RESUMEN
The composition and structure of gut microbiota plays an important role in obesity induced by a high-fat diet (HFD) and related metabolic syndrome (MetS). Previous studies have shown that galacto-oligosaccharides (GOSs) have an effective anti-obesity effect. In this study, we aimed to investigate the effect of enzymatically synthesized α-galacto-oligosaccharides (ES-α-GOSs) on MetS and gut microbiota dysbiosis in HFD-fed mice, and to further investigate whether the attenuation of MetS is associated with the modulation of gut microbiota. Our results indicated that ES-α-GOS could notably ameliorate obesity-related MetS, including hyperlipidemia, insulin resistance and mild inflammation. The subsequent analysis of gut microbiota further showed that ES-α-GOS supplements can significantly modulate the overall composition of the gut microbiota and reverse the gut microbiota disorder caused by HFD feeding. Moreover, Spearman correlation analysis showed that 40 key bacteria reversed by ES-α-GOS were highly associated with metabolic parameters. These results suggested that ES-α-GOSs could serve as a potential candidate for preventing obesity-induced MetS in association with the modulation of gut microbiota.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Microbioma Gastrointestinal/efectos de los fármacos , Microbioma Gastrointestinal/fisiología , Síndrome Metabólico/microbiología , Oligosacáridos/farmacología , Tejido Adiposo/patología , Animales , Aspergillus niger/metabolismo , Bacterias , Glucemia , Suplementos Dietéticos , Disbiosis , Dislipidemias , Galactosidasas/metabolismo , Hiperlipidemias , Inflamación , Resistencia a la Insulina , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismoRESUMEN
Prebiotics are defined as selectively fermented food ingredients that induce specific changes in the composition and/or activity in the gastrointestinal microbiota beneficial to the host well-being and health. The aim of the presented experiment was to investigate the effect of a prebiotic applied alone or in combination with Hyppocastani extractum siccum, and Lini oleum virginale in rats with dimethylhydrazine induced colon cancer. Wistar albino rats were fed high fat diet supplemented with the prebiotic alone or in combination with Horse chestnut and flaxseed oil. The activity of faecal glycolytic enzymes, lipid parameters, bile acids, short chain fatty acids and counts of coliforms and lactobacilli were determined. Treatment with the prebiotic alone and in combination with selected substances significantly decreased the activity of glycolytic bacterial enzyme ß-glucuronidase (P<0.001) and increased activities of ß-galactosidase and ß-glucosidase. Bile acids concentration was significantly decreased (P<0.01) except for the combination of the prebiotic with Horse chestnut. The prebiotic alone decreased the lipid parameters (P<0.001) and enhanced production of short chain fatty acids. Application of prebiotic and bioactive natural substances significantly reduced number of coliforms (P<0.05). Prebiotic alone significantly increased the count of lactobacilli (P<0.05). These results show that prebiotics have a protective effect and may be the useful for colon cancer prevention and treatment.
Asunto(s)
Colon/microbiología , Neoplasias del Colon/microbiología , Inulina/administración & dosificación , Lactobacillus/efectos de los fármacos , Aceite de Linaza/administración & dosificación , Extractos Vegetales/administración & dosificación , Prebióticos , 1,2-Dimetilhidrazina , Aesculus/química , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ácidos y Sales Biliares/sangre , Neoplasias del Colon/sangre , Neoplasias del Colon/inducido químicamente , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Heces/química , Heces/enzimología , Heces/microbiología , Galactosidasas/química , Galactosidasas/metabolismo , Glucosidasas/química , Glucosidasas/metabolismo , Glucuronidasa/química , Glucuronidasa/metabolismo , Concentración de Iones de Hidrógeno , Lactobacillus/enzimología , Lactobacillus/metabolismo , Lípidos/sangre , Ratas , Ratas WistarRESUMEN
We here describe a simple and efficient synthetic method for a non-hydrolysable precursor of a GDP-fucose analogue: The synthesis of the racemic aminofuranofucitol 3 from sorbic alcohol by nitroso-Diels-Alder reaction. This 'all-cis-pyrrolidine', with all substituents occupying a cis position, has been determined as a potent inhibitor of α-L-fucosidase and a moderate inhibitor of α- and ß-D-galactosidase. The good recognition of this fucose moiety analogue by specific enzymes is thus confirmed. The C-anomeric bond in this particular structure is in the ß-position and makes this compound an interesting candidate for further chemical modifications. Influence of the methyl and hydroxymethyl groups on the inhibition potency is discussed.
Asunto(s)
1-Desoxinojirimicina/análogos & derivados , Inhibidores Enzimáticos/síntesis química , Galactosidasas/antagonistas & inhibidores , Pirrolidinas/química , Alcoholes del Azúcar/síntesis química , alfa-L-Fucosidasa/antagonistas & inhibidores , 1-Desoxinojirimicina/síntesis química , 1-Desoxinojirimicina/química , 1-Desoxinojirimicina/farmacología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Galactosidasas/metabolismo , Isomerismo , Pirrolidinas/síntesis química , Pirrolidinas/farmacología , Alcoholes del Azúcar/química , Alcoholes del Azúcar/farmacología , alfa-L-Fucosidasa/metabolismoRESUMEN
A collection of new reversible glycosidase inhibitors of the iminoalditol type featuring N-substituents containing perfluorinated regions has been prepared for evaluation of physicochemical, biochemical and diagnostic properties. The vast variety of feasible oligofluoro moieties allows for modular approaches to customised structures according to the intended applications, which are influenced by the fluorine content as well as the distance of the fluorous moiety from the ring nitrogen. The first examples, in particular in the D-galacto series, exhibited excellent inhibitory activities. A preliminary screen with two human cell lines showed that, at subinhibitory concentrations, they are powerful pharmacological chaperones enhancing the activities of the catalytically handicapped lysosomal D-galactosidase mutants associated with GM1 gangliosidosis and Morquio B disease.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Galactosidasas/antagonistas & inhibidores , Gangliosidosis GM1/tratamiento farmacológico , Alcoholes del Azúcar/química , Alcoholes del Azúcar/farmacología , Línea Celular , Café/enzimología , Inhibidores Enzimáticos/uso terapéutico , Escherichia coli/enzimología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Galactosidasas/metabolismo , Halogenación , Humanos , Iminas/química , Iminas/farmacología , Iminas/uso terapéutico , Rhizobium/enzimología , Alcoholes del Azúcar/uso terapéuticoRESUMEN
Mature seeds of yellow lupine contained sucrose, raffinose family oligosaccharides (RFOs), and galactosyl cyclitols as major soluble carbohydrates. The study showed that RFOs dominated in lupine seeds (16% DW). The disappearance of both types of alpha-d-galactosides in germinating lupine seeds was strongly inhibited by the presence of jasmonic acid-methyl ester (JA-Me) at a concentration of 10(-3)M in the incubation medium. JA-Me inhibited the activity of alpha-D-galactosidase (fraction I) during seed germination. Anatomical studies of lupine roots have shown certain cell structure differences between control and JA-Me-treated seedlings. The cross-sections of plant roots treated with JA-Me showed a characteristic folding of the cell walls in all root tissues, starting from the rhyzodermis, cortex and vascular cylinder. In water-treated (control) plants, the cell walls were rounded with no folding.
Asunto(s)
Acetatos/farmacología , Carbohidratos/análisis , Ciclopentanos/farmacología , Germinación/efectos de los fármacos , Lupinus/crecimiento & desarrollo , Lupinus/metabolismo , Oxilipinas/farmacología , Semillas/efectos de los fármacos , Semillas/crecimiento & desarrollo , Galactosidasas/metabolismo , Lupinus/efectos de los fármacos , Extractos Vegetales/metabolismo , Raíces de Plantas/anatomía & histología , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Rafinosa/metabolismoAsunto(s)
Formación de Anticuerpos , Terapia Biológica , Enfermedad de Fabry/inmunología , Enfermedad de Fabry/terapia , Galactosidasas/administración & dosificación , Galactosidasas/inmunología , Relación Dosis-Respuesta a Droga , Enfermedad de Fabry/enzimología , Femenino , Galactosidasas/metabolismo , Humanos , MasculinoRESUMEN
OBJECTIVE: We compared the effects of grapefruit flavonoids and inulin, as single dietary components or in a combination, on cecal fermentation in rats adapted to a semipurified diet. METHODS: The experimental diets contained 0.3% flavonoid extract and 5% or 10% inulin and a combination of both supplements. The large bowel metabolism assessment was based on cecal parameters: bulk effect, pH, microbial enzymes activity, and short-chain fatty acid production. RESULTS: Both supplements induced significant enlargement of the cecal digesta weight. Acidification of cecal digesta was more pronounced, with a higher inulin addition to the diet. Cecal pH was the highest with the flavonoid-rich diets and lowest in the case of a simultaneous addition of flavonoids and a high content of inulin. The flavonoid extract applied as a single dietary supplement was observed to decrease the activity of bacterial beta-glucosidase and beta- and alpha-galactosidases in the cecal digesta. In contrast, addition of the grapefruit extract to inulin-containing diets increased the activity of alpha-glucosidase, alpha-galactosidase, and beta-galactosidase. Great accumulation of cecal digesta in rats consuming the flavonoid-diet caused a considerable increase in the short-chain fatty acid pool, mainly acetic acid. Inulin added to the diet decreased the excessive enlargement of digesta caused by dietary flavonoids. Dietary addition of inulin to the flavonoid-diet also normalized hydration of cecal digesta and significantly decreased the pH of digesta. The presence of polyphenols in the inulin-containing diets did not change total short-chain fatty acid production in the cecum of rats. CONCLUSION: Our results suggested that simultaneous intake of inulin and polyphenols can decrease the detrimental effects of the latter on cecal fermentation.
Asunto(s)
Ciego/metabolismo , Citrus paradisi/química , Digestión/efectos de los fármacos , Fermentación/efectos de los fármacos , Flavonoides/farmacología , Inulina/farmacología , Fenoles/farmacología , Adaptación Fisiológica , Animales , Ciego/química , Ciego/microbiología , Suplementos Dietéticos , Digestión/fisiología , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Ácidos Grasos Volátiles/biosíntesis , Fermentación/fisiología , Flavonoides/administración & dosificación , Galactosidasas/metabolismo , Concentración de Iones de Hidrógeno , Inulina/administración & dosificación , Masculino , Fenoles/administración & dosificación , Polifenoles , Distribución Aleatoria , Ratas , Ratas Wistar , beta-Glucosidasa/metabolismoRESUMEN
Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a beta-D-Galp-(1-->4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all (1)H and (13)C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a beta-(1-->3)-linkage instead of the expected beta-(1-->4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->4)-alpha/beta-Galp-OH and beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->3)-alpha/beta-Galp-OH. The fact that the deviating beta-(1-->3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The beta-(1-->3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion).
Asunto(s)
Disacáridos/análisis , Galactanos/química , Conformación de Carbohidratos , Cromatografía en Gel , Citrus/química , Galactosidasas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Oligosacáridos/aislamiento & purificación , Cebollas/química , Solanum tuberosum/química , Glycine max/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Golgi-enriched enzyme preparations prepared from etiolated pea epicotyls incorporated [U-(14)C]galactose from UDP-[U-(14)C]galactose into the 1,4-beta-galactan sidechains of a pectin-xyloglucan complex. This complex could bind to paper and was degraded both by pectin-degrading enzymes and by a xyloglucan-specific endoglucanase. Gel permeation chromatography was used to assess the molecular size of the complex and of enzymically-degraded, galactan-containing fragments of it. Etiolated pea stems were labelled with [U-(14)C]sucrose for 1 h, and the newly-synthesised cell wall polysaccharides were extracted with EDTA or NaOH and fractionated by ion-exchange chromatography. The NaOH-extracted, acidic radioactive polysaccharides obtained in this way were also degraded both by pectin-degrading enzymes and by xyloglucan-specific endoglucanase. Analysis of the radioactive sugar composition indicated that neutral sugars characteristic of both pectin and xyloglucan were present. Analysis of the total non-radioactive, neutral sugar composition of the NaOH-extracted, acidic cell-wall polysaccharides indicated that pectin-xyloglucan complexes were a general feature of the cell wall in this tissue.
Asunto(s)
Pared Celular/metabolismo , Glucanos/metabolismo , Pectinas/metabolismo , Pisum sativum/citología , Pisum sativum/metabolismo , Xilanos/metabolismo , Celulasa/metabolismo , Galactosidasas/metabolismo , Glucanos/química , Pectinas/química , Tallos de la Planta/citología , Tallos de la Planta/metabolismo , Poligalacturonasa/metabolismo , Factores de Tiempo , Xilanos/químicaRESUMEN
Three experiments were conducted to test the hypothesis that supplementing nursery pig diets with a mixture of carbohydrases (CS) will improve pig performance and nutrient digestibility. The CS used in these experiments contained 7 units/g of alpha-1,6-galactosidase, 22 units/g of beta-1,4-mannanase, beta-1,4 mannosidase, and trace amounts of other enzymes. In Exp. 1, 108 pigs weaned at d 21 of age were fed one of three diets containing 0 (control), 0.1, or 0.2% CS for 5 wk, based on a three-phase feeding program (1, 2, and 2 wk). Over the entire 35-d period, ADG was not affected (P > 0.05) by treatment, but supplementing 0.1% CS increased (P < 0.05) gain:feed by 9%. Experiment 2 used 10 gilts fitted with simple T-cannula in the terminal ileum at 3 wk of age. After cannulation, pigs were fed the same control Phase I and II diets, but the Phase III diet contained either 0 or 0.1% CS. Ileal samples were collected for the 3 d following the 5-d adjustment period during Phase III. Apparent ileal digestibility of GE, lysine, threonine, and tryptophan was greater (P < 0.05) in the CS diet. In Exp. 3, 90 pigs weaned at 21 d of age were fed the same control Phase I and II diets, but the Phase III diet contained either 0 or 0.1% CS. Phase III diets were fed for 3 wk. Average daily gain of the CS group was greater (P < 0.05) than the control group during wk 3. Gain:feed ratio was greater (P < 0.05) for the carbohydrase group during the entire Phase III period. Four pigs per treatment were killed at the end of Exp. 3 to measure villus height and to determine the concentration of raffinose and stachyose in different parts of the gastrointestinal tract. Average villus height was greater (P < 0.05) in pigs fed the CS diet. Carbohydrase supplementation decreased (P < 0.05) the concentration of stachyose in freeze-dried digesta from the proximal and distal small intestine. Raffinose concentration, on the other hand, was decreased (P < 0.05) by CS supplementation only in the distal small intestine. These lower concentrations suggest that CS improved the digestibility of carbohydrate in soybean meal. In conclusion, the addition of CS to Phase I and Phase II nursery diets containing low levels of soybean meal did not improve pig performance, but its addition to corn-soybean meal-based Phase III nursery diets improved gain:feed ratio and energy and AA digestibility.
Asunto(s)
Alimentación Animal , Digestión , Glicósido Hidrolasas/administración & dosificación , Porcinos/crecimiento & desarrollo , Aminoácidos/metabolismo , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Suplementos Dietéticos , Relación Dosis-Respuesta a Droga , Galactosidasas/administración & dosificación , Galactosidasas/metabolismo , Glicósido Hidrolasas/metabolismo , Distribución Aleatoria , Glycine max , Porcinos/metabolismo , Zea mays , beta-Manosidasa/administración & dosificación , beta-Manosidasa/metabolismoRESUMEN
A benzoquinone, named alopecuquinone, was isolated from the ethanol extract of the inflorescences of Cyperus alopecuroides. Its structure was primarily elucidated by spectroscopic analysis including 1H, 13C NMR, APT, HMQC, 1H-1H COSY and CIMS. The known flavonoids, vicenin 2, orientin, diosmetin, quercetin 3,3'-dimethyl ether and its 3,4'-dimethyl ether, were also isolated and characterized. The ethanol extract of the plant material showed moderate estrogenic activity using a strain of Saccharomyces cerevisiae.
Asunto(s)
Benzoquinonas/química , Benzoquinonas/aislamiento & purificación , Cyperus/química , Flavonoides/química , Flavonoides/aislamiento & purificación , Isoflavonas , Magnoliopsida/química , Benzoquinonas/farmacología , Egipto , Estrógenos no Esteroides/química , Estrógenos no Esteroides/aislamiento & purificación , Estrógenos no Esteroides/farmacología , Flavonoides/farmacología , Galactosidasas/metabolismo , Humanos , Espectrometría de Masas/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Aceites Volátiles/análisis , Aceites Volátiles/química , Fitoestrógenos , Extractos Vegetales , Preparaciones de Plantas , Plantas Medicinales , Plásmidos/biosíntesis , Plásmidos/metabolismo , Receptores de Estrógenos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/enzimología , Espectrofotometría UltravioletaRESUMEN
Canestrato Pugliese cheeses from ewe milk were produced according to a traditional protocol and by adding 7.0 log10 cfu of fresh cells per gram of Bifidobacterium bifidum Bb02, Bifidobacterium longum Bb46, or both species. The traditional technology was modified slightly to favor the survival of probiotic microorganisms. After 56 d of ripening, the survival of B. bifidum Bb02 and B. longum Bb46 was 6.0 and 5.0 log10 cfu/g, respectively. After 19 d cheeses contained ca. 7.0 log10 cfu/g of bifidobacteria. Compared to traditional cheese, the addition of bifidobacteria seemed to support the growth and survival of mesophilic lactobacilli and Streptococcus thermophilus, used as starter, during ripening. No significant differences were observed in the main chemical composition, and only a slightly higher concentration of acetic acid was found in cheeses with bifidobacteria added. On the contrary, alpha- and beta-galactosidase activities were markedly more pronounced in the presence of bifidobacteria, especially with B. bifidum Bb02. In contrast with traditional cheese, the lactose was completely hydrolyzed in cheeses made with bifidobacteria. Urea-PAGE electrophoresis of the pH 4.6-soluble and pH 4.6-insoluble N fractions did not show appreciable variations. Only the reversed-phase-HPLC analysis of the pH 4.6-soluble N showed a slightly more complex profile in the presence of bifidobacteria. This finding was in agreement with the higher value of the pH 4.6-soluble N/total N ratio and with the more pronounced amino-, imino-, and dipeptidase activities found in all the cheeses with the bifidobacteria added, especially B. bifidum Bb02. No differences were found in the free amino acid and free fatty acid contents. The amino acids glutamic acid, valine, proline, leucine, and lysine and the fatty acids butyric, caproic, capric, and oleic acids were found at the highest concentrations. The sensory evaluation did not show significant differences, and Canestrato Pugliese cheeses were characterized by small and uniformly distributed eyes, were pale yellow, had an elastic consistency and a Pecorino-like smell, were very salty, and tended to be moderately piquant.
Asunto(s)
Bifidobacterium/crecimiento & desarrollo , Queso/microbiología , Streptococcus/crecimiento & desarrollo , Aminoácidos/análisis , Animales , Bifidobacterium/enzimología , Queso/análisis , Cromatografía Líquida de Alta Presión , Recuento de Colonia Microbiana , Ácidos Grasos/análisis , Galactosidasas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Nitrógeno/análisis , Probióticos , Ovinos , Streptococcus/enzimología , GustoRESUMEN
Ehrlich ascites tumor cells and ascitic fluid were assayed for glycosidase activity. alpha-Galactosidase and beta-galactosidase, alpha- and beta-mannosidase, alpha-N-acetylgalactosaminidase, and beta-N-acetylglucosaminidase activities were detected using p-nitrophenyl glycosides as substrates. alpha-Galactosidase and alpha-N-acetylgalactosaminidase were isolated from Ehrlich ascites tumor cells on epsilon-aminocaproylgalactosylamine-Sepharose. alpha-Galactosidase was purified 160,000-fold and was free of other glycosidase activities. alpha-N-Acetylgalactosaminidase was also purified 160,000-fold but exhibited a weak alpha-galactosidase activity which appears to be inherent in this enzyme. Substrate specificity of the alpha-galactosidase was investigated with 12 substrates and compared with that of the corresponding coffee bean enzyme. The pH optimum of the Ehrlich cell alpha-galactosidase centered near 4.5, irrespective of substrate, whereas the pH optimum of the coffee bean enzyme for PNP-alpha-Gal was 6.0, which is 1.5 pH units higher than that for other substrates of the coffee bean enzyme. The reverse was found for alpha-N-acetylgalactosaminidase: the pH optimum for the hydrolysis of PNP-alpha-GalNAc was 3.6, lower than the pH 4.5 required for the hydrolysis of GalNAc alpha 1,3Gal. Coffee bean alpha-galactosidase showed a relatively broad substrate specificity, suggesting that it is suited for cleaving many kinds of terminal alpha-galactosyl linkages. On the other hand, the substrate specificity of Ehrlich alpha-galactosidase appears to be quite narrow. This enzyme was highly active toward the terminal alpha-galactosyl linkages of Ehrlich glycoproteins and laminin, both of which possess Gal alpha 1, 3Gal beta 1,4GlcNAc beta-trisaccharide sequences. The alpha-N-acetylgalactosaminidase was found to be active toward the blood group type A disaccharide, and trisaccharide, and glycoproteins with type A-active carbohydrate chains.
Asunto(s)
Carcinoma de Ehrlich/enzimología , Galactosidasas/metabolismo , Hexosaminidasas/metabolismo , Semillas/enzimología , alfa-Galactosidasa/metabolismo , Animales , Ascitis/enzimología , Secuencia de Carbohidratos , Cromatografía de Afinidad , Café , Hexosaminidasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Cinética , Ratones , Datos de Secuencia Molecular , Especificidad por Sustrato , alfa-Galactosidasa/aislamiento & purificación , alfa-N-AcetilgalactosaminidasaRESUMEN
beta-D-galactosidase (beta-D-galactoside galactohydrolase, E.C. 3.2.1.23) activity was localised in the digestive tract of Setaria digitata. The enzyme extract shows maximum activity in the pH range between 3.5 and 5.0 and at 45 degrees C. The enzyme shows the Km value of 3.636 mM for the substrate 6-bromo-2-naphthyl beta-D-galactoside and Vmax of 28.57 nmol 6-bromo-2-naphthol liberated mg-1 protein min-1. Activation/inhibition of the enzyme by various ions, medicinal plants and drugs has been studied. Polyacrylamide gel electrophoresis revealed that the enzyme exists as single form. The medicinal plants and the drug filarin effectively inhibit the enzyme. The significance of these results are discussed in relation to chemotherapy.
Asunto(s)
Filarioidea/enzimología , Galactosidasas/metabolismo , beta-Galactosidasa/metabolismo , Animales , Dietilcarbamazina/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Galactósidos/metabolismo , Histocitoquímica , Concentración de Iones de Hidrógeno , Plantas Medicinales , Setariasis/parasitología , Temperatura , beta-Galactosidasa/antagonistas & inhibidoresRESUMEN
A fusion of DNA sequences encoding the SPO2 promoter, the alpha-amylase signal sequence from Bacillus amyloliquefaciens, and the mature part of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) was constructed on a Bacillus subtilis multicopy vector. Bacillus cells of the protease-deficient strain DB104 harboring this vector produced and secreted the plant enzyme alpha-galactosidase up to levels of 1,700 U/liter. A growth medium suppressing the residual proteolytic activity of strain DB104 was used to reach these levels in a fermentor. Purification of the secreted product followed by NH2-terminal amino acid sequencing showed that the alpha-amylase signal sequence had been processed correctly. The molecular mass of the product estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was slightly lower than that of the plant purified enzyme, which is most likely due to glycosylation of the latter. The alpha-galactosidase product was active both on the artificial substrate para-nitrophenyl-alpha-D-galactopyranoside and on the galactomannan substrate, guar gum. The activity of this Bacillus sp.-produced enzyme was similar to that of the glycosylated enzyme purified from guar seeds, indicating that glycosylation has no essential function for enzyme activity.
Asunto(s)
Bacillus subtilis/enzimología , Fabaceae/enzimología , Galactosidasas/metabolismo , Plantas Medicinales , alfa-Galactosidasa/metabolismo , Secuencia de Aminoácidos , Western Blotting , Fermentación , Datos de Secuencia Molecular , Plásmidos , Transformación Bacteriana , alfa-Galactosidasa/genéticaRESUMEN
The maximum conversion of glucose to fructose in lactase-hydrolyzed whey permeate by glucose isomerase was approximately 52% at .1 g enzyme/ml substrate after 7 h incubation at 60 degrees C. Removal of minerals from the substrate was essential for enzyme activity. The dependence of the enzyme on Mg++ and Co++ for activity in the presence of high ash concentration was demonstrated. Optimum Mg++ and Co++ additions were 250 and 100 ppm, respectively. The isomerization reaction was enhanced more when both 100 ppm Mg++ and 50 ppm Co++ were added. Hydrolyzed isomerized lactose whey syrup with sweetness equivalent to sucrose was successfully produced through enzymatic isomerization of glucose in lactase-hydrolyzed whey permeate after supplementation with pure glucose. Fructose in hydrolyzed isomerized lactose whey syrup was effectively separated from other sugars by Dowex 1X8-200 anion exchange resin in the bisulfite form.
Asunto(s)
Isomerasas Aldosa-Cetosa , Carbohidrato Epimerasas/metabolismo , Fructosa/metabolismo , Galactosidasas/metabolismo , Glucosa/metabolismo , Leche/metabolismo , beta-Galactosidasa/metabolismo , Animales , Bovinos , Cobalto/farmacología , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Lactosa/metabolismo , Magnesio/farmacologíaRESUMEN
The covalent attachment of polyethylene glycol (PEG) to beta-glucosidase from sweet almonds and alpha-galactosidase from green coffee beans results in alterations of their catalytic properties and masking of specific determinant sites on the enzymes. Both enzymes have increased Km and decreased Vmax values against their respective p-nitrophenyl substrate analogs after PEG attachment. When PEG is attached to 30% of alpha-galactosidase epsilon-amino groups, 12% activity remains against ceramide trihexoside, while its ability to convert type B erythrocytes to type H specificity is lost. However, it still is able to cleave terminal galactose residues from human saliva blood group substance B. PEG-beta-glucosidase (38%) did not elicit the production of complement-fixing antibodies, nor did it react with antibodies produced against the native enzyme. Antibody and lectin-specific binding were lost from both modified enzymes (PEG-beta-glucosidase and PEG-alpha-galactosidase). After conjugation with PEG, beta-glucosidase lost its ability to bind to concanavalin A-Sepharose. Antibodies directed against native alpha-galactosidase blocked its enzyme activity, but lost their ability to inhibit activity in progressively higher modified preparations of the enzyme. Antisera against PEG-alpha-galactosidase (53%) did not inhibit enzyme activity in any alpha-galactosidase or PEG-alpha-galactosidase preparation. These results indicate that PEG tends to cover lectin-specific carbohydrate moieties and antigenic determinants and that these sites probably remain cryptic during in vivo processing of PEG-enzymes.
Asunto(s)
Enzimas Inmovilizadas/metabolismo , Epítopos/análisis , Galactosidasas/metabolismo , Glucosidasas/metabolismo , alfa-Galactosidasa/metabolismo , beta-Glucosidasa/metabolismo , Complejo Antígeno-Anticuerpo , Café , Pruebas de Fijación del Complemento , Terapia Enzimática , Globósidos , Humanos , Sueros Inmunes , Cinética , Plantas/enzimología , Polietilenglicoles , alfa-Galactosidasa/inmunología , beta-Glucosidasa/inmunologíaRESUMEN
The effect of alpha-galactosidase, purified from Clostridium sporogenes (Maebashi), was examined on erythrocytes from rats, rabbits and gibbons. The amount of galactose released by alpha-galactosidase from Cl. sporogenes and from coffee beans was compared. The amount of sialic acid released by Vibrio cholera sialidase was also determined. Loss of blood group B specificity following treatment with alpha-galactosidase was demonstrated with anti-B lectin. In animal models, removal of all the alpha-galactosyl residues with the coffee bean or clostridial alpha-galactosidase resulted in no change in the sequestration pattern of the treated erythrocytes over a period of several days. In contrast, erythrocytes treated with sialidase were rapidly sequestered from the circulation.
Asunto(s)
Clostridium/enzimología , Café/enzimología , Envejecimiento Eritrocítico , Galactosidasas/metabolismo , alfa-Galactosidasa/metabolismo , Sistema del Grupo Sanguíneo ABO , Animales , Eritrocitos/metabolismo , Femenino , Galactosa , Galactosemias , Pruebas de Hemaglutinación , Hylobates , Masculino , Ácido N-Acetilneuramínico , Conejos , Ratas , Ácidos Siálicos/sangre , Especificidad por SustratoRESUMEN
alpha-Galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), an enzyme responsible for mobilizing the raffinose family of oligosaccharides in legume seeds, has been isolated from lentils (Lens culinaris) and purified about 4,000-fold. The Sephadex gel filtration profile showed the presence of two forms of the enzyme, alpha-galactosidase I with an apparent Mr = 160,000 and alpha-galactosidase II of Mr = 40,000. Enzyme II readily aggregates to form I when any attempt is made to concentrate the solution. Thus, only enzyme I was purified and its properties studied. The multistep purification procedure included affinity binding of the enzyme to concanavalin A-Sepharose, indicating its glycoprotein nature with glucose/mannose termini of the carbohydrate moieties. The amino acid and carbohydrate compositions of the native enzyme and that of the glycopeptide obtained from pronase-digested, denatured enzyme have been determined. Asparagine seems to be involved in forming the linkage with the carbohydrate moiety. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme I shows a single protein band with Mr = 40,000 which also stains with periodic acid-Schiff reagent. Thus, the enzyme consists of four identical glycoprotein subunits. The isoelectric point of the enzyme is 8.0. The pH optima of enzyme I and II are 6.1 and 4.7, respectively. The substrate specificity and the mode of substrate inhibition of enzyme I is discussed. The effect of temperature on Vmax and Km of the enzyme is presented; at pH 6.1 the energy of activation is 62.1 kJ/mol and the delta H value is -34.3 kJ/mol in the temperature range 20-50 degrees C. Preliminary studies show that enzyme I possesses hemagglutinating properties with glucose/mannose specificity.
Asunto(s)
Fabaceae/enzimología , Galactosidasas/metabolismo , Plantas Medicinales , Semillas/enzimología , alfa-Galactosidasa/metabolismo , Sustancias Macromoleculares , Matemática , Peso MolecularRESUMEN
The effect of phototherapy on intestinal mucosal bilirubin concentration and lactase activity in the Gunn rat was studied. Ten groups of six or seven animals each were studied. Heterozygous (Jj) and homozygous (jj) animals were given 24, 48, or 72 hr of continuous phototherapy, and Jj and jj animals were given 24- and 48-hr sham control treatments. Phototherapy reduced serum bilirubin concentration by 53% at 24 hr, 59% at 48 hr and 68% at 72 hr. The mucosal concentration of bilirubin appeared to parallel the declining serum concentration and was lower in treated than in control jj animals. The jejunal and ileal lactase activity, expressed per mg protein, was not depressed by phototherapy. The lactase activity of jejunum and of ileum of treated jj as compared to treated Jj and control jj at 24 and 48 hr and as compared to treated Jj at 72 hr was never reduced; i.e., jejunal lactase activity in jj treated for 72 hr was 15.1 +/- 4.2 (-x +/- S.D.) units/g protein as compared to 16.2 +/- 4.2 for Jj treated for 72 hr and 12.0 +/- 4.2 for jj 48-hr control animals. The ratio of lactase to sucrase activity demonstrated a significant increase in lactase activity relative to sucrase in all animals treated for 48 or 72 hr. This latter effect is potentially due to alteration in the circadian rhythm while under constant irradiance. These data convincingly demonstrate that the Gunn rat does not develop lactase deficiency consequent to phototherapy.