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1.
Braz. j. biol ; 84: e257071, 2024. graf, ilus
Artigo em Inglês | LILACS, VETINDEX | ID: biblio-1364496

RESUMO

In advanced biotechnology, the utilization of enzymes to achieve new or modified compounds with antibacterial, fungicidal, and anti-cancer specifications is crucial. Mushroom lactases are a hopeful biocatalyst for the synthesis and modification of different compounds. They are an accessible and inexpensive enzyme for the preparation of reaction objects and have recently received attention. Laccase purification was performed from basidiomycete Lentinus strigosus (LS) in several stages: Stage 1. On ion-exchange chromatography on TEAE Servacell 23 (400 ml), two distinctly separated laccase activity peaks were observed, eluted from the carrier at 0.21 and 0.27 M NaCl. In order to reduce the loss of enzymes, all fractions with laccase activity were collected, concentrated, and desalted using an ultrafiltration cell (Amicon, United States) with a UM-10 membrane. Stage 2. The resulting preparation with laccase activity was applied to a Q-Sepharose column (60 ml). Two well-separated peaks with laccase activity were obtained during the elution: laccase I (0.12 M NaCl) and laccase II (0.2 M NaCl). Stage 3. In the course of further purification of both enzymes, carried out on anion-exchange carrier Resource Q (6 ml), a broken gradient was used: 0 - 10%, 10 - 20%, and 20 - 100% with 1M NaCl. Stage 4. Both laccase I and laccase II, obtained after Resource Q, were desalted, concentrated to 1 ml each, and applied to a Superdex 75 gel filtration column. As a result, two laccases were obtained in a homogeneous form.


Na biotecnologia moderna, o uso de enzimas para obter compostos novos ou modificados com propriedades antibacterianas, antifúngicas e anticancerígenas é crucial. Lactases de cogumelos são biocatalisadores promissores para síntese e modificação de diferentes compostos, por serem enzimas baratas e disponíveis para a preparação de componentes de reação, e vem recebendo a devida atenção recentemente. A purificação da lacase foi realizada a partir do basidiomiceto Lentinus strigosus em vários estágios: Etapa 1 - na cromatografia de troca iônica em TEAE Servacell 23 (400 ml), foram observados dois picos de atividade da lacase distintamente separados, com eluição do transportador a 0,21 e 0,27 M de NaCl. Para reduzir a perda de enzimas, todas as frações com atividade de lacase foram coletadas, concentradas e dessalinizadas em uma célula de ultrafiltração (Amicon, Estados Unidos) com membrana UM-10; Etapa 2 - a preparação resultante com atividade de lacase foi aplicada a uma coluna Q-Sepharose (60 ml). Durante a eluição, foram obtidos dois picos bem separados com atividade de lacase: lacase I (NaCl 0,12 M) e lacase II (NaCl 0,2 M); Etapa 3 - no decurso da purificação adicional de ambas as enzimas, realizada no Recurso Q de transportador de troca aniônica (6 ml), um gradiente quebrado foi usado: 0-10%, 10-20% e 20-100% com NaCl 1M; Etapa 4 - tanto a lacase I como a lacase II, obtidas após o Recurso Q, foram dessalinizadas e concentradas para 1 ml cada e aplicadas a uma coluna de filtração em gel Superdex 75. Como resultado, duas lacases foram obtidas de forma homogênea.


Assuntos
Basidiomycota , Biotecnologia , Lacase , Enzimas , Antibacterianos
2.
Environ Pollut ; 319: 120999, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36608728

RESUMO

In the continual march to a predominantly urbanized civilization, anthropogenic activities have increased scrupulously, industrialization have occurred, economic growth has increased, and natural resources are being exploited, causing huge waste management problems, disposal issues, and the evolution of several pollutants. In order to have a sustainable environment, these pollutants need to be removed and degraded. Bioremediation employing microorganisms or enzymes can be used to treat the pollutants by degrading and/or transforming the pollutants into different form which is less or non-toxic to the environment. Laccase is a diverse enzyme/biocatalyst belonging to the oxidoreductase group of enzymes produced by microorganisms. Due to its low substrate specificity and monoelectronic oxidation of substrates in a wide range of complexes, it is most commonly used to degrade chemical pollutants. For degradation of emerging pollutants, laccase can be efficiently employed; however, large-scale application needs reusability, thermostability, and operational stability which necessitated strategies like immobilization and engineering of robust laccase possessing desirable properties. Immobilization of laccase for bioremediation, and treatment of wastewater for degrading emerging pollutants have been focussed for sustainable development. Challenges of employing biocatalysts for these applications as well as engineering robust laccase have been highlighted in this study.


Assuntos
Poluentes Ambientais , Lacase/química , Eliminação de Resíduos Líquidos , Enzimas Imobilizadas/metabolismo , Biodegradação Ambiental
3.
Sci Rep ; 13(1): 860, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36650163

RESUMO

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB1 and AFG2) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.


Assuntos
Aflatoxinas , Aflatoxinas/análise , Aflatoxina B1/química , Lacase/metabolismo , Simulação de Dinâmica Molecular , Contaminação de Alimentos/análise
4.
Sensors (Basel) ; 23(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36679398

RESUMO

This article presents a novel and selective electrochemical bioassay with antibody and laccase for the determination of free thyroid hormone (free triiodothyronine, fT3). The biosensor was based on a glassy carbon electrode modified with a Fe3O4@graphene nanocomposite with semiconducting properties, an antibody (anti-PDIA3) with high affinity for fT3, and laccase, which was responsible for catalyzing the redox reaction of fT3. The electrode modification procedure was investigated using a cyclic voltammetry technique, based on the response of the peak current after modifications. All characteristic working parameters of the developed biosensor were analyzed using differential pulse voltammetry. Obtained experimental results showed that the biosensor revealed a sensitive response to fT3 in a concentration range of 10-200 µM, a detection limit equal to 27 nM, and a limit of quantification equal to 45.9 nM. Additionally, the constructed biosensor was selective towards fT3, even in the presence of interference substances: ascorbic acid, tyrosine, and levothyroxine, and was applied for the analysis of fT3 in synthetic serum samples with excellent recovery results. The designed biosensor also exhibited good stability and can find application in future medical diagnostics.


Assuntos
Técnicas Biossensoriais , Grafite , Nanocompostos , Grafite/química , Lacase/química , Técnicas Eletroquímicas/métodos , Nanocompostos/química , Técnicas Biossensoriais/métodos , Hormônios Tireóideos , Eletrodos , Limite de Detecção
5.
Microb Cell Fact ; 22(1): 1, 2023 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-36593499

RESUMO

Two laccase isoenzymes (LacA and LacB) were isolated from a novel Trichoderma harzianum S7113 isolate employing ammonium sulfate precipitation, Sephadex G100, and DEAE Sepharose ion exchange chromatography. The molecular weights of the purified LacA and LacB laccases were estimated to be 63 and 48 kDa, respectively. The two isoenzymes had their optimum activities at the same temperature (50 °C), but at slightly different pH values (pH 3.0 for LacA and pH 2.5 for LacB). LacA and LacB had the same thermal stability at 40 °C and pH stability at pH 9.0. The two isoenzymes also showed a high level of specific activity toward ABTS, where the Km values of LacA and LacB were 0.100 and 0.065 mM, whereas their Vmax values were 0.603 and 0.182 µmol min-1, respectively. LacA and LacB catalytic activity was stimulated by Mg2+, Zn2+, K+, and Ni2+, whereas it was inhibited by Hg2+ and Pb2+, ß-mercaptoethanol, EDTA, and SDS, and completely inhibited by sodium azide. Our findings indicate that purified laccase has a promising capacity for bisphenol A (BPA) bioremediation across a broad pH range. This finding opens up new opportunities for the commercialization of this technique in a variety of biotechnology-based applications, particularly for removing endocrine chemicals from the environment.


Assuntos
Isoenzimas , Lacase , Lacase/metabolismo , Isoenzimas/metabolismo , Fenóis , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Temperatura
6.
Food Res Int ; 163: 112171, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36596117

RESUMO

Enzymatic crosslinking has gained attention in improving plant protein heat-induced gels, which are composed of weak network structures. The aim of the study was to investigate the effect of laccase crosslinking on the rheological and microstructural properties of heat-induced Bambara groundnut protein gels. The rheological properties of laccase-modified Bambara groundnut protein isolate (BPI1) gel formed in situ were investigated. Changes in viscoelastic properties were monitored during heating and cooling ramps and gel structure fingerprints were analyzed by frequency sweep. Laccase addition induced an initial protein structure breakdown (G″>G') at an enzyme dose-dependent (1-3 U/g) before gel formation and stabilization. Gel point temperatures were significantly decreased from 85°C to 29°C (∼3 folds) with increasing laccase activity (0 to 3 U/g protein, respectively). For laccase crosslinked gels, G' was substantially greater than G" (>1 log) with no dependency on angular frequency, which suggests the formation of relatively well-structured gels. The highest gel strength (tan δ of 0.09, G* of 555.51 kPa & An of 468.04 kPa) was recorded at a laccase activity of 2 U/g protein and the gels formed at this activity appeared homogeneous with compact lath sheet-like structure. The crosslinking effects of laccase were corroborated by the decrease in thiol and phenolic contents as well as the crosslinking of amino acids in model reactions. Overall, the use of laccase improved gel properties and significantly altered the gelation profile of BPI. Laccase-modified Bambara groundnut protein gels have potential to be used in food texture improvement and development of new food products. For instance, they can be used in plant-based milk products such as yoghurt and cheese.


Assuntos
Vigna , Lacase , Temperatura , Temperatura Alta , Géis
7.
Appl Microbiol Biotechnol ; 107(2-3): 719-733, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36602562

RESUMO

Laccase from Myceliophthora thermophila was immobilized using one-point and multi-point covalent attachment on both a native and a modified new commercial epoxy carrier (Immobead 150P). After 10 cycles of operation at pH 3.0 and temperature 70 °C, the multi-point covalently immobilized laccase on the modified Immobead 150P performed best in terms of immobilization characteristics, retaining 95% of its initial activity. Thermodynamic parameters of thermal inactivation emphasized the positive impact of the immobilization procedure. At 50 °C, the immobilized and free enzyme activity levels dropped by 27 and 73%, respectively, after 48 h of incubation. The immobilized enzyme enhanced its stability in alkaline conditions, resuming 95% of its original activity after 3 h at pH 9.0. Immobilization reduced substrate affinity because the free laccase's Km value was lower than that of the immobilized laccase. Finally, the application of immobilized laccase in an innovative wood treatment process was tested by grafting lauryl gallate (LG) in order to provide hydrophobic properties to the wood. The results showed a relative water contact angle of 85.7% for treated wood, whereas the control showed only 26.6%, after 4 min of contact between water and beechwood surface. KEY POINTS: • Multi-point covalent immobilization of a commercial laccase on a commercial support. • Enzymatic parameters generally improved by immobilization process. • New application of immobilized laccase: enzymatic-assisted wood hydrophobization.


Assuntos
Enzimas Imobilizadas , Lacase , Estabilidade Enzimática , Lacase/metabolismo , Concentração de Íons de Hidrogênio , Enzimas Imobilizadas/metabolismo , Água/química , Cinética
8.
Chemosphere ; 313: 137505, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36509189

RESUMO

No biodegradation methods are absolute in the treatment of all textile dyes, which leads to structure-dependent degradation. In this study, biodegradation of three azo dyes, reactive black 5 (RB5), acid blue 113 (AB113), and acid orange 7 (AO7), was investigated using an immobilized fungus, Trametes hirsuta D7. The degraded metabolites were identified using UPLC-PDA-FTICR MS and the biodegradation pathway followed was proposed. RB5 (92%) and AB113 (97%) were effectively degraded, whereas only 30% of AO7 was degraded. Molecular docking simulations were performed to determine the reason behind the poor degradation of AO7. Weak binding affinity, deficiency in H-bonding interactions, and the absence of interactions between the azo (-NN-) group and active residues of the model laccase enzyme were responsible for the low degradation efficiency of AO7. Furthermore, cytotoxicity and genotoxicity assays confirmed that the fungus-treated dye produced non-toxic metabolites. The observations of this study will be useful for understanding and further improving enzymatic dye biodegradation.


Assuntos
Compostos Azo , Trametes , Simulação de Acoplamento Molecular , Biodegradação Ambiental , Compostos Azo/toxicidade , Compostos Azo/metabolismo , Corantes/química , Lacase/química
9.
Chemosphere ; 313: 137478, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36513203

RESUMO

In spite of many works on the biodegradation of textile dyes and phenolic compounds, we propose a new, inexpensive, environmentally friendly, and sustainable material based on electrospun fiber and immobilized laccase. The polycaprolactone (PCL)/polyethyleneimine (PEI) electrospun fibers were optimized and prepared by electrospinning technique according to the operational parameters like PCL concentration (12 wt%), PEI concentration (10 wt%), voltage (16 kV), needle tip-collector distance (20 cm), and injection speed (0.7 mL/h). Next, characterization studies were performed to investigate the morphology and structure of the electrospun fibers without and with laccase. The crude laccase was obtained by cultivating the white rot fungus T. trogii (TT), and T. versicolor (TV). The resulting electrospun fibers showed a smooth surface with a mean diameter of around 560 nm, and larger diameters were observed after laccase immobilization. According to the results, immobilization increased the stability properties of laccase such as storage, and operational. For instance, the residual activity of the PCL/PEI/TTL and PCL/PEI/TVL after 10 repeated cycles, was 33.2 ± 0.2% and 26.0 ± 0.9%, respectively. After 3 weeks of storage, they retained around 30% of their original activity. Moreover, the PCL/PEI/TTL and PCL/PEI/TVL were found to possess high decolorization yield to remove Orange II and Malachite Green textile dyes from solutions imitating polluted waters. Among them, the PCL/PEI/TTL exhibited the highest decolorization efficiencies of Orange II and Malachite Green after 8 continuous uses at pH 5 and a temperature of 50 °C, reaching over 86%, and 46%, respectively. Moreover, PCL/PEI/TTL and PCL/PEI/TVL effectively degraded the 2,6-dichlorophenol phenolic compound at an optimal pH and temperature range and exhibited maximum removal efficiency of 52.6 ± 0.1% and 64.5 ± 7.6%, respectively. Our approach combines the advantageous properties of electrospun fiber material and immobilization strategy for the efficient use of industrial scale important enzymes such as laccase in various enzymatic applications.


Assuntos
Corantes , Lacase , Corantes/química , Lacase/química , Polietilenoimina , Têxteis , Enzimas Imobilizadas/química
10.
Chemosphere ; 313: 137612, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36563730

RESUMO

In this preset study, porous-cross-linked enzyme aggregates (CLEAs) of Pleurotus ostreatus laccase were utilized for the spontaneous decolorization and detoxification of triarylmethane and azo dyes, reactive blue 2 (RB) and malachite green (MG). The specific surface area and pore radius of the porous-CLEAs are 136.3 m2/g and 19.47 Ao, and the higher specific surface indicated greater biocatalytic efficiency, as increased mass transfer and dye interaction with the CLEAs laccase. CLEAs laccase decolorized 500 ppm of MG and RB with 98.12-58.33% efficiency after 120 min, at pH 5.0 and 50°C, without a mediator. Furthermore, the biotransformation of the MG and RB with immobilized laccase was confirmed with the help of UV-visible spectroscopy, high-performance liquid chromatography, and Fourier transform infrared spectroscopy. The reusability potential of CLEAs was assessed in batch mode for 10 cycles of dye decolorization. The decolorization activities for the immobilized laccase were 89% and 12% at the 6th cycle for MG and RB, respectively. This immobilized enzyme could effectively remove dyes from aqueous solution, and demonstrated significant detoxification in experimental plants (Triticum aestivum and Phaseolus mungo) and plant growth-promoting rhizobacteria (Azospirillum brasilense, Bacillus megaterium, Rhizobium leguminosarum, Bacillus subtilis, and Pseudomonas fluorescens). In conclusion, porous CLEAs laccase could be useful as a potential bioremediation tool for the detoxification and decolorization of dyeing wastewater in future.


Assuntos
Lacase , Pleurotus , Lacase/química , Pleurotus/metabolismo , Compostos Azo/metabolismo , Porosidade , Corantes/química
11.
J Biotechnol ; 361: 99-109, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36509383

RESUMO

Laccase production by fungal growth on agrifood waste is still poorly studied. Trametes versicolor K1 isolated from palm bark produced a yellow non glycosylated laccase from tomato waste based medium (TMT) and a blue glycosylated laccase on glucose medium (GLU). Lignocellulosic biomass, such as pinecones (PIN), palm leaves (PLM), olive pomace (OLV), and alfa stems (ALF) have also been used as growth medium for T. versicolor K1. In these conditions, very low or no laccase production was observed. When peptone was supplied in TMT medium, the laccase activity increased from 4170 U/L to 8618 U/L. By increasing the culture volume up to 1 L, laccase production on TMT was 9929 U/L. The yellow laccase (TmtLac) was purified from the supernatant TMT medium and has shown similar characteristics with the blue laccase (GluLac) purified from the GLU medium. Their apparent protein size was 63 kDa. Catalytic activities of the yellow form were not very different from those of the blue form, but specific activity of the purified yellow laccase produced on tomato waste was much higher. The Km and Vm values for four substrates, ABTS, DMP, guaiacol, and pyrogallol were almost similar for both isoenzymes. The optimum pH and temperature were respectively 4.0 and 50 °C. Although the level of glycosylation is clearly different, the thermostability of TmtLac and GluLac are quite similar. TmtLac is even slightly more tolerant at 60 °C for 24 h than GluLac. Moreover TmtLac showed greater stability at alkaline pH after 24 h compared to that of GluLac.We demonstrate that activity of the yellow TmtLac is not significantly affected compared to the blue laccase and that tomato waste is a simple and interesting lignocellulosic substrate to the laccase producer Trametes sp.


Assuntos
Trametes , Lacase/metabolismo
12.
Bioresour Technol ; 370: 128526, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36572161

RESUMO

Lignin degradation represents a significant challenge in biological valorization, but it is suffering from insufficiency, putting barriers to efficient lignin conversion. Herein, the study first develops a highly efficient laccase secretion apparatus, enabling high enzyme activity of 184 U/mL, complementing the biochemical limits on lignin depolymerization well in Halomonas sp. Y3. Further engineering of PHA biosynthesis produces a significantly high PHA titer of 286, 742, and 868 mg/L from alkaline lignin, catechol, and protocatechuate, respectively. The integration of laccase-secretion and PHA production modules enables a record titer of 693 and 1209 mg/L in converting lignin and lignin-containing stream to PHA, respectively. The titer is improved furtherly to 740 and 1314 mg/L by developing a non-sterilized fermentation. This study advances a cheaper and greener production of valuable chemicals from lignin by constructing a biosynthetic platform for PHA production and provides novel insight into the lignin conversion by extremophilic microbes.


Assuntos
Halomonas , Poli-Hidroxialcanoatos , Lignina/metabolismo , Halomonas/genética , Halomonas/metabolismo , Lacase , Engenharia Metabólica
13.
Bioresour Technol ; 370: 128544, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36584721

RESUMO

The production of hydroxyl radicals (OH) has been documented during composting. However, the effect of OH on composting efficiency remains unclear. Here, iron mineral supplemented thermophilic composting (imTC) is proposed and demonstrated for enhancing OH production and accelerating the maturation of composting. The results indicated that the maximum OH production of imTC was 1922.74 µmol·kg-1, which increased by 1.39 times than that of ordinary thermophilic composting (oTC). Importantly, the increase of OH could greatly enhance organic matter degradation and humic substances formation during imTC, resulting in shorting the maturity time by 25 %. Enrichment of laccase-producing bacteria resulted in higher laccase activity (31.85 U·g-1) in imTC compared with oTC (23.82 U·g-1), which may have contributed to the higher level of humification in imTC treatment. This work, for the first time, proposes a feasible strategy for improving composting efficiency through the regulation of OH production during aerobic composting.


Assuntos
Compostagem , Solo , Espécies Reativas de Oxigênio , Esgotos , Ferro , Lacase , Substâncias Húmicas/análise , Minerais
14.
Enzyme Microb Technol ; 163: 110168, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36481541

RESUMO

Glycerol is an important starting material for the synthesis of many chemical compounds and its selective oxidation represents an efficient way to produce value-added compounds. Glyceric acid, one of these selective oxidation products, is an important intermediate in the food, medicine, cosmetics, and light industries. In this work, four commercially available native laccases were screened for glycerol oxidation using different initiators, and the two most efficient biocatalysts were covalently immobilized on functionalized magnetic and polymethacrylate (Lifetech™) solid supports. Apart from the mostly employed Fe3O4 magnetic particles, in this work Ni-Zn or Ni-Zn-Co spinel ferrite (MFe2O4) microparticles were used. Particularly, the utilization (for the first time for laccase immobilization) of Ni-Zn ferrite support Ni0.7Zn0.3Fe2O4 functionalized with 3-aminopropyl-trimethoxysilane, via crosslinking by glutaraldehyde and reduction with NaBH4 led to excellent biocatalytic efficiency and stability. These results confirm the feasibility of Trametes versicolor laccase for covalent bonding, as presumed by computational modelling. The resulted enzymatic preparations were characterized in detail in terms of stability and reusability, demonstrating enhanced storage, pH and thermal stability compared to the native enzymes. The most active biocatalysts (790.93 [U/g]) were successfully used for glycerol oxidation and the specific conversion in glyceric acid exceeded 50%.


Assuntos
Lacase , Trametes , Lacase/química , Glicerol , Enzimas Imobilizadas/química , Concentração de Íons de Hidrogênio
15.
Appl Microbiol Biotechnol ; 107(1): 273-286, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36477928

RESUMO

Thermoalkaliphilic laccase (CtLac) from the Caldalkalibacillus thermarum strain TA2.A1 has advantageous properties with potential industrial applications, such as high enzyme activity and stability at 70 °C and pH 8.0. In the present study, a directed evolution approach using a combination of random and site-directed mutagenesis was adopted to enhance the laccase activity of CtLac. Spectrophotometric assay and real-time oxygen measurement techniques were employed to compare and evaluate the enzyme activity among mutants. V243 was targeted for site-directed mutagenesis based on library screening. V243D showed a 25-35% higher laccase activity than wild-type CtLac in the spectrophotometric assay and oxygen consumption measurement results. V243D also showed higher catalytic efficiency than wild-type CtLac with decreased Km and increased kcat values. In addition, V243D enhanced oxidative degradation of the lignin model compound, guaiacylglycerol-ß-guaiacyl ether (GGGE), by 10% and produced a 5-30% increase in high-value aldehydes than wild-type CtLac under optimal enzymatic conditions (i.e., 70 °C and pH 8.0). Considering the lack of protein structural information, we used the directed evolution approach to predict Val at the 243 position of CtLac as one of the critical amino acids contributing to the catalytic efficiency of the enzyme. Moreover, it found that the real-time oxygen measurement technique could overcome the limitations of the spectrophotometric assay, and apply to evaluate oxidase activity in mutagenesis research. KEY POINTS: • CtLac was engineered for enhanced laccase activity through directed evolution approach • V243D showed higher catalytic efficiency (kcat/Km) than wild-type CtLac • V243D produced higher amounts of high-value aldehydes from rice straw than wild-type CtLac.


Assuntos
Lacase , Lignina , Lacase/metabolismo , Lignina/metabolismo , Mutagênese Sítio-Dirigida , Aldeídos , Oxigênio
16.
Sci Total Environ ; 864: 161137, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36566870

RESUMO

An easily recyclable biocatalyst (Lac@CDI-MCNFs) was synthesized by immobilizing laccase on rice straw-derived carbonyldiimidazole mediated magnetized cellulose nanofibers (MCNFs). Lac@CDI-MCNFs were utilized for bioremediation of cefixime antibiotic (CT), carbofuran pesticide (CF) and safranin O dye (SO) via oxidation-reduction reactions in wastewater. MCNFs provided enhanced pH, temperature and storage stability to laccase and allowed reusability for up to 25 cycles with mere 20 % decline in efficacy. The Lac@CDI-MCNFs effectively degraded 98.2 % CT and 96.8 % CF into benign metabolites within 20 h and completely degraded SO in just 7 h. Response surface modelling (RSM) was employed based on the Box Behnken Design to evaluate the effect of various parameters i.e. pH, catalyst dosage and the pollutants concentration which was further validated with experimental studies. The degradation products were identified using LCMS, which allowed the degradation pathway of the pollutants to be determined. The degradation of all pollutants followed first- order kinetics with rate constants of 0.1775, 0.0832 and 0.958 h-1 and half-life of 3.9, 5.0 and 0.723 h for CT, CF and SO, respectively. Lac@CDI-MCNFs was demonstrated to be an effective catalyst for the degradation of multifarious pollutants.


Assuntos
Poluentes Ambientais , Nanofibras , Biodegradação Ambiental , Celulose , Enzimas Imobilizadas/metabolismo , Lacase/metabolismo
17.
Molecules ; 27(23)2022 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-36500612

RESUMO

In this study, polyethyleneimine was combined with magnetic Fe3O4 nanoparticles through the bridging of carboxyl-functionalized ionic liquid, and laccase was loaded onto the carrier by Cu2+ chelation to achieve laccase immobilization (MCIL-PEI-Cu-lac). The carrier was characterized by Fourier transform infrared spectroscopy, scanning electron microscope, thermogravimetric analysis, X-ray diffraction analysis, magnetic hysteresis loop and so on. MCIL-PEI-Cu-lac has good immobilization ability; its loading and activity retention could reach 52.19 mg/g and 91.65%, respectively. Compared with free laccase, its thermal stability and storage stability have been significantly improved, as well. After 6 h of storage at 60 °C, 51.45% of the laccase activity could still be retained, and 81.13% of the laccase activity remained after 1 month of storage at 3 °C. In the pollutants removal test, the removal rate of 2,4-dichlorophenol (10 mg/L) by MCIL-PEI-Cu-lac could reach 100% within 10 h, and the removal efficiency could still be maintained 60.21% after repeated use for 8 times. In addition, MCIL-PEI-Cu-lac also has a good removal effect on other phenolic pollutants (such as bisphenol A, phenol, 4-chlorophenol, etc.). Research results indicated that an efficient strategy for laccase immobilization to biodegrade phenolic pollutants was developed.


Assuntos
Poluentes Ambientais , Líquidos Iônicos , Lacase/química , Enzimas Imobilizadas/química , Polietilenoimina , Fenômenos Magnéticos
18.
Molecules ; 27(23)2022 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-36500632

RESUMO

The aims of this article were to investigate Bacillus safensis HL3 spore for its capacity to degrade and detoxify indigo carmine and to provide an effective biological agent for the treatment of isatin dye wastewater. Bacillus safensis HL3 spore was found to decolorize indigo carmine by 97% in the presence of acetosyringone within 2 h. Significantly increased activities of spore laccase, intracellular tyrosinase, and lignin peroxidase upon exposure to indigo carmine were observed. The results of RT-qPCR also showed that the expression of laccase gene was significantly increased. The spore has the ability to degrade indigo carmine through oxidization. Furthermore, the pathway by which indigo carmine is degraded was investigated using liquid chromatography-mass spectrometry analysis to identify the biodegradation products. A detailed pathway of indigo carmine degradation by bacterial spores was proposed for the first time. Toxicity tests indicated that the biodegradation products of indigo carmine are non-toxic to Nicotiana tabacum seeds and are less hazardous to human erythrocytes than the original dye. Indigo carmine is a typical recalcitrant dye and severely jeopardizes human health. The results demonstrate the utility of the spore from Bacillus safensis HL3 for the degradation of indigo carmine and simultaneous reduction of its toxicity.


Assuntos
Bacillus , Índigo Carmim , Humanos , Bacillus/metabolismo , Biodegradação Ambiental , Lacase/metabolismo , Corantes/química
19.
Sci Rep ; 12(1): 22326, 2022 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-36567332

RESUMO

A mild and efficient method was developed for the synthesis of new derivatives of thiazolo[3,2-a] pyrimidin-3(2H)-ones from available starting materials based on the oxidation of catechols to ortho-quinone by Myceliophthora thermophila laccase (Novozym 51,003) and 1,4-addition of active methylene carbon to these in situ generated intermediates in moderate to good yields (35-93%). The structure of the products was confirmed through 1H NMR, 13C NMR, HMBC, HSQC, DEPT-135, and mass spectroscopy techniques. These novel compounds were evaluated as active antitumor agents against human colorectal adenocarcinoma and liver adenocarcinoma cell lines. All compounds displayed potent inhibition activities against the HT-29 cell line with IC50 values of 9.8-35.9 µM, superior to the positive control doxorubicin, and most showed potent anticancer activities against the HepG2 cell line.


Assuntos
Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Lacase , Pirimidinas , Tiazóis , Humanos , Antineoplásicos/química , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Hep G2 , Células HT29 , Lacase/química , Estrutura Molecular , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia
20.
Sci Rep ; 12(1): 21602, 2022 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-36517502

RESUMO

Production of value-added compounds from waste materials is of utmost importance for the development of a sustainable society especially regarding their use as catalysts in industrially relevant synthetic reactions. Herein, we show the production of laccases from four white-rot fungi, which were grown on agricultural residues, specifically Trametes versicolor 11269, Pleurotus ostreatus 1020, Panus tigrinus 707 and Lentinula edodes SC-495. The produced laccases were tested on a laccase-mediator system (LMS) for the biocatalytic oxidation of the model substrate benzyl alcohol into benzaldehyde. The LMS was carried out in the presence both of tetrahydrofuran as co-solvent and of the mediator 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) due to its high redox potential and its ability to perform the oxidation. Tolerance studies showed that the dialyzed solutions were able to tolerate 1% (99:1 v/v) of co-solvent, whereas a concentration of 10% v/v had a detrimental activity. Performances in the biocatalytic oxidation of laccase solutions from different purification steps were compared. Similar conversion was observed for laccase in dialysis (raw) and gel filtration (GF) product versus commercial T. versicolor laccase. The latter oxidized almost 99% of substrate while the other laccase solutions were able to reach a conversion from 91% for the laccase solution from P. tigrinus 707 after dialysis, to 50% for the laccase solution from P. ostreatus 1020 after gel filtration. This work highlights the potential of unpurified laccase solutions to be used as catalysts in synthetic reactions.


Assuntos
Lacase , Trametes , Lacase/metabolismo , Trametes/metabolismo , Álcool Benzílico , Diálise Renal , Oxirredução , Solventes
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