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1.
Carbohydr Polym ; 229: 115471, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31826427

RESUMO

Micro/nano celluloses (MC)/NC) were magnetized by nanoγ-Fe2O3 into the nanoγ-Fe2O3@MC (NMMC) and nanoγ-Fe2O3@NC (NMMC) which oxidized to NMMCD and NMNCD dialdehydes for Schiff-base immobilization of urease as NMMCD/urease and NMMCD/urease. The relative enzyme-activity of the immobilized ureases were comparable with the free-urease, although 75%-80% of the enzyme activity preserved for NMMCD/urease and NMNCD/urease after six cycles. The compared catalytic activities of the NMMCD/urease and NMMCD/urease in Biginelli/Hantzsch reactions in water at 60 °C surprised us by 100% selectivity for the Biginelli product 3,4-dihydropyrimidin-2(1H)-one (DHPM1). With the superiority of NMNCD/urease, this high selectivity using immobilized ureases is owing to the admirable urease inhibitory of the formed Biginelli product DHPM1 by urea condensation instead of urea hydrolysis. The robust enzyme inhibitory of the DHPM1 for free urease was also confirmed by phenol red test to show the deactivation of enzyme for enzymatic hydrolysis of urea and ammonia production in water.


Assuntos
Celulose/química , Enzimas Imobilizadas/química , Ureia/química , Urease/química , Aspergillus niger/enzimologia , Catálise , Celulose/isolamento & purificação , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Compostos Férricos/química , Gossypium/química , Concentração de Íons de Hidrogênio , Fenômenos Magnéticos , Estabilidade Proteica , Temperatura , Urease/antagonistas & inibidores
2.
Talanta ; 205: 120126, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450397

RESUMO

Inspired by the porous and fibrous structure of commercially available bamboo, herein we created an l-glutaminase enzyme reactor based on bamboo sticks. The enzyme was immobilized onto the bamboo sticks through a glutaraldehyde modification to achieve covalent bonding. The enzymatic hydrolysis efficiency of the prepared l-glutaminase@bamboo sticks based porous enzyme reactor was evaluated by chiral ligand exchange capillary electrochromatography using l-glutamine as the substrate. l-glutaminase@bamboo exhibited improved enzymatic hydrolysis performances, including high hydrolysis efficiency (maximum rate Vmax: two fold higher than the free enzyme), prolonged stability (14 days) and good reusability. l-Glutaminase@bamboo sticks also expanded application capability in pharmaceutical industry in enzyme inhibitor screening. These excellent properties could be attributed to the micropores of bamboo sticks, which led to the fast enzymatic kinetics. The results suggest that the pores of bamboo sticks played an important role in the proposed enzyme reactor during the hydrolysis of l-glutamine and l-glutaminase inhibitor screening.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Glutaminase/antagonistas & inibidores , Glutaminase/metabolismo , Poaceae/química , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glutaminase/química , Glutaral/metabolismo , Cinética , Porosidade , Propriedades de Superfície
3.
Int J Biol Macromol ; 138: 1-12, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31302127

RESUMO

In this study, a CotA laccase from Bacillus subtilis cjp3 was successfully immobilized onto magnetic graphene oxide (MGO) nanomaterials via covalent bonding with hydrochloride/N-hydroxysuccinimide (EDC/NHS). The morphology, structure, and properties of the MGO-laccase were then characterized by scanning-electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray-photoelectron spectroscopy (XPS), and a magnetic-property-measurement system (MPMS). The magnetic composite exhibited an extremely high binding capacity of ~145.04mg/g and maintained maximal relative enzyme activities at 25°C, pH7, and a reaction time of 2h. The pH, thermal, operational, and storage stabilities of MGO-laccase were significantly improved over those of free laccase. Moreover, MGO-laccase exhibited a higher tolerance than that of free laccase in the presence of organic solvents, inhibitors, metal ions, and salts. Furthermore, MGO-laccase showed good decolorization performance of malachite green (MG), with decolorization rates reaching 99% after 5h of reaction at 30°C and pH6. In addition, the maximum saturation magnetization of MGO-laccase was 27.7emu/g, allowing for rapid magnetic separation. Accordingly, magnetic separation allowed MGO-laccase to maintain 75% of its activity after ten consecutive decolorization cycles.


Assuntos
Bacillus subtilis/enzimologia , Grafite/química , Lacase/química , Lacase/metabolismo , Imãs/química , Corantes de Rosanilina/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cinética , Lacase/antagonistas & inibidores , Metais/farmacologia , Corantes de Rosanilina/isolamento & purificação , Sais/farmacologia , Solventes/farmacologia , Succinimidas/química
4.
Int J Biol Macromol ; 136: 1133-1141, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31220494

RESUMO

ß-glucosidases (BGLs) hydrolyze short-chain cellulooligosaccharides. Some BGLs can hydrolyze anthocyanins and be applied in the clarification process of food industries, especially grape juice and wine. Enzyme immobilization is a valuable tool to increase enzyme stabilization. In this work, Malbranchea pulchella BGL was immobilized on Monoaminoethyl-N-ethyl-agarose ionic support, MANAE-agarose, and Concanavalin A-Sepharose affinity support, Con-A-Sepharose. The formed biocatalysts, denominated BLG-MANAE and BLG-ConA, were applied in the grape juice and red wine clarification. BGL-MANAE and BGL-ConA hyperactivated M. pulchella BGL 10- and 3-fold, respectively. Both biocatalysts showed at least 70% activity at pH range 2-11, until 24 h incubation. BGL-MANAE and BGL-ConA showed activity of 60% and 100%, respectively, at 50 °C, up to 24 h. Both biocatalysts were efficiently reused 20-fold. They were stable in the presence of up to 0.1 M glucose for 24 h incubation, and with 5%, 10% and 15% ethanol kept up to 70% activity. BGL-MANAE biocatalyst was 11% and 25% more efficient than BGL-ConA in clarification of concentrate and diluted wines, respectively. Likewise, BGL-MANAE biocatalysts were 14% and 33% more efficient than the BGL-ConA in clarification of diluted and concentrated juices, respectively. Therefore, the BGL-MANAE biocatalyst was especially effective in red wine and grape juice clarification.


Assuntos
Antocianinas/metabolismo , Ascomicetos/enzimologia , Sucos de Frutas e Vegetais/análise , Sefarose/análogos & derivados , Vitis/química , Vinho/análise , beta-Glucosidase/metabolismo , Biocatálise , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise , Sefarose/química , Temperatura , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/química
5.
Artif Cells Nanomed Biotechnol ; 47(1): 1075-1084, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942622

RESUMO

In this study, an attempt has been made to evaluate the effect of products of ß-galactosidase (ßGS) catalyzed reaction i.e. glucose and galactose and their structurally related compound vitamin C (VC) on the catalytic activity of native and PANI-CS-NC and PANI-CS-Ag-NC adsorbed ßGS. Results indicated a decline in catalytic activity of soluble enzyme in the presence of all investigated compounds. The order of inhibition was found to be VC < glucose < galactose. However, the immobilized preparations were found more resistant to inactivation caused by the added compounds. About 48% activity was retained by PANI-CS-Ag-NC-ßGS in the presence of galactose (5%, w/v), while the native enzyme exhibited only 18% of its original activity. A significant decrease in absorbance and fluorescence intensity was evaluated in soluble enzyme incubated in the presence of all investigated compounds. Three-dimensional fluorescence graphs, CD and FT-IR spectroscopic studies illustrated noteworthy conformational changes in the secondary structure and microenvironment of the soluble enzyme in the presence of VC and tested sugars. These results suggest that both PANI-CS-NC and PANI-CS-Ag-NC bound ßGS are more resistant to the exposure caused by the higher concentration of added glucose, galactose, and VC and, therefore, can be effectively utilized for the production of a hassle-free lactose nano-biosensor.


Assuntos
Compostos de Anilina/química , Quitosana/química , Inibidores Enzimáticos/farmacologia , Nanocompostos/química , Prata/química , beta-Galactosidase/antagonistas & inibidores , beta-Galactosidase/química , Ácido Ascórbico/farmacologia , Catálise , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Galactose/farmacologia , Glucose/farmacologia , Ligação Proteica , beta-Galactosidase/metabolismo
6.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1110-1111: 67-73, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30780013

RESUMO

A capillary electrophoresis (CE)-based beta-glucosidase (beta-Glu) immobilized enzyme microreactor (IMER) was constructed for enzyme kinetics study and inhibitor screening with the aid of polydopamine coating. The enzyme kinetic and inhibition studies of beta-Glu were comprehensively evaluated using p-nitrophenyl beta-d-glucopyranoside as a model substrate and castanospermine as a model inhibitor. The Michaelis-Menten constant value of the immobilized beta-Glu in the developed IMER was calculated to be 2.79 mmol/L. The half-maximal inhibitory concentration and inhibition constant of castanospermine were 13.22 µg/mL and 1.54 µg/mL, respectively. In addition, after 50 consecutive runs, the IMER activity was remained at 89.5% of the initial immobilized beta-Glu activity, which showed that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen beta-Glu inhibitors from twelve flavonoids. Four flavonoids include genistein, baicalein, epicatechin gallate and epigallocatechin gallate had significant inhibitory effect on beta-Glu, and their binding mode with enzyme was further verified via the molecular docking analysis. In summary, the developed CE based beta-Glu-IMER is a reliable method for screening beta-Glu inhibitors from natural products.


Assuntos
Reatores Biológicos , Eletroforese Capilar/instrumentação , Enzimas Imobilizadas/metabolismo , beta-Glucosidase/metabolismo , Eletroforese Capilar/métodos , Ensaios Enzimáticos/instrumentação , Ensaios Enzimáticos/métodos , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Flavonoides/química , Flavonoides/metabolismo , Cinética , Simulação de Acoplamento Molecular , beta-Glucosidase/antagonistas & inibidores , beta-Glucosidase/química
7.
Int J Biol Macromol ; 129: 672-678, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30772412

RESUMO

Three ß-glucosidases (Pectinex Ultra SP-L, Pectinex Ultra Clear and homemade preparation from Aspergillus niger) were immobilized using different strategies: ionic adsorption on aminated (MANAE)-agarose beads at pH 5, 7, and 9, followed by biocatalysts modification with glutaraldehyde, or on glutaraldehyde pre-activated supports. The pH of the immobilization was altered to allow different enzyme molecule orientations on the support surface. The biocatalysts from Pectinex Ultra SP-L showed the highest thermal and operational stabilities when immobilized on MANAE-agarose-glutaraldehyde at pH 7. The ß-glucosidase from Pectinex Ultra Clear and from A. niger produced best results when immobilized on MANAE-agarose beads at pH 5 and 7, respectively, which was later treated with glutaraldehyde. The best immobilization results using pre-activated supports were observed for the enzyme present in Pectinex Ultra SP-L, to which the highest thermal stabilities were obtained. Remarkably, the enzyme from A. niger, immobilized on MANAE-agarose at pH 9 and subsequently treated with glutaraldehyde, produced the highest stabilization (approximately 560 times more stable than soluble enzyme at 60 °C). Results showed that optimal protocol for ß-glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type of enzyme.


Assuntos
Enzimas Imobilizadas/química , Glutaral/química , beta-Glucosidase/química , Aspergillus niger/enzimologia , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Glucose/farmacologia , Temperatura , beta-Glucosidase/antagonistas & inibidores
8.
Int J Biol Macromol ; 128: 681-691, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30711566

RESUMO

This investigation may be of interest for researchers working on the determination of several biocatalytic properties of the laccase from Trametes versicolor. So, We will treated the effects of pH, temperature, several organic components and heavy metals by performing enzyme assays in the presence of a 2,6 dimethoxyphenol (DMP) as substrate on the laccase activity. The optimum activity and temperature are 4 and 40 °C, respectively. The maximum rate of the reaction is 124.53 U/mg and the Michaelis constant is in order of 1.23 mM. The effect of metal ions on the laccase activity with a final concentrations range varying from 1 to 5 mM show that the Cu2+ ions increase the activity for concentration inferiors to 4 mM and the other metal ions have a relative influence on the laccase activity. Four tri-block copolymers based on poly(ethylene oxide) and poly(propylene oxide) and two polyethylene glycols are used to study the synthetic polymers effects on the enzymatic activity. Also, we have demonstrated that the laccase keeps 95% of its initial activity at 60 °C in the PEGDA8000 and PEGDA6000 gel matrix. The maximum rate of the immobilized laccase is approximately around 21.03 and 47.22% smaller than the free one.


Assuntos
Biocatálise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lacase/química , Lacase/metabolismo , Trametes/enzimologia , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Concentração de Íons de Hidrogênio , Cinética , Lacase/antagonistas & inibidores , Metais Pesados/farmacologia , Modelos Moleculares , Polímeros/química , Propilenoglicóis/química , Conformação Proteica , Temperatura
9.
J Sep Sci ; 42(5): 1067-1076, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30663871

RESUMO

Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of ß-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized ß-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Reatores Biológicos , Inibidores Enzimáticos/farmacologia , Peptídeos/farmacologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Eletroforese Capilar , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Células HEK293 , Humanos , Cinética , Peptídeos/química
10.
Int J Biol Macromol ; 122: 298-305, 2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30401648

RESUMO

In the present study, α-glucosidase (α-Glu) was immobilized on the chitosan-modified cellulose filter paper (CFP/CS). For α-Glu immobilization, glutaraldehyde (GA) was used as a coupling agent. CFP, environmentally friendly and commercially available with low cost, will avoid the tedious procedure for synthesizing immobilization carrier. In addition, the CFP/CS-immobilized α-Glu can be directly taken out from the reaction mixture after an enzymatic reaction. This makes the instantaneous separation of immobilized enzyme comes true and is convenient to the subsequent study. Combined with capillary electrophoresis (CE), the CFP/CS-immobilized α-Glu was then used for enzyme kinetic and inhibition study. The CFP/CS-immobilized α-Glu exhibited enhanced pH and temperature tolerance. In addition, the performance of the CFP/CS-immobilized α-Glu was studied. Immobilized α-Glu exhibited excellent batch-to-batch reproducibility (RSD = 6.7%, n = 5) and improved reusability (71.0% of its initial activity after 10 repeated cycles). Finally, immobilized α-Glu was used to screen enzyme inhibitors from 11 traditional Chinese medicines (TCMs). The results showed that CFP/CS has potential development prospects as a novel enzyme immobilization carrier.


Assuntos
Celulose/química , Quitosana/química , Enzimas Imobilizadas/química , Filtração , Papel , alfa-Glucosidases/química , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/metabolismo , Inibidores de Glicosídeo Hidrolases/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Reprodutibilidade dos Testes , Temperatura , alfa-Glucosidases/metabolismo
11.
Curr Pharm Biotechnol ; 19(11): 925-931, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30370843

RESUMO

BACKGROUND: Glutathione transferases (GSTs) catalyze the conjugation of glutathione (GSH) to endogenous and xenobiotic electrophilic compounds and have been involved in the development of resistance toward cancer chemotherapeutic drugs and in the etiology, pathology and progression of several other diseases. In the present work, the human isoenzyme GSTA1-1 (hGSTA1-1) was used to assemble a microplate-based platform for high-throughput screening of natural productbased inhibitors from plant extracts. METHODS: The enzyme was immobilized using sol-gel chemistry and deposited as a layer at the bottom surface of 96-well format ELISA microplate. The sensing signal was based on the inhibition of the colorimetric reaction between 1-chloro-dinitrobenzene (CDNB) and GSH, catalyzed by the sol-gel entrapped enzyme. RESULTS: As a proof of concept, the system was used for screening aqueous extracts from medicinal and aromatic plants with excellent reproducibility (approximately 95%). CONCLUSION: The operational simplicity and accuracy of this system, suggest that it can be explored as a bioanalytical tool with potential use in drug design and development efforts for finding new sources of GST inhibitors useful in chemomodulation of cancer drugs.


Assuntos
Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Glutationa Transferase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/isolamento & purificação , Enzimas Imobilizadas/química , Glutationa Transferase/química , Ensaios de Triagem em Larga Escala , Humanos , Extratos Vegetais/isolamento & purificação , Plantas Medicinais/química , Reprodutibilidade dos Testes
12.
Appl Microbiol Biotechnol ; 102(23): 9937-9948, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30276711

RESUMO

Chitinolytic enzymes are capable to catalyze the chitin hydrolysis. Due to their biomedical and biotechnological applications, nowadays chitinolytic enzymes have attracted worldwide attention. Chitinolytic enzymes have provided numerous useful materials in many different industries, such as food, pharmaceutical, cosmetic, or biomedical industry. Marine enzymes are commonly employed in industry because they display better operational properties than animal, plant, or bacterial homologs. In this mini-review, we want to describe marine chitinolytic enzymes as versatile enzymes in different biotechnological fields. In this regard, interesting comments about their biological role, reaction mechanism, production, functional characterization, immobilization, and biotechnological application are shown in this work.


Assuntos
Biotecnologia , Quitinases/metabolismo , Oceanos e Mares , Archaea/enzimologia , Bactérias/enzimologia , Quitina/química , Quitinases/antagonistas & inibidores , Cianobactérias/enzimologia , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/metabolismo , Fungos/enzimologia , Microalgas/enzimologia , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Microbiologia da Água
13.
Anal Chim Acta ; 1006: 90-98, 2018 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-30016268

RESUMO

In the present study, the immobilization method of physical adsorption combined with covalent crosslinking was developed to avoid the shortcomings of both the noncovalent and covalent coupling methods. For the first time, tyrosinase (TYR) was immobilized on the surface of aminated magnetic nanoparticles (Fe3O4-NH2) by the developed method. TYR was firstly adsorbed on the surface of Fe3O4-NH2 by electrostatic interaction, and then by covalent crosslinking with glutaraldehyde (GA), TYR was firmly immobilized on the supports. The immobilized TYR showed enhanced pH and temperature endurances at the optimum pH of 7.0 and temperature of 35 °C. TYR reusability was significantly improved after immobilization and it retained 61.4 ±â€¯2.3% of its initial activity after 5 repeated cycles. Immobilized TYR also showed improved storage stability (73.2 ±â€¯1.1% after 30 days of storage at 4 °C). In addition, the immobilized TYR showed a higher biological affinity to substrate owing to the stabilization of TYR in its active conformation by electrostatic interaction prior to covalent crosslinking. Finally, the immobilized TYR was used to screen of enzyme inhibitors from 11 traditional Chinese medicines (TCMs) to validate whether this method can be used for enzyme inhibitor screening or not.


Assuntos
Biocatálise , Reagentes para Ligações Cruzadas/química , Enzimas Imobilizadas/metabolismo , Nanopartículas de Magnetita/química , Monofenol Mono-Oxigenase/metabolismo , Adsorção , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Medicina Tradicional Chinesa , Monofenol Mono-Oxigenase/antagonistas & inibidores , Monofenol Mono-Oxigenase/química , Eletricidade Estática
14.
Talanta ; 184: 388-393, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29674059

RESUMO

A novel and simple biosensor for the determination of bisphenol A (BPA) based on xanthine oxidase (XOD) enzymatic inhibition has been developed. The biosensor was prepared from xanthine oxidase immobilised by crosslinking with glutaraldehyde, with hypoxanthine as enzyme substrate, and was successfully applied to the determination of BPA using fixed potential amperometry. Biosensor performance was optimised with respect to the applied potential, influence of pH of the electrolyte solution, XOD loading and the substrate concentration. The enzyme inhibition mechanism was evaluated from Cornish-Bowden plus Dixon plots and was found to be reversible and competitive with an apparent inhibition constant of 8.15 nM. Under optimised conditions, the determination of BPA can be achieved in the linear range up to 41 nM with a detection limit of 1.0 nM, which is equal to the lowest reported in the literature, with very good repeatability and reproducibility. The selectivity of the biosensor was evaluated by performing an interference study and found to be excellent; and stability was investigated. It was successfully applied to the detection of BPA in mineral water and in river water.


Assuntos
Compostos Benzidrílicos/análise , Técnicas Biossensoriais , Técnicas Eletroquímicas , Inibidores Enzimáticos/análise , Fenóis/análise , Poluentes Químicos da Água/análise , Xantina Oxidase/antagonistas & inibidores , Compostos Benzidrílicos/farmacologia , Carbono/química , Eletrodos , Eletrólitos/química , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/metabolismo , Vidro/química , Glutaral/química , Glutaral/metabolismo , Concentração de Íons de Hidrogênio , Hipoxantina/química , Hipoxantina/metabolismo , Minerais/análise , Fenóis/farmacologia , Rios/química , Poluentes Químicos da Água/farmacologia , Xantina Oxidase/metabolismo
15.
Talanta ; 182: 600-605, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29501199

RESUMO

Alanine aminotransferase (ALT) plays significant role in biological and clinical research. In this study, a unique ALT enzyme reactor based on multifunctional polymer@magnetic nanoparticles has been constructed for the first time and the enzymolysis efficiency has been evaluated by chiral ligand exchange capillary electrophoresis technique. Poly(N-acryloxysuccinimide) has been synthesized by reversible addition-fragmentation chain transfer polymerization method and immobilized on the magnetic nanoparticles via the succinimide group in the polymer. Interestingly, the enzyme also could easily react with the succinimide group, which enables of ALT covalent bonding onto the polymer. The enzyme amount immobilized and the immobilization time have been investigated. Comparing with free ALT in solution (Vmax of free enzyme = 0.6 mM min-1), the resultant enzyme reactor has exhibited good reusability and stability, and displayed about five times enhanced enzymolysis efficiency with L-alanine as the substrate (Vmax of enzyme reactor = 3.4 mM min-1). Furthermore, the prepared enzyme reactor has been applied in ALT inhibitors screening. The enzyme reactors based on the multifunctional polymer@magnetic nanoparticles have depicted great potential in anti-liver drugs development, liver diseases study and ALT related biological process inspect.


Assuntos
Alanina Transaminase/química , Alanina/química , Reatores Biológicos , Enzimas Imobilizadas/química , Nanopartículas de Magnetita/química , Acrilatos/química , Alanina Transaminase/antagonistas & inibidores , Biocatálise , Eletroforese Capilar/métodos , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Enzimas Imobilizadas/antagonistas & inibidores , Reutilização de Equipamento , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Nanopartículas de Magnetita/ultraestrutura , Polimerização , Succinimidas/química
16.
Anal Biochem ; 549: 53-57, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29550345

RESUMO

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are key cholinesterase enzymes responsible for the hydrolysis of acetylcholine into choline and acetic acid, an essential process for the restoration of the cholinergic neuron. The loss of cholinergic function in the central nervous system contributes to the cognitive decline associated with advanced age and Alzheimer's disease (AD). Inhibitions assays represent a significant role in the drug discovery process. Herein, we describe an improved label free method to screen and characterize new BChE ligands. The liquid chromatography system uses an immobilized capillary enzyme reactor (ICER) as a low affinity and high selectivity column coupled to a mass spectrometer (MS). The enzyme activity was evaluated by monitoring the choline's precursor ion [M + H]+m/z 104 for a brief period. The method was validated using two known cholinesterase inhibitors tacrine and galanthamine. The IC50 values were 0.03 ±â€¯0.006 µM and 0.88 ±â€¯0.2 for tacrine and galanthamine respectively, and Ki was 0.11 ±â€¯0.2 for galanthamine. The efficient combination of the huBChE-ICER with sensitive enzymatic assay detection such as MS, improved the reliable, fast identification of new ligands. Moreover, specific direct quantitation of the product contributes to the reduction of false positive and negative results.


Assuntos
Butirilcolinesterase/química , Inibidores da Colinesterase/química , Enzimas Imobilizadas , Galantamina/química , Espectrometria de Massas , Tacrina/química , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Humanos , Ligantes
17.
J Pharm Biomed Anal ; 151: 252-259, 2018 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-29367161

RESUMO

The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (KM = 81.9 ±â€¯7.49 µmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts.


Assuntos
Catepsina D/antagonistas & inibidores , Inibidores Enzimáticos/análise , Enzimas Imobilizadas/antagonistas & inibidores , Extratos Vegetais/análise , Catepsina D/química , Catepsina D/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Avaliação Pré-Clínica de Medicamentos/instrumentação , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Reprodutibilidade dos Testes , Dióxido de Silício/química , Especificidade por Substrato
18.
J Biol Chem ; 293(8): 2640-2649, 2018 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-29305423

RESUMO

Transglutaminase 2 (TG2) is a ubiquitously expressed, intracellular as well as extracellular protein with multiple modes of post-translational regulation, including an allosteric disulfide bond between Cys-370-Cys-371 that renders the enzyme inactive in the extracellular matrix. Although recent studies have established that extracellular TG2 is switched "on" by the redox cofactor protein thioredoxin-1 (TRX), it is unclear how TG2 is switched "off." Here, we demonstrate that TG2 oxidation by small-molecule biological oxidants, including glutathione, cystine, and hydrogen peroxide, is unlikely to be the inactivation mechanism. Instead, endoplasmic reticulum (ER)-resident protein 57 (ERp57), a protein in the ER that promotes folding of nascent proteins and is also present in the extracellular environment, has the cellular and biochemical characteristics for inactivating TG2. We found that ERp57 colocalizes with extracellular TG2 in cultured human umbilical vein endothelial cells (HUVECs). ERp57 oxidized TG2 with a rate constant that was 400-2000-fold higher than those of the aforementioned small molecule oxidants. Moreover, its specificity for TG2 was also markedly higher than those of other secreted redox proteins, including protein disulfide isomerase (PDI), ERp72, TRX, and quiescin sulfhydryl oxidase 1 (QSOX1). Lastly, siRNA-mediated ERp57 knockdown in HUVECs increased TG2-catalyzed transamidation in the extracellular environment. We conclude that, to the best of our knowledge, the disulfide bond switch in human TG2 represents the first example of a post-translational redox regulatory mechanism that is reversibly and allosterically modulated by two distinct proteins (ERp57 and TRX).


Assuntos
Matriz Extracelular/enzimologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Transglutaminases/antagonistas & inibidores , Regulação Alostérica/efeitos dos fármacos , Biocatálise/efeitos dos fármacos , Células Cultivadas , Cistina/metabolismo , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Glutationa/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacologia , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/química , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Transglutaminases/química , Transglutaminases/genética , Transglutaminases/metabolismo
19.
Int J Biol Macromol ; 108: 32-40, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29174355

RESUMO

A potentiometric biosensor based on agarose-guar gum (A-G) entrapped bio-nanoconjugate of urease with gold nanoparticles (AUNps), has been reported for the first time for glyphosate detection. The biosensor is based on inhibition of urease activity by glyphosate, which was measured by direct potentiometry using ammonium ion selective electrode covered with A-G-urease nanoconjugate membrane. TEM and FTIR analysis revealed nanoconjugate formation and its immobilization in A-G matrix respectively. The composite biopolymer employed for immobilization yields thin, transparent, flexible membrane having superior mechanical strength and stability. It retains the maximum activity (92%) of urease with negligible leaching. The conjugation of urease with AUNps allows improvement in response characteristics for potentiometric measurement. The biosensor shows a linear response in the glyphosate concentration range from 0.5ppm-50ppm, with limit of detection at 0.5ppm, which covers maximum residual limit set by WHO for drinking water. The inhibition of catalytic activity of urease nanoconjugate by gyphosate was confirmed by FTIR analysis. The response of fabricated biosensor is selective towards glyphosate as against various other pesticides. The biosensor exhibits good performance in terms of reproducibility and prolonged storage stability of 180days. Thus, the present biosensor provides an alternative method for simple, selective and cost effective detection of glyphosate based on urease inhibition.


Assuntos
Técnicas Biossensoriais/métodos , Glicina/análogos & derivados , Membranas Artificiais , Nanoconjugados/química , Urease/antagonistas & inibidores , Urease/química , Dolichos/enzimologia , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Glicina/análise , Glicina/farmacologia , Potenciometria , Água/química
20.
Enzyme Microb Technol ; 106: 67-74, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28859812

RESUMO

Phospholipase Lecitase Ultra (LU) and lipase from Thermomyces lanuginosus (TLL) have been immobilized under conditions that favor either enzyme crowding or enzyme dispersion. Highly loaded LU was more stable than low loaded biocatalyst under all studied conditions. Using TLL, the results depended on the inactivation conditions, e.g., crowding was positive at pH 5 and negative at pH 7. Then, all preparations were treated with glutaraldehyde (Glu), polyethyleneimine (PEI) or sequentially with Glu and PEI. These treatments may permit to stabilize the physically immobilized lipases by avoiding enzyme desorption via intermolecular crosslinking. Moreover, immobilizing a second enzyme on the lipase-glutaraldehyde-PEI has been proposed as a strategy without risks of PEI desorption by incubation in high ion strength solutions. The treatments altered the enzyme activity slightly but produced significant enzyme stabilization. This enzyme stabilization was more significant when using the highly loaded preparations, where intermolecular crosslinking was easier to obtain. SDS-PAGE analyses confirmed that crowded enzyme preparations were intermolecular crosslinked using Glu plus PEI, but some molecules still remained non-crosslinked. In general, PEI treatment was the most effective in increasing enzyme stability, while glutaraldehyde had a milder stabilization effect.


Assuntos
Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lipase/química , Lipase/metabolismo , Ascomicetos/enzimologia , Biotecnologia , Reagentes para Ligações Cruzadas/química , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glutaral/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Lipase/antagonistas & inibidores , Fosfolipases/antagonistas & inibidores , Fosfolipases/química , Fosfolipases/metabolismo , Polietilenoimina/química
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