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1.
J Enzyme Inhib Med Chem ; 34(1): 1474-1480, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31414611

RESUMO

The discovery of allosteric modulators is a multi-disciplinary approach, which is time- and cost-intensive. High-throughput screening combined with novel computational tools can reduce these factors. Thus, we developed an enzyme activity assay, which can be included in the drug discovery work-flow subsequent to the in-silico library screening. While the in-silico screening yields in the identification of potential allosteric modulators, the developed in-vitro assay allows for the characterisation of them. Candida rugosa lipase (CRL), a glyceride hydrolysing enzyme, has been selected for the pilot development. The assay conditions were adjusted to CRL's properties including pH, temperature and substrate specificity for two different substrates. The optimised assay conditions were validated and were used to characterise Tropolone, which was identified as an allosteric modulator. In conclusion, the assay is a reliable, reproducible, and robust tool, which can be streamlined with in-silico screening and incorporated in an automated high-throughput screening workflow.


Assuntos
Lipase/metabolismo , Miniaturização , Regulação Alostérica , Candida/enzimologia , Cristalografia por Raios X , Estabilidade Enzimática , Ensaios de Triagem em Larga Escala , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Limite de Detecção , Lipase/química , Reprodutibilidade dos Testes , Especificidade por Substrato , Temperatura Ambiente
2.
J Microbiol Biotechnol ; 29(7): 1043-1052, 2019 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-31353877

RESUMO

Active lipase-producing bacterium Burkholderia gladioli Bps-1 was rapidly isolated using a modified trypan blue and tetracycline, ampicillin (TB-TA) plate. The electro-phoretically pure enzyme was obtained by purification using ethanol precipitation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight was 34.6 kDa and the specific activity was determined to be 443.9 U/mg. The purified lipase showed the highest activity after hydrolysis with p-NPC16 at a pH of 8.5 and 50°C, and the Km, kcat, and kcat/Km values were 1.05, 292.95 s-1 and 279 s-1mM-1, respectively. The lipase was highly stable at 7.5 ≤ pH ≤ 10.0. K+ and Na+ exerted activation effects on the lipase which had favorable tolerance to short-chain alcohols with its residual enzyme activity being 110% after being maintained in 30% ethanol for 1 h. The results demonstrated that the lipase produced by the strain B. gladioli Bps-1 has high enzyme activity and is an alkaline lipase. The lipase has promising chemical properties for a range of applications in the food-processing and detergent industries, and has particularly high potential for use in the manufacture of biodiesel.


Assuntos
Burkholderia gladioli/enzimologia , Burkholderia gladioli/isolamento & purificação , Lipase/metabolismo , Biocatálise , Biocombustíveis , Burkholderia gladioli/crescimento & desenvolvimento , Burkholderia gladioli/metabolismo , Meios de Cultura , Detergentes , Estabilidade Enzimática , Etanol/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Lipase/química , Lipase/isolamento & purificação , Peso Molecular , Especificidade por Substrato , Temperatura Ambiente
3.
Food Chem ; 298: 125002, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31260958

RESUMO

Lipids are only minor wheat flour constituents but play major roles in bread making (BM). Here, the importance of a well-balanced lipid population in BM was studied by applying a lipase from Fusarium oxysporum in the process. Monogalactosyldiacylglycerols and N-acyl phosphatidylethanolamines were the most accessible lipase substrates. Hydrolysis thereof into their corresponding lysolipids was largely if not entirely responsible for loaf volume increases upon lipase application. Degradation of endogenously present lipids and enzymatically released lysolipids caused loaf volume to decrease, confirming that an appropriate balance between different types of lipids is crucial in BM. For optimal dough gas cell stability, the level of lipids promoting lamellar mesophases and, thus, liquid condensed monolayers needs to be maximal while maintaining an appropriate balance between lipids promoting hexagonal I phases, non-polar lipids and lipids promoting hexagonal II or cubic phases.


Assuntos
Pão , Farinha , Lipase/metabolismo , Lipídeos/química , Triticum , Fermentação , Fusarium/enzimologia , Galactolipídeos/química , Galactolipídeos/metabolismo , Hidrólise , Lipase/química , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Triticum/química , Triticum/metabolismo
4.
Nat Commun ; 10(1): 2375, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147543

RESUMO

Human antigen R (HuR) is a member of the Hu family of RNA-binding proteins and is involved in many physiological processes. Obesity, as a worldwide healthcare problem, has attracted more and more attention. To investigate the role of adipose HuR, we generate adipose-specific HuR knockout (HuRAKO) mice. As compared with control mice, HuRAKO mice show obesity when induced with a high-fat diet, along with insulin resistance, glucose intolerance, hypercholesterolemia and increased inflammation in adipose tissue. The obesity of HuRAKO mice is attributed to adipocyte hypertrophy in white adipose tissue due to decreased expression of adipose triglyceride lipase (ATGL). HuR positively regulates ATGL expression by promoting the mRNA stability and translation of ATGL. Consistently, the expression of HuR in adipose tissue is reduced in obese humans. This study suggests that adipose HuR may be a critical regulator of ATGL expression and lipolysis and thereby controls obesity and metabolic syndrome.


Assuntos
Tecido Adiposo Branco/metabolismo , Proteína Semelhante a ELAV 1/genética , Intolerância à Glucose/genética , Hipercolesterolemia/genética , Resistência à Insulina/genética , Lipase/genética , Obesidade/genética , Adipócitos/patologia , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/imunologia , Animais , Crescimento Celular , Dieta Hiperlipídica , Proteína Semelhante a ELAV 1/metabolismo , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Intolerância à Glucose/metabolismo , Humanos , Hipercolesterolemia/metabolismo , Hipertrofia , Inflamação/imunologia , Lipase/metabolismo , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA/genética , Gordura Subcutânea/metabolismo
5.
Nat Commun ; 10(1): 2756, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31227702

RESUMO

Flight loss in birds is as characteristic of the class Aves as flight itself. Although morphological and physiological differences are recognized in flight-degenerate bird species, their contributions to recurrent flight degeneration events across modern birds and underlying genetic mechanisms remain unclear. Here, in an analysis of 295 million nucleotides from 48 bird genomes, we identify two convergent sites causing amino acid changes in ATGLSer321Gly and ACOT7Ala197Val in flight-degenerate birds, which to our knowledge have not previously been implicated in loss of flight. Functional assays suggest that Ser321Gly reduces lipid hydrolytic ability of ATGL, and Ala197Val enhances acyl-CoA hydrolytic activity of ACOT7. Modeling simulations suggest a switch of main energy sources from lipids to carbohydrates in flight-degenerate birds. Our results thus suggest that physiological convergence plays an important role in flight degeneration, and anatomical convergence often invoked may not.


Assuntos
Evolução Biológica , Aves/fisiologia , Metabolismo Energético/genética , Voo Animal/fisiologia , Genoma/genética , Animais , Metabolismo dos Carboidratos/fisiologia , Genômica/métodos , Lipase/genética , Lipase/metabolismo , Lipólise/fisiologia , Palmitoil-CoA Hidrolase/genética , Palmitoil-CoA Hidrolase/metabolismo , Filogenia
6.
Food Chem ; 296: 1-8, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31202292

RESUMO

Immobilized lipases are excellent biocatalysts for the enzymatic synthesis of short- and medium-chain fatty esters used as food flavor compounds. Herein a new approach for a magnetic core-shell biocatalyst by immobilization of Candida antarctica B lipase is reported, coating single-core magnetic nanoparticles with an organic shell, preferably poly(benzofurane-co-arylacetic acid), followed by the covalent attachment of the enzyme and embedment of the primary biocatalyst in a silica layer. Although covalent and sol-gel immobilization were efficient on their own, their combination can ensure additional operational stability through multi-point linkages. Moreover, silanes holding glycidoxy groups, which can also form covalent linkages, have been successfully used as precursors for the silica coating layer. The structural, magnetic and morphological characteristics were assessed by TEM, SEM-EDX, X-ray photoelectron spectroscopy and vibrating sample magnetometry. The new biocatalysts demonstrated high catalytic efficiency in the solventless synthesis of isoamyl esters of natural carboxylic acids, also in multiple reaction cycles.


Assuntos
Ésteres/metabolismo , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Nanopartículas de Magnetita/química , Biocatálise , Ácidos Carboxílicos/química , Ácidos Carboxílicos/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Ésteres/análise , Proteínas Fúngicas/química , Cromatografia Gasosa-Espectrometria de Massas , Lipase/química , Dióxido de Silício/química
7.
Food Chem ; 297: 124925, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253266

RESUMO

A novel lipase gene (McLipB) was cloned from a thermophilic fungus Malbranchea cinnamomea and expressed in Pichia pastoris. The deduced amino acid sequence of the lipase (McLipB) shared the highest identity of 46% with the Candida rugosa lipase LIP4. The extracellular lipase activity of 4304 U/mL with protein concentration of 7.7 mg/mL was achieved in a 5-L fermentor. The optimal pH and temperature of McLipB were 7.5 and 40 °C, respectively. The lipase showed high specificity towards triglycerides with short and medium chain fatty acids, and had non-position specificity. McLipB hydrolyzed butter to produce mainly butyric acid, hexanoic acid and a small amount of octanoic acid and decanoic acid. Furthermore, it degraded more than 90% dipropyl phthalate, dibutyl phthalate and dihexyl phthalate to their corresponding monoalkyl phthalates. The properties of McLipB indicate that it has great application potential for production of lipolyzed milkfat flavor and biodegradation of phthalate esters.


Assuntos
Ascomicetos/enzimologia , Lipase/metabolismo , Sequência de Aminoácidos , Técnicas de Cultura Celular por Lotes , Candida/enzimologia , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/química , Lipase/genética , Ácidos Ftálicos/química , Ácidos Ftálicos/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Especificidade por Substrato , Temperatura Ambiente , Triglicerídeos/metabolismo
8.
Food Chem ; 297: 124988, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253285

RESUMO

Agaricus blazei, Auricularia fuscosuccinea and Pleurotus albidus mycelia were obtained in solid-state cultivation (SSC), using grains (brown rice, canjica corn and wheat) as raw material. Colonized grain flours were analysed for their nutritional, physical and physico-chemical characteristics and biological activity in vitro. Wheat flour with P. albidus showed higher values for protein (18.34 g/100 g), ergosterol (0.60 mg/g), mycelial biomass (183 mg/g) and total amino acids (58.34 mg/g). Corn flour with A. fuscosuccinea showed the highest total phenolic content (2.38 mg GAE/g), antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) (8.90 µmol TEAC/g) and 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (16.52 µmol TEAC/g) assay. Wheat flour with P. albidus were more effective at inhibiting of pancreatic lipase (74.5%) and of α-glucosidase (98.2%). In conclusion, grains colonized by macrofungi mycelia through SSC can enrich the nutritional value and the biological activity of the flours, which presents a potential for functional foods.


Assuntos
Agaricus/fisiologia , Farinha/análise , Valor Nutritivo , Pleurotus/fisiologia , Aminoácidos/química , Antioxidantes/química , Biomassa , Ergosterol/análise , Lipase/antagonistas & inibidores , Lipase/metabolismo , Oryza/metabolismo , Fenóis/análise , Triticum/metabolismo , Zea mays/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo
9.
Food Chem ; 297: 124955, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31253342

RESUMO

This study aimed to investigate the effect of hydrogen peroxide (H2O2) on membrane lipids metabolism and its relation to pulp breakdown development of longan fruit during postharvest storage. Compared to the control longans, H2O2-treated longans showed higher pulp breakdown index, cell membrane permeability, and activities of phospholipase D (PLD), lipase and lipoxygenase (LOX). Moreover, H2O2-treated longans maintained higher levels of pulp phosphatidic acid (PA) and saturated fatty acids (SFA). However, H2O2-treated longans exhibited lower levels of pulp phosphatidylcholine (PC), phosphatidylinositol (PI) and unsaturated fatty acids (USFA), lower index of unsaturated fatty acids (IUFA), and lower ratio of USFA to SFA (U/S). These findings demonstrated that H2O2 caused the increased activities of enzymes involving in membrane lipids degradation and the accelerated decompositions of membrane USFA and phospholipids in longan pulp, which eventually triggered the destruction of the pulp cell membrane structure and the development of pulp breakdown in longans during storage.


Assuntos
Enzimas/metabolismo , Frutas/química , Peróxido de Hidrogênio/farmacologia , Lipídeos de Membrana/metabolismo , Sapindaceae/química , Enzimas/química , Ácidos Graxos/análise , Ácidos Graxos/química , Armazenamento de Alimentos , Frutas/efeitos dos fármacos , Frutas/metabolismo , Peróxido de Hidrogênio/química , Lipase/química , Lipase/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoxigenase/química , Lipoxigenase/metabolismo , Lipídeos de Membrana/química , Fosfolipase D/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sapindaceae/efeitos dos fármacos , Sapindaceae/metabolismo
10.
Chem Commun (Camb) ; 55(45): 6417-6420, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-31094364

RESUMO

An AIE dual-reactive supramolecular probe has been devised for the first time to simultaneously measure endogenous lipase and α-amylase activity in a homogeneous system. Fluorescence quantitative analysis of lipase and α-amylase in real biological samples enables rapid and accurate diagnosis of diseases.


Assuntos
Lipase/análise , Sondas Moleculares/química , alfa-Amilases/análise , Fluorescência , Humanos , Lipase/metabolismo , Substâncias Macromoleculares/química , alfa-Amilases/metabolismo
11.
Int J Nanomedicine ; 14: 3235-3244, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31118633

RESUMO

Purpose: Here, we present the successful preparation of a highly efficient gallic acid resin grafted with magnetic nanoparticles (MNPs) and containing a branched brush polymeric shell. Methods: Using a convenient co-precipitation method, we prepared Fe3O4 nanoparticles stabilized by citric acid. These nanoparticles underwent further silica modification and amino functionalization followed by gallic acid functionalization on their surface. Under alkaline conditions, we used a condensation reaction that combined formaldehyde and gallic, to graft the gallic acid-formaldehyde resin on the surface. We then evaluated the polymer-grafted MNPs to assay the Candida Antarctica B lipase(Cal-B) immobilization via physical adsorption. Conclusion: Furthermore, during optimization of parameters that defined conditions of immobilization, we found that the optimum immobilization was achieved in 15 mins. Also, optimal immobilization temperature and pH were 38ºC and 7.5, respectively. In addition, the reusability study of immobilized lipase polymer-grafted MNPs was done by isolating the MNPs from the reaction medium using magnetic separation, which showed that grafted MNPs reached 5 cycles with 91% activity retention.


Assuntos
Enzimas Imobilizadas/metabolismo , Compostos Férricos/química , Proteínas Fúngicas/metabolismo , Ácido Gálico/química , Lipase/metabolismo , Nanopartículas de Magnetita/química , Resinas Sintéticas/química , Adsorção , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Nanopartículas de Magnetita/ultraestrutura , Polímeros , Dióxido de Silício/química , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura Ambiente , Termogravimetria , Fatores de Tempo , Difração de Raios X
12.
J Food Sci ; 84(6): 1331-1339, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31132153

RESUMO

Animal fat plays a key role in the structure, quality, and acceptability of emulsified meat products. However, a high consumption of saturated fat has been related to several health problems. Fat encapsulation with a nondigestible carbohydrate, such as pectin, may offer a promising alternative to reduce fat intake from a meat product, by preventing its digestion and absorption. The objective of this study was to develop a meat sausage with pectin-encapsulated-fat (PEF) to decrease its lipid digestibility, without compromising its acceptability. Pork fat particles encapsulation by emulsification with a 4% pectin solution, and also stability during meat processing and cooking, was confirmed by confocal microscopy. No changes (P > 0.05) compared to Control (C) were found on thermal stability and composition of sausages formulated with direct addition of pectin (T1) and with incorporation of PEF (T2). However, in comparison with C, pH, color, and texture of T1 and T2 samples were affected (P < 0.05). Nevertheless, these changes had no influence (P > 0.05) on sensory acceptability of treated samples, and actually improved (P < 0.05) their texture acceptance. In vitro digestive degradation of triacylglycerols was decreased (P < 0.05) by 20% on T2 samples compared to control and it was superior (P < 0.05) to T1 (8%). Confocal images confirmed lipid digestibility reduction of T2 samples. Incorporation of PEF in a meat sausage offers a better protection against the hydrolytic action of lipases over triaclyglycerides, than a direct addition of pectin, without affecting its sensory acceptability. Therefore, it can be a potential strategy to reduce fat intake from meat products. PRACTICAL APPLICATION: Reduction or replacement strategies tested to modify or decrease fat content in meat products usually leads to nondesirable sensory attributes. However, decreasing lipid digestibility by encapsulating animal fat with nondigestible pectin offers a new approach to reduce fat intake from full-animal-fat meat products, without affecting their sensory acceptability.


Assuntos
Comportamento do Consumidor , Emulsões/química , Gorduras/metabolismo , Manipulação de Alimentos/métodos , Produtos da Carne/análise , Pectinas , Animais , Cor , Culinária , Dieta com Restrição de Gorduras , Digestão , Humanos , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Carne , Produtos da Carne/normas , Carne Vermelha , Triglicerídeos/metabolismo
13.
J Microbiol Biotechnol ; 29(6): 944-951, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31091867

RESUMO

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of 80°C at pH 11.0. The optimum substrate for enzyme activity was C10, which is highly stable in methanol solvent. The enzyme was strongly activated by Ca2+, Mg2+, and strongly inhibited by Fe2+ and Zn2+. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.


Assuntos
Proteínas de Bactérias/genética , Burkholderia/enzimologia , Burkholderia/genética , Expressão Gênica , Lipase/genética , Chaperonas Moleculares/genética , Microbiologia do Solo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Ativadores de Enzimas , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solventes , Especificidade por Substrato , Tensoativos , Temperatura Ambiente
14.
Microb Pathog ; 132: 222-229, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059755

RESUMO

The emerging incidence of antibiotic resistance trait among the bacteria populating poultry presents a devastating public health issue. On the other hand, at present, diabetes and obesity are the most serious public health issues and are increasing subsequently at alarming rate. In view of this, the present in vitro context was aimed to investigate the antibacterial activities of Momordica charantia (M. charantia) fruits extracts against poultry associated Bacillus spp. and to assess further its phytoconstituents, alpha-(α)-glucosidase activities, and anti-obesity properties. The anti-pathogenic attributes of M. charantia fruit extracts were carried out using disc diffusion assay and results showed the pronounced antibacterial trait of ethanolic extract with maximum zone of inhibition of 28.3 ±â€¯1.2 mm against Bacillus licheniformis. The qualitative phytochemical analyses of fruit extracts illustrated the presence of diverse phytoconstituents. The α-glucosidase inhibition assay for the extracts was performed according to the α-glucosidase activity kit. The results depicted the lowest α-glucosidase activity (57.13 ±â€¯2.3 to 18.14 ±â€¯1.3 U/L) in the presence of ethanolic extract at varied concentrations. The anti-obesity potentialities of fruit extracts were demonstrated in terms of porcine pancreatic lipase (PPL type II) activity using p-nitro-phenyl butyrate (p-NPB) as a substrate. The ethanolic extract of M. charantia fruits was observed to exhibit maximum inhibition of pancreatic lipase ranging from 20.12 ±â€¯2.3 to 68.34 ±â€¯1.3% in a dose dependent manner with an IC50 value of 607.6 ±â€¯1.3 µg/mL. FTIR and GC-MS results indicated the presence of distinct compounds in the ethanol extract and major bioactive constituents were found to be Dimethyl sulfone (35.24%), 9-octadecanamide (20.52%), Pentadecanoic acid (6.64%), Lanost-9 (11)-en-18-oic acid, 23-(acetylxyl)-3-(4-bromobenzoyl) oxyl-20-hydroxyl-gamma-lactone (2.6%), and 2,2-sulfonyldiethanol (2.46%). In conclusion, M. charantia fruits could be of great concern in pharmaceutical industries due to its adequate biological properties and may also help in the management of poultry associated bacterial pathogens.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Frutas/química , Momordica charantia/química , Extratos Vegetais/farmacologia , Animais , Fármacos Antiobesidade/farmacologia , Bacillus/efeitos dos fármacos , Lipase/metabolismo , Testes de Sensibilidade Microbiana , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Extratos Vegetais/química , Aves Domésticas , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/microbiologia , Suínos , alfa-Glucosidases/metabolismo
15.
Chem Commun (Camb) ; 55(50): 7155-7158, 2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31134258

RESUMO

Herein, the first example of crackled organosilica nanocapsules (CONs) is reported to directly immobilize enzymes without any further chemical modification. Enzymes are adsorbed on both the exterior and interior surfaces of CONs, integrating the merits of adsorption and encapsulation. When used for Candida rugosa lipase (CRL) immobilization, the CONs displayed higher enzyme loading, lower enzyme leaching, and elevated enzyme activity, compared to the conventional non-crackled nanocapsules/particles.


Assuntos
Candida/enzimologia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lipase/metabolismo , Nanocápsulas/química , Compostos de Silício/química , Lipase/química
16.
Chemistry ; 25(43): 10156-10164, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31136019

RESUMO

A new protocol based on lipase-catalyzed tandem reaction toward α,ß-enones/enoesters is presented. For the synthesis of the desired products the tandem process based on enzyme-catalyzed hydrolysis and Knoevenagel reaction starting from enol acetates and aldehyde is developed. The relevant impact of the reaction conditions including organic solvent, enzyme type, and temperature on the course of the reaction was revealed. It was shown that controllable release of the active methylene compound from the corresponding enol carboxylate ensured by enzymatic reaction diminishes significantly the formation of the unwanted co-products. Furthermore, this protocol was extended by including a second tandem chemoenzymatic transformation engaging various aldehyde precursors. After a careful optimization of the reaction conditions, the target products were obtained with yields up to 86 % and with excellent E/Z-selectivity.


Assuntos
Aldeídos/metabolismo , Lipase/metabolismo , Aldeídos/química , Animais , Biocatálise , Hidrólise , Lipase/química , Pancrelipase/metabolismo , Pentanonas/química , Pentanonas/metabolismo , Estereoisomerismo , Suínos
17.
Mol Med Rep ; 19(6): 4673-4684, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30957185

RESUMO

Non­alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, and has high rates of morbidity and mortality worldwide. Daphnetin (DAP) possesses notable antioxidative, anti­inflammatory and anticoagulant activities; DAP is an active ingredient extracted from Daphne Koreana Nakai. To investigate the effects and the underlying mechanism of DAP on NAFLD, we treated HepG2 cells with oleic acid (OA) and DAP simultaneously and non­simultaneously. In the simultaneous treatment condition, HepG2 cells were co­treated with 0.5 mM OA and DAP (5, 20, and 50 µM) for 24 h. In the non­simultaneous treatment conditions, HepG2 cells were pretreated with 0.5 mM OA for 24 h, and then treated with DAP (5, 20 and 50 µM) for 24 h. Following the aforementioned treatments, the biochemical indexes associated with NAFLD were measured as follows: i) The intracellular contents of triglyceride (TG), reactive oxygen species (ROS) and fluorescent glucose 2­[N­(7­nitrobenz­2­oxa­1,3­diazol­4­yl) amino]­2­deoxyglucose were analyzed with corresponding detection kits; and ii) the cellular expression levels of glycolipid metabolism­ and oxidative stress­related genes, including 5'AMP­activated protein kinase (AMPK), sterol regulatory element­binding protein­1C (SREBP­1C), patatin­like phospholipase domain­containing protein 3 (PNPLA3), peroxisome proliferator­activated receptor α (PPARα), phosphoinositide 3­kinase (PI3K), protein kinase B (AKT), nuclear factor­like 2 (Nrf2), cytochrome P450 (CYP) 2E1 and CYP4A were determined by reverse transcription­quantitative polymerase chain reaction and western blotting. The results revealed the potential mechanism underlying the effects of DAP on NAFLD in vitro: i) By increasing the phosphorylation of AMPK, DAP inhibited the expression of SREBP­1C and PNPLA3, and induced that of PPARα. Lipid accumulation within hepatocytes was reduced; ii) by upregulating PI3K expression and pAKT/AKT levels, DAP may alleviate insulin resistance and promote hepatocellular glucose uptake; and iii) by upregulating the expression of Nrf2, DAP downregulated the expression of CYP2E1 and CYP4A, and the levels of reactive oxygen species in hepatocytes.


Assuntos
Resistência à Insulina , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Oleico/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Umbeliferonas/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP4A/genética , Citocromo P-450 CYP4A/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipase/antagonistas & inibidores , Lipase/genética , Lipase/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , PPAR alfa/genética , PPAR alfa/metabolismo , Fosfatidilinositol 3-Quinase/genética , Fosfatidilinositol 3-Quinase/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismo
18.
Prep Biochem Biotechnol ; 49(7): 727-734, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31017519

RESUMO

The accurate estimation of kinetic parameters is of fundamental importance for biochemical studies for research and industry. In this paper, we demonstrate the application of a modular microfluidic system for execution of enzyme assays that allow determining the kinetic parameters of the enzymatic reactions such as Vmax - the maximum rate of reaction and KM - the Michaelis constant. For experiments, the fluorogenic carbonate as a probe for a rapid determination of the kinetic parameters of hydrolases, such as lipases and esterases, was used. The microfluidic system together with the method described yields the kinetic constants calculated from the concentration of enzymatic product changes via a Michaelis-Menten model using the Lambert function W(x). This modular microfluidic system was validated on three selected enzymes (hydrolases).


Assuntos
Ensaios Enzimáticos/instrumentação , Esterases/metabolismo , Dispositivos Lab-On-A-Chip , Lipase/metabolismo , Carbonatos/análise , Carbonatos/metabolismo , Desenho de Equipamento , Esterases/análise , Fluorescência , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Cinética , Lipase/análise
19.
Nanoscale ; 11(17): 8226-8236, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30973556

RESUMO

Carbon dots (CDs) have recently garnered significant attention owing to their excellent luminescence properties, thereby demonstrating a variety of applications in in vitro and in vivo imaging. Understanding the long-term metabolic fate of these agents in a biological environment is the focus of this work. Here we show that the CDs undergo peroxide catalysed degradation in the presence of lipase. Our results indicate that differently charged CD species exhibit unique degradation kinetics upon being subjected to enzyme oxidation. Furthermore, this decomposition correlates with the relative accessibility of the enzymatic molecule. Using multiple physico-chemical characterization studies and molecular modelling, we confirmed the interaction of passivating surface abundant molecules with the enzyme. Finally, we have identified hydroxymethyl furfural as a metabolic by-product of the CDs used here. Our results indicate the possibility and a likely mechanism for complete CD degradation in living systems that can pave the way for a variety of biomedical applications.


Assuntos
Carbono/química , Enzimas/metabolismo , Pontos Quânticos/química , Animais , Biocatálise , Feminino , Peróxido de Hidrogênio/química , Lipase/metabolismo , Camundongos , Camundongos Nus , Oxirredução , Espectroscopia Fotoeletrônica , Polietilenoimina/química , Pontos Quânticos/metabolismo
20.
Enzyme Microb Technol ; 126: 18-23, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000160

RESUMO

A solvent-mediated method (SMM) was used to prepare supersaturated sugar solutions in hydrophobic and mixture of hydrophilic/hydrophobic ionic liquids (ILs), namely, [Bmim][Tf2N] and [Bmim][TfO]/[Bmim][Tf2N], respectively. In this method, sugars were first solubilized in a mixture of organic solvent and water (i.e. methanol:water, 1:1 v/v), and then added to [Bmim][Tf2N] and/or [Bmim][TfO]/[Bmim][Tf2N] mixture. Supersaturated sugar solution in ILs were obtained by removing organic solvents and water under vacuum evaporation. Sugar solubilities in ILs, especially in hydrophobic IL ([Bmim][Tf2N]) and in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM were greater than in ILs prepared using water-mediated method (WMM), which suggested methanol aided sugar solvation in hydrophobic media. In addition, interactions between glucose molecules and between glucose and methanol, water, and IL were investigated by all-atom molecular dynamics (MD) simulation. The MD simulation results showed that initial water and water/methanol molecules around glucose were gradually replaced by IL anions. Notably, SMM resulted in stronger interaction between IL anions and glucose than WMM, which was attributed to greater solubility of sugar in ILs prepared by SMM. Resultantly, the productivity of lipase-catalyzed production of glucose laurate using supersaturated glucose solution in [Bmim][TfO]/[Bmim][Tf2N] mixture prepared by SMM was at least 1.76-fold greater than that obtained in IL mixture prepared by WMM.


Assuntos
Ésteres/síntese química , Ácidos Graxos/síntese química , Proteínas Fúngicas/metabolismo , Glucose/química , Líquidos Iônicos/química , Lipase/metabolismo , Catálise , Esterificação , Proteínas Fúngicas/química , Interações Hidrofóbicas e Hidrofílicas , Lipase/química , Simulação de Dinâmica Molecular , Solubilidade
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