RESUMEN
Soluble starch-synthesizing enzymes, starch synthase (SSS) and starch-branching enzyme (SBE), were isolated, fractionated, and purified from white potato tubers (Solanum tuberosum) on a large scale. Five steps were used: potato tuber extract from 2 kg of peeled potatoes, two acetone precipitations, and two fractionations on a large ultrafiltration polysulfone hollow fiber 100 kDa cartridge. Three kinds of fractions were obtained: (1) mixtures of SSS and SBE; (2) SSS, free of SBE; and (3) SBE, free of SSS. Contaminating enzymes (amylase, phosphorylase, and disproportionating enzyme) and carbohydrates were absent from the 2nd acetone precipitate and from the column fractions, as judged by the Molisch test and starch triiodide test. Activity yields of 122% (300,000-400,000 units) of SSS fractions and 187% (40,000-50,000 units) of SBE fractions were routinely obtained from the cartridge. Addition of 0.04% (w/v) polyvinyl alcohol 50K and 1 mM dithiothreitol to the glycine buffer (pH 8.4) gave long-term stability and higher yields of SSS and SBE, due to activation of inactive enzymes. Several SSS and SBE fractions from the two fractionations had very high specific activities, indicating high degrees of purification. Polyacrylamide gel electrophoresis of selected SSS and SBE fractions gave two to five SSS and/or SBE activity bands, corresponding to the one to five protein bands present in the 2nd acetone precipitate.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/aislamiento & purificación , Fraccionamiento Químico/métodos , Tubérculos de la Planta/enzimología , Solanum tuberosum/enzimología , Almidón Sintasa/aislamiento & purificación , Almidón/biosíntesis , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Acetona/química , Secuencia de Carbohidratos , Precipitación Química , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Polímeros/química , Solubilidad , Almidón Sintasa/química , Almidón Sintasa/metabolismo , Especificidad por Sustrato , Sulfonas/química , UltrafiltraciónRESUMEN
L-DOPA alpha-glycosides were synthesized by reaction of L-DOPA with sucrose, catalyzed by four different glucansucrases from Leuconostoc mesenteroides B-512FMC, B-742CB, B-1299A, and B-1355C. The glucansucrases catalyzed the transfer of d-glucose from sucrose to the phenolic hydroxyl position-3 and -4 of L-DOPA. The glycosides were fractionated and purified by Bio-Gel P-2 column chromatography, and the structures were determined by (1)H NMR spectroscopy. The major glycoside was 4-O-alpha-d-glucopyranosyl L-DOPA, and the minor glycoside was 3-O-alpha-D-glucopyranosyl L-DOPA. The two glycosides were formed by all four of the glucansucrases. The ratio of the 4-O-alpha-glycoside to the 3-O-alpha-glycoside produced by the B-512FMC dextransucrase was higher than that for the other three glucansucrases. The glycosylation of L-DOPA significantly reduced the oxidation of the phenolic hydroxyl groups, which prevents their methylation, potentially increasing the use of L-DOPA in the treatment of Parkinson's disease. The use of one enzyme, glucansucrase, and sucrose as the D-glucosyl donor makes the synthesis considerably simpler and cheaper than the formerly published procedure using cyclomaltodextrin and cyclomaltodextrin glucanyltransferase, followed by glucoamylase, and beta-amylase hydrolysis.
Asunto(s)
Glicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Leuconostoc/enzimología , Levodopa/biosíntesis , Sacarosa/metabolismo , Biocatálisis , Glicósidos/metabolismo , Glicosilación , Glicosiltransferasas/química , Levodopa/metabolismoRESUMEN
Dopamine-HCl and L-DOPA-alpha-glycosides were prepared by reaction with cyclomaltohexaose, catalyzed by Bacillus macerans cyclomaltodextrin glucanyltransferase. The reaction gave maltodextrins attached to dopamine and L-DOPA; the maltodextrins were trimmed by reactions with glucoamylase and beta-amylase to produce alpha-glucosyl- and alpha-maltosyl-glycosides, respectively. The glucoamylase- or beta-amylase-treated dopamine- and L-DOPA-alpha-glycosides were fractionated and purified by BioGel P-2 gel-filtration column chromatography and preparative descending paper chromatography. Analysis by MALDI-TOF mass spectrometry and one- and two-dimensional NMR showed that the purified glycosides of dopamine and L-DOPA were glycosylated at the hydroxyl groups of positions 3 and 4 of the catechol ring. The major product was found to be 4-O-alpha-glycopyranosyl L-DOPA, and it was shown to be more resistant to oxidative tolerance experiments, involving hydrogen peroxide and ferrous ion, than L-DOPA. L-DOPA-alpha-glycosides are possibly more effective substitutes for L-DOPA in treating Parkinson's disease in that they are more resistant to oxidation and methylation, which renders L-DOPA ineffective and deleterious.
Asunto(s)
Dopamina/síntesis química , Glucosiltransferasas/metabolismo , Glicósidos/síntesis química , Levodopa/análogos & derivados , Bacillus/enzimología , Dopamina/metabolismo , Levodopa/síntesis química , Levodopa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Polisacáridos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Remaud-Simeon and co-workers [Moulis, C.; Joucla, G.; Harrison, D.; Fabre, E.; Potocki-Veronese, G.; Monsan, P.; Remaud-Simeon, M. J. Biol. Chem., 2006, 281, 31254-31267] have recently proposed that a truncated Escherichia coli recombinant B-512F dextransucrase uses sucrose and the hydrolysis product of sucrose, D-glucose, as initiator primers for the nonreducing-end synthesis of dextran. Using (14)C-labeled D-glucose in a dextransucrase-sucrose digest, it was found that <0.02% of the D-glucose appears in a dextran of M(n) 84,420, showing that D-glucose is not an initiator primer, and when the dextran was treated with 0.01 M HCl at 80 degrees C for 90 min and a separate sample with invertase at 50 degrees C for 24h, no D-fructose was formed, indicating that sucrose is not present at the reducing-end of dextran, showing that sucrose also was not an initiator primer. It is further shown that both d-glucose and dextran are covalently attached to B-512FMC dextransucrase at the active site during polymerization. A pulse reaction with [(14)C]-sucrose and a chase reaction with nonlabeled sucrose, followed by dextran isolation, reduction, and acid hydrolysis, gave (14)C-glucitol in the pulsed dextran, which was significantly decreased in the chased dextran, showing that the D-glucose moieties of sucrose are added to the reducing-ends of the covalently linked growing dextran chains. The molecular size of dextran is shown to be inversely proportional to the concentration of the enzyme, indicating a highly processive mechanism in which D-glucose is rapidly added to the reducing-ends of the growing chains, which are extruded from the active site of dextransucrase. It is also shown how the three conserved amino acids (Asp551, Glu589, and Asp 622) at the active sites of glucansucrases participate in the polymerization of dextran and related glucans from a single active site by the addition of the D-glucose moiety of sucrose to the reducing-ends of the covalently linked glucan chains in a two catalytic-site, insertion mechanism.
Asunto(s)
Dextranos/biosíntesis , Glucosiltransferasas/metabolismo , Leuconostoc/enzimología , Biocatálisis , Dominio Catalítico , Secuencia Conservada , Dextranos/química , Durapatita/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/metabolismo , Escherichia coli/genética , Glucosa/química , Glucosa/metabolismo , Glucosiltransferasas/química , Glucosiltransferasas/genética , Hidrólisis , Cinética , Peso Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Sacarosa/metabolismoRESUMEN
Porcine pancreatic and Bacillus amyloliquefaciens alpha-amylases were examined for the formation of covalent carbohydrate intermediates during reaction. The enzymes were precipitated and denatured by adding 10 volumes of acetone. When these denatured enzymes were mixed with methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and chromatographed on BioGel P-2, no carbohydrate was found in the protein void volume peak. When the enzymes were added to the methyl alpha-6-[(3)H]-maltooligosaccharide glycosides and allowed to react for 15s at 1 degrees C and then precipitated and denatured with 10 volumes of acetone, (3)H-labeled carbohydrates were found in the BioGel P-2 protein void volume peak, indicating the formation of enzyme-carbohydrate covalent intermediates. (1)H NMR analysis of the denatured enzyme from the reaction with methyl alpha-maltooligosaccharide glycosides confirmed that carbohydrate was attached to the denatured enzyme. (1)H NMR saturation-transfer analysis further showed that the carbohydrate was attached to the denatured enzyme by a beta-configuration. This configuration is what would be expected for an enzyme that catalyzes the hydrolysis of alpha-(1-->4) glycosidic linkages by a two-step, S(N)2 double-displacement reaction to give retention of the alpha-configuration of the substrates at the reducing-end of the products.
Asunto(s)
Bacillus/enzimología , Glicósidos/metabolismo , Oligosacáridos/metabolismo , Páncreas/enzimología , alfa-Amilasas/metabolismo , Animales , Conformación de Carbohidratos , Catálisis , Hidrólisis , Espectroscopía de Resonancia Magnética , PorcinosRESUMEN
Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and methyl alpha-D-glucopyranoside, methyl beta-D-glucopyranoside, phenyl alpha-D-glucopyranoside, and phenyl beta-D-glucopyranoside gave four kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual d-glucopyranosides to cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%, depending on the particular D-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5-1.0 gave a wide range of sizes from d.p. 2-17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p. 9-17, decreased, and the small and intermediate sizes, d.p. 2-8, increased; as the molar ratios were increased further from 3.0 to 5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4-8, decreased, and the small sizes, d.p. 2 and 3 became predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios of d-glucose to cyclomaltohexaose.
Asunto(s)
Bacillus/enzimología , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Glicósidos/biosíntesis , Polisacáridos/biosíntesis , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Glucosa/química , Glucosa/metabolismo , Glicósidos/química , Datos de Secuencia Molecular , Polisacáridos/químicaRESUMEN
Beta-Salicin is a naturally occurring glycoside found in the bark of poplar and willow trees. Ancient man used it as an analgesic and antipyretic. It has a D-glucopyranose unit attached by a beta-linkage to the phenolic hydroxyl of salicyl alcohol. Two new salicin analogues have been enzymatically synthesized by transglycosylation reactions: (a) by the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and salicyl alcohol, followed by reactions with alpha amylase and glucoamylase to give D-glucopyranose attached by an alpha-linkage to the phenolic hydroxyl of salicyl alcohol as the major product, alpha-salicin; and (b) by the reaction of Leuconostoc mesenteroides B-742CB dextransucrase with sucrose and salicyl alcohol, followed by reactions with dextranase and glucoamylase to give alpha-d-glucopyranose attached to the primary alcohol hydroxyl of salicyl alcohol as the major product, alpha-isosalicin.
Asunto(s)
Bacillus/enzimología , Alcoholes Bencílicos/química , Alcoholes Bencílicos/metabolismo , Glucosiltransferasas/metabolismo , Leuconostoc/enzimología , Alcoholes Bencílicos/aislamiento & purificación , Secuencia de Carbohidratos , Cromatografía en Capa Delgada , Glucano 1,4-alfa-Glucosidasa/metabolismo , Glucósidos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Peso Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sacarosa/metabolismo , alfa-Amilasas/metabolismoRESUMEN
Acarbose analogues, 4IV-maltohexaosyl acarbose (G6-Aca) and 4IV-maltododecaosyl acarbose (G12-Aca), were prepared by the reaction of cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and acarbose. The inhibition kinetics of acarbose and the two acarbose analogues were studied for four different alpha-amylases: Aspergillus oryzae, Bacillus amyloliquefaciens, human salivary, and porcine pancreatic alpha-amylases. The three inhibitors showed mixed, noncompetitive inhibition, for all four alpha-amylases. The acarbose inhibition constants, Ki, for the four alpha-amylases were 270, 13, 1.27, and 0.80 microM, respectively; the Ki values for G6-Aca were 33, 37, 14, and 7 nM, respectively; and the G12-Aca Ki constants were 59, 81, 18, and 11 nM, respectively. The G6-Aca and G12-Aca analogues are the most potent alpha-amylase inhibitors observed, with Ki values one to three orders of magnitude more potent than acarbose, which itself was one to three orders of magnitude more potent than other known alpha-amylase inhibitors.
Asunto(s)
Acarbosa/análogos & derivados , Acarbosa/farmacología , Inhibidores Enzimáticos/farmacología , alfa-Amilasas/antagonistas & inhibidores , Animales , Bacillus/enzimología , Cromatografía en Capa Delgada , Glucosa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Páncreas/enzimología , Saliva/enzimología , Porcinos , alfa-Amilasas/metabolismoRESUMEN
The specificity of Saccharomyces cerevisiae yeast on the removal of carbohydrates by fermentation was studied. The common monosaccharides, D-glucose, D-fructose, D-mannose, and D-galactose were completely removed; D-glucuronic acid and D-ribose were partially removed; but D-xylose, D-rhamnose, and L-sorbose were not removed and were completely resistant. Of four glycosides, methyl and phenyl alpha- and beta-D-glucopyranosides, three of the four were partially removed and methyl beta-D-glucopyranoside was not removed. The disaccharides, maltose, sucrose, and turanose were completely removed, while cellobiose, lactose, and melibiose were completely resistant. Isomaltose and alpha,alpha-trehalose were partially removed. Maltotriose and raffinose were partially removed, but isomaltotriose and melezitose were completely resistant. The tetrasaccharides, maltotetraose, isomaltotetraose, and acarbose, were completely resistant. Further, the yeast enzymes did not alter any of the resistant carbohydrates by transglycosylation or condensation reactions or by any other types of reactions.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Fermentación , Saccharomyces cerevisiae/metabolismo , Cromatografía en Capa Delgada/métodos , Monosacáridos/metabolismo , Oligosacáridos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologíaRESUMEN
Two new acarbose analogues were synthesized by the reaction of acarbose with sucrose and dextransucrases from Leuconostoc mesenteroides B-512FMC and B-742CB. The major products for each reaction were subjected to yeast fermentation, and then separated and purified by Bio-Gel P2 gel permeation chromatography and descending paper chromatography. The structures of the products were determined by one- and two-dimensional 1H and 13C NMR spectroscopy and by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). B-512FMC-dextransucrase produced one major acarbose product, 2(I)-alpha-D-glucopyranosylacarbose and B-742CB-dextransucrase produced two major acarbose products, 2(I)-alpha-D-glucopyranosylacarbose and 3(IV)-alpha-D-glucopyranosylacarbose.
Asunto(s)
Acarbosa/análogos & derivados , Glucosiltransferasas/metabolismo , Leuconostoc/enzimología , Acarbosa/síntesis química , Acarbosa/aislamiento & purificación , Cromatografía , Fermentación , Glicosilación , Resonancia Magnética Nuclear Biomolecular , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sacarosa/químicaRESUMEN
It was found that Bacillus macerans cyclomaltodextrin glucanotransferase (CGTase) reacts with cyclomaltohexaose (alpha-cyclodextrin, alpha-CD) to give a series of cyclomaltooligosaccharides (cyclomaltodextrins, CDs), having seven to more than 20 D-glucose residues and maltooligosaccharides (maltodextrins, MDs) from G5 to G12+. When D-glucose (Glc) was added to the alpha-CD at very low molar ratios (1:100) of Glc to alpha-CD, the predominant products (95%) were CDs, some of which were macrocyclic MDs with 20-60 D-glucose residues, along with MDs that also had high molecular weights, containing 10-75 D-glucose residues and gave a blue iodine-iodide color. As the molar ratio of Glc to alpha-CD was increased, the amount of CDs progressively decreased and MDs proportionately increased in the range of G2-G12. At 25 mM alpha-CD and Glc to alpha-CD molar ratio of 1:1, a 75% yield of MDs, G1-G12, each in approximately equal amounts, was obtained; and at 20 mM and a 5:1 ratio, a 97% yield of MDs, G2-G9, was obtained but in unequal amounts. At higher ratios (10:1), the CDs completely disappeared, and at very high ratios (50:1 to 100:1) only low-molecular-weight MDs, G2-G4, were formed.
Asunto(s)
Bacillus/metabolismo , Glucosiltransferasas/metabolismo , alfa-Ciclodextrinas , Bacillus/enzimología , Cromatografía en Capa Delgada , Ciclodextrinas/análisis , Ciclodextrinas/biosíntesis , Ciclodextrinas/metabolismo , Glucosa/metabolismo , Glicosilación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
New kinds of acarbose analogues were synthesized by the reaction of acarbose with cyclomaltohexaose and cyclomaltodextrin glucanyltransferase (CGTase). Three major CGTase coupling products were separated and purified by Bio-Gel P2 gel-permeation chromatography. Digestion of the three products by beta-amylase and glucoamylase showed that they were composed of maltohexaose (G6), maltododecaose (G12), and maltooctadecaose (G18), respectively, attached to the nonreducing-end of acarbose. 13C NMR of the glucoamylase product (D-glucopyranosyl-acarbose) showed that the D-glucose moiety was attached alpha- to the C-4-OH group of the nonreducing-end cyclohexene ring of acarbose, indicating that the maltodextrins were attached alpha-(1-->4) to the nonreducing-end cyclohexene of acarbose.