RESUMO
Umami peptides are valuable taste substances due to their exceptional taste and beneficial properties. In this study, purification of fermented goose bone broth was performed using continuous chromatography and sensory analysis, and after identification through nano-LC-MS/MS, four umami peptides were screened out by umami activity prediction and molecular docking, which are VGYDAE, GATGRDGAR, GETGEAGER, and GETGEAGERG derived from collagen. Sensory analysis indicated that they were also umami-enhancing, with thresholds ranging from 0.41 to 1.15 mmol/L, among which GER9 was the best. Combining the results of docking and molecular dynamics simulation, it was known that hydrogen bond and electrostatic interactions were vital in driving the umami formation. Moreover, Glu, Ser, and Asp of umami receptor T1R1/T1R3 were the key residues for the binding between four umami peptides and T1R1/T1R3. These findings provide novel insights into the high-value utilization of goose bones and offer profound theoretical guidance for understanding the umami mechanism.
Assuntos
Gansos , Receptores Acoplados a Proteínas G , Animais , Simulação de Acoplamento Molecular , Receptores Acoplados a Proteínas G/metabolismo , Gansos/metabolismo , Simulação de Dinâmica Molecular , Paladar , Espectrometria de Massas em Tandem , Peptídeos/químicaRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are stable carcinogens that are widely distributed in the environment and food, and humans are exposed to PAHs primarily through the respiratory tracts, dermal contact, and dietary intake. Meat products are an essential part of the human diet, and the formation of PAHs during meat processing is unavoidable. Therefore, a comprehensive understanding of PAHs in meat products can be a contribution to the minimization of human exposure dose. The aim of this review is to provide a comprehensive description of the toxicological analysis of PAHs intake and the various production pathways. The distribution of different PAHs in various meat products, including poultry and aquatic products, is analyzed. The discussion focuses on controlling PAHs through the use of endogenous marinades and antioxidants as well as reducing exogenous particulate matter-PAHs attachment. In addition, potential strategies for PAHs reduction and possible directions for future research are proposed.
Assuntos
Produtos da Carne , Hidrocarbonetos Policíclicos Aromáticos , Humanos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/análise , Contaminação de Alimentos/análise , Dieta , Carne/análiseRESUMO
Benzo[a]pyrene (BaP), which is emitted during the processing of smoked sausages, accumulates in sausages and poses a serious threat to human health. This study focused on the removal of BaP from sausages and accompanying particulate matter (PM) during the smoking of sausages by films formed by combining corn starch (CS) with K-carrageenan (KC)/sodium alginate (SA). Initially, the effects of different additions of KC and SA on the rheological analysis, thermogravimetric analysis (TGA) and film-forming properties of the composite films were investigated. The BaP reduction capacities of CS-KC and CS-SA composite films in sausage were 41.1%-47.0% and 54.2%-56.5%, respectively, because the three-dimensional mesh structure of the composite films provided a large number of adsorption sites. Finally, kinetic studies demonstrated that BaP control in composite films is mainly achieved by intraparticle diffusion. Therefore, due to its excellent recyclability and biodegradability, composite starch film has a promising application in smoked meat products.
Assuntos
Benzo(a)pireno , Produtos da Carne , Humanos , Benzo(a)pireno/análise , Produtos da Carne/análise , Fumaça , Amido , CinéticaRESUMO
The increasing global meat demand raises concerns regarding the spoilage of meat caused by microbial invasion and oxidative decomposition. Natural substances, as a gift from nature to humanity, possess broad-spectrum bioactivity and have been utilized for meat preservation. However, their limited stability, solubility, and availability hinder their further development. To address this predicament, advanced organic nanocarriers provide an effective shelter for the formation of nano-natural substances (NNS). This review comprehensively presents various natural substances derived from plants, animals, and microorganisms, along with the challenges they face. Subsequently, the potential of organic nanocarriers is explored, highlighting their distinct features and applicability, in addressing these challenges. The review methodically examines the application of NNS in meat preservation, with a focus on their pathways of action and preservation mechanisms. Furthermore, the outlook and future trends for NNS applications in meat preservation are concluded. The theory and practice summary of NNS is expected to serve as a catalyst for advancements that enhance meat security, promote human health, and contribute to sustainable development.
Diversified organic nanocarriers conquer the limitations of natural substancesNNS based on organic nanocarriers are a reliable and health-promoting optionNNS can manifest their effectiveness through diverse pathways and mechanismsThe utilization of NNS in meat preservation represents a transformative strategy.
RESUMO
BACKGROUND: Ultraviolet (UV) irradiation has been widely employed to disinfect food, however, the efficacy of UV irradiation in degrading polycyclic aromatic hydrocarbons (PAHs) in smoked sausages has not been explored. In this article, the UV degradation ability of PAHs in smoked sausages was investigated with different UV irradiation conditions, including different irradiation powers, durations and wavelengths. The effects of UV radiation on the quality of sausages were also evaluated, and potential degradation mechanisms were elucidated. RESULTS: The results showed that the irradiation duration was the primary determinant of PAHs degradation, achieving 84.4% and 84.2% degradation rates at 16 W and 32 W power for 30 min, respectively. Among the three UV wavelengths assessed, 254 nm demonstrated a significantly higher degradation rate for benzo[a]pyrene (BaP), PAH4 and PAHs compared to 365 nm and 310 nm. To further explore the degradation mechanism, UV irradiation was combined with water, 0.1 mol/L hydrogen peroxide (H2 O2 ) and 0.1 mol/L ascorbic acid (vitamin C) coatings. The 0.1 mol/L H2 O2 coating exhibited the most pronounced degradation effect, suggesting that the highly reactive oxygen hydroxyl radicals (·OH) generated by UV irradiation played a critical role in initiating redox reactions. CONCLUSION: This systematic investigation paves the way for developing novel strategies to eliminate PAHs or other organic contaminants from smoked sausages. © 2023 Society of Chemical Industry.
Assuntos
Produtos da Carne , Hidrocarbonetos Policíclicos Aromáticos , Hidrocarbonetos Policíclicos Aromáticos/análise , Raios Ultravioleta , Fumaça , Produtos da Carne/análiseRESUMO
Reactive oxygen species-mediated therapeutic strategies, including chemodynamic therapy (CDT) and photodynamic therapy (PDT), have exhibited translational promise for effective cancer management. However, monotherapy often ends up with the incomplete elimination of the entire tumor due to inherent limitations. Herein, we report a core-shell-structured Pd1.7Bi@CeO2-ICG (PBCI) nanoplatform constructed by a facile and effective strategy for synergistic CDT, PDT, and photothermal therapy. In the system, both Pd1.7Bi and CeO2 constituents exhibit peroxidase- and catalase-like characteristics, which not only generate cytotoxic hydroxyl radicals (â¢OH) for CDT but also produce O2 in situ and relieve tumor hypoxia for enhanced PDT. Furthermore, upon 808 nm laser irradiation, Pd1.7Bi@CeO2 and indocyanine green (ICG) coordinately prompt favorable photothermia, resulting in thermodynamically amplified catalytic activities. Meanwhile, PBCI is a contrast agent for near-infrared fluorescence imaging to determine the optimal laser therapeutic window in vivo. Consequently, effective tumor elimination was realized through the above-combined functions. The as-synthesized unitary PBCI theranostic nanoplatform represents a potential one-size-fits-all approach in multimodal synergistic therapy of hypoxic tumors.
Assuntos
Neoplasias , Fotoquimioterapia , Humanos , Fotoquimioterapia/métodos , Terapia Fototérmica , Neoplasias/tratamento farmacológico , Terapia Combinada , Hipóxia/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Advanced glycation end products (AGEs) and nitrosamines (NAs) in sausage are associated with pathogenic and carcinogenic risks. However, the multiple reaction parameters affecting the production of AGEs and NAs during sausage processing remain unclear. This experiment evaluated the effects of processing parameters, food additives and fat ratios on the formation of AGEs and NAs in sausages. The results showed a 2-3-fold increase in Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) when the sausage processing temperature was increased from 90 °C to 130 °C, and N-nitrosodimethylamine (NDEA) increased from 3.68 ng/g to 6.41 ng/g. The addition of salt inhibited the formation of AGEs and NAs, and the inhibitory ability of 2 g/100 g of salt was 63.6% for CML and 36.5% for N-nitrosodimethylamine (NDMA). The addition of 10 mg/kg nitrite to sausages reduced CML formation by 43.9%, however, nitrite had a significant contribution to the formation of NAs. The addition of fat only slightly contributed to the production of CML. In addition, the relationship between α-dicarbonyl compounds and the formation of AGEs was investigated by measuring the changes in α-dicarbonyl compounds in sausages. The results showed two trends of AGEs and α-dicarbonyl compounds: AGEs increased with the increase in α-dicarbonyl compounds and AGE level increased but α-dicarbonyl compound level decreased.
RESUMO
Various harmful Maillard reaction products such as lactulosyl-lysine (furosine), furfurals, and advanced glycation end products (AGEs) could be formed during the thermal processing of dairy products, which could lead to various chronic diseases. In this review, the furosine, furfurals, and AGEs formation, occurrence, analysis methods, and toxicological and health aspects in various dairy products were summarized to better monitor and control the levels of harmful Maillard reaction products in processed dairy products. It was observed that all types of dairy products, including raw milk, contain harmful Maillard reaction products, with the highest in whey cheese and condensed milk. High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the common method for the determination of furosine and furfurals and AGEs in dairy products, respectively. However, the simple, rapid, environment-friendly, and accurate methods of determination are still to be developed. Incorporating resveratrol, pectin oligosaccharides (POS) in milk are effective methods to inhibit AGEs formation. This review provides a guide not only for consumers regarding the selection and consumption of dairy products, but also for monitoring and controlling the quality of dairy products.
Assuntos
Produtos Finais de Glicação Avançada , Reação de Maillard , Animais , Cromatografia Líquida , Leite , Espectrometria de Massas em TandemRESUMO
The influence of acidity regulators and buffers on the formation of acrylamide (AA) and 5-hydroxymethylfurfural (5-HMF) in French fries and the underlying mechanism were evaluated. Prior to frying, the potato strips were dipped in the corresponding acidity regulator solutions or buffers for 30 min at room temperature. The results showed that acids inhibited AA formation, but increased 5-HMF levels. The AA level decreased and 5-HMF level increased with decreasing pH of potato strips. Interestingly, increasing concentration of acid radical ions resulted in AA increase and 5-HMF decrease, which was opposite to the acidification effect of citric acid and acetic acid. Both pH and acid radical ion were important factors for AA and 5-HMF formation. Moreover, acidity regulators might impact AA formation by acting on the generation of methylglyoxal (MGO) and glyoxal (GO) and impact 5-HMF formation by acting on the generation of 3-deoxyglucosone (3-DG).
Assuntos
Acrilamida , Solanum tuberosum , Furaldeído/análogos & derivados , Temperatura Alta , Concentração de Íons de Hidrogênio , ÍonsRESUMO
Isobutanol (IBT) has attracted much attention from researchers as a next generation drop-in biofuel. Ralstonia eutropha is a gram-negative bacterium which naturally produces polyhydroxybutyrate (PHB), and has been reported to produce IBT after metabolic engineering. Similar to other microbes, R. eutropha experiences toxicity from branched-chain alcohols and is unable to grow in the presence of IBT concentrations higher than 0.5% (v v(-1)). Such low tolerance greatly limits the ability of R. eutropha to grow and produce IBT. In order to study toxicity to the cells, IBT-tolerant strains were developed by experimental evolution, revealing that two genes, previously described as being related to IBT tolerance in Escherichia coli (acrA and acrA6), also presented mutations in R. eutropha evolved strains. The effect on the physiology of the cells of in-frame deletions of each of these genes was assessed in wild type and engineered IBT-producing strains in an attempt to reproduce a tolerant phenotype. The mutant strains' ability to tolerate, consume, and produce IBT were also analyzed. Although deletions of acrA6 and acrA did not significantly improve R. eutropha growth in the presence of IBT, these deletions improved cell survival in the presence of high concentrations of IBT in the extracellular milieu. Moreover, an in-frame acrA deletion in an engineered IBT-producing R. eutropha enhanced the strain's ability to produce IBT, which could potentially be associated with enhanced survival at high IBT concentrations.
Assuntos
Butanóis/farmacologia , Cupriavidus necator/efeitos dos fármacos , Cupriavidus necator/genética , Evolução Molecular Direcionada , Deleção de Genes , Técnicas de Inativação de Genes , Viabilidade Microbiana/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Butanóis/toxicidade , Cupriavidus necator/crescimento & desenvolvimento , Cupriavidus necator/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , MutagêneseRESUMO
2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 µM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.
Assuntos
Acetolactato Sintase/metabolismo , Cupriavidus necator/enzimologia , Hidroliases/metabolismo , Cetoácidos/metabolismo , Cetol-Ácido Redutoisomerase/metabolismo , Acetolactato Sintase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Butiratos/metabolismo , Meios de Cultura , Cupriavidus necator/genética , DNA Bacteriano/genética , Hemiterpenos , Hidroliases/genética , Isoleucina/biossíntese , Cetol-Ácido Redutoisomerase/genética , Leucina/biossíntese , Mutagênese Sítio-Dirigida , Valina/biossínteseRESUMO
Alleviating our society's dependence on petroleum-based chemicals has been highly emphasized due to fossil fuel shortages and increasing greenhouse gas emissions. Isopropanol is a molecule of high potential to replace some petroleum-based chemicals, which can be produced through biological platforms from renewable waste carbon streams such as carbohydrates, fatty acids, or CO2. In this study, for the first time, the heterologous expression of engineered isopropanol pathways were evaluated in a Cupriavidus necator strain Re2133, which was incapable of producing poly-3-hydroxybutyrate [P(3HB)]. These synthetic production pathways were rationally designed through codon optimization, gene placement, and gene dosage in order to efficiently divert carbon flow from P(3HB) precursors toward isopropanol. Among the constructed pathways, Re2133/pEG7c overexpressing native C. necator genes encoding a ß-ketothiolase, a CoA-transferase, and codon-optimized Clostridium genes encoding an acetoacetate decarboxylase and an alcohol dehydrogenase produced up to 3.44 g l(-1) isopropanol in batch culture, from fructose as a sole carbon source, with only 0.82 g l(-1) of biomass. The intrinsic performance of this strain (maximum specific production rate 0.093 g g(-1) h(-1), yield 0.32 Cmole Cmole(-1)) corresponded to more than 60 % of the respective theoretical performance. Moreover, the overall isopropanol production yield (0.24 Cmole Cmole(-1)) and the overall specific productivity (0.044 g g(-1) h(-1)) were higher than the values reported in the literature to date for heterologously engineered isopropanol production strains in batch culture. Strain Re2133/pEG7c presents good potential for scale-up production of isopropanol from various substrates in high cell density cultures.
Assuntos
2-Propanol/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Engenharia Metabólica , Técnicas de Cultura Celular por Lotes , Biomassa , Clostridium/enzimologia , Clostridium/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Enzimas/biossíntese , Enzimas/genética , Frutose/metabolismo , Dosagem de Genes , Expressão Gênica , Redes e Vias Metabólicas/genética , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Análise de Sequência de DNARESUMO
Carbonic anhydrase (CA) enzymes catalyze the interconversion of CO2 and bicarbonate. These enzymes play important roles in cellular metabolism, CO2 transport, ion transport, and internal pH regulation. Understanding the metabolic role of CAs in the chemolithoautotropic bacterium Ralstonia eutropha is important for the development of high performance fermentation processes based on the bacterium's capability to fix carbon using the Calvin-Benson-Bassham (CBB) cycle. Analysis of the R. eutropha H16 genome sequence revealed the presence of four CA genes: can, can2, caa and cag. We evaluated the importance of each of the CAs in the metabolism of R. eutropha by examination of growth and enzyme activity in gene deletion, complementation, and overexpression strains. All four purified CAs were capable of performing the interconversion of CO2 and HCO3-, although the equilibrium towards the formation of CO2 or HCO3- differs with each CA. Deletion of can, encoding a ß-CA, affected the growth of R. eutropha; however the growth defect could be compensated by adding CO2 to the culture. Deletion of the caa, encoding an α-CA, had the strongest deleterious influence on cell growth. Strains with deletion or overexpression of can2 or cag genes exhibited similar behavior to wild type under most of the conditions tested. In this work, Caa was studied in greater detail using microscopy and complementation experiments, which helped confirm its periplasmic localization and determine its importance for robust growth of R. eutropha. A hypothesis for the coordinated role of these four enzymes in the metabolism of R. eutropha is proposed.
RESUMO
Lipid and fatty acid metabolism has been well studied in model microbial organisms like Escherichia coli and Bacillus subtilis. The major precursor of fatty acid biosynthesis is also the major product of fatty acid degradation (ß-oxidation), acetyl-CoA, which is a key metabolite for all organisms. Controlling carbon flux to fatty acid biosynthesis and from ß-oxidation allows for the biosynthesis of natural products of biotechnological importance. Ralstonia eutropha can utilize acetyl-CoA from fatty acid metabolism to produce intracellular polyhydroxyalkanoate (PHA). R. eutropha can also be engineered to utilize fatty acid metabolism intermediates to produce different PHA precursors. Metabolism of lipids and fatty acids can be rerouted to convert carbon into other value-added compounds like biofuels. This review discusses the lipid and fatty acid metabolic pathways in R. eutropha and how they can be used to construct reagents for the biosynthesis of products of industrial importance. Specifically, how the use of lipids or fatty acids as the sole carbon source in R. eutropha cultures adds value to these biotechnological products will be discussed here.
Assuntos
Biotecnologia , Cupriavidus necator/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Poli-Hidroxialcanoatos/metabolismoRESUMO
Lipase enzymes catalyze the reversible hydrolysis of triacylglycerol to fatty acids and glycerol at the lipid-water interface. The metabolically versatile Ralstonia eutropha strain H16 is capable of utilizing various molecules containing long carbon chains such as plant oil, organic acids, or Tween as its sole carbon source for growth. Global gene expression analysis revealed an upregulation of two putative lipase genes during growth on trioleate. Through analysis of growth and activity using strains with gene deletions and complementations, the extracellular lipase (encoded by the lipA gene, locus tag H16_A1322) and lipase-specific chaperone (encoded by the lipB gene, locus tag H16_A1323) produced by R. eutropha H16 was identified. Increase in gene dosage of lipA not only resulted in an increase of the extracellular lipase activity, but also reduced the lag phase during growth on palm oil. LipA is a non-specific lipase that can completely hydrolyze triacylglycerol into its corresponding free fatty acids and glycerol. Although LipA is active over a temperature range from 10 °C to 70 °C, it exhibited optimal activity at 50 °C. While R. eutropha H16 prefers a growth pH of 6.8, its extracellular lipase LipA is most active between pH 7 and 8. Cofactors are not required for lipase activity; however, EDTA and EGTA inhibited LipA activity by 83 %. Metal ions Mg(2+), Ca(2+), and Mn(2+) were found to stimulate LipA activity and relieve chelator inhibition. Certain detergents are found to improve solubility of the lipid substrate or increase lipase-lipid aggregation, as a result SDS and Triton X-100 were able to increase lipase activity by 20 % to 500 %. R. eutropha extracellular LipA activity can be hyper-increased, making the overexpression strain a potential candidate for commercial lipase production or in fermentations using plant oils as the sole carbon source.
Assuntos
Cupriavidus necator/enzimologia , Lipase/genética , Lipase/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Chaperonas Moleculares/metabolismo , Cátions Bivalentes/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/crescimento & desenvolvimento , Cupriavidus necator/metabolismo , Detergentes/metabolismo , Ativadores de Enzimas , Estabilidade Enzimática , Deleção de Genes , Teste de Complementação Genética , Concentração de Íons de Hidrogênio , Lipase/química , Lipase/isolamento & purificação , Metais/metabolismo , Óleo de Palmeira , Óleos de Plantas/metabolismo , TemperaturaRESUMO
Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.
Assuntos
Butanóis/metabolismo , Cupriavidus necator/genética , Cupriavidus necator/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Pentanóis/metabolismo , Butanóis/toxicidade , Cupriavidus necator/crescimento & desenvolvimento , Hidroxibutiratos/metabolismo , Plasmídeos , Poliésteres/metabolismoRESUMO
The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a ß-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ΔphaB1 ΔphaB2 ΔphaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Cupriavidus necator/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Oxirredutases do Álcool/classificação , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Meios de Cultura/química , Cupriavidus necator/classificação , Cupriavidus necator/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Teste de Complementação Genética , Genoma Bacteriano , Genótipo , MutaçãoRESUMO
Leptin is best known as a key satiety factor and it is now appreciated that leptin has many additional biological functions. Our previous study suggested that leptin-resistant obesity might exacerbate 1-methyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity in vivo. Here, we ask whether leptin might protect neuronal cells against 1-methyl-4-pyridinium (MPP+)-induced cell death. We used differentiated SH-SY5Y cells and investigated plausible cytoprotective signalling mechanisms. When SH-SY5Y cells were maintained under serum-free conditions for 48 h, MPP+ (1 mM) reduced cell viability to 66.8% of the drug-free control, and leptin significantly inhibited cell death in a dose-dependent manner. Among inhibitors of known leptin signalling pathways, a PI-3K inhibitor inhibited the protective effect of leptin during MPP+ exposure, whereas inhibitors affecting the Janus kinase/signal transducers and activators of transcription (JAK/STAT) or mitogen-activated protein kinase (MAPK) pathways did not influence cell viability. We used immunoblotting to show that the PI-3K/Akt pathway was involved in the effect of leptin on cell viability. In conclusion, our results show that leptin exercises a cytoprotective effect against MPP+ -induced cell death and that this effect is dependent on activation of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in SH-SY5Y cells. The data tend to support our previous results in vivo.