Showing papers on "Melibiose published in 1999"

Gustatory Response by the Hymenopteran Parasitoid Cotesia glomerata to a Range of Nectar and Honeydew Sugars

Abstract: The feeding response of food-deprived Cotesia glomerata to solutions of 14 naturally occurring sugars was determined. Glucose, fructose, sucrose, maltose, erlose, melezitose, trehalose, and stachyose all elicited a feeding response. The sugars differed, however, with respect to the lowest concentration at which they were accepted (acceptance threshold). The parasitoids showed no feeding response when presented with 2 M solutions of galactose, mannose, rhamnose, lactose, raffinose, and melibiose. Sugars from the latter group did not show a deterrent effect when offered to water-deprived parasitoids. When mannose, rhamnose, melibiose, or raffinose were combined with low molar solutions of either fructose or sucrose, sucrose acceptance was affected by mannose and raffinose, whereas no negative interactions were found in mixtures with fructose. Compared to acceptance thresholds reported in other insect systems, the responses of C. glomerata differ considerably with respect to both the range of saccharides accepted as well as the acceptance thresholds. The novel finding that the parasitoid accepts a number of sugars that fail to elicit a feeding response in its herbivorous hosts is of particular interest to the use of (selective) food supplements in biological control programs.

Cloning of the Gene Encoding a Novel Thermostable α-Galactosidase from Thermus brockianus ITI360

Abstract: An a-galactosidase gene from Thermus brockianus ITI360 was cloned, sequenced, and expressed in Escherichia coli, and the recombinant protein was purified. The gene, designated agaT, codes for a 476-residue polypeptide with a calculated molecular mass of 53,810 Da. The native structure of the recombinant enzyme (AgaT) was estimated to be a tetramer. AgaT displays amino acid sequence similarity to the a-galactosidases of Thermotoga neapolitana and Thermotoga maritima and a low-level sequence similarity to a-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme is thermostable, with a temperature optimum of activity at 93°C with para-nitrophenyl-a-galactopyranoside as a substrate. Half-lives of inactivation at 92 and 80°C are 100 min and 17 h, respectively. The pH optimum is between 5.5 and 6.5. The enzyme displayed high affinity for oligomeric substrates. The Kms for melibiose and raffinose at 80°C were determined as 4.1 and 11.0 mM, respectively. The a-galactosidase gene in T. brockianus ITI360 was inactivated by integrational mutagenesis. Consequently, no a-galactosidase activity was detectable in crude extracts of the mutant strain, and it was unable to use melibiose or raffinose as a single carbohydrate source.

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 alpha-galactosidase.

Abstract: Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an α-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083T. It was found to act with retention of configuration (α→α), releasing α-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As α-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of α-galactosides using the transgalactosylation activity of Bi. adolescentisα-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 °C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp and tetrasaccharide α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride α-d-Galp-(1 → 6)-α-d-Galp-(1 → 6)-d-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.

Cell death by overload of the elastin-laminin receptor on human activated lymphocytes: protection by lactose and melibiose.

Abstract: Background Activated human lymphocytes were shown to express the elastin–laminin receptor in vitro and also in vivo in atherosclerotic plaques. In the presence of the agonist, elastin peptides, this receptor was shown to mediate an increased cell proliferation and an increased synthesis and excretion of an elastase-type serine endopeptidase. In this study, we investigated the variation of the above reaction as a function of agonist concentration. Materials and methods Human lymphocytes were obtained by tonsillectomy and cultured in the presence of phytohaemagglutinin and elastin peptides. Cell viability was evaluated by vital dye exclusion. Elastase and cathepsin G activities were determined in culture supernates and cell lysates using synthetic substrates. Apoptotic cells were identified by the TUNEL method and by electron microscopy. Results At increasing concentrations of elastin peptides, a dose-dependent increase in cell death was observed. Up to 100 μg mL−1 elastin peptides and an increasing fraction of lymphocytes were found permeable to trypan blue, and a large proportion was in apoptosis. Elastin peptide-induced cell death was inhibited by 1 μg mL−1 lactose and melibiose. Conclusion We describe here cell death of human activated lymphocytes expressing the elastin–laminin receptor in the presence of increasing concentrations of elastin peptides, agonists of the receptor. The mechanism of cell death appears to be related to the triggering of the release of elastase and free radicals mediated by the elastin–laminin receptor. Antagonists of this receptor, lactose and melibiose, protected the lymphocytes from the receptor-mediated cell death.

Thermostable α‐galactosidase from Bacillus stearothermophilus NUB3621: cloning, sequencing and characterization

Abstract: An α-galactosidase gene from the thermophilic bacterium Bacillus stearothermophilus NUB3621 was cloned, sequenced, expressed in Escherichia coli and the recombinant protein was purified. The Bacillus enzyme, designated AgaN, is similar to α-galactosidases of family 36 in the classification of glycosyl hydrolases. The enzyme was estimated to be a tetramer with a molecular mass of subunits 80.3 kDa. The purified AgaN is thermostable and has a temperature optimum of activity at 75°C and a half-life of inactivation of 19 h at 70°C. AgaN displays high affinity for oligomeric substrates such as melibiose and raffinose and is able to hydrolyze raffinose in the presence of 60% sucrose with high efficiency.

Arg-52 in the melibiose carrier of Escherichia coli is important for cation-coupled sugar transport and participates in an intrahelical salt bridge.

Abstract: Bacterial secondary active transporters capture free energy from the movement of cations down their electrochemical gradient and use it to drive the transport of solutes such as sugars, amino acids, Krebs cycle intermediates, antibiotics, and inorganic ions across the cell membrane (27, 31, 33). The melibiose carrier (MelB) of Escherichia coli is a cation/substrate symporter which couples transport of Na+ and melibiose across the bacterial inner membrane (for reviews see references 22, 32, and 38). In addition to melibiose, MelB transports a variety of sugar substrates including α- and β-galactosides as well as some monosaccharides (46, 50). An interesting feature of MelB is its ability to couple sugar transport to three different cations, Na+, Li+, and H+, depending on the configuration of the transported sugar (44–46, 50). Sugar binding studies using membrane vesicles have shown that the presence of Na+ and Li+ ions increases the carrier’s affinity for galactosides and that the cations compete for a single binding site (6, 8). The melB gene has been cloned (18) and sequenced (52). The primary amino acid sequence deduced from the gene sequence predicts a hydrophobic protein (70% apolar) with a molecular mass of 52 kDa (52). The results of hydropathy analysis and melB-phoA fusions have provided good evidence for a two-dimensional structure where the protein forms 12 α-helical transmembrane domains connected by hydrophilic loops (1, 34, 52). E. coli MelB is a member of the galactoside-pentose-hexuronide family of bacterial transport proteins (32). The MelB subfamily consists of melibiose carriers from E. coli, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes. Although a high degree (78 to 85%) of amino acid identity exists among carriers in the MelB subfamily (30, 32), there are distinct differences in cation selectivity. For example, the MelB of E. coli couples H+, Na+, and Li+ to sugar transport, while the K. pneumoniae carrier couples either H+ or Li+, but not Na+ (16). The amino acid residues responsible for Na+ recognition were localized by constructing chimeras of the E. coli and K. pneumoniae melibiose carriers (15). Replacement of the first 81 amino acids of the K. pneumoniae carrier with those of the E. coli MelB was sufficient to allow the K. pneumoniae carrier to couple Na+ and sugar transport (15). Interestingly, a single-amino-acid substitution in helix II of K. pneumoniae, Ala-58→Asn, also resulted in Na+-coupled sugar transport (17). Cation recognition in E. coli MelB has also been investigated by site-directed mutagenesis. Studies have focused primarily on acidic residues that reside on membrane-spanning helices in the amino-terminal portion of the carrier. Neutral amino acid substitutions for Asp-19 (helix I), Asp-55 (helix II), Asp-59 (helix II), and Asp-124 (helix IV) cause the loss of Na+-coupled sugar transport (32, 35, 36, 53). In these mutants, sugar binding is comparable to that of wild-type MelB in the absence of Na+, but this binding is no longer stimulated by Na+. Taken together, the results of these studies have led to a model in which Asp residues at positions 19, 55, 59, and 124 provide part of a network for the coordination of cations in E. coli MelB (22, 32, 36, 54). The studies mentioned above show that the acidic amino acids on transmembrane helix II of the E. coli MelB, Asp-tt and Asp-59, are important for cation recognition. While the roles of these aspartates have been studied thoroughly, less is known about the positively charged residue Arg-52 in this helix. It has been reported that a substitution of Ala for Arg-52 leads to a 95% loss in carrier activity but that the remaining activity is still stimulated by Na+ and Li+ (54). In the present study, we further investigate the role of Arg-52. We use site-directed mutagenesis to substitute Gln, Val, and Ser for Arg-52 (R52Q, R52V, and R52S, respectively). We show that substitution of Arg-52 causes a dramatic loss of melibiose transport, with only a small amount of Na+-stimulated activity remaining. Subsequently, we use the Val-52, Ser-52, and Gln-52 mutant strains to isolate revertant strains which regain the ability to transport melibiose. Sequence analyses reveal that revertant mutations, with one exception, are found at locations other than position 52. The majority of these mutations result in substitution of amino acids located on transmembrane domains in both the amino and carboxyl halves of the protein. Our analysis of the melibiose transport properties in the strains with site-directed or second-site revertant mutations provide significant new information about the functional role of Arg-52. On the basis of our data, we suggest that specific transmembrane helices are close to one another in the three-dimensional structure of the protein. The data also suggest that Arg-52 is involved in an intrahelical salt bridge with Asp-55 and possibly in an interhelical salt bridge with Asp-19.

Comparison of melibiose utilizing baker's yeast strains produced by genetic engineering and classical breeding.

Abstract: S. F. VINCENT, P. J. L. BELL, P. BISSINGER AND K. M. H. NEVALAINEN 1999.Yeast strains currently used in the baking industry cannot fully utilize the trisaccharide raffinose found in beet molasses due to the absence of melibiase (a‐ galactosidase) activity. To overcome this deficiency, the MEL1 gene encoding melibiase enzyme was introduced into baker’s yeast by both classical breeding and recombinant DNA technology. Both types of yeast strains were capable of vigorous fermentation in the presence of high levels of sucrose, making them suitable for the rapidly developing Asian markets where high levels of sugar are used in bread manufacture. Melibiase expression appeared to be dosage-dependent, with relatively low expression sufficient for complete melibiose utilization in a model fermentation system.

Production and characterization of raffinose-hydrolysing and invertase activities of Aspergillus fumigatus

Abstract: Raffinose-type galactose oligosaccharides constitute a substantial part (40%) of the soluble sugars present in soybean seeds and are responsible for flatulence following ingestion of soybean and other legumes. Enzymic hydrolysis of these oligosaccharides would improve the nutritional value of soybean milk.Aspergillus fumigatus produces substantial raffinose-hydrolysing and invertase activities when grown on wheat straw. Three proteins displaying maximal activity at pH 4.5–5.5 and 55–60°C and having molar mass of 66.8, 50.3 and 30.2 kDa were purified. Raffinose and sucrose were hydrolyzed with equivalent affinities by each protein. Nevertheless, theK m andV lim values determined for hydrolysis of sucrose by the 66.8 kDa enzyme differed from those determined with the 50.3 kDa protein. Glucose was produced when sucrose was the substrate. The three proteins hydrolyzed also stachyose but not melibiose, maltose, inulin or 4-nitrophenyl α-d-galactopyranoside.A. fumigatus enzymes may be candidates for processing of soybean milk to reduce its flatulence potential.

Quantitative Model for Gal4p-Mediated Expression of the Galactose/Melibiose Regulon in Saccharomyces cerevisiae

Abstract: A mathematical model based on equilibrium binding between Gal4p and its specific DNA binding site has been developed. A model for GAL gene expression solely due to cooperativity, as a function of Gal4p concentration, has been developed for a gal80 mutant. The above model was extended to include other known regulatory molecules, namely Gal80p and Gal3p. Parameters determined from the above simulation were then used to represent a physiological status of gene expression in response to glucose (in terms of Gal4p concentration) and galactose in a wild-type strain. We demonstrate that in a wild-type strain glucose repression is more stringent due to cooperativity and autogenous regulation, while the induction response to galactose is only through autogenous regulation. The biological significance of autogenous regulation in Saccharomyces cerevisiae is discussed vis-a-vis the lactose operon of Escherichia coli.

Phenotypic Consequences Resulting from a Methionine-to-Valine Substitution at Position 48 in the HPr Protein of Streptococcus salivarius

Abstract: In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His 15 ) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser 46 ) by an ATP-dependent protein kinase (His∼P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His 15 by EI nor the phosphorylation at Ser 46 by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His∼P) than the wild-type strain, while the levels of free HPr and HPr(His∼P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of α-galactosidase, β-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.

Changes in the Carbohydrate Contents of Rehmanniae Radix during Processing

Abstract: 18 types of Rehmanniae Radix Preparatas, 9 types(from 1st to 9th steamed) of sun-dried and 9 types of oven-dried, were prepared, and the carbohydrates of Rehmanniae Radix Preparata was analized by HPLC. The results were: 1. The Rehmanniae Radix had several carbohydrates such as rhamnose, fucose, glucose, galactose, raffinose and stachyose, and the content of stachyose was the of total carbohydrates. 2. In the course of the processing, the oligosaccharides, such as stachyose and rafiinose, were rapidly degraded to melibiose and sucrose at first and second processing step. 3. Sucrose and melibiose gradually degraded with each additional processing. 4. The monosaccharides, such as rhamnose, fructose, glucose and galactose, was continuously degraded during the processing. These results suggested that Rehmannia radix preparata sufficiently developed only during second processing.

Production of glucosyltransferases by wild-type Leuconostoc mesenteroides in media containing sugars other than sucrose

Abstract: produces glucosyltransferases (GTFs) and fructosyltransferases (FTFs) which are inducible enzymes which respectively synthesize dextrans and levans from sucrose. Except for a few mutant strains which produce high activities in glucose medium, L. mesenteroides is thought not to produce GTFs and FTFs unless sucrose is present. We show here that cultures of eight strains produced low, but detectable GTF activity when glucose, maltose or melibiose replaced sucrose as the growth substrate. Four strains also produced FTFs of approximately 130 kDa in medium with or without sucrose. The GTFs and FTFs produced on sugars other than sucrose could be detected as bands on SDS gels even when not detected by other methods. Except for strain B-523, the number, sizes and relative intensities of the bands were independent of the sugar used for growing the cultures. Alternansucrase from strains B-1355 and B-1501 in glucose or maltose medium was almost entirely associated with the cell fraction, ruling out binding to glucans as the cause of the association.

Enzymatic regioselective deprotection of peracetylated mono- and disaccharides

Abstract: Selective enzymatic hydrolysis of the peracetylated disaccharides, namely cellobiose, lactose, maltose and melibiose, with lipase from Asperilligus niger in aqueous buffer and organic solvent for 30 min afforded exclusively the corresponding heptaacetates with a free hydroxyl group at C-1 in high yield. Prolonged reaction of the β-1,4 linked cellobiose and lactose peracetates afforded selectively their hexaacetates with free hydroxyl groups at C-1,2, whereas the α-1,4 linked disaccharides maltose and melibiose peracetate gave a complex mixture of products. The reaction of 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetylglucopyranose (11) for 22 h afforded as the major product the diacetate 12 with free hydroxyl groups at C-1,4.

O-glycopeptide analogues of enkephalin: FMOC-amino acid glycoside synthesis, solid-phase glycopeptide synthesis and optimizations, and pharmacology

Abstract: The synthesis of a series of N-9-fluorenylmethoxycarbonyl (N-FMOC) protected amino acid glycosides is reported. These (1-2)-trans glycosides came directly from Koenigs-Knorr type glycosylations under Hanessian's silver triflate conditions, except for the synthesis of N-acetylgalactosamine FMOC amino acid in which silver perchlorate conditions were used to promote α-glycoside formation. The effect of D-amino acid aglycones was investigated under glucosylation conditions, and a yield dependence on amino protection was seen in the enantiomers of threonine. Due to this match vs. mismatch dichotomy, both O'Donnell Schiff bases and FMOC-amino aglycones were used in the subsequent glycosylation reactions. Glycosides were made using the monosaccharides xylose, mannose, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, and disaccharides lactose [galactose-β-(1-4)-glucose], cellobiose [glucose-β-(1-4)-glucose] and melibiose [galactose-α-(1-6)-glucose]. All glycosides were converted to their respective FMOC-amino acid forms for direct use in solid-phase glycopeptide synthesis (SPGPS) using established methodology. A strategy into the synthesis of an FMOC-amino acid trisaccharide of Lewis ˣ (Leˣ) was also investigated in an effort to expand on the established glycoside methodology. Preliminary work with D-glucosamine and L-fucose is reported. Our synthetic rationale was based on retaining the peptide pharmacophore or message sequence constant as DCDCE (D-cys²ʼ⁵-enkephalin) with a serine-glycine tether, and making changes only in the environment of the amino-acid glycoside. Changes in amino acid, amino acid chirality, and in the sugar moiety itself would provide a stereochemical investigation into the requisite orientation and electronics for optimum blood-brain barrier (BBB) penetration, opiate receptor binding, and analgesia. Several glycopeptides were synthesized, and all were purified in both reduced and oxidized forms (if containing cysteine). A highly optimized glycopeptide synthetic strategy has been developed and will be presented and critiqued. Pharmacological analysis involving serum stability studies, BBB-penetration studies, GPI/MVD physicochemical studies and mu/delta-opiate receptor studies were completed on all glycopeptides. SAM-1095, the most potent of the glycopeptides synthesized, was resynthesized on a large scale, and this compound was assessed for in vivo pharmacology, along with the non-glycosylated version SAM-995. Preliminary results demonstrate an analgesic effect similar to that of the narcotic morphine. Assessment of all pharmacology will afford a platform for future SAR-based glycopeptide investigations.

Bacillus stearothermophilus var. calidolactis C953'ten α-Galaktosidaz Üretimi*

Abstract: An intracellular, thermostable a-galactosidase (α-D-galactoside galactohydrolase EC 3.2.1.22.) was produced from a strain of Bacillus stearothermophilus var. calidolactis C953. It was found that the presence of NH 4 Cl in the fermentation medium affected microbial growth and that optimum enzyme production occurred with peptone in various sources of nitrogen where NH 4 Cl was used as the main inorganic nitrogen source. After peptone, in decreasing order of enzyme productivity, came tryptone, soybean meal, soya-neutralized peptone and casamino acid. It was determined that, in various carbon sources, raffinose produced the highest α-galactosidase activity, followed by melibiose, D-galactose and molasses, and that in the medium with soybean meal extract the second highest level of activity occurred. Soybean oligosaccharides (raffinose-like sugars), because of their present low commercial value, could be of pratical use in the production of α-galactosidase.