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1.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1386-1394, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748596

RESUMO

We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.


Assuntos
Sistemas CRISPR-Cas , Lipólise , Perilipina-1 , Células 3T3-L1 , Adipócitos/metabolismo , Animais , Técnicas de Inativação de Genes , Lipase/metabolismo , Lipólise/genética , Camundongos , Perilipina-1/genética , Perilipina-1/metabolismo
2.
Food Chem ; 333: 127528, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32682231

RESUMO

Endogenous lipase and lipoxygenase play important roles in accelerating lipid oxidation. Polyphenols are a series of commonly used chemicals for preserving fish and seafood products, due to their positive inhibitory effects on lipid oxidation. However, the mechanism involved is still unknown. The inhibitory effects of chlorogenic acid (CGA) on lipase and lipoxygenase were investigated and explored with multi- spectroscopic and molecular docking approaches. Results showed that CGA could inhibit the activities of lipase and lipoxygenase with concentration increased in a highly dose-dependent manner. CGA quenched intrinsic fluorescence intensities of enzymes by static quenching and binding with CGA which led to changes in 3D structures of enzymes. Results of the molecular docking confirmed binding modes, binding sites and major interaction forces between CGA and enzymes, which reduced the corresponding activity. Thus, this study could provide basic mechanisms of the inhibitory effects of polyphenols on lipid oxidation during food preservation.


Assuntos
Ácido Clorogênico/metabolismo , Ácido Clorogênico/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Inibidores de Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/farmacologia , Simulação de Acoplamento Molecular , Animais , Sítios de Ligação , Conservação de Alimentos , Lipase/antagonistas & inibidores , Lipase/química , Lipase/metabolismo , Lipoxigenase/química , Lipoxigenase/metabolismo , Oxirredução/efeitos dos fármacos , Polifenóis/farmacologia , Espectrometria de Fluorescência
3.
PLoS Pathog ; 16(6): e1008554, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32542055

RESUMO

Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/ß hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis.


Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Hepacivirus/fisiologia , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise , Montagem de Vírus/fisiologia , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , Linhagem Celular Tumoral , Ativação Enzimática , Células HEK293 , Humanos , Lipase/genética , Gotículas Lipídicas/virologia , Triglicerídeos/genética , Triglicerídeos/metabolismo
4.
Food Chem ; 330: 127225, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32569931

RESUMO

The whole genome of Streptomyces violascens (=ATCC 27968) was sequenced and the cloning and expression of OUC-Lipase 6 were conducted in Bacillus subtilis WB800. The recombinant enzyme belongs to the lipolytic enzymes family V. OUC-Lipase 6 showed optimal activity at 30 °C and pH 9.0, and retained 90.2% of its activity in an alkaline buffer (pH 8.0, 30 °C and 96 h). OUC-Lipase 6 showed good stability under medium temperature conditions (residual activity of 68.8%, pH 8.0, 45 °C and 96 h). OUC-Lipase 6 could selectively hydrolyze fatty acids on the glyceride backbone, thus improving the contents of DHA and EPA in codfish oil. OUC-Lipase 6 also showed regioselectivity, resulting in a better enrichment efficiency for EPA than DHA. After hydrolyzing for 36 h via OUC-Lipase 6, the contents of EPA and DHA were improved to 3.24-fold and 1.98-fold, respectively.


Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Lipase/metabolismo , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Genoma Bacteriano , Glicerídeos/química , Hidrólise , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismo , Especificidade por Substrato
5.
Arch Biochem Biophys ; 689: 108475, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585312

RESUMO

The unconventional yeast Yarrowia lipolytica is known as a producer of extracellular lipases. Here we overexpressed extracellular lipase (YlLip2) in yeast strain Y. lipolytica AJD ΔXΔA-Lip2 harboring the overexpression cassette of the YALI0A20350 gene under the strong hybrid promoter UAS1B16-TEF. To maintain a high level of YlLip2 production, two extracellular proteases of Y. lipolytica, AEPp and AXPp, were deleted. The purified recombinant YlLip2 presented optimal catalytic activities at 37 °C and pH 8.0. The effect of two lipopeptide biosurfactants, i.e., amphisin produced by Pseudomonas fluorescens DSS73 and viscosinamide secreted by P. fluorescens DR54, on the conformation and activity of YlLip2 was evaluated using spectral methods, surface tension, and the enzyme activity assay. YlLip2 demonstrated high tolerance of the tested biosurfactants and had greater activity retention after incubation with both biosurfactants. Finally, we observed that intrinsic fluorescence intensity of YlLip2 decreased significantly with increasing lipopeptides concentration ranging from 2.5 to 60 µM. Our results showed that both biosurfactants improve enzymatic activity of YlLip2 and might suggest better interaction of the substrate with the active site. These favorable characteristics make YlLip2 a prospective additive in the pharmaceutical, food, cosmetic, and detergent industries.


Assuntos
Lipase/metabolismo , Lipopeptídeos/metabolismo , Yarrowia/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lipase/genética , Pseudomonas fluorescens/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Regulação para Cima , Yarrowia/genética , Yarrowia/metabolismo
6.
Int J Nanomedicine ; 15: 2935-2945, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425525

RESUMO

Background: New anticancer agents that rely on natural/healthy, not synthetic/toxic, components are very much needed. Methods: Ricinoleyl hydroxamic acid (RHA) was synthesized from castor oil and hydroxylamine using Lipozyme TL IM as a catalyst. To optimize the conversion, the effects of the following parameters were investigated: type of organic solvent, period of reaction, amount of enzyme, the molar ratio of reactants and temperature. The highest conversion was obtained when the reaction was carried out under the following conditions: hexane as a solvent; reaction period of 48 hours; 120 mg of Lipozyme TL IM/3 mmol oil; HA-oil ratio of 19 mmol HA/3 mmol oil; and temperature of 40°C. The cytotoxicity of the synthesized RHA was assessed using human dermal fibroblasts (HDF), and its application towards fighting cancer was assessed using melanoma and glioblastoma cancer cells over a duration of 24 and 48 hours. Results: RHA was successfully synthesized  and it demonstrated strong anticancer activity against glioblastoma and melanoma cells at as low as a 1 µg/mL concentration while it did not demonstrate any toxicity against HDF cells. Conclusion: This is the first report on the synthesis of RHA with great potential to be used as a new anticancer agent.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Óleo de Rícino/química , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Catálise , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Hexanos/química , Humanos , Hidroxilamina/química , Lipase/química , Lipase/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Solventes/química
7.
Food Chem ; 327: 127061, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454271

RESUMO

This study mainly investigated the effect of different salt concentrations (1, 3, or 5%) on triglycerides (TG) hydrolysis in muscle during salting by analyzing moisture distribution, TG hydrolysis, TG hydrolase activity, native and phosphorylated adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein content, lipid droplets morphology, and muscle microstructure. The results showed that increasing salt concentration could significantly decrease T21 moisture proportion and relaxation time (p < 0.05), which was more beneficial to the lipase activity. The TG hydrolase activity increased first and then decreased with the salt concentration increasing during dry-salting process, and 3% salt concentration was the point of inflection. Western blot (WB) analysis detected both ATGL, HSL and their phosphorylated proteins, which were increased with the salt content increase. The microstructure analysis showed that the lipid droplets were split into small lipid droplets with the increase of salt content, which was more conducive to the triglycerides hydrolysis.


Assuntos
Tecido Adiposo/metabolismo , Músculos Isquiossurais/metabolismo , Lipase/metabolismo , Gotículas Lipídicas/metabolismo , Cloreto de Sódio/farmacologia , Esterol Esterase/metabolismo , Triglicerídeos/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Músculos Isquiossurais/efeitos dos fármacos , Hidrólise , Gotículas Lipídicas/efeitos dos fármacos , Fosforilação , Suínos
8.
Food Chem ; 326: 126785, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32438224

RESUMO

Obesity and oxidative damage are two important risk factors associated closely with metabolic syndrome. Utilization of functional food ingredients is considered as a feasible way to tackle these challenges. In the present study, eight representative species of citrus peel extracts (CPEs) were evaluated and compared for their flavonoid profiles, antioxidant activities, and pancreatic lipase (PL) inhibitory capacities and mechanisms. Results indicated that hesperidin, naringin, neohesperidin, narirutin and eriocitrin were the five major flavonoids in CPEs, among which hesperidin was the main active PL inhibitor. Moreover, hesperidin could interact with PL by hydrogen bonds and van der Waals forces, and the interaction would not obviously change the secondary structure of PL. Overall, ponkan peel extract, having the strongest overall antioxidant activity, the highest content of hesperidin and total phenolic compounds among all tested CPEs, is a promising natural ingredient to scavenge free radicals and manage obesity.


Assuntos
Antioxidantes/farmacologia , Citrus/química , Flavonoides/química , Lipase/metabolismo , Pâncreas/enzimologia , Extratos Vegetais/química , Animais , Fenóis/química , Suínos
9.
Food Chem ; 326: 126895, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32438227

RESUMO

This investigation reports the effect of droplet size behavior on the overall lipolysis profile and molecular lipolysis mechanisms under in vitro gastric conditions. O/W emulsions (5% triolein, 1% sodium taurodeoxycholate) with different initial droplet sizes (fine: 0.58 µm; medium: 1.82 µm; and large: 4.00 µm) were subjected to static in vitro digestion. For the first time, multiple lipolysis products including diolein and monoolein regioisomers were quantified within a single HPLC run. An inverse relation was found between the droplet size and the initial rate and final extent of lipolysis based on the digested triolein. Furthermore, a mechanistic gastric lipolysis model was established based on a reaction scheme including enzymatic and chemical isomerization conversions. The estimated rate of the sn-1/3 hydrolysis was around two- to thirty-fold faster compared to the rates of sn-2 cleavage and isomerization, respectively. These findings resulted in a profound insight in in vitro gastric molecular lipolysis mechanisms.


Assuntos
Lipase/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Estômago/química , Animais , Digestão , Emulsões/química , Hidrólise , Lipólise , Tamanho da Partícula , Coelhos
10.
Int J Food Microbiol ; 327: 108654, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32416377

RESUMO

A soup stock made from katsuobushi is an important element of, and the basic seasoning responsible for the taste of, traditional Japanese cuisine. Fermented and ripened katsuobushi, called karebushi, is manufactured via a repeated molding process on the katsuobushi surface. Our aim was to characterize the surface Aspergillus community and their enzymes involved in the fermentation and ripening. Five dominant Aspergillus species isolated from the karebushi surface were identified-A. amstelodami, A. chevalieri, A. pseudoglaucus, A. ruber, and A. sydowii. Analyses were performed on final molding stage-samples from different manufacturers, and 1st to 4th molding stage-samples from the same manufacturer. The composition ratios of the five Aspergillus spp. varied according to the manufacturer of the karebushi. A. amstelodami and A. chevalieri tended to be detected as dominant species when the water content of the karebushi fillet was >15% and the fat content was >3.5%, respectively. In samples from a given manufacturer, the dominant species in the final molding stage tended to be A. chevalieri and A. pseudoglaucus. Mixed molds were cultured by solid-state fermentation using katsuobushi powder medium at two different water activity (aw) levels. Crude extracts from each culture showed lipase, aminopeptidase, carboxypeptidase, and protease activities. Notably, the crude extracts cultivated at 0.85 aw showed higher protease activity toward hemoglobin and lipase activity toward p-nitrophenyl palmitate than those at 0.95 aw. These hydrolytic enzymes are probably involved in decolorization of katsuobushi and lipid degradation during the long fermentative and ripening period. In addition, mixed cultures could transform 2,6-dimethoxyphenol into 1,2,3-trimethoxybenzene, previously reported as an attractive and mild flavor component. Our results may help promote the use of desirable Aspergillus spp. as starter cultures for manufacturers to stabilize and improve the quality of fermented and ripened karebushi.


Assuntos
Aspergillus/fisiologia , Fermentação , Alimentos e Bebidas Fermentados/microbiologia , Produtos Pesqueiros/microbiologia , Microbiologia de Alimentos , Animais , Produtos Pesqueiros/normas , Hidrólise , Lipase/metabolismo
11.
Med Sci Monit ; 26: e920684, 2020 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-32283546

RESUMO

BACKGROUND Acute pancreatitis (AP) is a symptom of sudden pancreas inflammation, which causes patients severe suffering. In general, fibroblast growth factor (FGF) levels are increased and amylase and lipase activities are elevated during AP pathogenesis, but protein concentration are low. However, the mechanism through which FGF signaling regulates AP pathogenesis remains elusive. MATERIAL AND METHODS The concentrations of PGE2, TNF-alpha, sCRP, FGF1, and FGF2 in the serum samples of the AP group and healthy control group were detected by enzyme-linked immunosorbent assay. In addition, IkappaBalpha and p-IkappaBalpha levels were analyzed in the serum samples. Subsequently, the AP rat model was established, and FGF1, FGF2, anti-FGF1, and anti-FGF2 antibodies and Bay11-7082 were injected into AP rats. TNF-alpha, PAI-1 JNK, p-JNK, IkappaBalpha, and p-IkappaBalpha levels were also examined. RESULTS Results showed that levels of PGE2, TNF-alpha, sCRP, p-IkappaBalpha, FGF1, and FGF2, as well as amylase and lipase activity were increased in patients with AP compared with those in healthy people. In addition, protein concentrations were lower in patients with AP than in the healthy group. Activation of FGF signaling by injecting FGF1 or FGF2 also inhibited AP-induced inflammation response in the pancreas and increased amylase and lipase activities, as well as protein concentration. However, the injection of FGF1 and FGF2 antibodies accelerated AP-mediated inflammation responses in the serum. In addition, Bay11-7082 injection inhibited AP activation of inflammation response and amylase and lipase activities. Protein concentration were also increased in AP rats. CONCLUSIONS FGF signaling protects against AP-mediated damage by inhibition of AP-activating inflammatory responses.


Assuntos
Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Inflamação/metabolismo , Pancreatite/patologia , Transdução de Sinais , Doença Aguda , Adulto , Amilases/metabolismo , Animais , Proteína C-Reativa/análise , Estudos de Casos e Controles , Dinoprostona/sangue , Feminino , Fator 1 de Crescimento de Fibroblastos/sangue , Fator 2 de Crescimento de Fibroblastos/sangue , Humanos , Inflamação/patologia , Lipase/metabolismo , Masculino , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa/sangue , Nitrilos/farmacologia , Ratos , Ratos Sprague-Dawley , Sulfonas/farmacologia , Fator de Necrose Tumoral alfa/sangue
12.
PLoS One ; 15(4): e0231177, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32271820

RESUMO

Enrichment of omega-3 fatty acids (É·-3 FAs) in natural oils is important to realize their health benefits. Lipases are promising catalysts to perform this enrichment, however, fatty acid specificity of lipases is poor. We attempted to improve the fatty acid selectivity of a lipase from Geobacillus thermoleovorans (GTL) by two approaches. In a semi-rational approach, amino acid positions critical for binding were identified by docking the substrate to the GTL and best substitutes at these positions were identified by site saturation mutagenesis followed by screening to obtain a variant of GTL (CM-GTL). In the second approach based on rational design, a variant of GTL was designed (DM-GTL) wherein the active site was narrowed by incorporating two heavier amino acids in the lining of acyl-binding pocket to hinder access to bulky É·-3 FAs. The affinities DM-GTL with designed substrates were evaluated in silico. Both, CM-GTL and DM-GTL have shown excellent ability to discriminate against the É·-3 FAs during hydrolysis of oils. Engineering the binding pocket of an enzyme of a complex substrate, such as a triglyceride, by incorporating the information on substrate structure and computationally derived binding modes, has resulted in designing two efficient lipase variants with improved substrate selectivity.


Assuntos
Ácidos Graxos Ômega-3/metabolismo , Geobacillus/enzimologia , Lipase/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Aminoácidos/metabolismo , Domínio Catalítico , Simulação por Computador , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/química , Lipase/isolamento & purificação , Metilação , Modelos Moleculares , Proteínas Mutantes/análise , Proteínas Mutantes/química , Mutação/genética , Dobramento de Proteína , Especificidade por Substrato , Temperatura
13.
PLoS One ; 15(4): e0224853, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298262

RESUMO

Diets rich in flavonoids have been related with low obesity rates, which could be related with their potential to inhibit pancreatic lipase, the main enzyme of fat assimilation. Some flavonoids can aggregate in aqueous medium suggesting that the inhibition mechanism could occur on both molecular and colloidal levels. This study investigates the interaction of two flavonoid aggregates, quercetin and epigallocatechin gallate (EGCG), with pancreatic lipase under simplified intestinal conditions. The stability and the morphology of these flavonoid aggregates were studied in four different solutions: Control (water), salt, low lipase concentration and high lipase concentration. Particles were found by optical microscopy in almost all the solutions tested, except EGCG-control. The results show that the precipitation rate decreases for quercetin and increases for EGCG in salt solution and that lipase stabilize quercetin aggregates. In addition, both flavonoids were shown to precipitate together with pancreatic lipase resulting in a sequestering of the enzyme.


Assuntos
Antioxidantes/farmacologia , Catequina/análogos & derivados , Mucosa Intestinal/metabolismo , Lipase/metabolismo , Quercetina/farmacologia , Animais , Antioxidantes/metabolismo , Catequina/metabolismo , Catequina/farmacologia , Dimerização , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/enzimologia , Lipase/antagonistas & inibidores , Quercetina/metabolismo , Suínos
14.
Food Chem ; 322: 126738, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32283361

RESUMO

Quality characteristics of whole wheat flour with superheated steam treatment and storage stability of semi-dried whole wheat noodle were investigated, as well as texture and cooking properties of cooking noodles. Superheated steam treatment significantly reduced the total plate count and the activities of lipase, lipoxygenase and polyphenol oxidase in whole wheat flour (p < 0.05), and decreased the free phenolic content. When treated at 155-170 °C, the thermomechanical properties of whole wheat dough improved with the increase of the development time, stability and C2 torque. Superheated steam treatment showed no obviously detrimental effects on hardness and cooking loss of noodle. During storage, treated semi-dried whole wheat noodle exhibited slower rate of microbial growth. Superheated steam treatment significantly reduced the acidity, inhibited the formation of free fatty acid, and decreased the activities of enzyme during storage. This would delay the deterioration and improve the storage stability of semi-dried whole wheat noodle.


Assuntos
Culinária , Farinha/análise , Armazenamento de Alimentos , Triticum/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Dureza , Temperatura Alta , Concentração de Íons de Hidrogênio , Lipase/metabolismo , Lipoxigenase/metabolismo , Fenol/análise , Paladar
15.
Food Chem ; 318: 126482, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32145543

RESUMO

We have examined the trans-resveratrol/lipase interaction by quantitative and qualitative analyses of fluorescence spectra, molecular docking and quantum-chemical calculations at DFT level. Interactions of CpLIP2 from C. parapsilosis CBS 604 and trans-resveratrol were confirmed with a major contribution of tryptophan residues to fluorescence quenching. A thermodynamic study across a wide temperature range was consistent with the presence of a single binding site with a binding free energy of -24 kJ/mol. Nevertheless, trans-resveratrol competitively inhibited CpLIP2 activity. Molecular docking and quantum-chemical calculations were consistent with a strong binding of trans-resveratrol to the CpLIP2 catalytic site via electrostatic and hydrophobic forces. The structural analysis quantitatively revealed an energy transfer from W51 and W350 to trans-resveratrol with a distance of 32 Å. Precise understanding of trans-resveratrol/CpLIP2 interactions has important implications on lipases for screening of stilbenoid.


Assuntos
Candida parapsilosis/enzimologia , Lipase/metabolismo , Resveratrol/metabolismo , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Fluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Lipase/antagonistas & inibidores , Lipase/química , Simulação de Acoplamento Molecular , Resveratrol/química , Resveratrol/farmacocinética , Termodinâmica
16.
Arch Microbiol ; 202(6): 1497-1506, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32219482

RESUMO

Lipases with high tolerance to temperature play a significant role in industry from food manufacturing to waste management systems. Thus, there is a need to investigate these enzymes from different geographical areas to look out for a more thermo-stable one. Characterization of lipases through experimental approaches is time consuming process and sometimes the results are ambiguous due to errors. However, integration of computational technologies is quite useful for prediction of optimized conditions. Such technologies can be applied as synthetic biology, which has many major applications in engineered biological approaches for accurate prediction of effects of different physical and chemical parameters on the system. In this study, cloning and expression of a lipase gene from Bacillus amyloliquefaciens, isolated from a novel geographical region of Pakistan, in Escherichia coli DH5α cells followed by sequencing was carried out. To isolate thermostable lipase producing strains, all the samples were kept at 50 °C. Genomic DNA was isolated and signal peptide (1-32 residues) sequence was chopped (ΔSPLipase). The ΔSPLipase was amplified and expressed in Linearized p15TV-L vector. The purified lipase appeared as single band of approximately 26 kDa. Suitable conditions of factors required for maximum lipase activity such as temperature, pH, substrate, organic solvent, detergents and metal ions were predicted through synthetic biology approach and further confirmed in wet lab. The predicted suitable factors for enzyme were almost similar to those determined experimentally. The optimum enzyme activity was recorded at pH 8 and 50 °C temperature. Interestingly, the activity of enzyme was found on a number of solvents, metal ions, detergents, and surfactants. The predicted optimum values and their experimental confirmations highlights the importance of integrated synthetic biology approaches in wet lab experiments. The characterized lipase of B. amyloliquefaciens at molecular level from Pakistani strains displayed good activity on a range of factors that implies this strain to be used for application in industrial level production.


Assuntos
Bacillus amyloliquefaciens/enzimologia , Bacillus amyloliquefaciens/metabolismo , Proteínas de Bactérias/metabolismo , Lipase/metabolismo , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/isolamento & purificação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/genética , Paquistão , Solventes , Especificidade por Substrato , Biologia Sintética , Temperatura
17.
Food Chem ; 319: 126533, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32172046

RESUMO

Lipase-catalyzed acylation of a hydrophilic tripeptide-KHA (TP-KHA; amino acid sequence Lys-His-Ala) with a lipophilic lauric acid was performed to produce a multi-functional compound, lauroyl tripeptide-KHA (TPL-KHA), with surface, antibacterial, and antioxidant activities. The significant acylation reaction parameters were optimized as follows: organic solvent of 2-methyl-2-butanol, reaction temperature at 55 °C, substrate molar ratio (lauric acid:TP-KHA) of 4.0, and reaction time for 72 h. Structural analyses by LC-ESI-MS and 1H NMR identified that Nε-lauroyl tripeptide-KHA was chemo-selectively synthesized by the acylation reaction under the optimum conditions. TPL-KHA showed the surface activity at the air-water interface with critical micelle concentration (CMC) of 2.71 mM and γCMC of 30.44 mN/m. TPL-KHA exhibited bacteriostatic and bactericidal effects on Gram-positive and Gram-negative foodborne pathogens (minimum inhibitory concentrations: 2.83-4.00 mM, minimum bactericidal concentrations: 3.17-5.83 mM). Moreover, it was demonstrated that TPL-KHA had the ability to scavenge ABTS+ radicals and inhibit the lipid oxidation.


Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Ácidos Láuricos/química , Lipase/metabolismo , Acilação , Antibacterianos/química , Antioxidantes/metabolismo , Biocatálise , Solventes/química , Água/química
18.
Genes Dev ; 34(7-8): 495-510, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32139423

RESUMO

Obesity-induced diabetes affects >400 million people worldwide. Uncontrolled lipolysis (free fatty acid release from adipocytes) can contribute to diabetes and obesity. To identify future therapeutic avenues targeting this pathway, we performed a high-throughput screen and identified the extracellular-regulated kinase 3 (ERK3) as a hit. We demonstrated that ß-adrenergic stimulation stabilizes ERK3, leading to the formation of a complex with the cofactor MAP kinase-activated protein kinase 5 (MK5), thereby driving lipolysis. Mechanistically, we identified a downstream target of the ERK3/MK5 pathway, the transcription factor FOXO1, which promotes the expression of the major lipolytic enzyme ATGL. Finally, we provide evidence that targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, but elevates energy dissipation, promoting lean phenotype and ameliorating diabetes. Thus, ERK3/MK5 represents a previously unrecognized signaling axis in adipose tissue and an attractive target for future therapies aiming to combat obesity-induced diabetes.


Assuntos
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Metabolismo Energético/genética , Lipólise/genética , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Obesidade/complicações , Células 3T3 , Tecido Adiposo/enzimologia , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Proteína Forkhead Box O1/metabolismo , Deleção de Genes , Células HEK293 , Humanos , Hipoglicemiantes/uso terapêutico , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipase/genética , Lipase/metabolismo , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética
19.
Nature ; 579(7798): 279-283, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32132708

RESUMO

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.


Assuntos
Glucagon/farmacologia , Gluconeogênese/efeitos dos fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fígado/efeitos dos fármacos , Acetilcoenzima A/metabolismo , Tecido Adiposo/efeitos dos fármacos , Animais , Diabetes Mellitus Tipo 2/fisiopatologia , Ativação Enzimática/efeitos dos fármacos , Glucagon/sangue , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipase/metabolismo , Lipólise/efeitos dos fármacos , Lipólise/genética , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Oxirredução/efeitos dos fármacos
20.
J Biosci Bioeng ; 129(6): 679-686, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32089433

RESUMO

Nanoflowers are recently developed flower-shaped nanoparticles consisting of several layers of petals to improve surface reaction and stability. Among them organic-inorganic hybrid nanoflowers have been spotlighted, since they can overcome enzyme-related limitations, including low stability, high production cost, substrate/product inhibition and difficult recovery. In this study, triple enzyme-inorganic hybrid nanoflowers (TrpE@ihNFs) were synthesized using a mixture of three enzymes (α-amylase, lipase and protease) and Cu2+ ions. TrpE@ihNFs were characterized by their morphology and chemical point of view by using different techniques including SEM, FTIR, EDX, and XRD. Afterwards we compared the enzyme activity and stability of TrpE@ihNFs with the free enzymes, including lipase, amylase and protease at the different pH and temperatures spectrophotometrically. Our data reveal that enzyme activities and stability of TrpE@ihNFs were significantly higher compared to the each free enzyme. In conclusion, we showed TrpE@ihNFs which can be used for the treatment of wastewater, biosensors, biocatalysts, and bio-related devices in the future.


Assuntos
Endopeptidases/metabolismo , Lipase/metabolismo , Nanopartículas , alfa-Amilases/metabolismo , Concentração de Íons de Hidrogênio , Lipase/química , Pâncreas , Temperatura
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