RESUMO
The efficiency of a versatile in vivo cascade involving a promiscuous alcohol dehydrogenase, obtained from a biodiversity search, and a Baeyer-Villiger monooxygenase was enhanced by the independent control of the production level of each enzyme to produce ε-caprolactone and 3,4-dihydrocoumarin. This goal was achieved by adjusting the copy number per cell of Escherichia coli plasmids. We started from the observation that this number generally correlates with the amount of produced enzyme and demonstrated that an in vivo multi-enzymatic system can be improved by the judicious choice of plasmid, the lower activity of the enzyme that drives the limiting step being counter-balanced by a higher concentration. Using a preconception-free approach to the choice of the plasmid type, we observed positive and negative synergetic effects, sometimes unexpected and depending on the enzyme and plasmid combinations. Experimental optimization of the culture conditions allowed us to obtain the complete conversion of cyclohexanol (16 mM) and 1-indanol (7.5 mM) at a 0.5-L scale. The yield for the conversion of cyclohexanol was 80% (0.7 g ε-caprolactone, for the productivity of 244 mg·L -1 ·h -1 ) and that for 1-indanol 60% (0.3 g 3,4-dihydrocoumarin, for the productivity of 140 mg·L -1 ·h -1 ).
Assuntos
Caproatos/metabolismo , Cumarínicos/metabolismo , Escherichia coli/metabolismo , Lactonas/metabolismo , Engenharia Metabólica , Catálise , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genéticaRESUMO
The metabolic effects of an oral supplementation with a Curcuma longa extract, at a dose nutritionally relevant with common human use, on hepatic metabolism in rats fed a high fructose and saturated fatty acid (HFS) diet was evaluated. High-resolution magic-angle spinning NMR and GC/MS in combination with multivariate analysis have been employed to characterize the NMR metabolite profiles and fatty acid composition of liver tissue respectively. The results showed a clear discrimination between HFS groups and controls involving metabolites such as glucose, glycogen, amino acids, acetate, choline, lysophosphatidylcholine, phosphatidylethanolamine, and ß-hydroxybutyrate as well as an increase of MUFAs and a decrease of n-6 and n-3 PUFAs. Although the administration of CL did not counteract deleterious effects of the HFS diet, some metabolites, namely some n-6 PUFA and n-3 PUFA, and betaine were found to increase significantly in liver samples from rats having received extract of curcuma compared to those fed the HFS diet alone. This result suggests that curcuminoids may affect the transmethylation pathway and/or osmotic regulation. CL extract supplementation in rats appears to increase some of the natural defences preventing the development of fatty liver by acting on the choline metabolism to increase fat export from the liver.
Assuntos
Suplementos Nutricionais , Fígado/metabolismo , Extratos Vegetais/farmacologia , Animais , Betaína/metabolismo , Colina/metabolismo , Curcuma , Dieta Hiperlipídica , Análise Discriminante , Ácidos Graxos , Frutose , Glutationa/metabolismo , Análise dos Mínimos Quadrados , Masculino , Malondialdeído/metabolismo , Análise Multivariada , Espectroscopia de Prótons por Ressonância Magnética , Ratos Sprague-Dawley , Triglicerídeos/metabolismoRESUMO
We explored, using nuclear magnetic resonance (NMR) metabolomics and fatty acids profiling, the effects of a common nutritional complement, Curcuma longa, at a nutritionally relevant dose with human use, administered in conjunction with an unbalanced diet. Indeed, traditional food supplements have been long used to counter metabolic impairments induced by unbalanced diets. Here, rats were fed either a standard diet, a high level of fructose and saturated fatty acid (HFS) diet, a diet common to western countries and that certainly contributes to the epidemic of insulin resistance (IR) syndrome, or a HFS diet with a Curcuma longa extract (1% of curcuminoids in the extract) for ten weeks. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) on the serum NMR profiles and fatty acid composition (determined by GC/MS) showed a clear discrimination between HFS groups and controls. This discrimination involved metabolites such as glucose, amino acids, pyruvate, creatine, phosphocholine/glycerophosphocholine, ketone bodies and glycoproteins as well as an increase of monounsaturated fatty acids (MUFAs) and a decrease of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Although the administration of Curcuma longa did not prevent the observed increase of glucose, triglycerides, cholesterol and insulin levels, discriminating metabolites were observed between groups fed HFS alone or with addition of a Curcuma longa extract, namely some MUFA and n-3 PUFA, glycoproteins, glutamine, and methanol, suggesting that curcuminoids may act respectively on the fatty acid metabolism, the hexosamine biosynthesis pathway and alcohol oxidation. Curcuma longa extract supplementation appears to be beneficial in these metabolic pathways in rats. This metabolomic approach highlights important serum metabolites that could help in understanding further the metabolic mechanisms leading to IR.
Assuntos
Curcuma/metabolismo , Ácidos Graxos/farmacologia , Xarope de Milho Rico em Frutose/farmacologia , Redes e Vias Metabólicas/fisiologia , Extratos Vegetais/farmacologia , Animais , Antioxidantes/metabolismo , Análise Química do Sangue , Glicemia/análise , Colesterol/sangue , Dieta , Gorduras na Dieta , Suplementos Nutricionais , Ácidos Graxos/administração & dosagem , Ácidos Graxos/sangue , Frutose/administração & dosagem , Xarope de Milho Rico em Frutose/administração & dosagem , Insulina/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Espectroscopia de Ressonância Magnética , Masculino , Redes e Vias Metabólicas/efeitos dos fármacos , Metabolômica , Estresse Oxidativo/fisiologia , Extratos Vegetais/administração & dosagem , Ratos , Ratos Sprague-Dawley , Triglicerídeos/sangueRESUMO
Out of 107 fungal strains belonging to three phyla (Ascomycota, Basidiomycota and Zygomycota) and 46 genera, 86 exhibited Baeyer-Villiger monooxygenase (BVMO) activity against racemic bicyclo[3.2.0]heptenone. The strains were classified into three "profiles" based on regio- and enantioselectivity. Statistical analyses of our results, extended by literature data, showed that these profiles could be related to the taxonomic classification of the strains, and suggest that the BVMOs from the Zygomycota phylum may be different in their primary structures from established ones.
Assuntos
Ascomicetos , Basidiomycota , Compostos Bicíclicos com Pontes/química , Oxigenases de Função Mista/metabolismo , Ascomicetos/enzimologia , Ascomicetos/genética , Ascomicetos/metabolismo , Basidiomycota/enzimologia , Basidiomycota/genética , Basidiomycota/metabolismo , Genômica , Prevalência , Eslovênia , Especificidade por SubstratoRESUMO
As the consumption of fructose and saturated fatty acids (FAs) has greatly increased in western diets and is linked with an increased risk of metabolic syndrome, the aim of this study was to investigate the effects of a moderate (10 weeks) and a prolonged (30 weeks) high fructose and saturated fatty acid (HFS) diet on plasma FA composition in rats. The effects of a few weeks of HFS diet had already been described, but in this paper we tried to establish whether these effects persist or if they are modified after 10 or 30 weeks. We hypothesized that the plasma FA profile would be altered between 10 and 30 weeks of the HFS diet. Rats fed with either the HFS or a standard diet were tested after 10 weeks and again after 30 weeks. After 10 weeks of feeding, HFS-fed rats developed the metabolic syndrome, as manifested by an increase in fasting insulinemia, total cholesterol and triglyceride levels, as well as by impaired glucose tolerance. Furthermore, the plasma FA profile of the HFS group showed higher proportions of monounsaturated FAs like palmitoleic acid [16:1(n-7)] and oleic acid [18:1(n-9)], whereas the proportions of some polyunsaturated n-6 FAs, such as linoleic acid [18:2(n-6)] and arachidonic acid [20:4(n-6)], were lower than those in the control group. After 30 weeks of the HFS diet, we observed changes mainly in the levels of 16:1(n-7) (decreased) and 20:4(n-6) (increased). Together, our results suggest that an HFS diet could lead to an adaptive response of the plasma FA profile over time, in association with the development of the metabolic syndrome.
Assuntos
Gorduras na Dieta/metabolismo , Sacarose Alimentar/metabolismo , Ácidos Graxos/sangue , Ácidos Graxos/metabolismo , Frutose/metabolismo , Administração Oral , Animais , Ácidos Graxos/administração & dosagem , Frutose/administração & dosagem , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.
Assuntos
Membrana Celular/química , Microdomínios da Membrana/química , Obesidade/metabolismo , Pâncreas Exócrino/química , Proteômica , Animais , Membrana Celular/ultraestrutura , Dieta , Masculino , Microdomínios da Membrana/metabolismo , Pâncreas Exócrino/metabolismo , Proteínas/análise , Ratos , Ratos Sprague-DawleyRESUMO
To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.