RESUMEN
Enzymatically modified isoquercitrin (EMIQ), oligoglucosyl naringenin-7-(glucose [G]), and oligoglucosyl hesperetin (H)-7-G were produced via oligoglucosylation of quercetin-3-glucose, naringenin-7-G (prunin), and H-7-G, respectively, by cyclodextrin glucosyltransferase from Bacillus macerans. The aim was to explore the oligoglucosylation and the resulting changes in physicochemical properties. Water solubility of EMIQ, oligoglucosyl prunin, and oligoglucosyl H-7-G enormously increased in comparison with that of their aglycones. Glycosylation of an aglycone generally enhances its solubility. Resistance of the aglycones to oxidative degradation by the Cu2+ ion was strongly increased by the oligoglucosylation. This is probably because oligoglucosylation may protect sensitive parts of an aglycones molecule from the Cu2+ oxidation. Only EMIQ maintained its structure during thermal treatment much longer than quercetin did. Degradation of flavonoid aglycones by ultraviolet light C irradiation at 254nm was not affected, and their antioxidant activities gradually decreased with the greater extent of oligoglucosylation.
Asunto(s)
Bacillus/química , Ciclodextrinas/química , Flavanonas/química , Flavonoides/química , SolubilidadRESUMEN
The C-terminal domain of the three-domain-comprising trehalose synthase from Thermus thermophilus was truncated in order to study the effect on the enzyme's activity and substrate specificity. Compared with the wild-type (WT) enzyme, the two truncated enzymes (DM1 and DM2) showed lower maltose- and trehalose-converting activities and a different transglycosylation reaction mechanism. In the mutants, the glucose moiety cleaved from the maltose substrate was released from the enzyme and intercepted by external glucose oxidase, preventing the production of trehalose. The WT enzyme, however, retained the glucose in the active site to effectively produce trehalose. In addition, DM1 synthesized much higher amounts of mannose-containing disaccharide trehalose analog (Man-TA) than did the WT and DM2. The results suggest that the C-terminal domain in the WT enzyme is important for retaining the glucose moiety within the active site. The mutant enzymes could be used to produce Man-TA, a postulated inhibitor of gut disaccharidases.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Thermus thermophilus/enzimología , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Disacáridos/metabolismo , Glucosiltransferasas/genética , Glicosilación , Microbiología Industrial , Cinética , Maltosa/metabolismo , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Especificidad por Sustrato , Thermus thermophilus/genética , Trehalosa/metabolismoRESUMEN
To control the surface properties of a commonly used polymer, poly(methyl methacrylate) (PMMA), poly(perfluoromethyl methacrylate)s (PFMMAs) with short perfluorinated side groups (i.e., -CF3, -CF2CF3, -(CF3)2, -CF2CF2CF3) were used as blend components because of their good solubility in organic solvents, low surface energies, and high optical transmittance. The surface energies of the blend films of PFMMA with the -CF3 group and PMMA increased continuously with increasing PMMA contents from 17.6 to 26.0 mN/m, whereas those of the other polymer blend films remained at very low levels (10.2-12.6 mN/m), similar to those of pure PFMMAs, even when the blends contained 90 wt %PMMA. Surface morphology and composition measurements revealed that this result originated from the different blend structures, such as lateral and vertical phase separations. We expect that these PFMMAs will be useful in widening the applicable window of PMMA.
RESUMEN
A gene encoding an endo-ß-1,3-glucanase from Streptomyces matensis ATCC 23935 (SmßG) was characterised by cloning and expressing it in Escherichiacoli. The purified enzyme produced ß-1,3-glucooligosaccharides, mainly laminaripentaose, from insoluble curdlan. The optimum pH and temperature were 6.0 and 55 °C, respectively. SmßG inhibited the growth of Candida albicans, which indicates that this enzyme could be potentially used as an anti-fungal agent to control invasive Candida infections. The results suggest that SmßG may be a useful bioavailable ß-1,3-glucanase.
Asunto(s)
Antifúngicos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clonación Molecular , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Streptomyces/enzimología , Secuencia de Aminoácidos , Antifúngicos/metabolismo , Antifúngicos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Estabilidad de Enzimas , Glicósido Hidrolasas/metabolismo , Glicósido Hidrolasas/farmacología , Streptomyces/química , Streptomyces/genética , Especificidad por SustratoRESUMEN
Useful nucleoside diphosphate (NDP)-sugars and α-galacto-oligosaccharides were synthesized by recombinant Escherichia coli whole cells and compared to those produced by enzyme-coupling. Production yields of NDP-glucoses (Glcs) by whole cells harboring trehalose synthase (TS) were 60% for ADP-Glc, 82% for GDP-Glc, and 27% for UDP-Glc, based on NDP used. Yield of UDP-galactose (Gal) by the whole-cell harboring a UDP-Gal 4-epimerase (pGALE) was 26% of the quantity of UDP-Glc. α-Galacto-oligosaccharides, α-Gal epitope (Galα-3Galß-4Glu) and globotriose (Galα-4Galß-4Glu), were produced by the combination of three recombinant whole cells harboring TS, pGALE, and α-galactosyltransferase, with production yields of 48% and 54%, based on UDP, respectively. Production yields of NDP-sugars and α-galacto-oligosaccharides by recombinant whole-cell reactions were approximately 1.5 times greater than those of enzyme-coupled reactions. These results suggest that a recombinant whole-cell system using cells harboring TS with trehalose as a substrate may be used as an alternative and practical method for the production of NDP-sugars and α-galacto-oligosaccharides.
Asunto(s)
Escherichia coli/metabolismo , Oligosacáridos/biosíntesis , Biotecnología , Escherichia coli/genética , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Azúcares de Nucleósido Difosfato/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trehalosa/metabolismoRESUMEN
A new sugar nucleotide cycling (SNC) process was established in a one-pot three enzyme-coupled reaction using disaccharide trehalose. Trehalose synthase from Pyrococcus horikoshii could be applied to the SNC process for the synthesis of functional α-galactosyl oligosaccharides, α-galactose (Gal) epitopes and globotriose, using the effective regeneration of UDP-Gal. The α-Gal epitopes and globotriose were found to attach to the cell-surface of enteropathogenic Escherichia coli O127 (EPEC) which were bound to human Caco-2 cells. These α-galactosyl oligosaccharides were able to prohibit the attachment of EPEC, which could have resulted in colonization and disease. The α-Gal epitope III with a lactulose acceptor showed the most inhibitory activity of anti-adhesion. The results suggest that the α-galactosyl oligosaccharides may be alternative anti-adhesion molecules that overcome antibiotic resistance.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Biotecnología/economía , Biotecnología/métodos , Glucosiltransferasas/metabolismo , Oligosacáridos/biosíntesis , Pyrococcus horikoshii/enzimología , Trehalosa/metabolismo , Animales , Células CACO-2 , Bovinos , Análisis Costo-Beneficio , Epítopos/metabolismo , Galactosa/biosíntesis , Humanos , Oligosacáridos/farmacología , Trisacáridos/biosíntesisRESUMEN
UNLABELLED: Enzymatic bioconversion of rutin to quercetin-3-O-glucoside (Q-3-G) by Penicillium decumbens naringinase was increased with reaction pH increased approximately to pH 6.0. It resulted in greater than 92% production of Q-3-G due to the removal of the terminal rhamnose at the controlled pH 6.0. The enzymatic bioconversion of rutin to Q-3-G was repetitively performed, yielding 84% after 5 batches with little quercetin formation. Interestingly, the water solubility of Q-3-G was enhanced 69- and 328-fold over those of rutin and quercetin, which may make Q-3-G more bioavailable in food. Q-3-G was approximately 6- and 1.4-fold more potent than rutin as an inhibitor of human intestinal maltase and human DL-3-hydroxy-3-methylglutalyl coenzyme A reductase. Q-3-G was less potent (16- and 1.3-fold, respectively) than quercetin as an inhibitor of these enzymes. However, the results suggest that Q-3-G may be confirmed more effective and bioavailable food component than rutin and even quercetin because of its enhanced solubility and inhibitory properties. PRACTICAL APPLICATION: Bioconverted intermediate, quercetin-3-O-glucoside (Q-3-G), was found and confirmed to be largely more soluble than rutin and quercetin in water solution, which might make it more bioavailable as food ingredient. In addition, Q-3-G inhibited mildly the intestinal maltase, which might act as antidiabetic substance by modulating the adsorption of glucose in the intestine.
Asunto(s)
Flavonoides/biosíntesis , Complejos Multienzimáticos/metabolismo , Penicillium/metabolismo , Rutina/química , beta-Glucosidasa/metabolismo , Disponibilidad Biológica , Flavonoides/farmacología , Glucósidos , Inhibidores de Glicósido Hidrolasas , Humanos , Concentración de Iones de Hidrógeno , Hidroximetilglutaril-CoA Reductasas/biosíntesis , Quercetina/análogos & derivados , Ramnosa/metabolismo , SolubilidadRESUMEN
The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21(WAF1) was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21(WAF1) function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21(WAF1) is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Interleucinas/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Ciclo Celular , Línea Celular Tumoral , Movimiento Celular , Citocinas/metabolismo , Humanos , Microscopía Confocal/métodos , Modelos Biológicos , FN-kappa B/metabolismo , Nanopartículas/química , Invasividad Neoplásica , Regiones Promotoras Genéticas , Factores de TiempoRESUMEN
Hesperetin-7-O-glucoside (Hes-7-G) was produced by the enzymatic conversion of hesperidin by Aspergillus sojae naringinase due to the removal of the terminal rhamnose. Extracts from orange juice and peel containing the hesperidin were so treated by this enzyme that the hesperidin could also be converted to Hes-7-G. The solubility of Hes-7-G in 10% ethanol was enhanced 55- and 88-fold over those of hesperidin and hesperetin, respectively, which may make Hes-7-G more bioavailable. Hes-7-G was 1.7- and 2.4-fold better than hesperidin and hesperetin, respectively, in the inhibition of human intestinal maltase. Hes-7-G was more potent by 2- and 4-fold than hesperidin in the inhibition of human HMG-CoA reductase. Additionally, Hes-7-G exhibited more effective inhibition of the growth of Helicobacter pylori than hesperetin, while its effectiveness was similar to that of hesperidin. Therefore, the results suggest that bioconverted Hes-7-G is more effective and bioavailable than hesperidin, as it has enhanced inhibitory and solubility properties.
Asunto(s)
Aspergillus/enzimología , Citrus/química , Inhibidores Enzimáticos/química , Helicobacter pylori/efectos de los fármacos , Hesperidina/análogos & derivados , Hesperidina/química , Intestinos/enzimología , Extractos Vegetales/química , Biocatálisis , Biotransformación , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/química , Inhibidores de Glicósido Hidrolasas , Infecciones por Helicobacter/microbiología , Helicobacter pylori/crecimiento & desarrollo , Hesperidina/farmacología , Humanos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Intestinos/efectos de los fármacos , Estructura Molecular , Complejos Multienzimáticos/química , Extractos Vegetales/farmacología , alfa-Glucosidasas/metabolismo , beta-Glucosidasa/químicaRESUMEN
A gene encoding a putative UDP-glucose 4-epimerase (pGALE) in Pyrococcus horikoshii was cloned and expressed in Escherichia coli. The purified enzyme could reversibly catalyze both the synthesis of UDP-Gal and UDP-Glc but preferred the binding of UDP-Gal by approximately 10-fold. The optimum pH and temperature were 6.5 and 65°C. The enzyme acted effectively without the addition of nicotinamide adenine dinucleotide (NAD(+)), possibly due to the presence of tightly bound NAD(+). In particular, pGALE could be coupled with trehalose synthase (TreT) from P. horikoshii to regenerate UDP-Gal from UDP. The possible byproduct of glycosyltransferase, UDP, was capable of being converted to UDP-Glc with trehalose by TreT, and UDP-Glc was simultaneously converted to UDP-Gal by pGALE. Conclusively, the results suggest that pGALE and TreT with trehalose is an effective one-pot two-enzyme system for the regeneration of UDP-Gal, a high-cost substrate of galactosyltransferase, to complete a sugar nucleotide cycle.
Asunto(s)
Pyrococcus horikoshii/enzimología , Trehalosa/metabolismo , UDPglucosa 4-Epimerasa/metabolismo , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato/metabolismo , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Cartilla de ADN , Concentración de Iones de Hidrógeno , Reacción en Cadena de la Polimerasa , Especificidad por Sustrato , TemperaturaRESUMEN
Many microorganisms produce trehalose for stability and survival against various environmental stresses. Unlike the widely distributed trehalose-biosynthetic pathway, which utilizes uridine diphosphate glucose and glucose-6-phosphate, the newly identified enzyme trehalose glycosyltransferring synthase (TreT) from hyperthermophilic bacteria and archaea synthesizes an α,α-trehalose from nucleoside diphosphate glucose and glucose. In the present study, we determined the crystal structure of TreT from Pyrococcus horikoshii at 2.3 Å resolution to understand the detailed mechanism of this novel trehalose synthase. The conservation of essential residues in TreT and the high overall structural similarity of the N-terminal domain to that of trehalose phosphate synthase (TPS) imply that the catalytic reaction of TreT for trehalose synthesis would follow a similar mechanism to that of TPS. The acceptor binding site of TreT shows a wide and commodious groove and lacks the long flexible loop that plays a gating role in ligand binding in TPS. The observation of a wide space at the fissure between two domains and the relative shift of the N-domain in one of the crystal forms suggest that an interactive conformational change between two domains would occur, allowing a more compact architecture for catalysis. The structural analysis and biochemical data in this study provide a molecular basis for understanding the synthetic mechanism of trehalose, or the nucleotide sugar in reverse reaction of the TreT, in extremophiles that may have important industrial implications.
Asunto(s)
Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Pyrococcus horikoshii/enzimología , Trehalosa/biosíntesis , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico/genética , Cristalografía por Rayos X , Dimerización , Glucosiltransferasas/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Conformación Proteica , Estructura Cuaternaria de Proteína , Pyrococcus horikoshii/genética , Pyrococcus horikoshii/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
An extracellular enzyme (RMEBE) possessing alpha- (1-->4)-(1-->6)-transferring activity was purified to homogeneity from Rhodothermus marinus by combination of ammonium sulfate precipitation, Q-Sepharose ion-exchange, and Superdex- 200 gel filtration chromatographies, and preparative native polyacrylamide gel electrophoresis. The purified enzyme had an optimum pH of 6.0 and was highly thermostable with a maximal activity at 80 degrees . Its half-life was determined to be 73.7 and 16.7 min at 80 and 85 degrees , respectively. The enzyme was also halophilic and highly halotolerant up to about 2 M NaCl, with a maximal activity at 0.5M. The substrate specificity of RMEBE suggested that it possesses partial characteristics of both glucan branching enzyme and neopullulanase. RMEBE clearly produced branched glucans from amylose, with partial alpha-(1-->4)-hydrolysis of amylose and starch. At the same time, it hydrolyzed pullulan partly to panose, and exhibited alpha-(1-->4)-(1-->6)-transferase activity for small maltooligosaccharides, producing disproportionated alpha-(1-->6)-branched maltooligosaccharides. The enzyme preferred maltopentaose and maltohexaose to smaller maltooligosaccharides for production of longer branched products. Thus, the results suggest that RMEBE might be applied for production of branched oligosaccharides from small maltodextrins at high temperature or even at high salinity.
Asunto(s)
Amilasas/química , Amilasas/aislamiento & purificación , Espacio Extracelular/enzimología , Rhodothermus/enzimología , Agua de Mar/microbiología , Amilasas/genética , Amilasas/metabolismo , Amilopectina , Amilosa/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Peso Molecular , Oligosacáridos/metabolismo , Almidón/metabolismo , Especificidad por Sustrato , Temperatura , Transferasas/química , Transferasas/genética , Transferasas/aislamiento & purificación , Transferasas/metabolismoRESUMEN
Naringin, an active flavonoid found in citrus fruit extracts, has pharmacological utility. The present study identified a novel mechanism of the anticancer effects of naringin in urinary bladder cancer cells. Naringin treatment resulted in significant dose-dependent growth inhibition together with G(1)-phase cell-cycle arrest at a dose of 100 microM (the half maximal inhibitory concentration) in 5637 cells. In addition, naringin treatment strongly induced p21WAF1 expression, independent of the p53 pathway, and downregulated expression of cyclins and cyclin dependent kinases (CDKs). Moreover, treatment with naringin induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. Among the pathways examined, only PD98059, an ERK-specific inhibitor, blocked naringin-dependent p21WAF1 expression. Consistently, blockade of ERK function reversed naringin-mediated inhibition of cell proliferation and decreased cell-cycle proteins. Furthermore, naringin treatment increased both Ras and Raf activation. Transfection of cells with dominant-negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, the naringin-induced reduction in cell proliferation and cell-cycle proteins also was abolished in the presence of RasN17 and RafS621A mutant genes. These data demonstrate that the Ras/Raf/ERK pathway participates in p21WAF1 induction, subsequently leading to a decrease in the levels of cyclin D1/CDK4 and cyclin E-CDK2 complexes and naringin-dependent inhibition of cell growth. Overall, these unexpected findings concerning the molecular mechanisms of naringin in 5637 cancer cells provide a theoretical basis for the therapeutic use of flavonoids to treat malignancies.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Flavanonas/farmacología , Fase G1/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Ciclina D , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ciclinas/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genes Dominantes , Humanos , Inmunoprecipitación , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Oncogénicas/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/farmacología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
A gene encoding a putative glycogen branching enzyme (SmGBE) in Streptococcus mutans was expressed in Escherichia coli and purified. The biochemical properties of the purified enzyme were examined relative to its branching specificity for amylose and starch. The activity of the approximately 75kDa enzyme was optimal at pH 5.0, and stable up to 40°C. The enzyme predominantly transferred short maltooligosyl chains with a degree of polymerization (dp) of 6 and 7 throughout the branching process for amylose. When incubated with rice starch, the enzyme modified its optimal branch chain-length from dp 12 to 6 with large reductions in the longer chains, and simultaneously increased its branching points. The results indicate that SmGBE can make a modified starch with much shorter branches and a more branched structure than to native starch. In addition, starch retrogradation due to low temperature storage was significantly retarded along with the enzyme reaction.
RESUMEN
A gene encoding a putative glycogen-debranching enzyme in Sulfolobus shibatae (abbreviated as SSGDE) was cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by heat treatment and Ni-NTA affinity chromatography. The recombinant SSGDE was extremely thermostable, with an optimal temperature at 85 degrees C. The enzyme had an optimum pH of 5.5 and was highly stable from pH 4.5 to 6.5. The substrate specificity of SSGDE suggested that it possesses characteristics of both amylo-1,6-glucosidase and alpha-1,4-glucanotransferase. SSGDE clearly hydrolyzed pullulan to maltotrlose, and 6-O-alpha-maltosyl-beta-cyclodextrin (G2-beta-CD) to maltose and beta-cyclodextrin. At the same time, SSGDE transferred maltooligosyl residues to the maltooligosaccharides employed, and maltosyl residues to G2-beta-CD. The enzyme preferentially hydrolyzed amylopectin, followed in a decreasing order by glycogen, pullulan, and amylose. Therefore, the present results suggest that the glycogen-debranching enzyme from S. shibatae may have industrial application for the efficient debranching and modification of starch to dextrins at a high temperature.
Asunto(s)
Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Sulfolobus/enzimología , Amilopectina/metabolismo , Amilosa/metabolismo , Fraccionamiento Químico , Cromatografía de Afinidad , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Expresión Génica , Glucanos/metabolismo , Glucógeno/metabolismo , Concentración de Iones de Hidrógeno , Maltosa/metabolismo , Oligosacáridos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Sulfolobus/genética , Temperatura , beta-Ciclodextrinas/metabolismoRESUMEN
Magnolol, an active component extracted from Magnolia officinalis, has been reported to inhibit the development of atherosclerotic disease. However, it is not known whether magnolol exerts similar cardioprotective effects in cells treated with TNF-alpha. In the present study, magnolol treatment was found to show potent inhibitory effects on cell proliferation in cultured VSMC in the presence of TNF-alpha. These inhibitory effects were associated with reduced extracellular signal-regulated kinase (ERK) 1/2 activity and G1 cell cycle arrest. Magnolol treatment strongly induced the expression of p21WAF1, but resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins involved in G1 progression. In addition to G1 cell cycle arrest and growth inhibition in VSMC, magnolol also caused the strong inhibition of TNF-alpha-induced matrix metalloproteinase-9 (MMP-9) expression in a dose-dependent manner as determined by zymography and immunoblot. Moreover, magnolol treatment strongly decreased MMP-9 promoter activity in response to TNF-alpha. We further demonstrated that magnolol reduced the transcriptional activity of NF-kappaB and activation protein-1 (AP-1), two important nuclear transcription factors that are involved in MMP-9 expression. Collectively, these results show that magnolol inhibits cell proliferation, G1 to S phase cell cycle progress and MMP-9 expression through the transcription factors NF-kappaB and AP-1 in TNF-alpha-induced VSMC. The findings of the present study reveal a potential mechanism that explains the anti-atherogenic activity of magnolol.
Asunto(s)
Compuestos de Bifenilo/farmacología , Proliferación Celular/efectos de los fármacos , Lignanos/farmacología , Metaloproteinasa 9 de la Matriz/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Compuestos de Bifenilo/química , Células Cultivadas , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Relación Dosis-Respuesta a Droga , Fase G1/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Immunoblotting , Lignanos/química , Magnolia/química , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Extractos Vegetales/química , Extractos Vegetales/farmacología , Factor de Transcripción AP-1/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
beta-Galactosyl-trehalose oligosaccharides (beta-GTOs) were enzymatically prepared as a mixture of 6-beta-galactosyl-trehalose (1) and 4-beta-galactosyl-trehalose (2) with a 9:1 ratio (w/w). The beta-GTO mixture showed a highly enhanced hygroscopicity as compared to those of trehalose and other sugars used. At 72 h of incubation under 90% relative humidity and room temperature, it had a large increase in weight due to its moisture absorption, which was five times larger than that of trehalose, 1.9 times larger than that of sucrose, and 1.5 times larger than that of maltotriose. It was very effective in the growth promotion of Bifidobacteria, such as Bifidobacterium longum and Bifidobacterium bifidum, which was better than the growth promotion in the cases of trehalose and galactooligosaccharide. It also showed a highly anticariogenic property; it had only 10% cell proliferation of Streptococcus sobrinus for that of the sucrose control and 60% inhibition of insoluble glucan synthesis. Its effectiveness of inhibition was two and 1.5 times better than that of trehalose and one and two times than xylitol, respectively, against cell growth and glucan synthesis. Conclusively, the functionality of the beta-GTO in terms of hygroscopicity, bifidogenicity, and anticariogenicity was considerably improved as compared to that of trehalose. It is thus suggested that the beta-GTO might be applied as an effective humectant and prebiotic substitute with enhanced noncariogenicity in food applications.
Asunto(s)
Anticarcinógenos/farmacología , Galactosa/metabolismo , Oligosacáridos/biosíntesis , Oligosacáridos/farmacología , Trehalosa/metabolismo , Bifidobacterium/efectos de los fármacos , Bifidobacterium/crecimiento & desarrollo , Fenómenos Químicos , Química Física , Reacción de Maillard , Oligosacáridos/química , Streptococcus sobrinus/efectos de los fármacos , Streptococcus sobrinus/crecimiento & desarrolloRESUMEN
The gene encoding beta-glucosidase of the marine hyperthermophilic eubacterium Thermotoga neapolitana (bglA) was subcloned and expressed in Escherichia coli. The recombinant BglA (rBglA) was efficiently purified by heat treatment at 75 degrees C, and a Ni-NTA affinity chromatography and its molecular mass were determined to be 56.2 kDa by mass spectrometry (MS). At 100 degrees C, the enzyme showed more than 94% of its optimal activity. The half-life of the enzyme was 3.6 h and 12 min at 100 and 105 degrees C, respectively. rBglA was active toward artificial (p-nitrophenyl beta-D: -glucoside) and natural substrates (cellobiose and lactose). The enzyme also exhibited activity with positional isomers of cellobiose: sophorose, laminaribiose, and gentiobiose. Kinetic studies of the enzyme revealed that the enzyme showed biphasic behavior with p-nitrophenyl beta-D: -glucoside as the substrate. Whereas metal ions did not show any significant effect on its activity, dithiothreitol and beta-mercaptoethanol markedly increased enzymatic activity. When arbutin and cellobiose were used as an acceptor and a donor, respectively, three distinct intermolecular transfer products were found by thin-layer chromatography and recycling preparative high-performance liquid chromatography. Structural analysis of three arbutin transfer products by MS and nuclear magnetic resonance indicated that glucose from cellobiose was transferred to the C-3, C-4, and C-6 in the glucose unit of acceptor, respectively.
Asunto(s)
Thermotoga neapolitana/enzimología , beta-Glucosidasa/metabolismo , Secuencia de Aminoácidos , Biotecnología/métodos , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Glicosilación , Calor , Cinética , Datos de Secuencia Molecular , Agua de Mar/microbiología , Especificidad por Sustrato , Thermotoga neapolitana/genética , beta-Glucosidasa/genéticaRESUMEN
The gene encoding beta-glycosidase of the hyperthermophilic archaea Sulfolobus shibatae (SSG) was expressed in Escherichia coli. Recombinant SSG (referred to as rSSG hereafter) was efficiently purified, and its transglycosylation activity was tested with lactose as a donor and various sugars as acceptors. When sucrose was used as an acceptor, we found a distinct intermolecular transglycosylation product and confirmed its presence by TLC and high performance anion exchange chromatography (HPAEC). The sucrose transglycosylation product was isolated by paper chromatography, and its chemical structure was determined by 1H and 13C NMR. The sucrose transfer product was determined to be beta-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-beta-d-fructofuranoside with a galactose molecule linked to sucrose via a beta-(1-->6)-glycosidic bond.
Asunto(s)
Glucosidasas/química , Glucosidasas/metabolismo , Sacarosa/análogos & derivados , Sacarosa/metabolismo , Sulfolobus solfataricus/enzimología , Cromatografía en Capa Delgada , Glucosidasas/genética , Glucosidasas/aislamiento & purificación , Glicosilación , Espectroscopía de Resonancia Magnética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sacarosa/química , Sulfolobus solfataricus/genéticaRESUMEN
A gene (ORF PH1035), annotated to encode an uncharacterized hypothetical protein in Pyrococcus horikoshii, was first cloned and expressed in Escherichia coli. The recombinant enzyme was purified to homogeneity by Ni-NTA affinity chromatography and its molecular mass was determined to be 49,871Da by MALDI-TOF mass spectrometry. When the purified enzyme was reacted with nucleoside diphosphate-glucoses including UDP-glucose as a donor and glucose, rather than glucose-6-phosphate, as an acceptor, it specifically created a free trehalose. The enzyme was also able to partly hydrolyze the trehalose to glucose. The optimum pH was 5.5 and the enzyme was highly stable from pH 6 to 8. The deduced amino acid sequence showed a high homology with that of the glycosyl transferase group 1 (Pfam00534) in the BLAST search. The results suggest that the enzyme is a novel glycosyltransferase catalyzing the synthesis of the trehalose in the archaeon.