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1.
J Environ Sci (China) ; 147: 74-82, 2025 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39003085

RESUMO

Endocrine disruptors such as bisphenol A (BPA) adversely affect the environment and human health. Laccases are used for the efficient biodegradation of various persistent organic pollutants in an environmentally safe manner. However, the direct application of free laccases is generally hindered by short enzyme lifetimes, non-reusability, and the high cost of a single use. In this study, laccases were immobilized on a novel magnetic three-dimensional poly(ethylene glycol) diacrylate (PEGDA)-chitosan (CS) inverse opal hydrogel (LAC@MPEGDA@CS@IOH). The immobilized laccase showed significant improvement in the BPA degradation performance and superior storage stability compared with the free laccase. 91.1% of 100 mg/L BPA was removed by the LAC@MPEGDA@CS@IOH in 3 hr, whereas only 50.6% of BPA was removed by the same amount of the free laccase. Compared with the laccase, the outstanding BPA degradation efficiency of the LAC@MPEGDA@CS@IOH was maintained over a wider range of pH values and temperatures. Moreover, its relative activity of was maintained at 70.4% after 10 cycles, and the system performed well in actual water matrices. This efficient method for preparing immobilized laccases is simple and green, and it can be used to further develop ecofriendly biocatalysts to remove organic pollutants from wastewater.


Assuntos
Compostos Benzidrílicos , Enzimas Imobilizadas , Lacase , Fenóis , Polietilenoglicóis , Poluentes Químicos da Água , Lacase/química , Lacase/metabolismo , Fenóis/química , Poluentes Químicos da Água/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Polietilenoglicóis/química , Quitosana/química , Hidrogéis/química , Biodegradação Ambiental , Disruptores Endócrinos/química
2.
Environ Geochem Health ; 46(8): 282, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38963450

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants with carcinogenic, mutagenic and teratogenic effects. The white-rot fungi in the fungal group have significant degradation ability for high molecular weight organic pollutants. However, exogenous fungi are easily antagonized by indigenous microorganisms. Low molecular weight organic acids, a small molecular organic matter secreted by plants, can provide carbon sources for soil microorganisms. Combining organic acids with white rot fungi may improve the nutritional environment of fungi. In this study, immobilized Trametes versicolor was used to degrade benzo[a]pyrene in soil, and its effect on removing benzo[a]pyrene in soil mediated by different low molecular weight organic acids was investigated. The results showed that when the degradation was 35 days, the removal effect of the experimental group with citric acid was the best, reaching 43.7%. The degradation effect of Trametes versicolor on benzo[a]pyrene was further investigated in the liquid medium when citric acid was added, and the effects of citric acid on the biomass, extracellular protein concentration and laccase activity of Trametes versicolor were investigated by controlling different concentrations of citric acid. In general, citric acid can act as a carbon source for Trametes versicolor and promote its extracellular protein secretion and laccase activity, thereby accelerating the mineralization of benzo[a]pyrene by Trametes versicolor. Therefore, citric acid can be used as a biostimulant in the remediation of PAHs contaminated soil with Trametes versicolor.


Assuntos
Benzo(a)pireno , Biodegradação Ambiental , Ácido Cítrico , Poluentes do Solo , Benzo(a)pireno/toxicidade , Benzo(a)pireno/metabolismo , Ácido Cítrico/metabolismo , Poluentes do Solo/metabolismo , Poluentes do Solo/toxicidade , Lacase/metabolismo , Microbiologia do Solo , Polyporaceae/metabolismo , Trametes/metabolismo , Biomassa
3.
Braz J Biol ; 84: e282099, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38985070

RESUMO

The present study explored the potential of leaf litter as a source of fungi able to produce ligninolytic enzymes for the biodegradation of anthraquinone dyes. Within the colonies isolated from the leaf litter, only three colonies of two species Trametes were selected based on the detection of oxidation and decolorization halos in Petri dishes with PDA (potato-dextrose-agar) + Guaicol and PDA + RBBR (Remazol Brilliant Blue R). The identification of the colonies was done through sequencing of the ITS region. The enzymatic activity of Lac (lacase), MnP (manganês peroxidase) and LiP (lignina peroxidase) was analyzed by spectrophotometry during fermentation in PD+RBBR imedium. Isolates A1SSI01 and A1SSI02 were identified as Trametes flavida, while A5SS01 was identified as Trametes sp. Laccase showed the highest enzymatic activity, reaching 452.13 IU.L-1 (A1SSI01, 0.05% RBBR) after 96h. Isolate A1SSI02 reached the highest percentage of decolorization, achieving 89.28% in seven days. The results imply that these Trametes isolates can be highly effective in waste treatment systems containing toxic anthraquinone dyes. Keywords: laccase, peroxidases, basidiomycete, litter and biodecolorization.


Assuntos
Biodegradação Ambiental , Lacase , Peroxidases , Folhas de Planta , Trametes , Folhas de Planta/química , Folhas de Planta/microbiologia , Trametes/enzimologia , Peroxidases/metabolismo , Lacase/metabolismo , Florestas , Antraquinonas/metabolismo , Corantes , Lignina/metabolismo , Brasil
4.
Physiol Plant ; 176(4): e14415, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38962818

RESUMO

The monotonicity of color type in naturally colored cottons (NCCs) has become the main limiting factor to their widespread use, simultaneously coexisting with poor fiber quality. The synchronous improvement of fiber quality and color become more urgent and crucial as the demand for sustainable development increases. The homologous gene of wild cotton Gossypium stocksii LAC15 in G. hirsutum, GhLAC15, was also dominantly expressed in the developing fibers of brown cotton XC20 from 5 DPA (day post anthesis) to 25 DPA, especially at the secondary cell wall thickening stage (20 DPA and 25 DPA). In XC20 plants with downregulated GhLAC15 (GhLAC15i), a remarkable reduction in proanthocyanidins (PAs) and lignin contents was observed. Some of the key genes in the phenylpropane and flavonoid biosynthesis pathway were down-regulated in GhLAC15i plants. Notably, the fiber length of GhLAC15i plants showed an obvious increase and the fiber color was lightened. Moreover, we found that the thickness of cotton fiber cell wall was decreased in GhLAC15i plants and the fiber surface became smoother compared to that of WT. Taken together, this study revealed that GhLAC15 played an important role in PAs and lignin biosynthesis in naturally colored cotton fibers. It might mediate fiber color and fiber quality by catalyzing PAs oxidation and lignin polymerization, ultimately regulating fiber colouration and development.


Assuntos
Fibra de Algodão , Regulação da Expressão Gênica de Plantas , Gossypium , Lacase , Lignina , Proteínas de Plantas , Gossypium/genética , Gossypium/metabolismo , Gossypium/enzimologia , Lacase/metabolismo , Lacase/genética , Lignina/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Parede Celular/metabolismo , Proantocianidinas/metabolismo , Cor , Pigmentação/genética
5.
Arch Microbiol ; 206(8): 355, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-39017938

RESUMO

Cryptococcus neoformans is an opportunistic pathogenic fungus that produces melanin during infection, an important virulence factor in Cryptococcal infections that enhances the ability of the fungus to resist immune defense. This fungus can synthesize melanin from a variety of substrates, including L-DOPA (L-3,4-dihydroxyphenylalanine). Since melanin protects the fungus from various stress factors such as oxidative, nitrosative, extreme heat and cold stress; we investigated the effects of environmental conditions on melanin production and survival. In this study, we investigated the effects of different pH values (5.6, 7.0 and 8.5) and temperatures (30 °C and 37 °C) on melanization and cell survival using a microtiter plate-based melanin production assay and an oxidative stress assay, respectively. In addition, the efficacy of compounds known to inhibit laccase involved in melanin synthesis, i.e., tunicamycin, ß-mercaptoethanol, dithiothreitol, sodium azide and caspofungin on melanization was evaluated and their sensitivity to temperature and pH changes was measured. The results showed that melanin content correlated with pH and temperature changes and that pH 8.5 and 30 °C, were best for melanin production. Besides that, melanin production protects the fungal cells from oxidative stress induced by hydrogen peroxide. Thus, changes in pH and temperature drastically alter melanin production in C. neoformans and it correlates with the fungal survival. Due to the limited antifungal repertoire and the development of resistance in cryptococcal infections, the investigation of environmental conditions in the regulation of melanization and survival of C. neoformans could be useful for future research and clinical phasing.


Assuntos
Cryptococcus neoformans , Melaninas , Estresse Oxidativo , Temperatura , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/efeitos dos fármacos , Melaninas/metabolismo , Concentração de Íons de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Lacase/metabolismo , Tunicamicina/farmacologia , Caspofungina/farmacologia , Azida Sódica/farmacologia , Mercaptoetanol/farmacologia , Ditiotreitol/farmacologia , Criptococose/microbiologia , Viabilidade Microbiana/efeitos dos fármacos , Lipopeptídeos/farmacologia , Lipopeptídeos/metabolismo
6.
Environ Sci Pollut Res Int ; 31(32): 45441-45451, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38951392

RESUMO

Bisphenol A diglycidyl ether (BADGE), a derivative of the well-known endocrine disruptor Bisphenol A (BPA), is a potential threat to long-term environmental health due to its prevalence as a micropollutant. This study addresses the previously unexplored area of BADGE toxicity and removal. We investigated, for the first time, the biodegradation potential of laccase isolated from Geobacillus thermophilic bacteria against BADGE. The laccase-mediated degradation process was optimized using a combination of response surface methodology (RSM) and machine learning models. Degradation of BADGE was analyzed by various techniques, including UV-Vis spectrophotometry, high-performance liquid chromatography (HPLC), Fourier transform infrared (FTIR) spectroscopy, and gas chromatography-mass spectrometry (GC-MS). Laccase from Geobacillus stearothermophilus strain MB600 achieved a degradation rate of 93.28% within 30 min, while laccase from Geobacillus thermoparafinivorans strain MB606 reached 94% degradation within 90 min. RSM analysis predicted the optimal degradation conditions to be 60 min reaction time, 80°C temperature, and pH 4.5. Furthermore, CB-Dock simulations revealed good binding interactions between laccase enzymes and BADGE, with an initial binding mode selected for a cavity size of 263 and a Vina score of -5.5, which confirmed the observed biodegradation potential of laccase. These findings highlight the biocatalytic potential of laccases derived from thermophilic Geobacillus strains, notably MB600, for enzymatic decontamination of BADGE-contaminated environments.


Assuntos
Compostos Benzidrílicos , Biodegradação Ambiental , Geobacillus stearothermophilus , Geobacillus , Lacase , Lacase/metabolismo , Geobacillus stearothermophilus/enzimologia , Geobacillus/enzimologia , Compostos Benzidrílicos/metabolismo , Fenóis/metabolismo , Compostos de Epóxi/metabolismo
7.
Genes (Basel) ; 15(6)2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38927685

RESUMO

Laccase (LAC) is a diverse group of genes found throughout the plant genome essential for plant growth and the response to stress by converting monolignin into intricate lignin formations. However, a comprehensive investigation of maize laccase has not yet been documented. A bioinformatics approach was utilized in this research to conduct a thorough examination of maize (Zea mays L.), resulting in the identification and categorization of 22 laccase genes (ZmLAC) into six subfamilies. The gene structure and motifs of each subgroup were largely consistent. The distribution of the 22 LAC genes was uneven among the maize chromosomes, with the exception of chromosome 9. The differentiation of the genes was based on fragment replication, and the differentiation time was about 33.37 million years ago. ZmLAC proteins are primarily acidic proteins. There are 18 cis-acting elements in the promoter sequences of the maize LAC gene family associated with growth and development, stress, hormones, light response, and stress response. The analysis of tissue-specific expression revealed a high expression of the maize LAC gene family prior to the V9 stage, with minimal expression at post-V9. Upon reviewing the RNA-seq information from the publicly available transcriptome, it was discovered that ZmLAC5, ZmLAC10, and ZmLAC17 exhibited significant expression levels when exposed to various biotic and abiotic stress factors, suggesting their crucial involvement in stress responses and potential value for further research. This study offers an understanding of the functions of the LAC genes in maize's response to biotic and abiotic stress, along with a theoretical basis for comprehending the molecular processes at play.


Assuntos
Regulação da Expressão Gênica de Plantas , Lacase , Família Multigênica , Proteínas de Plantas , Estresse Fisiológico , Zea mays , Zea mays/genética , Zea mays/crescimento & desenvolvimento , Estresse Fisiológico/genética , Lacase/genética , Lacase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Filogenia , Regiões Promotoras Genéticas , Cromossomos de Plantas/genética
8.
Int J Mol Sci ; 25(11)2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38892114

RESUMO

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.


Assuntos
Ácidos Cafeicos , Proliferação de Células , Dopamina , Lacase , Melaninas , Melanócitos , Poliestirenos , Humanos , Lacase/metabolismo , Melanócitos/metabolismo , Melanócitos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Poliestirenos/química , Ácidos Cafeicos/farmacologia , Ácidos Cafeicos/química , Dopamina/metabolismo , Melaninas/metabolismo , Adesão Celular/efeitos dos fármacos , Levodopa/farmacologia , Levodopa/metabolismo , Levodopa/química , Propriedades de Superfície , Linhagem Celular Tumoral , Células-Tronco de Carcinoma Embrionário/metabolismo , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos
9.
Sci Rep ; 14(1): 14303, 2024 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-38906902

RESUMO

Dopamine is one of the significant neurotransmitters and its monitoring in biological fluids is a critical issue in healthcare and modern biomedical technology. Here, we have developed a dopamine biosensor based on surface plasmon resonance (SPR). For this purpose, the carboxymethyl dextran SPR chip was used as a surface to immobilize laccase as a bioaffinity recognition element. Data analysis exhibited that the acidic pH value is the optimal condition for dopamine interaction. Calculated kinetic affinity (KD) (48,545 nM), obtained from a molecular docking study, showed strong association of dopamine with the active site of laccase. The biosensor exhibited a linearity from 0.01 to 189 µg/ml and a lower detection limit of 0.1 ng/ml (signal-to-noise ratio (S/N) = 3) that is significantly higher than the most direct dopamine detecting sensors reported so far. Experiments for specificity in the presence of compounds that can co-exist with dopamine detection such as ascorbic acid, urea and L-dopa showed no significant interference. The current dopamine biosensor with high sensitivity and specificity, represent a novel detection tool that offers a label-free, simple procedure and cost effective monitoring system.


Assuntos
Técnicas Biossensoriais , Dopamina , Simulação de Acoplamento Molecular , Ressonância de Plasmônio de Superfície , Ressonância de Plasmônio de Superfície/métodos , Dopamina/análise , Dopamina/metabolismo , Técnicas Biossensoriais/métodos , Lacase/metabolismo , Lacase/química , Limite de Detecção , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cinética , Concentração de Íons de Hidrogênio , Dextranos/química
10.
J Hazard Mater ; 474: 134779, 2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38850935

RESUMO

Bisphenol A (BPA), an endocrine disrupter with estrogen activity, can infiltrate animal and human bodies through the food chain. Enzymatic degradation of BPA holds promise as an environmentally friendly approach while it is limited due to lower stability and recycling challenges. In this study, laccase from Bacillus pumilus TCCC 11568 was expressed in Pichia pastoris (fLAC). The optimal catalytic conditions for fLAC were at pH 6.0 and 80 °C, with a half-life T1/2 of 120 min at 70 °C. fLAC achieved a 46 % degradation rate of BPA, and possible degradation pathways were proposed based on identified products and reported intermediates of BPA degradation. To improve its stability and degradation capacity, a whole-cell biocatalyst (WCB) was developed by displaying LAC (dLAC) on the surface of P. pastoris GS115. The functionally displayed LAC demonstrated enhanced thermostability and pH stability along with an improved BPA degradation ability, achieving a 91 % degradation rate. Additionally, dLAC maintained a degradation rate of over 50 % after the fourth successive cycles. This work provides a powerful catalyst for degrading BPA, which might decontaminate endocrine disruptor-contaminated water through nine possible pathways.


Assuntos
Bacillus pumilus , Compostos Benzidrílicos , Biodegradação Ambiental , Disruptores Endócrinos , Lacase , Fenóis , Compostos Benzidrílicos/metabolismo , Lacase/metabolismo , Lacase/genética , Fenóis/metabolismo , Bacillus pumilus/enzimologia , Bacillus pumilus/genética , Bacillus pumilus/metabolismo , Disruptores Endócrinos/metabolismo , Concentração de Íons de Hidrogênio , Saccharomycetales/metabolismo , Saccharomycetales/genética
11.
Biosens Bioelectron ; 261: 116501, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38905858

RESUMO

A novel laccase mimic enzyme Cu-Mn with excellent photothermal properties was firstly prepared via a combination of hydrothermal and in situ synthesis. Cu-Mn nanozymes could catalyze the typical laccase substrate 2,4-dichlorophenol (2,4-DP) to generate the red quinone imine. Further, loading the MnO2 nanosheets with photothermal properties, Cu-Mn nanozymes possessed not only excellent laccase catalytic activity, but also high photothermal conversion efficiency. The presence of glutathione S-transferase (GST) recovered the glutathione (GSH)-induced weakness of the laccase activity and photothermal properties of Cu-Mn. Hence, a GST enzyme-regulated dual-mode sensing strategy was established based on Cu-Mn nanozymes. The detection limits of GST monitoring based on colorimetric and photothermal methods were 0.092 and 0.087 U/L with response times of 20 min and 8 min, respectively. Furthermore, the proposed method enabled the measuring of GST levels in human serum and was successfully employed in the primary evaluation of hepatitis patients. Another attraction, the impressive photothermal behavior also endowed the Cu-Mn nanozymes with promising antimicrobial properties, which exhibited significant antimicrobial effects against Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus). Unsurprisingly, multifunctional Cu-Mn nanozymes certainly explore new paths in biochemical analysis and antimicrobial applications.


Assuntos
Antibacterianos , Técnicas Biossensoriais , Cobre , Escherichia coli , Glutationa Transferase , Lacase , Staphylococcus aureus , Lacase/química , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Glutationa Transferase/química , Escherichia coli/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Cobre/química , Cobre/farmacologia , Catálise , Oxirredução , Limite de Detecção , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Clorofenóis/farmacologia , Clorofenóis/química , Colorimetria/métodos , Óxidos/química , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Nanoestruturas/química
12.
Food Chem ; 455: 139862, 2024 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-38833866

RESUMO

Laccase mediators possess advantage of oxidizing substrates with high redox potentials, such as aflatoxin B1 (AFB1). High costs of chemically synthesized mediators limit laccase industrial application. In this study, thin stillage extract (TSE), a byproduct of corn-based ethanol fermentation was investigated as the potential natural mediator of laccases. Ferulic acid, p-coumaric acid, and vanillic acid were identified as the predominant phenolic compounds of TSE. With the assistance of 0.05 mM TSE, AFB1 degradation activity of novel laccase Glac1 increased by 17 times. The promoting efficiency of TSE was similar to ferulic acid, but superior to vanillic acid and p-coumaric acid, with 1.2- and 1.3-fold increases, respectively. After Glac1-TSE treatment, two oxidation products were identified. Ames test showed AFB1 degradation products lost mutagenicity. Meanwhile, TSE also showed 1.3-3.0 times promoting effect on laccase degradation activity in cereal flours. Collectively, a safe and highly efficient natural mediator was obtained for aflatoxin detoxification.


Assuntos
Lacase , Zea mays , Lacase/metabolismo , Lacase/química , Zea mays/química , Zea mays/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Aflatoxina B1/química , Aflatoxina B1/metabolismo , Oxirredução , Extratos Vegetais/química , Fermentação , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo
13.
ACS Appl Mater Interfaces ; 16(27): 34538-34548, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38940445

RESUMO

Biothiol assays offer vital insights into health assessment and facilitate the early detection of potential health issues, thereby enabling timely and effective interventions. In this study, we developed ultrasmall CuMn-Histidine (His) nanozymes with multiple enzymatic activities. CuMn-His enhanced peroxidase (POD)-like activity at neutral pH was achieved through hydrogen bonding and electrostatic effects. In addition, CuMn-His possesses laccase (LAC)-like and superoxide dismutase (SOD)-like activities at neutral pH. Based on three different enzyme mimetic activities of CuMn-His at neutral pH, the colorimetric sensing array without changing the buffer solution was successfully constructed. The array was successfully used for the identification of three biothiols, glutathione (GSH), cysteine (Cys), and homocysteine (Hcy). Subsequently, excellent application results were shown in complex serum and cellular level analyses. This study provides an innovative strategy for the development of ultrasmall bimetallic nanozymes with multiple enzymatic activities and the construction of colorimetric sensing arrays.


Assuntos
Colorimetria , Colorimetria/métodos , Concentração de Íons de Hidrogênio , Humanos , Histidina/química , Glutationa/sangue , Glutationa/química , Glutationa/análise , Homocisteína/sangue , Homocisteína/análise , Compostos de Sulfidrila/química , Nanoestruturas/química , Cisteína/sangue , Cisteína/análise , Cisteína/química , Superóxido Dismutase/química , Técnicas Biossensoriais/métodos , Lacase/química , Lacase/metabolismo
14.
ACS Appl Bio Mater ; 7(7): 4760-4771, 2024 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-38916249

RESUMO

Laccase is an oxidase of great industrial interest due to its ability to catalyze oxidation processes of phenols and persistent organic pollutants. However, it is susceptible to denaturation at high temperatures, sensitive to pH, and unstable in the presence of high concentrations of solvents, which is a issue for industrial use. To solve this problem, this work develops the synthesis in an aqueous medium of a new Mn metalloenzyme with laccase oxidase mimetic catalytic activity. Geobacillus thermocatenulatus lipase (GTL) was used as a scaffold enzyme, mixed with a manganese salt at 50 °C in an aqueous medium. This leads to the in situ formation of manganese(IV) oxide nanowires that interact with the enzyme, yielding a GTL-Mn bionanohybrid. On the other hand, its oxidative activity was evaluated using the ABTS assay, obtaining a catalytic efficiency 300 times higher than that of Trametes versicolor laccase. This new Mn metalloenzyme was 2 times more stable at 40 °C, 3 times more stable in the presence of 10% acetonitrile, and 10 times more stable in 20% acetonitrile than Novozym 51003 laccase. Furthermore, the site-selective immobilized GTL-Mn showed a much higher stability than the soluble form. The oxidase-like activity of this Mn metalloenzyme was successfully demonstrated against other substrates, such as l-DOPA or phloridzin, in oligomerization reactions.


Assuntos
Lacase , Manganês , Lacase/metabolismo , Lacase/química , Manganês/química , Teste de Materiais , Geobacillus/enzimologia , Tamanho da Partícula , Materiais Biocompatíveis/química , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/metabolismo , Lipase/metabolismo , Lipase/química
15.
Biodegradation ; 35(5): 769-787, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38822999

RESUMO

The persistence and ubiquity of polycyclic aromatic hydrocarbons (PAHs) in the environment necessitate effective remediation strategies. Hence, this study investigated the potential of purified Laccases, TlFLU1L and TpFLU12L, from two indigenous fungi Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12), respectively for the oxidation and detoxification of anthracene. Anthracene was degraded with vmax values of 3.51 ± 0.06 mg/L/h and 3.44 ± 0.06 mg/L/h, and Km values of 173.2 ± 0.06 mg/L and 73.3 ± 0.07 mg/L by TlFLU1L and TpFLU12L, respectively. The addition of a mediator compound 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to the reaction system significantly increased the degradation of anthracene, with up to a 2.9-fold increase in vmax value and up to threefold decrease in Km values of TlFLU1L and TpFLU12L. The GC-MS analysis of the metabolites suggests that anthracene degradation follows one new pathway unique to the ABTS system-hydroxylation and carboxylation of C-1 and C-2 position of anthracene to form 3-hydroxy-2-naphthoic acid, before undergoing dioxygenation and side chain removal to form chromone which was later converted into benzoic acid and CO2. This pathway contrasts with the common dioxygenation route observed in the free Laccase system, which is observed in the second degradation pathways. Furthermore, toxicity tests using V. parahaemolyticus and HT-22 cells, respectively, demonstrated the non-toxic nature of Laccase-ABTS-mediated metabolites. Intriguingly, analysis of the expression level of Alzheimer's related genes in HT-22 cells exposed to degradation products revealed no induction of neurotoxicity unlike untreated cells. These findings propose a paradigm shift for bioremediation by highlighting the Laccase-ABTS system as a promising green technology due to its efficiency with the discovery of a potentially less harmful degradation pathway, and the production of non-toxic metabolites.


Assuntos
Antracenos , Biodegradação Ambiental , Lacase , Talaromyces , Lacase/metabolismo , Antracenos/metabolismo , Talaromyces/enzimologia , Trichoderma/enzimologia , Animais , Proteínas Fúngicas/metabolismo
16.
Microb Cell Fact ; 23(1): 167, 2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38849849

RESUMO

BACKGROUND: White-rot fungi are known to naturally produce high quantities of laccase, which exhibit commendable stability and catalytic efficiency. However, their laccase production does not meet the demands for industrial-scale applications. To address this limitation, it is crucial to optimize the conditions for laccase production. However, the regulatory mechanisms underlying different conditions remain unclear. This knowledge gap hinders the cost-effective application of laccases. RESULTS: In this study, we utilized transcriptomic and metabolomic data to investigate a promising laccase producer, Cerrena unicolor 87613, cultivated with fructose as the carbon source. Our comprehensive analysis of differentially expressed genes (DEGs) and differentially abundant metabolites (DAMs) aimed to identify changes in cellular processes that could affect laccase production. As a result, we discovered a complex metabolic network primarily involving carbon metabolism and amino acid metabolism, which exhibited contrasting changes between transcription and metabolic patterns. Within this network, we identified five biomarkers, including succinate, serine, methionine, glutamate and reduced glutathione, that played crucial roles in co-determining laccase production levels. CONCLUSIONS: Our study proposed a complex metabolic network and identified key biomarkers that determine the production level of laccase in the commercially promising Cerrena unicolor 87613. These findings not only shed light on the regulatory mechanisms of carbon sources in laccase production, but also provide a theoretical foundation for enhancing laccase production through strategic reprogramming of metabolic pathways, especially related to the citrate cycle and specific amino acid metabolism.


Assuntos
Lacase , Redes e Vias Metabólicas , Lacase/metabolismo , Lacase/genética , Biomarcadores/metabolismo , Carbono/metabolismo , Regulação Fúngica da Expressão Gênica , Transcriptoma , Polyporaceae/enzimologia , Polyporaceae/genética , Polyporaceae/metabolismo , Frutose/metabolismo , Metabolômica , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética
17.
Int J Biol Macromol ; 273(Pt 1): 133115, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38871108

RESUMO

Aflatoxin B1 (AFB1) contamination of oils is a serious concern for the safety of edible oil consumers. Enzyme-assisted detoxification of AFB1 is an efficient and safe method for decontaminating oils, but pristine enzymes are unstable in oils and require modifications before use. Therefore, we designed a novel and magnetically separable laccase-carrying biocatalyst containing spent-mushroom-substrate (SMS)-derived biochar (BF). Laccase was immobilized on NH2-activated magnetic biochar (BF-NH2) through covalent crosslinking, which provided physicochemical stability to the immobilized enzyme. After 30 days of storage at 4 °C, the immobilized laccase (product named "BF-NH2-Lac") retained ~95 % of its initial activity, while after five repeated cycles of ABTS oxidation, ~85 % activity retention was observed. BF-NH2-Lac was investigated for the oxidative degradation of AFB1, which exhibited superior performance compared to free laccase. Among many tested natural compounds as mediators, p-coumaric acid proved the most efficient in activating laccase for AFB1 degradation. BF-NH2-Lac demonstrated >90 % removal of AFB1 within 5.0 h, while the observed degradation efficiency in corn oil and buffer was comparable. An insight into the adsorptive and degradative removal of AFB1 revealed that AFB1 removal was governed mainly by degradation. The coexistence of multi-mycotoxins did not significantly affect the AFB1 degradation capability of BF-NH2-Lac. Investigation of the degradation products revealed the transformation of AFB1 into non-toxic AFQ1, while corn oil quality remained unaffected after BF-NH2-Lac treatment. Hence, this study holds practical importance for the research, knowledge-base and industrial application of newly proposed immobilized enzyme products.


Assuntos
Aflatoxina B1 , Carvão Vegetal , Óleo de Milho , Enzimas Imobilizadas , Lacase , Lacase/metabolismo , Lacase/química , Enzimas Imobilizadas/metabolismo , Enzimas Imobilizadas/química , Carvão Vegetal/química , Aflatoxina B1/química , Aflatoxina B1/metabolismo , Óleo de Milho/química , Porosidade , Reciclagem
18.
Sci Rep ; 14(1): 13371, 2024 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-38862560

RESUMO

Broad-spectrum biocatalysts enzymes, Laccases, have been implicated in the complete degradation of harmful pollutants into less-toxic compounds. In this study, two extracellularly produced Laccases were purified to homogeneity from two different Ascomycetes spp. Trichoderma lixii FLU1 (TlFLU1) and Talaromyces pinophilus FLU12 (TpFLU12). The purified enzymes are monomeric units, with a molecular mass of 44 kDa and 68.7 kDa for TlFLU1 and TpFLU12, respectively, on SDS-PAGE and zymogram. It reveals distinct properties beyond classic protein absorption at 270-280 nm, with TlFLU1's peak at 270 nm aligning with this typical range of type II Cu site (white Laccase), while TpFLU12's unique 600 nm peak signifies a type I Cu2+ site (blue Laccase), highlighting the diverse spectral fingerprints within the Laccase family. The Km and kcat values revealed that ABTS is the most suitable substrate as compared to 2,6-dimethoxyphenol, caffeic acid and guaiacol for both Laccases. The bioinformatics analysis revealed critical His, Ile, and Arg residues for copper binding at active sites, deviating from the traditional two His and a Cys motif in some Laccases. The predicted biological functions of the Laccases include oxidation-reduction, lignin metabolism, cellular metal ion homeostasis, phenylpropanoid catabolism, aromatic compound metabolism, cellulose metabolism, and biological adhesion. Additionally, investigation of degradation of polycyclic aromatic hydrocarbons (PAHs) by purified Laccases show significant reductions in residual concentrations of fluoranthene and anthracene after a 96-h incubation period. TlFLU1 Laccase achieved 39.0% and 44.9% transformation of fluoranthene and anthracene, respectively, while TpFLU12 Laccase achieved 47.2% and 50.0% transformation, respectively. The enzyme structure-function relationship study provided insights into the catalytic mechanism of these Laccases for possible biotechnological and industrial applications.


Assuntos
Lacase , Talaromyces , Trichoderma , Talaromyces/enzimologia , Lacase/metabolismo , Lacase/química , Lacase/isolamento & purificação , Lacase/genética , Trichoderma/enzimologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/genética , Especificidade por Substrato , Cobre/metabolismo , Cinética , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredutases/isolamento & purificação , Domínio Catalítico
19.
Int J Mol Sci ; 25(12)2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38928160

RESUMO

Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.


Assuntos
Aflatoxina B1 , Bacillus licheniformis , Proteínas de Bactérias , Lacase , Aflatoxina B1/metabolismo , Bacillus licheniformis/genética , Bacillus licheniformis/metabolismo , Bacillus licheniformis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Lacase/metabolismo , Lacase/genética , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Saccharomycetales
20.
Proc Natl Acad Sci U S A ; 121(19): e2403049121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38691587

RESUMO

Molecular chaperones assist in protein refolding by selectively binding to proteins in their nonnative states. Despite progress in creating artificial chaperones, these designs often have a limited range of substrates they can work with. In this paper, we present molecularly imprinted flexible polymer nanoparticles (nanoMIPs) designed as customizable biomimetic chaperones. We used model proteins such as cytochrome c, laccase, and lipase to screen polymeric monomers and identify the most effective formulations, offering tunable charge and hydrophobic properties. Utilizing a dispersed phase imprinting approach, we employed magnetic beads modified with destabilized whole-protein as solid-phase templates. This process involves medium exchange facilitated by magnetic pulldowns, resulting in the synthesis of nanoMIPs featuring imprinted sites that effectively mimic chaperone cavities. These nanoMIPs were able to selectively refold denatured enzymes, achieving up to 86.7% recovery of their activity, significantly outperforming control samples. Mechanistic studies confirmed that nanoMIPs preferentially bind denatured rather than native enzymes, mimicking natural chaperone interactions. Multifaceted analyses support the functionality of nanoMIPs, which emulate the protective roles of chaperones by selectively engaging with denatured proteins to inhibit aggregation and facilitate refolding. This approach shows promise for widespread use in protein recovery within biocatalysis and biomedicine.


Assuntos
Chaperonas Moleculares , Nanopartículas , Polímeros , Desnaturação Proteica , Nanopartículas/química , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Polímeros/química , Redobramento de Proteína , Dobramento de Proteína , Citocromos c/química , Citocromos c/metabolismo , Lacase/química , Lacase/metabolismo , Lipase/química , Lipase/metabolismo
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