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1.
Artigo em Inglês | MEDLINE | ID: mdl-32181694

RESUMO

In the past decade, Mexican coasts have received an enormous influx of macroalgae species, producing serious environmental and public health concerns. Here, we developed a green methodology to generate a new polymer from the lignin contained in the macroalgae. The methodology consists in lignin extraction-by-boiling and its subsequent polymerization with a laccase-like enzyme from the green algae Tetraselmis gracilis (Tg-laccase). Mass spectrometry revealed the presence of guaiacyl (G), p-hydroxyphenyl (H), and sinapyl alcohol as the main monolignols in the lignin from Sargassum sp. On the other hand, MALDI-TOF spectra shows an increase in the size of the lignin chain after enzymatic polymerization process with Tg-laccase. Besides, the characterization of the novel polymer -using 1H NMR, FTIR, SEC-FPLC, and UV/Vis- allowed establishing that during the polymerization process there is a decrease in the number of phenolic groups as well as loss of aromatic protons, which allowed proposing a polimerizacion mechanism. This methodology could be promising in the development of a new lignin-based polymer and would open a new direction for the environmental management of the macroalgae on the Mexican beaches.


Assuntos
Clorófitas/enzimologia , Química Verde/métodos , Lacase/química , Lignina/síntese química , Polimerização , Alga Marinha/química , Lignina/química , Peso Molecular , Fenóis/química
2.
Food Chem ; 315: 126233, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32018078

RESUMO

Botrytized sweet wines are made with berries infected by the fungus Botrytis cinerea. The aim of this study was to identify biomarkers of B. cinerea infection in sweet wines with a focus on laccases which are exocellular oxidase enzymes produced by this fungus during fruit contamination. Total proteins from six commercial sweet wines, including three naturally botrytized wines and three non-botrytized wines were analysed by LC-QTOF-MS. Five laccases, namely laccase-1-BcLCC1, laccase-2-BcLCC2, laccase-3-BcLCC7, laccase-8-BcLCC8 and laccase-12-BcLCC12, were identified in both types of wine. Then, a targeted proteomic approach by LC-MRM was used to semi-quantify laccase-2-BcLCC2 and laccase-3-BcLCC7, in the six samples. LC-MRM targeted analysis of the two enzymes allowed the discrimination of botrytized versus non-botrytized sweet white wines.


Assuntos
Biomarcadores/análise , Botrytis/enzimologia , Lacase/metabolismo , Proteômica , Vinho/análise , Cromatografia Líquida , Frutas/química , Lacase/química , Vitis/metabolismo
3.
Electron. j. biotechnol ; 43: 1-7, Jan. 2020. tab, graf, ilus
Artigo em Inglês | LILACS | ID: biblio-1087520

RESUMO

Background: Textile industry not only plays a vital role in our daily life but also a prominent factor in improving global economy. One of the environmental concern is it releases huge quantities of toxic dyes in the water leading to severe environmental pollution. Bacterial laccase and azoreductase successfully oxidize complex chemical structure of nitrogen group-containing azo dyes. Additionally, the presence of textile dye infuriates bacterial peroxidase to act as a dye degrading enzyme. Our present study deals with three textile dye degrading enzymes laccase, azoreductase, and peroxidase through analyzing their structural and functional properties using standard computational tools. Result: According to the comparative analysis of physicochemical characteristics, it was clear that laccase was mostly made up of basic amino acids whereas azoreductase and peroxidase both comprised of acidic amino acids. Higher aliphatic index ascertained the thermostability of all these three enzymes. Negative GRAVY value of the enzymes confirmed better water interaction of the enzymes. Instability index depicted that compared to laccase and preoxidase, azoreductase was more stable in nature. It was also observed that the three model proteins had more than 90% of total amino acids in the favored region of Ramachandran plot. Functional analysis revealed laccase as multicopper oxidase type enzyme and azoreductase as FMN dependent enzyme, while peroxidase consisted of α-ß barrel with additional haem group. Conclusion: Present study aims to provide knowledge on industrial dye degrading enzymes, choosing the suitable enzyme for industrial set up and to help in understanding the experimental laboratory requirements as well.


Assuntos
Compostos Azo/metabolismo , Peroxidase/química , Lacase/química , NADH NADPH Oxirredutases/química , Temperatura , Compostos Azo/química , Indústria Têxtil , Biodegradação Ambiental , Simulação por Computador , Estabilidade Enzimática , Peroxidase/metabolismo , Lactase/metabolismo , Corantes/metabolismo , NADH NADPH Oxirredutases/metabolismo
4.
Chemosphere ; 246: 125676, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31918078

RESUMO

Covalent-immobilization of the laccase enzyme onto the iron oxide nanoparticles was achieved using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) as cross-linkers. The presence of sulphur moeity in the laccase immobilized nanoparticles (LNPs) observed through Scanning Electron Microscopy- Energy dispersive X-ray spectroscopy (SEM-EDS) spectra confirmed the immobilization of laccase enzyme. The TEM analysis of iron oxide nanoparticles (FNPs), chitosan coated iron nanoparticles (CNPs) and laccase immobilized nanoparticles (LNPs) confirmed their sizes around 12, 15 and 20 nm, respectively. The effect of LNPs in degrading chlorpyrifos under field conditions was studied by simulating the conditions in a column. Column A, which was used as control showed more leaching of chlorpyrifos as compared to column B containing LNPs. The sorption coefficient (Kd) value obtained for control (column A) and LNPs containing column B were 21.6 and 112.3 L/kg, respectively. LNPs altered the Kd values of soil thereby showing lesser leaching potential. Higher the Kd value, lesser will be the leaching potential in the ground water. Copper in laccase enzyme resulted in hydrolysis of chlorpyrifos. Chitosan used for coating on FNPs and soil organic matter resulted in the adsoption of chlorpyrifos. Current results will allow a better assessment of the role of LNPs as a competent deterrent in chlorpyrifos mobility and degradation.


Assuntos
Clorpirifos/análise , Lacase/química , Nanopartículas/química , Poluentes do Solo/química , Quitosana/química , Clorpirifos/metabolismo , Cobre , Enzimas Imobilizadas/química , Compostos Férricos , Lacase/metabolismo , Solo
5.
Chemosphere ; 248: 125931, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32000035

RESUMO

Soil microbial enzymes may induce lignin decomposition, accompanied by generation of free radicals. The evolution of environmentally persistent free radicals (EPFRs) and reactive oxygen species (ROS) during laccase-catalyzed lignin decomposition remains unclear. Characterization by electron paramagnetic resonance spectroscopy revealed gradually increased concentration of EPFRs, with maximum levels within 6 h that remained constant, accompanied by the increase in g-factor from 2.0037 to 2.0041. The results suggested the generation of oxygen-centered radicals on lignin. The EPFRs produced on solid samples slowly decreased by 17.2% over 17 d. ROS were also detected to have a similar trend as that of the evolution of EPFRs. Scanning electron microscopy, attenuated total reflectance-Fourier transform infrared spectroscopy, gel permeation chromatography and nuclear magnetic resonance analyses suggested the demethylation and oxidation of lignin. We clarify the biogeochemical transformation of lignin and potential contributions to the generation of EPFRs and ROS in soil.


Assuntos
Radicais Livres/química , Lignina/química , Espectroscopia de Ressonância de Spin Eletrônica , Lacase/química , Oxirredução , Espécies Reativas de Oxigênio/química , Solo
6.
Appl Biochem Biotechnol ; 190(3): 1035-1048, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31659712

RESUMO

Laccases are a group of enzymes with a critical activity in the degradation process of both phenolic and non-phenolic compounds. These enzymes present in a diverse array of species, including fungi and bacteria. Since this enzyme is in the market for different usages from industry to medicine, having a better knowledge of its structures and properties from diverse sources will be useful to select the most appropriate candidate for different purposes. In the current study, sequence- and structure-based characteristics of these enzymes from fungi and bacteria, including pseudo amino acid composition (PseAAC), physicochemical characteristics, and their secondary structures, are being compared and classified. Autodock 4 software was used for docking analysis between these laccases and some phenolic and non-phenolic compounds. The results indicated that features including molecular weight, aliphatic, extinction coefficient, and random coil percentage of these protein groups present high degrees of diversity in most cases. Categorization of these enzymes by the notion of PseAAC, showed over 96% accuracy. The binding free energy between fungal laccases and their substrates showed to be considerably higher than those of bacterial ones. According to the outcomes of the current study, data mining methods by using different machine learning algorithms, especially neural networks, could provide valuable information for a fair comparison between fungal and bacterial laccases. These results also suggested an association between efficacy and physicochemical features of laccase enzymes from different sources.


Assuntos
Aminoácidos/análise , Bactérias/enzimologia , Fungos/enzimologia , Lacase/química , Lacase/isolamento & purificação , Modelos Químicos , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Reprodutibilidade dos Testes
7.
Sci Total Environ ; 708: 134970, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31740057

RESUMO

Remediation of polycyclic aromatic hydrocarbons (PAHs) in oily sludge has become the focus of attention. UV spectrophotometer analysis showed that four types of PAHs were found in sample, which including phenanthrene, anthracene, benzo(a)anthracene and benzo(b)fluoranthene. In order to degrade PAH effectively, the laccase reverse micelles system was proposed. The system protects laccase from being affected by organic phase. Reverse micelles were prepared by using isooctane to simulate oil. The optimum water content W0 was 10 by measuring the electrical conductivity of the system. Under this condition, the effects of pH, temperature and ionic strength on the degradation rate of PAHs were investigated. Also, compared with that of non-immobilized laccase, the ratio between the secondary structures of laccase under different conditions was studied. The results showed that the highest laccase activity was obtained at pH 4.2 and 30 °C with 60 mmol/L KCl. Meanwhile, the structure of α-helix accounts for the largest proportion, and the ratio of α-helix in the laccase secondary structure in the laccase-reverse micelle system was higher than that of the non-immobilized one under this condition. Finally, predicting the reactive site of the degradation of polycyclic aromatic hydrocarbons was simulated by ORCA (Version 4.2.0). The application in oily sludge was further conducted. This study provides an effective method and basis for the degradation of PAHs in oily sludge.


Assuntos
Lacase/química , Micelas , Hidrocarbonetos Policíclicos Aromáticos , Temperatura
8.
Bioelectrochemistry ; 132: 107418, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31835109

RESUMO

A shell of nanostructured ferric tannates was spontaneously developed on the surface of naked maghemite nanoparticles (SAMNs, the core) by a simple wet reaction with tannic acid (TA). The as obtained core-shell nanomaterial (SAMN@TA) displays specific electrocatalytic and surface properties, which significantly differ from parent maghemite. Thanks to the known proclivity of TA to interact with proteins, SAMN@TA was proposed as a support for the direct immobilization of an enzyme. A ternary functional nanobioconjugate (SAMN@TA@TvL) was successfully self-assembled by incubating laccase from Trametes versicolor (TvL) and SAMN@TA. The SAMN@TA@TvL hybrid was kinetically characterized with respect to the native enzyme and applied for building an easy-to-use analytical device for the detection of polyphenols. The electrochemical biosensor allowed the determination of polyphenols by square wave voltammetry in mixed water-methanol solutions. The system sensitivity was 868.9 ±â€¯1.9nA µM-1, the LOD was 81 nM and the linearity range was comprised between 100 nM and 10 µM. The proposed approach was successfully applied to detect phenolics in blueberry extracts as real samples. Results suggest that SAMN@TA could be a promising, low cost and versatile tool for the creation of nano-bio-conjugates aimed at the development of new electrochemical sensing platforms.


Assuntos
Técnicas Eletroquímicas/métodos , Compostos Férricos/química , Lacase/química , Nanoestruturas/química , Fenóis/análise , Catálise
9.
Bioelectrochemistry ; 132: 107407, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31862537

RESUMO

The performance of electrochemical laccase-based biosensors can be improved by immobilizing the enzyme on composite Layer-by-Layer (LbL) supports in which materials with complementary functions are combined. LbL films are formed by layers combining an electrocatalytic material which favors electron transfer (sulfonated copper phthalocyanine, CuPcS(-)), an ionic liquid which enhances the electrical conductivity of the layers (1-butyl-3-methylimidazolium tetrafluoroborate, IL(+)) and a material able to promote enzyme immobilization (chitosan, CHI(+)). Composite films with different structures have been demonstrated to be efficient electrocatalysts, producing an increase in the magnitude of the responses towards catechol. The most intense and reproducible electrocatalytic effect was observed when a layer of the CuPcS(-) was placed on top of a layer formed by a mixture of CHI(+) + IL(+) to obtain [CHI(+) + IL(+)|CuPcS(-)]2 films. Biosensors with laccase immobilized on the surface of the LbL layers [CHI(+) + IL(+)|CuPcS(-)]2|Lac showed mediated electron transfer between the redox enzyme and the film and a reproducibility of device-to-device performance of 4.1%. The amperometric biosensor showed a sensitivity of 0.237 A·M-1 and a linear detection range from 2.4 µM to 26 µM for catechol. The excellent Limit of detection (LOD) of 8.96·10-10 M (3·σ /m) is one order of magnitude lower than that obtained in similar studies. A Michaelis-Menten constant of 3.16 µM confirms excellent enzyme-substrate affinity.


Assuntos
Catecóis/análise , Quitosana/química , Indóis/química , Líquidos Iônicos/química , Lacase/química , Compostos Organometálicos/química , Técnicas Biossensoriais , Catálise , Limite de Detecção , Reprodutibilidade dos Testes , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
10.
Biosens Bioelectron ; 151: 111961, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31868607

RESUMO

The direct electrical connection of laccase on the electrode surface is a key feature in the design of efficient and stable biocathodes. However, laccase can perform a direct electron transfer only when it is in the preferred orientation toward the electrode. Here we report the investigation of the orientation of laccase from white rot fungus on multi-walled carbon nanotube surface modified with a naphthalene group. Naphthylated multi wall carbon nanotubes were synthesized and the kinetics of laccase from white rot fungus adsorption and its direct electro-catalytic activity toward oxygen reduction was investigated by QCM and electrochemical techniques. Compared to pristine multi-walled carbon nanotubes laccase shows a high affinity to be adsorbed onto the surface of naphthylated carbon nanotubes at a very fast rate. The subsequent wiring to the naphthylated multi-walled carbon nanotubes is accompanied by a reorientation and arrangement of adsorbed laccase to create a composite biocathode that exhibits a high-performance for oxygen reduction by direct electron transfer with maximum current densities of 3 mA cm-2.


Assuntos
Lacase/química , Nanotubos de Carbono/química , Naftalenos/química , Técnicas Biossensoriais , Catálise , Técnicas Eletroquímicas , Eletrodos , Transporte de Elétrons , Cinética , Oxirredução , Oxigênio/química , Propriedades de Superfície
11.
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
12.
Chemosphere ; 240: 124882, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31726609

RESUMO

Biomimetic dynamic membrane (BDM) has been employed as a promising membrane separation technology regarding water/wastewater treatment (Model pollutant is methylene blue). Given its catalytic function on micro-pollutant removal and fouling control, detailed mechanism for impacts of fabrication method, carriers (CNT and GO) and laccase on the construction of biomimetic layer and enzyme immobilization have not been clear so far. In this work, the BDM performance with various fabrication methods, carriers and laccase were investigated and verified. The BDM fabrication tests demonstrated that BDM with mixed filtration method had better filtration performance (up to 120 L m-2 h-1 flux and 80% removal rate) than BDM with stepwise filtration method. Moreover, the laccases immobilized on GO exhibited a stronger laccase activity than those on CNT. Increasing CNT or GO dosage strengthened removal rate, but lowered flux, meanwhile flux and removal rate exhibited a significant fluctuation with certain laccase dosage. At 25 g m-2 CNT or GO dosage and 50 g m-2 laccase dosage, the optimized flux and removal rate values were obtained. Further study investigated the surface morphology and property of BDM, showing that BDM with mixed filtration method turned out to be the optimized enzyme immobilization mechanism and fabrication method. In addition, during multiple filtration cycles, with the optimized conditions, the removal rate, flux and laccase activity of BDM could maintain at high levels. On account of the finding of the present study, selecting a suitable fabrication method, appropriate CNT or GO dosage and laccase dosage can indeed optimize the structure of biomimetic layer and enzyme immobilization, expanding its possibility on sustainable operation.


Assuntos
Biomimética/métodos , Enzimas Imobilizadas/química , Filtração/métodos , Lacase/química , Membranas Artificiais , Purificação da Água/métodos , Catálise , Concentração de Íons de Hidrogênio , Azul de Metileno/análise , Águas Residuárias/química , Poluentes Químicos da Água/análise
13.
Microb Cell Fact ; 18(1): 200, 2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31727078

RESUMO

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.


Assuntos
Biocatálise , Poluentes Ambientais/metabolismo , Lacase , Biodegradação Ambiental , Ecossistema , Fungos/enzimologia , Lacase/química , Lacase/metabolismo , Plantas/enzimologia , Água/análise , Água/química , Purificação da Água
14.
Molecules ; 24(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671660

RESUMO

Many dyes and pigments are used in textile and printing industries, and their wastewater has been classed as a top source of pollution. Biodegradation of dyes by fungal laccase has great potential. In this work, the influence of reaction time, pH, temperature, dye concentration, metal ions, and mediators on laccase-catalyzed Remazol Brilliant Blue R dye (RBBR) decolorization were investigated in vitro using crude laccase from the white-rot fungus Ganoderma lucidum. The optimal decolorization percentage (50.3%) was achieved at 35 °C, pH 4.0, and 200 ppm RBBR in 30 min. The mediator effects from syringaldehyde, 1-hydroxybenzotriazole, and vanillin were compared, and 0.1 mM vanillin was found to obviously increase the decolorization percentage of RBBR to 98.7%. Laccase-mediated decolorization percentages significantly increased in the presence of 5 mM Na+ and Cu2+, and decolorization percentages reached 62.4% and 62.2%, respectively. Real-time fluorescence-quantitative PCR (RT-PCR) and protein mass spectrometry results showed that among the 15 laccase isoenzyme genes, Glac1 was the main laccase-contributing gene, contributing the most to the laccase enzyme activity and decolorization process. These results also indicate that under optimal conditions, G. lucidum laccases, especially Glac1, have a strong potential to remove RBBR from reactive dye effluent.


Assuntos
Antraquinonas/metabolismo , Corantes/metabolismo , Lacase/genética , Reishi/enzimologia , Biodegradação Ambiental , Cor , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos , Concentração de Íons de Hidrogênio , Isoenzimas/metabolismo , Lacase/química , Lacase/metabolismo , Metais/farmacologia , Reishi/genética , Temperatura , Fatores de Tempo , Transcrição Genética
15.
ACS Appl Mater Interfaces ; 11(43): 39436-39448, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31580644

RESUMO

Laccases are multi-copper oxidase enzymes having widespread applications in various biotechnological fields. However, low stability of free enzymes restricts their industrial use. Development of effective methods to preserve and even increase the enzymatic activity is critical to maximize their use, though this remains a challenge. In the present study we immobilized Trametes versicolor laccase on pH-responsive (and charge-switchable) Pluronic-stabilized silver nanoparticles (AgNPsTrp). Our results demonstrate that colloidal stabilization of AgNPsTrp with the amphiphilic copolymer Pluronic F127 enhances enzyme activity (AgNPsTrpF1 + Lac6) by changing the active site microenvironment, which is confirmed by circular dichroism (CD) and fluorescence spectroscopy. Detailed kinetic and thermodynamic studies reveal a facile strategy to improve the protein quality by lowering the activation energy and expanding the temperature window for substrate hydrolysis. The immobilized nanocomposite did not show any change in flow behavior which indirectly suggests that the enzyme stability is maintained, and the enzyme did not aggregate or unfold upon immobilization. Finally, assessing the anticancer efficacy of this nanocomposite in breast cancer MCF-7 cells shows the inhibition of cell proliferation through ß-estradiol degradation and cells apoptosis. To understand the molecular mechanism involved in this process, semi qRT-PCR experiments were performed, which indicated significant decrease in the mRNA levels of anti-apoptotic genes, for example, BCL-2 and NF-kß, and increase in the mRNA level of pro-apoptotic genes like p53 in treated cells, compared to control. Overall, this study offers a completely new strategy for tailoring nano-bio-interfaces with improved activity and stability of laccase.


Assuntos
Neoplasias da Mama , Enzimas Imobilizadas , Proteínas Fúngicas , Lacase , Poloxâmero , Polyporaceae/enzimologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Feminino , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lacase/química , Lacase/farmacologia , Células MCF-7 , Proteínas de Neoplasias/biossíntese , Poloxâmero/química , Poloxâmero/farmacologia
16.
Genes (Basel) ; 10(9)2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514462

RESUMO

Laccase is a widely used industrial oxidase for food processing, dye synthesis, paper making, and pollution remediation. At present, laccases used by industries come mainly from fungi. Plants contain numerous genes encoding laccase enzymes that show properties which are distinct from that of the fungal laccases. These plant-specific laccases may have better potential for industrial purposes. The aim of this work was to conduct a genome-wide search for the soybean laccase genes and analyze their characteristics and specific functions. A total of 93 putative laccase genes (GmLac) were identified from the soybean genome. All 93 GmLac enzymes contain three typical Cu-oxidase domains, and they were classified into five groups based on phylogenetic analysis. Although adjacent members on the tree showed highly similar exon/intron organization and motif composition, there were differences among the members within a class for both conserved and differentiated functions. Based on the expression patterns, some members of laccase were expressed in specific tissues/organs, while some exhibited a constitutive expression pattern. Analysis of the transcriptome revealed that some laccase genes might be involved in providing resistance to oomycetes. Analysis of the selective pressures acting on the laccase gene family in the process of soybean domestication revealed that 10 genes could have been under artificial selection during the domestication process. Four of these genes may have contributed to the transition of the soft and thin stem of wild soybean species into strong, thick, and erect stems of the cultivated soybean species. Our study provides a foundation for future functional studies of the soybean laccase gene family.


Assuntos
Evolução Molecular , Lacase/genética , Proteínas de Plantas/genética , Caules de Planta/genética , Seleção Genética , Soja/genética , Resistência à Doença , Lacase/química , Lacase/metabolismo , Família Multigênica , Melhoramento Vegetal/métodos , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Caules de Planta/fisiologia , Soja/enzimologia , Soja/microbiologia
17.
Ecotoxicol Environ Saf ; 184: 109670, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31526924

RESUMO

Due to the abundant binding sites and high stability, a synthesized meso-MIL-53(Al) was selected as the backbone and used for immobilizing laccase (Lac-MIL-53(Al)) to catalytically degrade of TCS. XRD, BET and FTIR analyses proved that the carboxyl groups on PTA of meso-MIL-53(Al) could provide sufficient adsorption sites for physically immobilizing laccase through hydrogen bonds and electrostatic interactions. Although the catalytic efficiency of Vmax/Km slightly decreased from 785 to 607 min-1 due to the mass transfer limitation upon immobilized, Lac-MIL-53(Al) showed high activity recovery (93.8%) and stability. The conformational analysis indicated the laccase could partially enter into the MOF by conformational changes without impairing laccase, although the laccase molecular (6.5 nm × 5.5 nm × 4.5 nm) was larger than the mesopore sizes of the MOF (4 nm). The kinetics indicated that Lac-MIL-53(Al) could remove 99.24% of TCS within 120 min due to the synergy effect of the adsorption of meso-MIL-53(Al) and catalytic degradation of laccase. Meanwhile, Lac-MIL-53(Al) could remain approximately 60% of activity for up to 8 times reuse without desorption. The GC/MS and LC/MS/MS analyses further confirmed that TCS could be transformed to 2, 4-DCP by laccase via the breakage of the ether bond, or to passivated dimers, trimers and tetramers by the self-coupling and oxidization of the phenoxyl radicals, and finally removed by precipitation. In summary, enzyme-MOF composite might be a potential strategy to control the micropollutants in the wastewater.


Assuntos
Alumínio/química , Enzimas Imobilizadas/química , Lacase/química , Estruturas Metalorgânicas/química , Triclosan/análise , Poluentes Químicos da Água/análise , Adsorção , Catálise , Enzimas Imobilizadas/metabolismo , Cinética , Lacase/metabolismo , Oxirredução , Triclosan/metabolismo , Águas Residuárias/química , Poluentes Químicos da Água/metabolismo , Purificação da Água/métodos
18.
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
19.
Int J Biol Macromol ; 141: 855-867, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31505206

RESUMO

Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70 °C = 1.2 h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2 h at 100 °C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.


Assuntos
Aspergillus/enzimologia , Lacase/química , Modelos Moleculares , Conformação Proteica , Sequência de Aminoácidos , Catálise , Fenômenos Químicos , Estabilidade Enzimática , Glicosilação , Concentração de Íons de Hidrogênio , Lacase/biossíntese , Lacase/isolamento & purificação , Peso Molecular , Oxirredução , Relação Estrutura-Atividade , Especificidade por Substrato , Difração de Raios X
20.
Acta Crystallogr D Struct Biol ; 75(Pt 9): 804-816, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478903

RESUMO

Laccases are enzymes that catalyze the oxidation of a wide range of organic and inorganic substrates accompanied by the reduction of molecular oxygen to water. Here, a subatomic resolution X-ray crystallographic study of the mechanism of inhibition of the laccase from the basidiomycete fungus Steccherinum murashkinskyi by chloride and fluoride ions is presented. Three series of X-ray diffraction data sets were collected with increasing doses of absorbed X-ray radiation from a native S. murashkinskyi laccase crystal and from crystals of complexes of the laccase with chloride and fluoride ions. The data for the native laccase crystal confirmed the previously deduced enzymatic mechanism of molecular oxygen reduction. The structures of the complexes allowed the localization of chloride and fluoride ions in the channel near the T2 copper ion. These ions replace the oxygen ligand of the T2 copper ion in this channel and can play the role of this ligand in the enzymatic reaction. As follows from analysis of the structures from the increasing dose series, the inhibition of laccases by chloride and fluoride anions can be explained by the fact that the binding of these negatively charged ions at the position of the oxygen ligand of the T2 copper ion impedes the reduction of the T2 copper ion.


Assuntos
Cloretos/metabolismo , Cobre/metabolismo , Fluoretos/metabolismo , Lacase/química , Basidiomycota/enzimologia , Domínio Catalítico , Cristalografia por Raios X/métodos , Ligantes , Modelos Moleculares , Oxirredução , Oxigênio/metabolismo , Conformação Proteica , Imagem Individual de Molécula/métodos
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