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1.
Int J Nanomedicine ; 15: 4639-4657, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636623

RESUMO

Background: ß-Glucosidase (ß-Glu) can activate amygdalin to kill prostate cancer cells, but the poor specificity of this killing effect may cause severe general toxicity in vivo, limiting the practical clinical application of this approach. Materials and Methods: In this study, starch-coated magnetic nanoparticles (MNPs) were successively conjugated with ß-Glu and polyethylene glycol (PEG) by chemical coupling methods. Cell experiments were used to confirm the effects of immobilized ß-Glu on amygdalin-mediated prostate cancer cell death in vitro. Subcutaneous xenograft models were used to carry out the targeting experiment and magnetically directed enzyme/prodrug therapy (MDEPT) experiment in vivo. Results: Immobilized ß-Glu activated amygdalin-mediated prostate cancer cell death. Tumor-targeting studies showed that PEG modification increased the accumulation of ß-Glu-loaded nanoparticles in targeted tumor tissue subjected to an external magnetic field and decreased the accumulation of the nanoparticles in the liver and spleen. Based on an enzyme activity of up to 134.89 ± 14.18mU/g tissue in the targeted tumor tissue, PEG-ß-Glu-MNP/amygdalin combination therapy achieved targeted activation of amygdalin and tumor growth inhibition in C57BL/6 mice bearing RM1 xenografts. Safety evaluations showed that this strategy had some impact on liver and heart function but did not cause obvious organ damage. Conclusion: All findings indicate that this magnetically directed enzyme/prodrug therapy strategy has the potential to become a promising new approach for targeted therapy of prostate cancer.


Assuntos
Amigdalina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Nanopartículas/química , Neoplasias da Próstata/tratamento farmacológico , beta-Glucosidase/metabolismo , Animais , Linhagem Celular Tumoral , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Humanos , Campos Magnéticos , Fenômenos Magnéticos , Masculino , Camundongos Endogâmicos C57BL , Nanopartículas/uso terapêutico , Polietilenoglicóis/química , Pró-Fármacos/farmacologia , Neoplasias da Próstata/patologia , Amido/química , beta-Glucosidase/química
2.
Food Chem ; 331: 127322, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32569968

RESUMO

Here we report a novel strategy for the immobilization of invertase using amyloid-like fibrils as a support. Optimal conditions to get Tyr-Tyr covalent binding between invertase and the support were determined using a photocrosslinking approach. The biological fibrils with invertase activity turn into microstructured catalysts according to electron microscopy outcomes. Thermal and storage stability as well as optimal pH and temperature of the enzyme were conserved. Moreover, the immobilized enzyme recovered by low g-force centrifugation retained 83% of its initial enzymatic activity after 15 reuse cycles. Considering that enzyme cost is the most significant part of the overall fee of enzymatic biomass conversion, the highly efficient recovery/reuse strategy described herein becomes relevant. Besides, it can also be applied to the immobilization of other enzymes for industrial biocatalysis.


Assuntos
Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , beta-Frutofuranosidase/química , beta-Frutofuranosidase/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Agregados Proteicos , Temperatura
3.
PLoS One ; 15(6): e0229968, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497077

RESUMO

Although laccase has been recognized as a wonder molecule and green enzyme, the use of low yielding fungal strains, poor production, purification, and low enzyme kinetics have hampered its large-scale application. Thus,this study aims to select high yielding fungal strains and optimize the production, purification, and kinetics of laccase of Aspergillus sp. HB_RZ4. The results obtained indicated that Aspergillus sp. HB_RZ4 produced a significantly large amount of laccase under meso-acidophilic shaking conditions in a medium containing glucose and yeast extract. A 25 µM CuSO4 was observed to enhance the enzyme yield. The enzyme was best purified on a Sephadex G-100 column. The purified enzyme resembled laccase of A. flavus. The kinetics of the purified enzyme revealed high substrate specificity and good velocity of reaction,using ABTS as a substrate. The enzyme was observed to be stable over various pH values and temperatures. The peptide structure of the purified enzyme was found to resemble laccase of A. kawachii IFO 4308. The fungus was observed to decolorize various dyes independent of the requirement of a laccase mediator system.Aspergillus sp. HB_RZ4 was observed to be a potent natural producer of laccase, and it decolorized the dyes even in the absence of a laccase mediator system. Thus, it can be used for bioremediation of effluent that contains non-textile dyes.


Assuntos
Aspergillus/enzimologia , Biotecnologia/métodos , Corantes/metabolismo , Lacase/metabolismo , Casca de Planta/microbiologia , Biodegradação Ambiental , Corantes/isolamento & purificação , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Lacase/antagonistas & inibidores , Lacase/química
4.
Food Chem ; 328: 127099, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32474238

RESUMO

In this study, we developed a competitive colorimetric immunoassay for qualitative detection of DAN based on oxidation of iron (Ⅱ) (Fe2+) in the presence of glucose oxidase (GOx) and color change induced by Fe2+-phenanthroline (Phen) chromogenic system. Streptavidin (SA) acted as a linker between biotinylated anti-DAN-monoantibody (bio-mAb) and biotinylated GOx (bio-GOx) to form the immunocomplexes bio-mAb-SA-bio-GOx. In the absence of DAN, the immunocomplexes bio-mAb-SA-bio-GOx combining with coated DAN-ovalbumin (DAN-OVA) will be immobilized and catalyze glucose to produce H2O2. Fe2+ is oxidized to Fe3+ by H2O2, giving rise to a colorless result. In the presence of DAN, Fe2+ produces a chelation reaction with Phen, leading to orange-red color. Under optimal conditions, the detection limit (LOD) by naked eyes was 2.5 ng mL-1 in milk, chicken, beef, and pork samples. Low LOD, no matrix effect, and no signal reader requirement make it possibly applied to quickly screen DAN on site.


Assuntos
Técnicas Biossensoriais/métodos , Colorimetria/métodos , Fluoroquinolonas/análise , Glucose Oxidase/metabolismo , Imunoensaio/métodos , Quelantes de Ferro/química , Fenantrolinas/química , Biocatálise , Catálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Fluoroquinolonas/química , Glucose/química , Glucose Oxidase/química , Peróxido de Hidrogênio/química , Limite de Detecção
5.
Artigo em Inglês | MEDLINE | ID: mdl-32361468

RESUMO

The present study aimed to evaluate the effect of the immobilization method of trypsin on biochar on the hydrolysis of casein from different sources, when compared to the process using trypsin in native form, to obtain bioactive peptides. The modification of the surface of biochar with glutaraldehyde was effective, as shown by the results of FTIR assay and the texture profile of the materials. Both activated and functionalized biochar showed high immobilization efficiency (greater than 87%) and high binding capacity (greater than 91 mg/g). During hydrolysis, the biocatalyst obtained by enzyme immobilization on the functionalized biochar presented a higher hydrolysis capacity for the different caseins when compared to the enzyme immobilized by adsorption, with values of 3.05 and 2.73 U/mg for goat casein, 2.36 and 1.85 U/mg for bovine casein, and 2.60 and 2.37 U/mg for buffalo, casein, respectively, with 60 min of reaction. The results of inhibitory activity in this study ranged from 93.5% and 25.5% for trypsin in its free form and immobilized on functionalized activated carbon, respectively, under the same reaction conditions. The immobilization methods were efficient, presenting high immobilization capacity. The proteolytic activity of trypsin immobilized via covalent binding was higher when compared the immobilization by adsorption. Thus, the functionalized biochar has proven to be potential support for enzyme immobilization, and the biocatalyst can be reused for more than 4 cycles. Despite lower ACE inhibition values of hydrolyzed obtained with the immobilized enzymes compared to free enzymes, biocatalysts present advantage due to the possibility of reuse.


Assuntos
Caseínas/química , Carvão Vegetal/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Tripsina/química , Tripsina/metabolismo , Adsorção , Animais , Biocatálise , Bovinos , Estabilidade Enzimática , Glutaral/química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Ácidos Fosfóricos/química , Proteólise , Propriedades de Superfície , Temperatura
6.
Food Chem ; 324: 126889, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32353659

RESUMO

Pesticides have been extensively applied worldwide to protect crops from worms and insects; however, the continuous use of pesticides affects ecosystems, agricultural product safety, nontarget organisms, and human health. In this paper, we report a highly sensitive biosensor for the determination of pesticides based on tin sulfide (SnS2) and chitosan (CHIT) nanocomposites decorated with a unique British housefly acetylcholinesterase (AChE). The hydrothermally synthesized nano-SnS2 mixed with chitosan solution (CHIT-SnS2) was drop-casted onto a glassy carbon electrode (GCE). Subsequently, the British housefly AChE was immobilized on the CHIT/SnS2-coated GCE that was then employed for pesticide detection. The developed biosensor showed an ultra-high sensitivity and wide linear detection range from 0.02 nM to 20000 nM with a detection limit of 0.02 nM for the detection of chlorpyrifos as the model pesticide. Furthermore, the AChE/CHIT-SnS2/GCE exhibited acceptable storage stability, good reproducibility, and selectivity.


Assuntos
Acetilcolinesterase/metabolismo , Técnicas Biossensoriais/métodos , Quitosana/química , Moscas Domésticas/enzimologia , Compostos Organofosforados/análise , Sulfetos/química , Compostos de Estanho/química , Acetilcolinesterase/química , Animais , Carbono/química , Clorpirifos/análise , Técnicas Eletroquímicas , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Limite de Detecção , Nanocompostos/química , Praguicidas/análise , Reprodutibilidade dos Testes
7.
PLoS One ; 15(4): e0231981, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348360

RESUMO

An acetylcholinesterase biosensor modified with graphene and transition metal carbides was prepared to detect organophosphorus pesticides. Cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy were used to characterize the electrochemical catalysis of the biosensor: acetylcholinesterase/chitosan-transition metal carbides/graphene/glassy carbon electrode. With the joint modification of graphene and transition metal carbides, the biosensor has a good performance in detecting dichlorvos with a linear relationship from 11.31 µM to 22.6 nM and the limit of detection was 14.45 nM. Under the premise of parameter optimization, the biosensor showed a good catalytic performance for acetylcholine. Compared to the biosensors without modification, it expressed a better catalytic performance due to the excellent electrical properties, biocompatibility and high specific surface area of graphene, transition metal carbides. Finally, the biosensor exhibits good stability, which can be stored at room temperature for one month without significant performance degradation, and has practical potential for sample testing.


Assuntos
Acetilcolinesterase/metabolismo , Técnicas Biossensoriais/métodos , Grafite/química , Nanocompostos/química , Compostos Organofosforados/análise , Praguicidas/análise , Técnicas Eletroquímicas , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Limite de Detecção , Reprodutibilidade dos Testes , Titânio/química , Elementos de Transição/química
8.
J Chromatogr A ; 1619: 460948, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32059867

RESUMO

In this study, an online capillary electrophoresis (CE) based dual-enzyme (thrombin and factor Xa) co-immobilized microreactor (THR-FXa IMER) was constructed for studying enzyme kinetics and screening dual-target inhibitors against THR and FXa with the aid of the polydopamine/graphene oxide (PDA/GO) coating. Based on the developed THR-FXa IMER, the Michaelis-Menten constants (Km) of THR and FXa were calculated to be 187.26 and 48.80 µM, respectively. The inhibition constants (Ki) for two known inhibitors, argatroban and rivaroxaban, on THR and FXa were determined to be 14.73 and 0.41 nM, respectively. In addition, after 30 consecutive runs, the enzymes' activity was remained 98% of the initial immobilized activity for both THR and FXa, which shows that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen dual-target inhibitors against THR and FXa from 30 small molecular compounds. Among them, 10 compounds such as salvianolic acid C and epigallocatechin gallate (EGCG) have dual-enzyme inhibitory activity, and 2 compounds named saikosaponin A and oleuropein have single THR inhibitory activity, 5 compounds such as rosemary acid and salvianolic acid B have single FXa inhibitory activity. Finally, the molecular interactions between enzyme and potential inhibitors were further verified via the molecular docking, and a new compound with a theoretically good coagulation inhibition effect was designed by the scaffold hopping study. In summary, the developed THR-FXa IMER is a reliable method for screening THR and/or FXa inhibitors.


Assuntos
Eletroforese Capilar , Ensaios Enzimáticos , Inibidores Enzimáticos/análise , Fator Xa , Trombina/antagonistas & inibidores , Catequina/análogos & derivados , Catequina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/análise , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/metabolismo , Cinética , Simulação de Acoplamento Molecular , Ácidos Pipecólicos/farmacologia , Rivaroxabana/farmacologia
9.
Carbohydr Polym ; 234: 115914, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32070532

RESUMO

In this work, ionic liquids-modified magnetic carboxymethyl cellulose nanoparticles (IL-MCMC) were prepared and used as supports for enzyme immobilization. The specific activity of immobilized lipase PPL-IL-MCMC was 1.43 and 2.81 folds higher than that of free PPL and PPL-MCMC, respectively. Water contact angle analysis indicated that the introduction of ionic liquids increased the hydrophobicity of supports, which in tune induced the lid-opening of lipase, allowing its active sites to become more accessible. In addition, the affinity between lipase and substrate immobilized on the prepared supports was enhanced. The same method was also applied to analyze immobilize penicillin G acylase (PGA) to further investigate the general applicability of the method. The results showed that the immobilized PGA exhibited higher stability than many other reported PGAs. The developed composites may be utilized as excellent supports for enzyme immobilization in industrial application.


Assuntos
Celulose/metabolismo , Líquidos Iônicos/metabolismo , Lipase/metabolismo , Nanopartículas de Magnetita/química , Animais , Biocatálise , Celulose/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Líquidos Iônicos/química , Lipase/química , Estrutura Molecular , Pâncreas/enzimologia , Tamanho da Partícula , Propriedades de Superfície , Suínos
10.
Enzyme Microb Technol ; 134: 109486, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32044033

RESUMO

We report the effects of high hydrostatic pressure (HHP), immobilization in electrochemically generated poly-o-phenylenediamine nano-films, and reticulation with glutaraldehyde on the thermal stability of glucose oxidase (GOx). The pseudo-first-order rate constant of inactivation of immobilized GOx inactivated at 70 °C and atmospheric pressure was 20.6 times smaller than that of GOx in solution under the same conditions. Immobilized GOx inactivated at 70 °C and 180 MPa was 87.6 times more stable than GOx in solution inactivated at 70 °C and atmospheric pressure. However, applying high pressure during electropolymerization or cross-linking with glutaraldehyde only had minor influences on GOx thermal stability. The stabilizing effect of HHP was not retained upon depressurization.


Assuntos
Técnicas Biossensoriais/métodos , Glucose Oxidase/metabolismo , Glucose/metabolismo , Temperatura , Reagentes para Ligações Cruzadas , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Glutaral/metabolismo , Pressão Hidrostática , Cinética
11.
J Chromatogr A ; 1619: 460934, 2020 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-32029268

RESUMO

Peptide-N-glycosidase F (PNGase F) is the most frequently used enzyme to release N-glycan from glycoproteins in glycomics; however, the releasing process using PNGase F is tedious and can range in duration from hours to overnight. Recently, efforts have been made to accelerate this enzymatic reaction, and they include the use of microwave irradiation, ultrahigh pressure, enzyme immobilization, and other techniques. Here, we developed a novel method combining the oriented immobilization of PNGase F on magnetic particles and microwave-assisted enzymatic digestion techniques to achieve highly efficient release of N-glycans. The oriented immobilization of PNGase F on magnetic particles utilizes the affinity of its co-expressed His-tag towards iminodiacetic acid-Nickel modified magnetic particles. Compared with non-oriented immobilization, the oriented immobilization of PNGase F exhibits several advantages including tolerance to high temperature (52 °C) and the ability to retain strong activity after more than five reuses. When used in combination with microwave irradiation, efficient N-glycan removal from ribonuclease B was achieved within 5 min. The proposed strategy was also used to release glycan from fetuin and human serum and has proven to provide a promising deglycosylation method for the characterization of protein glycosylation.


Assuntos
Glicômica/métodos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/metabolismo , Polissacarídeos/metabolismo , Enzimas Imobilizadas/metabolismo , Fetuínas/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase/química , Micro-Ondas , Polissacarídeos/efeitos da radiação , Ribonucleases/metabolismo
12.
Talanta ; 209: 120554, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31892010

RESUMO

Lactate dehydrogenase (LDH), catalyzing the conversion of pyruvate to lactate during glycolysis, is overexpressed in cancer cells. LDH inhibitors are a promising approach for the treatment of cancer. But up till now, there is limited method for rapid screening of LDH inhibitors. Herein, the use of LDH functionalized magnetic nanoparticles as a drug discovery tool for the selective enrichment of LDH potential inhibitors from natural products was firstly reported in this study. Firstly, LDH was immobilized onto the surface of amino-modified magnetic nanoparticles via covalent binding. In order to obtain the maximum enzyme activity, the immobilization conditions including pH, time and LDH concentration were optimized. The amount of LDH immobilized on MNPs was about 49 µg enzyme/mg carrier under the optimized conditions. Subsequently, the ligand fishing assay was performed to validate the specificity and selectivity of immobilized LDH using a model mixture, which consisted of galloflavin, chlorogenic acid and verbascoside. Finally, the immobilized LDH approach combined with ultra-high performance liquid chromatography-tandem mass spectrometry technique (UHPLC-MS/MS) was applied to screen potential LDH inhibitors from two anthraquinone-rich natural products (Rhubarb and Polygonum cuspidatum). Nine and six compounds were identified from Rhubarb and Polygonum cuspidatum extracts respectively, of which three compounds were common to both. Our results have proven that LDH functionalized magnetic nanoparticles have a significant prospect for drug discovery from complex matrices.


Assuntos
Produtos Biológicos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Animais , Bovinos , Descoberta de Drogas/métodos , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Nanopartículas de Magnetita/química
13.
J Agric Food Chem ; 68(5): 1373-1381, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31927950

RESUMO

Most of the microorganisms can form biofilms, which makes biofilms an abundant bioresource to be exploited. Due to the limitations of the application of current immobilization methods for biofilms, we developed an immobilization method called the biofilm polysaccharide display (BPD) strategy while maintaining the native biofilm structure and catalytic microenvironment of Clostridium acetobutylicum B3. Lipase Lip181 showed significant improvements in stability after chemical immobilization. For example, immobilized Lip181 retained 74.23% of its original activity after incubation for 14 days, while free Lip181 was totally deactivated. In addition, immobilized Lip181 maintained high residual activity (pH 5.0-11.0), which showed improved resistance to pH changes. Notably, this method did not decrease but slightly increased the relative activity of Lip181 from 6.39 to 6.78 U/mg. Immobilized Lip181 was used to prepare cinnamyl acetate, and it showed a maximum yield of 85.09%. Overall, this biofilm immobilization method may promote the development of biocatalytic and biofilm materials.


Assuntos
Materiais Biocompatíveis/química , Biofilmes , Clostridium acetobutylicum/química , Lipase/metabolismo , Polissacarídeos/química , Biocatálise , Clostridium acetobutylicum/fisiologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Lipase/química , Polissacarídeos/metabolismo
14.
Chem Commun (Camb) ; 56(13): 2004-2007, 2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-31960849

RESUMO

The operation of wearable epidermal biofuel cells is prone to rapid irreversible deactivation effects under dynamic sweat pH changes from neutral to acidic. We demonstrate that the encapsulation of lactate-oxidase (LOx) within a hydrophobic protective carbon-paste anode imparts unusually high stability during dynamically changing pH fluctuations and allows the BFC to continue harvesting the lactate bioenergy even after long exposures to acidic conditions. The unique power-recovery ability of the carbon-paste BFC after its failure in harsh pH is attributed to the protective action of the non-polar paste environment.


Assuntos
Fontes de Energia Bioelétrica , Técnicas Biossensoriais/métodos , Glucose Oxidase/metabolismo , Carbono/química , Eletrodos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose Oxidase/química , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ácido Láctico/química , Suor/química , Dispositivos Eletrônicos Vestíveis
15.
Sensors (Basel) ; 20(2)2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31940833

RESUMO

A convenient electrochemical sensing pathway was investigated for neurotransmitter detection based on newly synthesized silole derivatives and laccase/horseradish-peroxidase-modified platinum (Pt)/gold (Au) electrodes. The miniature neurotransmitter's biosensors were designed and constructed via the immobilization of laccase in an electroactive layer of the Pt electrode coated with poly(2,6-bis(3,4-ethylenedioxythiophene)-4-methyl-4-octyl-dithienosilole) and laccase for serotonin (5-HT) detection, and a Au electrode modified with the electroconducting polymer poly(2,6-bis(selenophen-2-yl)-4-methyl-4-octyl-dithienosilole), along with horseradish peroxidase (HRP), for dopamine (DA) monitoring. These sensing arrangements utilized the catalytic oxidation of neurotransmitters to reactive quinone derivatives (the oxidation process was provided in the enzymes' presence). Under the optimized conditions, the analytical performance demonstrated a convenient degree of sensitivity: 0.0369 and 0.0256 µA mM-1 cm-2, selectivity in a broad linear range (0.1-200) × 10-6 M) with detection limits of ≈48 and ≈73 nM (for the serotonin and dopamine biosensors, respectively). Moreover, the method was successfully applied for neurotransmitter determination in the presence of interfering compounds (ascorbic acid, L-cysteine, and uric acid).


Assuntos
Peroxidase do Rábano Silvestre/metabolismo , Lacase/metabolismo , Neurotransmissores/análise , Técnicas Biossensoriais , Catálise , Dopamina/urina , Técnicas Eletroquímicas , Eletrodos , Enzimas Imobilizadas/metabolismo , Ouro/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Microscopia de Força Atômica , Oxirredução , Platina/química , Polímeros/química , Serotonina/urina , Compostos de Silício/química
16.
Carbohydr Res ; 488: 107904, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31901816

RESUMO

Starch hydrolysis to maltose by nano-magnetic combined cross-linked enzyme aggregates of α-amylase and maltogenic amylase (NM-Combi-CLEAs) is an important step to open new perspectives for special food and pharmaceutic production. Improvement of mass transfer, thermostability, functional specificity, and reusability of combined enzymes was performed. The obtained results exhibited that, 1:9 ratio of α-amylase/maltogenic amylase, use of tert-butanol as precipitant, 2 mM glutardialdehyde, 1:0.75 ratios of combined enzymes to lysine, 20 h crosslinking at 3-4 °C are well-suited conditions. The dynamic light scattering (DLS) results implied that the nanomagnetites diameter was about 81.9-88.9 nm, with polydispersity index (PDI) of 0.242 and a Ȥ-potential of -21 mV. Moreover, the particle size, PDI, and Ȥ-potential of NM-Combi-CLEAs were around 99.6 nm, 0.088, and -32 mV respectively. The NM-Combi-CLEAs kept 80.4% of its original activity after 10 cycles, its Km value exhibited about 1.5 folds reduction with about 1.5 times enhance in thermostability at 95 °C than free one. Immobilization activity yield revealed about 84% of activity retaining by NM-Combi-CLEAs strategy. Accordingly, this efficacious nanobiocatalyst with high thermostability and reusability recommended for starch conversion to maltose.


Assuntos
Glicosídeo Hidrolases/metabolismo , Maltose/metabolismo , Amido/química , alfa-Amilases/metabolismo , Difusão Dinâmica da Luz , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Hidrólise , Nanopartículas de Magnetita , Tamanho da Partícula , Termodinâmica
17.
Anal Chim Acta ; 1098: 148-154, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948578

RESUMO

An iron-nickel bimetallic metal-organic framework (FeNi-MOF) with inner peroxidase-like activity as a support for enzyme immobilization was synthesized for the first time. And the GOx-mediated synthesis of mimic multienzyme system (GOx/FeNi-MOF) was obtained by a one-step biomimetic mineralization. FeNi-MOF played a dual role of a peroxidase mimic and a protective coating. Therefore, the obtained multienzyme system showed both peroxidase-like activity of FeNi-MOF and the biological activity of natural enzyme. FeNi-MOF acted as a peroxidase mimic, showing a higher affinity for hydrogen peroxide (H2O2). The enhanced catalytic activity of the FeNi-MOF may result from synergistic effect between iron and nickel. In addition, the GOx/FeNi-MOF was selected as a proof of concept and succeeded in a one-step colorimetric detection of glucose by tandem catalysis. A small amount of the product was used for glucose detection, and the detection range was 0.3-35 mM, which indicated that our sensor can meet the clinical needs for diagnose diabetes.


Assuntos
Produtos Biológicos/química , Materiais Biomiméticos/química , Enzimas Imobilizadas/química , Glucose/análise , Peróxido de Hidrogênio/química , Ferro/química , Estruturas Metalorgânicas/química , Níquel/química , Peroxidases/química , Produtos Biológicos/metabolismo , Materiais Biomiméticos/metabolismo , Catálise , Enzimas Imobilizadas/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidases/metabolismo
18.
Anal Chim Acta ; 1098: 86-93, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948590

RESUMO

Cytochrome P450 (CYP450), and in particular CYP3A4, is the most abundantly expressed CYP450 isozyme implicated in many drug-drug and medicinal plant-drug interactions. Therefore, incorporation of CYP3A4 enzyme screening at an early stage of drug discovery is preferable in order to avoid enzymatic interactions. Here we present for the first time a paper-based CYP3A4 immobilized sol-gel-derived a platform using resorufin benzyl ether as a fluorogenic enzyme substrate used to investigate enzyme activity. The fluorescence intensity of the product can be simply quantified by using a handheld digital microscope and an image analysis software. The limit of quantitation was 0.35 µM with good precision (RSDs < 4.1%). Furthermore, the assay of CYP3A4 activity on the developed paper-based device provided comparable results with those obtained from conventional well-plates (p > 0.05), while offering simplicity and lower cost. Kinetic parameters of the immobilized CYP3A4 in sol-gel coated paper were calculated from the Lineweaver-Burk plot, including Michaelis constant (Km) and maximum velocity (Vmax), which were 2.71 ±â€¯0.35 µM and 0.43 ±â€¯0.05 µM/min, respectively. Moreover, a functional test of these devices was conducted by assessments of known CYP3A4 inhibitors (i.e. ketoconazole, itraconazole) and inducers (i.e. phenytoin, carbamazepine). To further demonstrate the broad range of uses, the devices were utilized to assay plant extracts i.e. Areca catechu seeds, Camellia sinensis leaves, Eclipta prostrata aerial part, providing results in good agreement with previous studies. Furthermore, the sol-gel immobilized enzyme stored at 4 °C can increase storage stability, offering the activity of 86.3 ±â€¯0.4% after 3-weeks storage, equivalent to the activity of the free enzyme solution after 1-week storage. The developed paper-based devices offer versatility, portability and low-cost.


Assuntos
Derivados de Benzeno/química , Sistema Enzimático do Citocromo P-450/análise , Enzimas Imobilizadas/análise , Éteres/química , Oxazinas/química , Papel , Sistema Enzimático do Citocromo P-450/metabolismo , Ativação Enzimática , Enzimas Imobilizadas/metabolismo , Géis/química , Humanos , Estrutura Molecular
19.
Food Chem ; 310: 125741, 2020 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31806389

RESUMO

Polyphenol oxidase (PPO), also known as tyrosinase and catechol oxidase, is the enzyme responsible for enzymatic browning in foods. It causes undesirable organoleptic, nutritional and colour changes. Here, we report the preparation of five nanomaterials and a study of their ability to modulate PPO enzyme activity. The materials consist of UVM-7 supports (a mesoporous silica material) modified with diverse functional groups (i.e. amine, carboxylic acid, isocyanate, alkane and pyridine). We also studied the PPO immobilisation capability of the materials. All the materials, except the carboxylic acid functionalised one, offer high PPO loading capabilities and the immobilisation speed increases with functionalisation. Nevertheless, only a minor effect of the inhibition of enzymatic browning was produced. Furthermore, the amine containing material was able to capture not only PPO, but also the oxidation products. Such behaviour was validated with fresh apple juice in which browning was avoided, even 90 min in the presence of oxygen at room temperature.


Assuntos
Catecol Oxidase/química , Catecol Oxidase/metabolismo , Sucos de Frutas e Vegetais , Nanoestruturas/química , Dióxido de Silício/química , Ácidos Carboxílicos/química , Ácido Edético/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Malus/química , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Piridinas/química , Propriedades de Superfície
20.
Appl Biochem Biotechnol ; 190(1): 218-231, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31332676

RESUMO

Yeast surface display has emerged as a viable approach for self-immobilization enzyme as whole-cell catalysts. Herein, we displayed Candida rugosa lipase 1 (CRL LIP1) on the cell wall of Pichia pastoris for docosahexaenoic acid (DHA) enrichment in algae oil. After a 96-h culture, the displayed CRL LIP1 achieved the highest activity (380 ± 2.8 U/g) for hydrolyzing olive oil under optimal pH (7.5) and temperature (45 °C) conditions. Additionally, we improved the thermal stability of displayed LIP1, enabling retention of 50% of its initial bioactivity following 6 h of incubation at 45 °C. Furthermore, the content of DHA enhanced from 40.61% in original algae oil to 50.44% in glyceride, resulting in a 1.24-fold increase in yield. The displayed CRL LIP1 exhibited an improved thermal stability and a high degree of bioactivity toward its native macromolecule substrates algae oil and olive oil, thereby expanding its potential for industrial applications in fields of food and pharmaceutical. These results suggested that surface display provides an effective strategy for simultaneous convenient expression and target protein immobilization.


Assuntos
Proteínas de Bactérias/metabolismo , Candida/enzimologia , Ácidos Docosa-Hexaenoicos/metabolismo , Temperatura Alta , Lipase/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo
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