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1.
Chem Commun (Camb) ; 55(61): 8935-8938, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31286126

RESUMO

The Eubacterium saburreum serine protease inhibitor from the human gut microbiota inhibits the eukaryotic pancreatic elastase associated with acute pancreatitis. Interestingly, the inhibition efficiency and stability are markedly increased by the para-sulphonato-calix[8]arene capped silver nanoparticles. Moreover, this enzyme is distinguishable by its high inhibitory effect at broad pH range between 2-10 and temperatures from 10 to 40 °C, in the presence of para-sulphonato-calix[8]arene capped silver nanoparticles the enzyme remains active even at 70 °C.


Assuntos
Calixarenos/química , Nanopartículas Metálicas/química , Elastase Pancreática/antagonistas & inibidores , Serpinas/química , Prata/química , Sequência de Aminoácidos , Animais , Ensaios Enzimáticos , Eubacterium/química , Concentração de Íons de Hidrogênio , Estabilidade Proteica , Alinhamento de Sequência , Serpinas/isolamento & purificação , Ácidos Sulfônicos/química , Suínos , Temperatura Ambiente
2.
Chem Commun (Camb) ; 55(61): 8963-8966, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290488

RESUMO

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , DNA Ligases/química , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Pontos Quânticos/química , Biotina/química , Carbocianinas/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Células HEK293 , Humanos , Limite de Detecção , Células MCF-7 , Hibridização de Ácido Nucleico , Imagem Individual de Molécula , Estreptavidina/química
3.
Anal Chim Acta ; 1078: 112-118, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358208

RESUMO

Herein, an array-based in situ fluorescence assay is proposed for high-throughput analysis and localization of multiplex matrix metalloproteinases (MMPs) activities in cell monolayers and tissue sections. Five specific MMPs (MMP-2, -3, -7, -9, and -14) peptide substrates containing FAM/Dabcyl fluorescent resonance energy transfer (FRET) pair are directly spotted on the surface of cell monolayers or tissue sections, and hydrolyzed by localized MMPs, resulting in fluorescence recovery of FAM. MMPs activities are determined by the fluorescence intensity of stained cells/tissues due to the cellular internalization of peptide fragments with FAM moiety. We demonstrate that the array-based in situ fluorescence assay is suitable for identifying the MMPs expression patterns of cells, as well as determining the secreted MMPs activities in cell monolayer with high sensitivity (as low as hundreds of cells per square centimeter). The feasibility of the assay is further confirmed by evaluating inhibition potencies of six compounds toward five MMPs. Profiling of five MMPs activities in the localized parts of 32 thyroid tissues is performed without separation or extraction procedures, demonstrating the good practicality of the method.


Assuntos
Ensaios Enzimáticos/métodos , Metaloproteinases da Matriz/análise , Microscopia de Fluorescência/métodos , Peptídeos/metabolismo , Linhagem Celular Tumoral , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Hidrólise , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Peptídeos/química , Estudo de Prova de Conceito , Glândula Tireoide/metabolismo
4.
Food Chem ; 299: 125038, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31284248

RESUMO

Wheat is one of the world's most widely consumed staple food. However, the number of people suffering from wheat-related disorders has increased drastically. Amylase-trypsin inhibitors (ATIs) have recently been identified as one of the main triggers of non-celiac wheat sensitivity (NCWS). In this study, an enzymatic assay for the determination of trypsin inhibition activity in hexaploid wheat was developed. This method was optimized with respect to several parameters, such as extraction and incubation procedures, and was validated according to international standards, concerning accuracy, precision and robustness of the method. Results revealed that linear inhibition and thus accuracy occurred only in a narrow concentration range. However, after optimization of settings the novel method was found to be satisfactory for accurate determination of trypsin inhibition in wheat. Purification of the wheat extract with immobilized trypsin beads led to the identification of CM inhibitors (chloroform/methanol soluble proteins) as main contributors of trypsin inhibition.


Assuntos
Amilases/farmacologia , Ensaios Enzimáticos/métodos , Triticum/enzimologia , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Alérgenos/farmacologia , Humanos
5.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
6.
J Chromatogr A ; 1602: 458-466, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31153601

RESUMO

Bioanalytical questions are more and more solved by bioassays directly in situ the planar separation. If compared to chemical derivatization in situ, several reagent applications on the same chromatogram make the workflow for enzymatic and biological assays more complex. Hence, if compared to piezoelectric spraying of chemical derivatization reagents, an assay transfer to the piezoelectric spraying technique was much more challenging. Important aspects were investigated, i.e., plate pre-wetting, spraying nozzle type and applied volumes for microorganism suspension as well as enzyme and substrate-chromogenic solutions. Finally, with the newly developed piezoelectric spraying procedures for the application of biological (Aliivibrio fischeri) and enzymatic (acetyl- and butyrylcholinesterase) assays, several obstacles of the state-of-the-art automated immersion were avoided such as the (1) required high volumes of solutions, (2) tailing of highly water-soluble zones upon slow plate withdrawal, (3) zone distortion or shift observed after previous buffer salt applications or long/slow immersion times/speeds, (4) gradual inactivation of the enzyme solution along with its ongoing re-use, and (5) lack of covering the whole plate surface. The benchmarking of both techniques also showed that simplicity remains the key argument for immersion. As proof of concept, piezoelectrically sprayed autograms were compared with those of immersion, by taking the example of Peganum harmala (P. h.) seed extract. The plate background and thus homogeneity of the applied solutions were found to be almost comparable. Three bands among the pronounced fluorescent bands were responsible for the most antibacterial activity of P. h. seed extract in the A. fischeri bioassay and were also inhibiting the AChE. These AChE and three further BChE inhibitors were detected, whereby the AChE inhibition was twice as strong as the BChE inhibition. By their in situ HRMS spectra, the active zones in the P. h. seed extract were assigned to be the AChE-inhibiting ß-carboline alkaloids, harmine, harmaline and ruine, as well as the BChE-inhibiting quinazoline alkaloids, vasicine and deoxyvasicine, and the ß-carboline alkaloid harmol. For the first time, the found inhibitors were calculated equivalently to the well-known ChE-inhibitor physostigmine, and thus, piezoelectric spraying was proven to be suited for quantifications.


Assuntos
Cromatografia/métodos , Eletricidade , Ensaios Enzimáticos/métodos , Sementes/química , Acetilcolinesterase/metabolismo , Aliivibrio fischeri/fisiologia , Automação , Bioensaio , Butirilcolinesterase/metabolismo , Cromatografia em Camada Delgada , Espectrometria de Massas , Peganum/química , Extratos Vegetais/química
7.
Microbiol Res ; 223-225: 13-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178046

RESUMO

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.


Assuntos
Celulases/biossíntese , Celulases/química , Flavobacterium/enzimologia , Flavobacterium/metabolismo , Regiões Antárticas , Sequência de Bases , Carbono/metabolismo , Ciclo do Carbono , Carboximetilcelulose Sódica/metabolismo , Domínio Catalítico , Celulase , Celulases/genética , Celulose , Ensaios Enzimáticos , Fermentação , Flavobacterium/genética , Flavobacterium/crescimento & desenvolvimento , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Especificidade por Substrato , Temperatura Ambiente , beta-Glucosidase/metabolismo
8.
Food Chem ; 293: 537-544, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151645

RESUMO

To verify the effect of protein phosphorylation on glycolysis and elucidate the regulatory mechanism from the perspective of enzyme activity, ovine muscle was treated with a kinase inhibitor, dimethyl sulfoxide, or a phosphatase inhibitor and the activities of glycogen phosphorylase, pyruvate kinase and phosphofructokinase were determined. The protein phosphorylation level was significantly different after incubation of muscle with kinase or phosphatase inhibitors. The pH value and lactate content revealed that a high phosphorylation level was the reason for the fast glycolysis. The glycogen phosphorylase, pyruvate kinase and phosphofructokinase activities were significantly higher in the phosphatase inhibitor group than in the other two groups (p < 0.05). Therefore, protein phosphorylation is involved in activating these three enzymes. In summary, protein phosphorylation plays a role in post-mortem glycolysis through the regulation of enzyme activity in ovine muscle.


Assuntos
Glicogênio Fosforilase/metabolismo , Músculos/enzimologia , Fosfofrutoquinases/metabolismo , Piruvato Quinase/metabolismo , Animais , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Glicólise/efeitos dos fármacos , Fosfofrutoquinases/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piruvato Quinase/antagonistas & inibidores , Ovinos
9.
Food Chem ; 295: 653-661, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174809

RESUMO

Although ß-xylosidases have a wide range of applications, cold-active ß-xylosidases have been poorly studied. In this study, a cold active ß-xylosidase gene (xyl) from Bacillus pumilus TCCC 11,350 was cloned and overexpressed in Escherichia coli. The recombinant XYL (rXYL) was revealed to be a bifunctional enzyme with both ß-xylosidase and α-l-arabinofuranosidase activities. Purified rXYL was most active at 30 °C, demonstrating 26% and 18% of its maximum activity at 4 °C and 0 °C, respectively. Meanwhile, rXYL showed a 52% activity in 200 mM xylose, indicating a relatively strong tolerance to xylose. Moreover, rXYL exhibited a high synergistic effect (11.14-fold and 16.21-fold) with endo-xylanase to degrade beechwood xylan in both sequential and simultaneous reactions at low temperatures. As the first report on the novel cold-adapted ß-xylosidase from B. pumilus, these results suggested rXYL had attractive properties for food industrial utilizations.


Assuntos
Bacillus pumilus/enzimologia , Xilosidases/metabolismo , Sequência de Aminoácidos , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Temperatura Ambiente , Xilanos/metabolismo , Xilose/metabolismo , Xilosidases/classificação , Xilosidases/genética
10.
Food Chem Toxicol ; 131: 110529, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150784

RESUMO

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 µmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.


Assuntos
Aldeídos/metabolismo , Fenóis/metabolismo , Proteômica/métodos , ATP Citrato (pro-S)-Liase/química , ATP Citrato (pro-S)-Liase/metabolismo , Aldeídos/química , Domínio Catalítico , Ensaios Enzimáticos , Epigenômica/métodos , Ontologia Genética/estatística & dados numéricos , Histona Desmetilases/química , Histona Desmetilases/metabolismo , Humanos , Informática/métodos , Metiltransferases/química , Metiltransferases/metabolismo , Simulação de Acoplamento Molecular , Nicotinamida N-Metiltransferase/química , Nicotinamida N-Metiltransferase/metabolismo , Olea/química , Azeite de Oliva/química , Fenóis/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Software
11.
Future Microbiol ; 14: 671-689, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31161792

RESUMO

Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem-cefoxitin combination and doripenem-tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem-cefoxitin treatment and by 35.54% after doripenem-tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem-cefoxitin and doripenem-tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Cefoxitina/farmacologia , Doripenem/farmacologia , Tetraciclina/farmacologia , beta-Lactamases/efeitos dos fármacos , Bactérias/enzimologia , Bactérias/metabolismo , Combinação de Medicamentos , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Sinergismo Farmacológico , Ensaios Enzimáticos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Cinética , Testes de Sensibilidade Microbiana , Inibidores da Síntese de Proteínas/farmacologia , Termodinâmica , beta-Lactamases/análise
12.
Anal Chim Acta ; 1072: 81-86, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31146868

RESUMO

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H]+m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay.


Assuntos
Acetilcolinesterase/análise , Reatores Biológicos , Butirilcolinesterase/análise , Ensaios Enzimáticos , Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Ensaios Enzimáticos/instrumentação , Galantamina/química , Galantamina/farmacologia , Humanos , Espectrometria de Massas/instrumentação
13.
Food Chem Toxicol ; 131: 110563, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31199992

RESUMO

Apple pomace (AP) utilised for analysis of triterpenic acids (TTAs) using HPLC-MS/MS. The methanol, ethanol and ethyl acetate extracts showed high phenolic content with significant antioxidant activity compared to chloroform and n-hexane. AP TTAs; ursolic acid, betulinic acid and maslinic acid showed potent antioxidant and enzyme inhibitory effects. The IC50 values were 13.2-30.8 µg/mL (tyrosinase), 19.6-42.5 µg/mL (xanthine oxidase) and 16.6-38.6 µg/mL (urease) for AP extracts and 8.4-25.8 µg/mL (tyrosinase), 12.6-30.2 µg/mL (xanthine oxidase) and 10.1-28.6 µg/mL (urease) for TTAs, compared to the positive controls; kojic acid (10.4 ±â€¯0.06 µg/mL), allopurinol (9.6 ±â€¯0.04 µg/mL) and thiourea (8.9 ±â€¯0.02 µg/mL) towards respective enzymes. UA showed a competitive type of inhibition for tyrosinase, while BA showed a noncompetitive type of inhibition towards xanthine oxidase. In addition, the AP extracts and TTAs exerted significant cytotoxic effects towards the proliferation of cancer cell lines. AP methanol extract (IC50 of 38.5 ±â€¯4.1, 47.1 ±â€¯3.5, 70.6 ±â€¯2.3, and 50.5 ±â€¯3.9 µg/mL) and ursolic acid (IC50 of 6.5 ±â€¯0.7, 15.5 ±â€¯1.4, 20.8 ±â€¯1.3, and 5.6 ±â€¯0.8 µg/mL) showed prominent anticancer activity on Hela, Skov-3, Caski, and NCL cancer cell lines, respectively. Thus, this study shows that the AP & TTAs could be utilized for functional food development and as a potent antioxidant, anticancer, skin whitening, and anti-urolithic agents.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Depuradores de Radicais Livres/farmacologia , Frutas/química , Malus/química , Triterpenos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Linhagem Celular Tumoral , Cães , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Depuradores de Radicais Livres/química , Depuradores de Radicais Livres/isolamento & purificação , Humanos , Células Madin Darby de Rim Canino , Monofenol Mono-Oxigenase/antagonistas & inibidores , Extratos Vegetais/análise , Extratos Vegetais/química , Extração em Fase Sólida , Triterpenos/química , Triterpenos/isolamento & purificação , Urease/antagonistas & inibidores , Xantina Oxidase/antagonistas & inibidores
14.
Anal Chim Acta ; 1076: 131-137, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203957

RESUMO

As an important biomarker, alkaline phosphatase (ALP) is one of the most commonly assayed enzymes in clinical practice. Here a novel turn-on fluorescent nanoswitch for ALP assay was suggested. The nanoswitch was easily constructed via two high-affinity ligands between GSH and Al3+ and PPi and Al3+ based on the difference in the affinity. The primary fluorescence of as-prepared GSH capped Cu nanoclusters (NCs) turned on first upon the Al3+ addition due to the Al3+ induced aggregation induced emission (AIE) enhancement based on the high affinity of GSH and Al3+. The presence of PPi then made Al3+ desorb from the surface of GSH capped Cu NCs due to the higher affinity of PPi and Al3+. As a result, the fluorescence of the Cu NCs was quenched. ALP could hydrolyze PPi into phosphate, destroying the PPi-Al3+ complex and releasing Al3+. Thus, the Al3+ binds to GSH again and the fluorescence was restored. The nanoswitch was demonstrated to be sensitive and selective for ALP assay and was successfully used for the ALP assay in the human serum.


Assuntos
Fosfatase Alcalina/sangue , Alumínio/química , Difosfatos/química , Ensaios Enzimáticos/métodos , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/métodos , Colorimetria/métodos , Cobre/química , Fluorescência , Glutationa/química , Humanos , Limite de Detecção
15.
Nat Commun ; 10(1): 2222, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110237

RESUMO

Substrates associate and products dissociate from enzyme catalytic sites rapidly, which hampers investigations of their trajectories. The high-resolution structure of the native Hordeum exo-hydrolase HvExoI isolated from seedlings reveals that non-covalently trapped glucose forms a stable enzyme-product complex. Here, we report that the alkyl ß-D-glucoside and methyl 6-thio-ß-gentiobioside substrate analogues perfused in crystalline HvExoI bind across the catalytic site after they displace glucose, while methyl 2-thio-ß-sophoroside attaches nearby. Structural analyses and multi-scale molecular modelling of nanoscale reactant movements in HvExoI reveal that upon productive binding of incoming substrates, the glucose product modifies its binding patterns and evokes the formation of a transient lateral cavity, which serves as a conduit for glucose departure to allow for the next catalytic round. This path enables substrate-product assisted processive catalysis through multiple hydrolytic events without HvExoI losing contact with oligo- or polymeric substrates. We anticipate that such enzyme plasticity could be prevalent among exo-hydrolases.


Assuntos
Domínio Catalítico , Glucosidases/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Biocatálise , Cristalografia por Raios X , Ensaios Enzimáticos/métodos , Glucosidases/química , Glucosidases/isolamento & purificação , Glicosídeos/metabolismo , Hordeum/metabolismo , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/metabolismo , Especificidade por Substrato
16.
Talanta ; 201: 450-454, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122448

RESUMO

A novel aggregation-induced emission (AIE) probe comprised of a hydrophilic protein kinase specific peptide and a hydrophobic tetraphenylethene (TPE) unit was synthesized through click reaction. The prepared TPE-peptide probe could be completely degraded by carboxypeptidase Y (CPY) to release hydrophobic TPE part, which aggregated in buffer solution and showed strong TPE emission. In the presence of casein kinase (CKII), the phosphorylation of peptide prevented the complete degradation by CPY producing the nonemissive probe. Thus, the developed probe can be used to detect CKII homogeneously and conveniently. This detection process can be finished within 1.5 h with high sensitivity (0.05 mU/µL) and good selectivity. The developed method can also be used to screen protein kinase inhibitor even in a complex biological system.


Assuntos
Técnicas Biossensoriais/métodos , Caseína Quinase II/análise , Ensaios Enzimáticos/métodos , Espectrometria de Fluorescência/métodos , Compostos de Benzilideno/síntese química , Compostos de Benzilideno/química , Caseína Quinase II/química , Catepsina A/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HeLa , Humanos , Oligopeptídeos/síntese química , Oligopeptídeos/química , Fosforilação , Inibidores de Proteínas Quinases/química , Proteólise , Triazóis/química
17.
Carbohydr Polym ; 216: 231-237, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31047062

RESUMO

Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana/química , Glucanos/síntese química , Enzima Ramificadora de 1,4-alfa-Glucana/genética , Enzima Ramificadora de 1,4-alfa-Glucana/isolamento & purificação , Amilopectina/química , Amilose/química , Ensaios Enzimáticos , Escherichia coli/genética , Hidrólise , Peso Molecular , Thermus thermophilus/enzimologia
18.
Biosens Bioelectron ; 138: 111308, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31103013

RESUMO

Poly(ADP-ribose) polymerase-1 (PARP-1), as an original tumor marker, has aroused wide attention in recent years. However, only a few researches have been done for PARP-1 activity detection because PARP-1 is lack of optical or electrochemical property. In this work, a label-free and high-sensitive photoelectrochemical (PEC) biosensor for PARP-1 activity detection based on poly[9,9-bis(6'-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) has been designed. To the best of our knowledge, it is the first time that PEC has been used to monitor PARP-1 activity. PARP-1 were activated under the function of activated dsDNA, as a result, branched polymers of ADP-ribose (PAR) with plentiful negative charge were formed in the presence of nicotinamide adenine dinucleotide (NAD+). Subsequently, positively charged PFP with good photoelectrochemical properties, were absorbed on PAR via electrostatic interaction. High photocurrent was produced under light induction, which was depended on the PARP-1 activity. The biosensor has a wide linear range from 0.01 to 2 U with a detection limit of 0.007 U. The strategy has been applied in breast and ovarian cancer cells to detection PARP-1 activity with approving results, which signifies that it is a promising tool for clinical diagnosis.


Assuntos
Biomarcadores Tumorais/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Eletrodos , Ensaios Enzimáticos/métodos , Humanos , Indóis/farmacologia , Limite de Detecção , NAD/farmacologia , Processos Fotoquímicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Eletricidade Estática
19.
Food Chem ; 294: 231-237, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126458

RESUMO

A fully mechanized Arduino-controlled multi-pumping flow analysis system and procedure for the determination of ß-galactosidase activity are proposed. The applied bioanalytical method is based on the determination of p-nitrophenol formed in the course of enzyme-catalyzed hydrolysis of p-nitrophenyl-galactopyranosides. The photometric detection is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system was applied for evaluation of optimal enzyme detection conditions (pH, temperature and reaction time), selection of appropriate substrate for the assays, comparison of enzymes of different origins (isoenzymes), detection of ß-galactosidase inhibitor and finally to the determination of enzyme activity in some dietary supplements dedicated for people suffering from lactose intolerance. Depending on measurements conditions the developed bioanalytical system allows determination of ß-galactosidase in the wide range of activity (up to 15 U/mL at detection limit ca 0.01 U/mL) with high sample flowthroughput (up to 30 detections per hour). Additionally, the potential utility of the developed analytical system for amyloglucosidase activity assays has been demonstrated.


Assuntos
Galactose/metabolismo , beta-Galactosidase/metabolismo , Biocatálise , Ensaios Enzimáticos , Galactose/química , Concentração de Íons de Hidrogênio , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Nitrofenóis/química , Temperatura Ambiente , beta-Galactosidase/antagonistas & inibidores
20.
World J Microbiol Biotechnol ; 35(6): 82, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134384

RESUMO

In developing countries, local enzyme production can help decrease the dependency of imported enzymes for bioconversion of e.g. cellulosic feedstocks, but the use of conventional nitrogen sources contributes significantly to such enzyme production cost. Use of local resources is therefore important to consider. Green seaweeds are marine macroalgae that are rich in nitrogen, but not exploited for their nitrogen content. Cellulase production was accomplished by using cocoa pod husk (CPH) and green seaweed (GS) (Ulva fasciata sp.) as growth substrates for Polyporus ciliatus CBS 366.74 in submerged cultivation. The nitrogen concentration of GS was comparable to that of CPH with 0.6% w/v peptone at 4% w/v substrate concentration. A decline of cellulase activity in peptone supplemented GS growth media indicated nitrogen sufficiency of GS to serve as a potential nitrogen source for the fungal growth and cellulase production. Comparison of enzyme production on CPH growth media supplemented with either GS or peptone based on equivalent carbon to nitrogen ratios was done for two Polyporus strains namely; P. ciliatus CBS 366.74 and P. brumalis CBS 470.77. Peptone could be substituted by up to 0.6% w/v with GS at inclusion levels of 50-100% of substrate concentration to attain satisfactory cellulase productivity. However, the cellulase productivity response varied among the two Polyporus species. This study demonstrated that green seaweeds may be used as alternative nitrogen sources for fungal cellulase production.


Assuntos
Celulase/biossíntese , Nitrogênio/metabolismo , Polyporus/metabolismo , Alga Marinha/química , Ulva/química , Cacau/química , Carbono/metabolismo , Celulose/metabolismo , Meios de Cultura/química , Ensaios Enzimáticos , Fermentação , Gana , Glicosídeo Hidrolases/metabolismo , Polyporus/enzimologia , Polyporus/crescimento & desenvolvimento , beta-Glucosidase/metabolismo
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