RESUMEN
Insulin resistance is frequently present in patients with glucocorticoid (GC) excess (Cushing's syndrome) or treated with high doses of GCs. Furthermore, others similarities between metabolic syndrome (visceral obesity, elevated blood glucose levels, dyslipidemia) and Cushing's syndrome suggest that GCs could play a role in obesity-linked complications. Here we reported that long-term corticosterone (CORT) exposure in mice induced weight gain, dyslipidemia as well as hyperglycaemia and systemic insulin resistance. CORT-treated mice exhibited an increased 11ß-Hsd1 expression and corticosterone levels in fat depots but a specific upregulation of glucocorticoid receptor (Gr) and hexose-6-phosphate dehydrogenase only in gonadal adipose tissue, suggesting that GC could act differentially on various fat depots. Despite fat accumulation in all depots, an increased expression of adipogenic (Pparγ, C/ebpα) and lipogenic (Acc, Fas) key genes was restricted to gonadal adipose tissue. Hypertrophied adipocytes observed in both visceral and subcutaneous depots also resulted from reduced lipolytic activity due to CORT treatment. Surprisingly, GC treatment promoted macrophage infiltration (F4/80, Cd68) within all adipose tissues along with predominant M2-like macrophage phenotype, and can directly act on macrophages to induce this phenotype. Moreover, macrophage infiltration preceded mass gain and adipocyte hypertrophy. Of note, specific macrophage depletion in gonadal fat preferentially reduced the M2-like macrophage content, and partially restored insulin sensitivity in mice with GC-induced obesity and insulin resistance. These data provide evidence that GCs act on adipose tissue in a depot-dependent manner and that gonadal adipose macrophages are key effectors of GC-associated insulin resistance.
Asunto(s)
Tejido Adiposo/metabolismo , Corticosterona/farmacología , Glucocorticoides/metabolismo , Resistencia a la Insulina/fisiología , Macrófagos/metabolismo , Obesidad/patología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , Adipocitos/patología , Tejido Adiposo/citología , Animales , Deshidrogenasas de Carbohidratos/biosíntesis , Células Cultivadas , Síndrome de Cushing/patología , Dislipidemias/inducido químicamente , Hiperglucemia/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de Glucocorticoides/biosíntesis , Aumento de Peso/fisiologíaRESUMEN
BACKGROUND: Increased adiposity in visceral depots is a crucial feature associated with glucocorticoid (GC) excess. The action of GCs in a target tissue is regulated by GC receptor (GR) and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) coupled with hexose-6-phosphate dehydrogenase (H6pdh). Glycogen synthase kinase-3ß (GSK3ß) is known to be a crucial mediator of ligand-dependent gene transcription. We hypothesized that the major effects of corticosteroids on adipose fat accumulation are in part mediated by changes in GSK3ß and H6pdh. METHODS: We characterized the alterations of GSK3ß and GC metabolic enzymes, and determined the impact of GR antagonist mifepristone on obesity-related genes and the expression of H6pdh and 11ß-HSD1 in adipose tissue of mice exposed to excess GC as well as in in vitro studies using 3T3-L1 adipocytes treated with GCs. RESULTS: Corticosterone (CORT) exposure increased abdominal fat mass and induced expression of lipid synthase acetyl-CoA carboxylase and ATP-citrate lyase with activation of GSK3ß phosphorylation in abdominal adipose tissue of C57BL/6J mice. Increased pSer(9) GSK3ß was correlated with the induction of H6pdh and 11ß-HSD1. In addition, mifepristone treatment reversed the production of H6pdh and attenuated CORT-mediated production of 11ß-HSD1 and lipogenic gene expression with reduction of pSer(9) GSK3ß, thereby leading to improvement of phenotype of adiposity within adipose tissue in mice treated with excess GCs. Suppression of pSer(9) GSK3ß by mifepristone was accompanied by activation of pThr(308) Akt and blockade of CORT-induced adipogenic transcriptor C/EBPα and PPARγ. In addition, mifepristone also attenuated CORT-mediated activation of IRE1α/XBP1. In addition, reduction of H6pdh by shRNA showed comparable effects to mifepristone on attenuating CORT-induced expression of GC metabolic enzymes and improved lipid accumulation in vitro in 3T3-L1 adipocytes. CONCLUSION: These findings suggest that elevated adipose GSK3ß and H6pdh expression contribute to 11ß-HSD1 mediating hypercortisolism associated with visceral adiposity.
Asunto(s)
Tejido Adiposo/enzimología , Adiposidad/efectos de los fármacos , Deshidrogenasas de Carbohidratos/metabolismo , Glucocorticoides/farmacología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Adipocitos/enzimología , Adipocitos/metabolismo , Adipogénesis/efectos de los fármacos , Adiposidad/genética , Animales , Deshidrogenasas de Carbohidratos/biosíntesis , Modelos Animales de Enfermedad , Glucógeno Sintasa Quinasa 3 beta/biosíntesis , Antagonistas de Hormonas/farmacología , Grasa Intraabdominal/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Mifepristona/farmacología , Obesidad Abdominal/enzimología , Obesidad Abdominal/genética , Obesidad Abdominal/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidoresRESUMEN
Three species of white rot fungi: Cerrena unicolor, Phlebia lindtneri and Pycnoporus sanguineus were cultured in two different media under five different lighting conditions: dark, white, red, blue, and green light. Laccase, cellobiose dehydrogenase, and protease activities were examined in the samples. Blue light efficiently boosted laccase synthesis in C. unicolor and P. sanguineus, whereas the highest activities (20 654 nkat/l) of P. lindtneri laccase were observed when this fungus was maintained in green light. On the contrary, the green light allowed obtaining the highest activities of cellobiose dehydrogenase of C. unicolor and P. lindtneri, while CDH of P. sanguineus seems to be dependent on white light. It is clearly visible that differences in protease activities are noticeable not only between the lights variants but also among the media used. However, high proteases activities are correlated with light variants inducing laccase in Lindeberg and Holm medium. Contrary to the cellulose-based medium, where they are weak in light variants that lead to high CDH activities.
Asunto(s)
Deshidrogenasas de Carbohidratos/biosíntesis , Proteínas Fúngicas/biosíntesis , Lacasa/biosíntesis , Péptido Hidrolasas/biosíntesis , Pycnoporus/enzimología , Adaptación Fisiológica , Inducción Enzimática/efectos de la radiación , Regulación Fúngica de la Expresión Génica/efectos de la radiación , Luz , Pycnoporus/efectos de la radiaciónRESUMEN
Agaricus bisporus is a litter degrading basidiomycete commonly found in humic-rich environments. It is used as model organism and cultivated in large scale for food industry. Due to its ecological niche it produces a variety of enzymes for detoxification and degradation of humified plant litter. One of these, pyranose dehydrogenase, is thought to play a role in detoxification and lignocellulose degradation. It is a member of the glucose-methanol-choline family of flavin-dependent enzymes and oxidizes a wide range of sugars with concomitant reduction of electron acceptors like quinones. In this work, transcription of pdh in A. bisporus was investigated with real-time PCR revealing influence of the carbon source on pdh expression levels. The gene was isolated and heterologously expressed in Pichia pastoris. Characterization of the recombinant enzyme showed a higher affinity towards disaccharides compared to other tested pyranose dehydrogenases from related Agariceae. Homology modeling and sequence alignments indicated that two loops of high sequence variability at substrate access site could play an important role in modulating these substrate specificities.
Asunto(s)
Agaricus/enzimología , Deshidrogenasas de Carbohidratos/genética , Proteínas Fúngicas/genética , Secuencia de Aminoácidos , Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/química , Dominio Catalítico , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología Estructural de Proteína , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Cellobiose dehydrogenase (CDH), a secreted flavocytochrome produced by a number of wood-degrading fungi, was detected in the culture supernatant of a biotechnologically important strain of Cerrena unicolor grown in a modified cellulose-based liquid medium. The enzyme was purified as two active fractions: CuCDH-FAD (flavin domain) (1.51-fold) with recovery of 8.35 % and CuCDH (flavo-heme enzyme) (21.21-fold) with recovery of 73.41 %. As CDH from other wood-rotting fungi, the intact form of cellobiose dehydrogenase of C. unicolor is a monomeric protein containing one flavin and one heme b with molecular mass 97 kDa and pI = 4.55. The enzyme is glycosylated (8.2 %) mainly with mannose and glucosamine residues. Moreover, the cellobiose dehydrogenase gene cdh1 and its corresponding cDNA from the fungus C. unicolor were isolated, cloned, and characterized. The 2316-bp full-length cDNA of cdh1 encoded a mature CDH protein containing 771 amino acids preceded by a signal peptide consisting of 18 amino acids. Moreover, both active fractions were characterized in terms of kinetics, temperature and pH optima, and antioxidant properties.
Asunto(s)
Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/química , Celulosa/química , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Polyporales/enzimología , Deshidrogenasas de Carbohidratos/genética , Celulosa/metabolismo , Clonación Molecular , ADN Complementario , Proteínas Fúngicas/genética , Polyporales/genéticaRESUMEN
Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5'-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5'-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28-34mg/l PN from 4-HO-Thr while the wild-type parent produced only 12mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.
Asunto(s)
Antimetabolitos/metabolismo , Bacillus subtilis , Ingeniería Metabólica , Piridoxina/biosíntesis , Treonina/análogos & derivados , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/genética , Oxidorreductasas/biosíntesis , Oxidorreductasas/genética , Treonina/biosíntesisRESUMEN
2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to L-sorbose. The optimum combination produced 4.9g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4g/L of 2-KLG after 168h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C.
Asunto(s)
Gluconobacter oxydans , Ingeniería Metabólica , Sorbitol/metabolismo , Azúcares Ácidos/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/genética , Gluconobacter oxydans/enzimología , Gluconobacter oxydans/genética , Rhodobacteraceae/enzimología , Rhodobacteraceae/genéticaRESUMEN
The study investigates the potential of substitution of the conventional carbohydrate nutrient (cellulose) in media with cheap agro-residues for cellobiose dehydrogenase production by Termitomyces clypeatus (CDHtc) under submerged conditions. Different agro-residues tested for enzyme production were characterized using FTIR and XRD analysis. As CDHtc production was highest with tamarind kernel powder (TKP), it was selected for process optimizations through shake-flask fermentations. The optimized parameters were then applied to batch cultures in a 5 L bioreactor that gave enzyme yield (57.4 U mL⻹) similar to that obtained under shake-flask fermentations (57.05 U mL⻹). The study also made an attempt to predict CDHtc production with respect to time of fermentation and mycelial growth. The specific growth rate and carrying capacity of the mycelia were also determined, and the values lie in the ranges of 0.024-0.027 h⻹ and 7.2-7.1 mg mL⻹, respectively.
Asunto(s)
Técnicas de Cultivo Celular por Lotes/métodos , Deshidrogenasas de Carbohidratos/biosíntesis , Proteínas Fúngicas/biosíntesis , Tamarindus/metabolismo , Termitomyces/enzimología , Técnicas de Cultivo Celular por Lotes/instrumentación , Reactores Biológicos/microbiología , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Fermentación , Semillas/química , Semillas/metabolismo , Semillas/microbiología , Tamarindus/química , Tamarindus/microbiología , Termitomyces/crecimiento & desarrollo , Termitomyces/metabolismo , Residuos/análisisRESUMEN
The expression levels of sorbose/sorbosone dehydrogenase genes (sdh and sndh) and the synthesis genes (pqqABCDEN) of the adjoint cofactor pyrroloquinoline quinone (PQQ) were genetically manipulated in Ketogulonigenium vulgare to increase the production of 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, in the consortium of K. vulgare and Bacillus cereus. We found that overexpression of sdh-sndh alone in K. vulgare could not significantly enhance the production of 2-KLG, revealing the cofactor PQQ was required for the biosynthesis of 2-KLG. Various expression levels of PQQ were achieved by differential expression of pqqA, pqqABCDE and pqqABCDEN, respectively. The combinatorial expression of sdh/sndh and pqqABCDEN in K. vulgare enabled a 20% increase in the production of 2-KLG (79.1±0.6gl(-1)) than that of the parental K. vulgare (65.9±0.4gl(-1)) in shaking flasks. Our results demonstrated the balanced co-expression of both the key enzymes and the related cofactors was an efficient strategy to increase chemicals' biosynthesis.
Asunto(s)
Bacillus cereus/metabolismo , Proteínas Bacterianas/biosíntesis , Deshidrogenasas de Carbohidratos/biosíntesis , Ingeniería Metabólica , Cofactor PQQ/metabolismo , Azúcares Ácidos/metabolismo , Ácido Ascórbico/biosíntesis , Ácido Ascórbico/genética , Bacillus cereus/genética , Proteínas Bacterianas/genética , Deshidrogenasas de Carbohidratos/genética , Regulación Bacteriana de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Cofactor PQQ/genética , Sorbosa/análogos & derivados , Sorbosa/genética , Sorbosa/metabolismoRESUMEN
Cellobiose dehydrogenases (CDHs) are extracellular glycosylated haemoflavoenzymes produced by many different wood-degrading and phytopathogenic fungi. Putative cellobiose dehydrogenase genes are recurrently discovered by genome sequencing projects in various phylogenetically distinct fungi. The genomes from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina were screened for candidate cdh genes, and one and three putative gene models were evidenced, respectively. Two putative cdh genes were selected and successfully expressed for the first time in Aspergillus niger. CDH activity was measured for both constructions (CDHcc and CDHpa), and both recombinant CDHs were purified to homogeneity and subsequently characterised. Kinetic constants were determined for several carbohydrates including ß-1,4-linked di- and oligosaccharides. Optimal temperature and pH were 60 °C and 5 for CDHcc and 65-70 °C and 6 for CDHpa. Both CDHs showed a broad range of pH stability between 4 and 8. The effect of both CDHs on saccharification of micronized wheat straw by an industrial Trichoderma reesei secretome was determined. The addition of each CDH systematically decreased the release of total reducing sugars, but to different extents and according to the CDH concentration. Analytical methods were carried out to quantify the release of glucose, xylose and gluconic acid. An increase of glucose and xylose was measured at a low CDHcc concentration. At moderated and high CDHcc and CDHpa concentrations, glucose was severely reduced with a concomitant increase of gluconic acid. In conclusion, these results give new insights into the physical and chemical parameters and diversity of basidiomycetous and ascomycetous CDHs. These findings also demonstrated that CDH drastically influenced the saccharification on a natural substrate, and thus, CDH origin, concentration and potential enzymatic partners should be carefully considered in future artificial secretomes for biofuel applications.
Asunto(s)
Agaricales/enzimología , Aspergillus niger/genética , Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/aislamiento & purificación , Podospora/enzimología , Polisacáridos/metabolismo , Triticum/química , Agaricales/genética , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/genética , Estabilidad de Enzimas , Expresión Génica , Concentración de Iones de Hidrógeno , Cinética , Tallos de la Planta/química , Podospora/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , TemperaturaRESUMEN
Human fetal membranes express 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which reduces biologically inert cortisone to active cortisol and may provide an extraadrenal source of cortisol mediating fetal development and parturition. The reductase activity of 11ß-HSD1 depends on the availability of the cofactor reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) derived from the enzymatic activity of hexose-6-phosphodehydrogenase (H6PD). Based on the feed-forward induction of 11ß-HSD1 by glucocorticoids in human fetal membranes, we hypothesize that glucocorticoids simultaneously induce H6PD in the fetal membranes. We found a parallel distribution of H6PD and 11ß-HSD1 in the amnion, chorion, and decidua. In cultured human amnion fibroblasts, small interfering RNA-mediated knockdown of H6PD expression significantly attenuated the conversion of cortisone to cortisol. Cortisol (0.01-1 µm) induced H6PD expression in a concentration-dependent manner, which was attenuated by glucocorticoid receptor (GR) antagonist RU486. Cortisol induced the expression of p300, a histone acetyltransferase, whereas C646, an inhibitor of p300, attenuated the induction of H6PD by cortisol. Coimmunoprecipitation revealed GR and p300 in the same nuclear protein complex upon cortisol stimulation. Chromatin immunoprecipitation showed that cortisol increased the binding of p300 and GR to H6PD promoter and the acetylation of histone 3 lysine 9 on the promoters. In conclusion, the induction of H6PD by cortisol requires the participation of GR and p300 as well as the acetylation of H3K9 by p300. This may be a prerequisite for the parallel induction of reductase activity of 11ß-HSD1 in human amnion fibroblasts in a feed-forward loop that may influence fetal development and the onset of parturition.
Asunto(s)
Amnios/metabolismo , Deshidrogenasas de Carbohidratos/biosíntesis , Proteína p300 Asociada a E1A/metabolismo , Fibroblastos/metabolismo , Hidrocortisona/metabolismo , Receptores de Glucocorticoides/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Membranas Extraembrionarias/metabolismo , Glucocorticoides/metabolismo , Histonas/metabolismo , Humanos , Hidrocortisona/farmacología , Lisina/química , Mifepristona/farmacología , NADP/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Interferente Pequeño/metabolismoRESUMEN
The presence of Pseudomonas aeruginosa in cutaneous wounds is of clinical significance and can lead to persistent infections. Manuka honey has gained ground in clinical settings due to its effective therapeutic action and broad spectrum of antibacterial activity. In this study, the effect of manuka honey on P. aeruginosa was investigated using MIC, MBC, growth kinetics, confocal microscopy, atomic force microscopy and real-time PCR. A bactericidal mode of action for manuka honey against P. aeruginosa was deduced (12â%, w/v, MIC; 16â%, w/v, MBC) and confirmed by confocal and atomic force microscopy, which showed extensive cell lysis after 60 min exposure to inhibitory concentrations of manuka honey. The inability of honey-treated cells to form microcolonies was demonstrated and investigated using Q-PCR for three key microcolony-forming genes: algD, lasR and oprF. The expression of algD increased 16-fold whereas oprF expression decreased 10-fold following honey treatment; lasR expression remained unaltered. These findings confirm that manuka honey is effective at inducing cell lysis and identify two targets, at the genetic level, that might be involved in this process.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/biosíntesis , Deshidrogenasas de Carbohidratos/biosíntesis , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Miel , Pseudomonas aeruginosa/efectos de los fármacos , Bacteriólisis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía de Fuerza Atómica , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la Polimerasa , Transactivadores/biosíntesisRESUMEN
Cellobiose dehydrogenase (CDH) is an emerging enzyme in the field of bioelectrocatalysis. Due to its flexible cytochrome domain, which acts as a built-in redox mediator, CDH is capable of direct electron transfer (DET) to electrode surfaces. This rare property is employed in mediatorless "third generation" biosensors. The ability of Corynascus thermophilus CDH to oxidize glucose under physiological conditions makes it a promising candidate for miniaturized glucose biosensors or glucose powered biofuel cell anodes. We report for the first time the electrochemical application and characterization of a recombinantly produced CDH in a glucose biosensor. Recombinant CDH from C. thermophilus (rCtCDH) was expressed by the methylotrophic yeast Pichia pastoris (376 U L(-1) , 132 mg L(-1) ). A comparative characterization of rCtCDH and CtCDH shows identical pH optima, K(M) values and heme b midpoint potentials. In contrast, the specific activity of rCtCDH (2.84 U mg(-1) ) and consequently the turnover numbers were ~five-times lower than for CtCDH, which was caused by a sub-stoichiometric occupation of catalytic sites with flavin-adenin-dinukleotid (FAD). The performance of rCtCDH-modified electrodes demonstrates the suitability for electrochemical studies. This opens the possibility to engineer the substrate specificity of C. thermophilus CDH for specific carbohydrates by rational engineering or directed evolution.
Asunto(s)
Fuentes de Energía Bioeléctrica , Técnicas Biosensibles/métodos , Deshidrogenasas de Carbohidratos/biosíntesis , Glucosa/metabolismo , Proteínas Recombinantes/biosíntesis , Sordariales/enzimología , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/genética , Celobiosa/metabolismo , Electrodos , Fermentación , Glucosa/química , Cinética , Peso Molecular , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sordariales/genéticaRESUMEN
The enzyme cellobiose dehydrogenase (CDH) is of considerable interest, not only for its biotechnological applications, but also its potential biological role in lignocellulosic biomass breakdown. The enzyme catalyzes the oxidation of cellobiose and other cellodextrins, utilizing a variety of one- and two-electron acceptors, although the electron acceptor employed in nature is still unknown. In this study we show that a CDH is present in the secretome of the thermophilic ascomycete Thielavia terrestris when grown with cellulose, along with a mixture of cellulases and hemicellulases capable of breaking down lignocellulosic biomass. We report the cloning of this T. terrestris CDH gene (cbdA), its recombinant expression in Aspergillus oryzae, and purification and characterization of the T. terrestris CDH protein (TtCDH). The TtCDH shows spectral properties and enzyme activity similar to other characterized CDH enzymes. Substrate specificity was determined for a number of carbohydrate electron donors in the presence of the two-electron acceptor 2,6-dichlorophenol-indophenol. The TtCDH also shows dramatic synergy with Thermoascus aurantiacus glycoside hydrolase family 61A protein in the presence of a ß-glucosidase for the cleavage of cellulose.
Asunto(s)
Deshidrogenasas de Carbohidratos/biosíntesis , Celulosa/metabolismo , Proteínas Fúngicas/biosíntesis , Sordariales/enzimología , Deshidrogenasas de Carbohidratos/química , Deshidrogenasas de Carbohidratos/genética , Deshidrogenasas de Carbohidratos/metabolismo , Celobiosa/química , Celulosa/química , Clonación Molecular , Medios de Cultivo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Cinética , Proteoma/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Cellobiose dehydrogenase (CDH) gene transcripts were quantified by reverse transcription-PCR (RT-PCR) in cultures of Phanerochaete chrysosporium supplemented with various cello- and xylooligosaccharides in order to elucidate the mechanism of enhanced CDH production in xylan/cellulose culture. Cellotriose and cellotetraose induced cdh expression, while xylobiose and xylotriose induced expression of cellobiohydrolase genes, especially cel7C.
Asunto(s)
Deshidrogenasas de Carbohidratos/biosíntesis , Regulación Fúngica de la Expresión Génica , Oligosacáridos/metabolismo , Phanerochaete/enzimología , Medios de Cultivo/química , Perfilación de la Expresión Génica , Phanerochaete/genética , Phanerochaete/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular hemoflavoenzyme produced by lignocellulose-degrading fungi including Pycnoporus cinnabarinus. We investigated the cellulolytic system of P. cinnabarinus, focusing on the involvement of CDH in the deconstruction of lignocellulosic biomass. RESULTS: First, P. cinnabarinus growth conditions were optimized for CDH production. Following growth under cellulolytic conditions, the main components secreted were cellulases, xylanases and CDH. To investigate the contribution of P. cinnabarinus secretome in saccharification processes, the Trichoderma reesei enzymatic cocktail was supplemented with the P. cinnabarinus secretome. A significant enhancement of the degradation of wheat straw was observed with (i) the production of a large amount of gluconic acid, (ii) increased hemicellulose degradation, and (iii) increased overall degradation of the lignocellulosic material. P. cinnabarinus CDH was heterologously expressed in Pichia pastoris to obtain large amounts of pure enzyme. In a bioreactor, the recombinant CDH (rCDH) expression level reached 7800 U/L. rCDH exhibited values of biochemical parameters similar to those of the natural enzyme, and was able to bind cellulose despite the absence of a carbohydrate-binding module (CBM). Following supplementation of purified rCDH to T. reesei enzymatic cocktail, formation of gluconic acid and increased hemicellulose degradation were observed, thus confirming the previous results observed with P. cinnabarinus secretome. CONCLUSIONS: We demonstrate that CDH offers an attractive tool for saccharification process enhancement due to gluconic acid production from raw lignocellulosic material.
Asunto(s)
Deshidrogenasas de Carbohidratos/biosíntesis , Pichia/enzimología , Pycnoporus/enzimología , Deshidrogenasas de Carbohidratos/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Lignina/metabolismo , Pichia/genética , Pycnoporus/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Adipose tissue glucocorticoid action relies on local enzymatic interconversion and glucocorticoid receptor (GR) availability. 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1), 2 (11ß-HSD2) and hexose-6-phosphate dehydrogenase (H6PDH) are likely involved in glucocorticoid activation/inactivation within adipose tissue. We examined adipose tissue mRNA expression of genes related to glucocorticoid action and their association with total and visceral adiposity. Messenger RNA was measured in paired subcutaneous and omental fat samples obtained from 56 women (age: 47.3 ± 4.8 years, BMI: 27.1 ± 5.2 kg/m(2)) undergoing gynaecological surgery. Expression levels of 11ß-HSD2, H6PDH and GRα were higher in omental adipose tissue while 11ß-HSD1 expression was similar between fat compartments. Subcutaneous and omental 11ß-HSD1 mRNA abundances were positively associated with total and visceral adiposity whereas omental H6PDH mRNA abundance was negatively associated with these measures. Only omental 11ß-HSD1 mRNA expression remained significantly associated with visceral adipose tissue area following statistical adjustment for fat mass, age and menopausal status. Omental 11ß-HSD1 mRNA expression explained 19.1% of the variance in visceral adipose tissue area. Omental fat tissue 11ß-HSD-1 protein and cortisol levels were higher in visceral obese women, supporting findings obtained with 11ß-HSD-1 mRNA. These results suggest that among the transcripts examined only omental 11ß-HSD1 is independently associated with visceral obesity in women.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Glucocorticoides/metabolismo , Grasa Intraabdominal/fisiología , Grasa Subcutánea/fisiología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 2/genética , Adulto , Western Blotting , Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/genética , Femenino , Humanos , Grasa Intraabdominal/enzimología , Grasa Intraabdominal/metabolismo , Modelos Lineales , Persona de Mediana Edad , ARN Mensajero/química , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Grasa Subcutánea/enzimología , Grasa Subcutánea/metabolismoRESUMEN
Cellobiose dehydrogenase (CDH; EC 1.1.99.18) is an extracellular glycosylated protein composed of two distinct domains, a C-terminal catalytic flavin domain and an N-terminal cytochrome-b-type heme domain, which transfers electrons from the flavin domain to external electron acceptors. The soluble flavin domain of the Phanerochaete chrysosporium CDH was successfully expressed in Escherichia coli. The enzyme showed dye-mediated CDH activity higher than that of the complete CDH, composed of flavin domain and heme domain, prepared using Pichia pastoris as the host microorganism. The ability to conveniently express the recombinant CDH flavin domain in E. coli provides great opportunities for the molecular engineering of the catalytic properties of CDH.
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Deshidrogenasas de Carbohidratos/biosíntesis , Deshidrogenasas de Carbohidratos/genética , Celobiosa/metabolismo , Escherichia coli/metabolismo , Expresión Génica , Phanerochaete/enzimología , Estabilidad de Enzimas , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Phanerochaete/genética , Pichia/genética , Pichia/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6-phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11beta-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/enzimología , Deshidrogenasas de Carbohidratos/genética , Diferenciación Celular , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/enzimología , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/biosíntesis , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/genética , Células 3T3-L1 , Adipogénesis , Animales , Deshidrogenasas de Carbohidratos/biosíntesis , Linaje de la Célula , Cortisona/metabolismo , Cortisona Reductasa/metabolismo , Inducción Enzimática , Humanos , Hidrocortisona/metabolismo , Ratones , Oxidación-Reducción , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We cloned and expressed a gene encoding a thermostable cellobiose dehydrogenase (CDH) from the thermophilic ascomycete Myriococcum thermophilum. The 2904bp long open reading frame contained six introns located either close to the 5'- or 3'-end of the ORF. The corresponding cDNA of 2487bp was cloned into the expression vector pPICZalphaB to achieve inducible heterologous expression and secretion of the recombinant flavocytochrome in the methylotrophic yeast Pichia pastoris. Transformants were selected on media with normal and 10-fold increased zeocin concentration, and selected clones were tested for inducible extracellular production of the recombinant oxidoreductase. The maximally obtained volumetric activity was 0.25U/ml in YPM (rich) medium and 2.15U/ml in production stage (minimal) medium in a fed-batch fermentation. Recombinant CDH was purified in two consecutive chromatographic steps leading to a final specific activity of up to 7.4U/mg protein at 40 degrees C. Kinetic properties of the recombinant CDH were characterized and the temperature optimum for the recombinant CDH was determined at 63 degrees C. Certain properties of the sequence of MtCDH are discussed in context with thermal and proteolytic stability.