. This finding reveals that BPA degradation by the crude laccase from T. versicolor starts from the C atoms between two benzene rings that connect BPA. Compared with expensive pure enzyme, the crude laccase solution prepared by T. versicolor showed greater efficiency in BPA degradation. This work provides theoretical references and experimental methods for the biological processing of harmful pollutants.


Assuntos
Compostos Benzidrílicos/metabolismo , Biodegradação Ambiental , Disruptores Endócrinos/metabolismo , Lacase/metabolismo , Fenóis/metabolismo , Trametes/metabolismo , Agaricales/metabolismo , Derivados de Benzeno/análise , Catálise , Poluentes Ambientais/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Simulação de Acoplamento Molecular , Trametes/enzimologia , Xilenos/análise
14.
Int J Biol Macromol ; 128: 681-691, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711566

RESUMO

This investigation may be of interest for researchers working on the determination of several biocatalytic properties of the laccase from Trametes versicolor. So, We will treated the effects of pH, temperature, several organic components and heavy metals by performing enzyme assays in the presence of a 2,6 dimethoxyphenol (DMP) as substrate on the laccase activity. The optimum activity and temperature are 4 and 40 °C, respectively. The maximum rate of the reaction is 124.53 U/mg and the Michaelis constant is in order of 1.23 mM. The effect of metal ions on the laccase activity with a final concentrations range varying from 1 to 5 mM show that the Cu2+ ions increase the activity for concentration inferiors to 4 mM and the other metal ions have a relative influence on the laccase activity. Four tri-block copolymers based on poly(ethylene oxide) and poly(propylene oxide) and two polyethylene glycols are used to study the synthetic polymers effects on the enzymatic activity. Also, we have demonstrated that the laccase keeps 95% of its initial activity at 60 °C in the PEGDA8000 and PEGDA6000 gel matrix. The maximum rate of the immobilized laccase is approximately around 21.03 and 47.22% smaller than the free one.


Assuntos
Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lacase/química , Lacase/metabolismo , Trametes/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Cinética , Lacase/antagonistas & inibidores , Metais Pesados/farmacologia , Modelos Moleculares , Polímeros/química , Propilenoglicóis/química , Conformação Proteica , Temperatura
15.
Chemosphere ; 222: 865-871, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30753965

RESUMO

Bentonite is a natural and environmentally clay mineral, and bentonite-derived mesoporous materials (BDMMs) were obtained conveniently from the alkali and acid treatment of bentonite. In the present study, BDMMs were explored for immobilization of laccase obtained from Trametes versicolor. As a result, bentonite-derived mesoporous materials-Laccase (BDMMs-Lac) was developed for the removal of tetracycline (TC). The enzyme immobilization process was carried out through physical adsorption contact (ion exchange adsorption, hydrogen bond adsorption, and Van der waals adsorption) between the BDMMs and laccase. The process of immobilization remarkably increased its operating temperature. The BDMMs-Lac exhibited over 60% removal efficiency for TC within 3 h in the presence of 1-hydroxybenzotriazole (HBT). In conclusion, BDMMs-Lac showed more promising potential than free laccase for practical continuous applications.


Assuntos
Bentonita/química , Enzimas Imobilizadas/química , Lacase/química , Tetraciclina/isolamento & purificação , Adsorção , Concentração de Íons de Hidrogênio , Minerais , Temperatura , Trametes/enzimologia
16.
Bioconjug Chem ; 30(3): 679-697, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30620558

RESUMO

Trametes versicolor can degrade bark as a source for carbon necessity. Therefore, it secretes lignin peroxidase, mangan peroxidase, and laccase. The laccase enzyme was produced in high yield at pH of 5 and glucose concentration of 10 g L-1. In optimized medium, the enzyme activity was between 200 and 250 U L-1 when an inducer was absent. It was seen that the activity reached 400 U L-1 when phenol was used as an inducer. The molecular weight of purified laccase was found to be 80 kDa with SDS-PAGE, and kinetic constant Km and Vmax values for 2,2'-azino-bis(3-ethylbenzthiazoline)-6-sulfonate were determined to be 3.66 × 10-4 µM and 1652 U L-1, respectively. Because of these properties, these enzymes are widely used, free or immobilized, in industrial areas. Laccase enzyme decolorization of six different dyes was carried out. A decolorization capacity of 50-99% was achieved by cultivation for 20 days using a beginning dye concentration of 20 ppm. The removal of color with an active enzyme was obtained around 90%. Also, the laccase enzyme was conjugated, amine-functionalized, low-symmetry phthalocyanine. This conjugate was examined by both photodynamic therapy and chemosensor application. This conjugate fluorescence had a quantum yield of 0.32 (lifetime 3.59 ns) and efficiently generated singlet oxygen (quantum yield 0.4). The conjugate successfully displayed photodamage in HeLa and HuH-7 cells in the photodynamic therapy application. These results indicate that the conjugate represents an interesting agent with potential applications in photodynamic therapy. In addition, the chemosensor behavior of this compound to different metal ions has been studied, and this conjugate is displayed as a fluorescence chemosensor for the determination of Fe3+ions.


Assuntos
Cobre/química , Indóis/química , Peroxidases/química , Linhagem Celular , Precipitação Química , Cromatografia por Troca Iônica , Cor , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Humanos , Lacase/química , Lacase/isolamento & purificação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Trametes/enzimologia
17.
Lab Chip ; 19(4): 634-640, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30644486

RESUMO

Chlorophenols have a strong medicinal smell and can be detected by the human nose at parts-per-million levels. Therefore, continuous monitoring of chlorophenols in water supplies is highly important. Herein, we reported a microfluidic sensor which can be used to detect 2,4-dichlorophenol (2,4-DCP) in real time with a limit of detection of around 0.1 ppm. The microfluidic sensor is a membrane-less galvanic cell which consists of two laminar flows running in parallel inside a straight channel. The sensor measures the potential difference between a solution containing 2,4-DCP and a reference solution containing acetate buffer. In a continuous-flow mode, the cell potential is proportional to the concentration of 2,4-DCP. To render specificity for the sensor, we incorporate a pre-treatment section where the incoming solution containing 2,4-DCP is split into two streams. One of the streams is brought into contact with cross-linked laccase aggregates (which catalyzes the hydrolysis of 2,4-DCP) and the second stream is taken as a reference solution. By comparing the potential difference between the two streams, we can determine the concentration of 2,4-DCP with high specificity. The microfluidic sensor platform is potentially useful for real-time detection of micropollutants present in aquatic systems with high sensitivity and specificity.


Assuntos
Clorofenóis/análise , Lacase/metabolismo , Técnicas Analíticas Microfluídicas , Clorofenóis/metabolismo , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lacase/química , Técnicas Analíticas Microfluídicas/instrumentação , Oxirredução , Tamanho da Partícula , Agregados Proteicos , Propriedades de Superfície , Trametes/enzimologia
18.
N Biotechnol ; 50: 44-51, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30668986

RESUMO

The high-molecular weight fraction of olive mill wastewater (HMW-OMW), a byproduct of olive oil biorefinery, was used at the reactor level as the basal medium for production of laccase and Mn-dependent peroxidase (MnP) by Trametes ochracea. Three reactor systems, namely stirred tank reactors equipped with either Rushton turbines or marine impeller and draft tube (STR and STR-MD, respectively) and an air-lift reactor (ALR) were compared for this purpose. Although inocula were supplied as intact pellets, in both STR-based systems fungal growth evolved rapidly into a dispersed form while the ALR enabled the maintenance of the pellet growth mode. STR was deemed to be the most promising system since it best supported the production MnP activity on the HMW-OMW-based medium and its performance in laccase production did not differ from that observed with the STR-MD. Among the stirring regimes considered (250, 400, 500 and 600 rpm), the best production in the STR was observed at 500 rpm and 1.0 vvm for both laccase (8850 ± 270 IU L-1 on day 15) and MnP (17,027.4 ± 87.2 IU L-1 on day 13). When the inocula were supplied to the STR in homogenized form, the MnP production peak (16,856 ± 1070 IU L-1) was attained 8 days earlier than the previous condition and that of laccase was nearly doubled (14,967 ± 907 IU L-1). When compared with literature data, T. ochracea MnP production and productivity on the HMW-OMW-based medium were the highest reported for a wild-type fungal strain.


Assuntos
Lacase/biossíntese , Lignina/metabolismo , Azeite de Oliva/metabolismo , Peroxidases/biossíntese , Trametes/metabolismo , Eliminação de Resíduos Líquidos , Águas Residuárias/química , Peso Molecular , Azeite de Oliva/química , Trametes/enzimologia
19.
Environ Sci Pollut Res Int ; 26(5): 5131-5139, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30607853

RESUMO

This study proposed the optimization of a laccase-mediator system to reduce pesticide levels (bentazone, carbofuran, diuron, clomazone, tebuconazole, and pyraclostrobin) on aqueous medium. Firstly, the mediator concentration of 1 mM was established (average removal of 36%). After that, seven redox-mediating compounds, namely, 2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, gallic acid, protocatechuic acid, and vanillin, were compared regarding their removal efficiency. The highest removal (77%) was achieved with the laccase-vanillin system. After this screening, the optimization was carried out by a 22 full factorial design. Variables under study were the enzyme (laccase) activity and vanillin concentration. Maximum removal (53-85%) was achieved with 0.95 U/mL laccase and 1.8 mM vanillin. Pesticide removal in reaction media was fitted to the first-order kinetics equation with an average half-time life of 2.2 h. This is the first study of the use of this natural compound as a mediator in the degradation of the pesticides under investigation. The results of this study contribute, with alternative methods, to decrease pesticide levels since they are highly persistent in aqueous samples and, as a result, mitigate the environmental impact.


Assuntos
Benzaldeídos/química , Lacase/metabolismo , Praguicidas/análise , Poluentes Químicos da Água/análise , Benzaldeídos/metabolismo , Biodegradação Ambiental , Catálise , Cinética , Oxirredução , Praguicidas/metabolismo , Trametes/enzimologia , Poluentes Químicos da Água/metabolismo
20.
Carbohydr Polym ; 207: 510-520, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30600034

RESUMO

Aerogels are obtained by laccase/2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-oxidation of galactomannans (GMs) from the leguminous plants fenugreek (Trigonella foenum-graecum), sesbania (Sesbania bispinosa) and guar (Cyamopsis tetragonolobus). GM oxidation in aqueous solutions causes a viscosity increase, resulting in structured and stable hydrogels. Upon lyophilization, water-insoluble aerogels are obtained, capable of water uptake up to several times their own weight. The materials derived from the three gums have been analyzed and compared. Chemical modifications have been studied by electrospray-ionization mass spectrometry (ESI-MS) and Fourier-transform infrared spectroscopy (FTIR). Polymer structure has been determined by liquid-state and semi-quantitative solid-state nuclear magnetic resonance (NMR) spectroscopy and chemical stability by incubation under variable conditions of pH, ionic strength and solvent nature. The results show that hydrogel formation is due to oxidation of primary hydroxyl groups to carbonyl and carboxyl groups and subsequent formation of hemiacetal and ester bonds. Fenugreek displays the highest stability, compared to guar and sesbania rehydrated aerogels. This feature could be interpreted by its higher degree of substitution (Gal:Man = 1:1) and consequently higher amount of galactose primary alcohols, leading to more extensive crosslinking.


Assuntos
Géis/química , Mananas/química , Galactanos/química , Lacase/química , Estrutura Molecular , Oxirredução , Gomas Vegetais/química , Sesbania/química , Trametes/enzimologia , Trigonella/química
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