Showing papers on "Melibiose published in 2003"
TL;DR: The specificity of Saccharomyces cerevisiae yeast on the removal of carbohydrates by fermentation was studied and the yeast enzymes did not alter any of the resistant carbohydrates by transglycosylation or condensation reactions or by any other types of reactions.
124 citations
TL;DR: Levansucrase from Microbacterium laevaniformans ATCC 15953 produced in a 3% sucrose medium was purified to homogeneity from cell-free extracts by ammonium sulfate precipitation, DEAE-Sepharose Fast Flow and Sephacryl S-100 HR chromatographies as mentioned in this paper.
65 citations
TL;DR: Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities and α-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase.
Abstract: Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, α-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 °C (K
M 0.55 mM, V
max 0.80 μmol min−1 mg−1 of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6′ α-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.
52 citations
TL;DR: It is suggested that the expression of the aga operon in lactococci is negatively controlled by GalR and induced by a metabolite derived from the metabolism of galactosides.
Abstract: Lactococcus raffinolactis, unlike most lactococci, is able to ferment alpha-galactosides, such as melibiose and raffinose. More than 12 kb of chromosomal DNA from L. raffinolactis ATCC 43920 was sequenced, including the alpha-galactosidase gene and genes involved in the Leloir pathway of galactose metabolism. These genes are organized into an operon containing aga (alpha-galactosidase), galK (galactokinase), and galT (galactose 1-phosphate uridylyltransferase). Northern blotting experiments revealed that this operon was induced by galactosides, such as lactose, melibiose, raffinose, and, to a lesser extent, galactose. Similarly, alpha-galactosidase activity was higher in lactose-, melibiose-, and raffinose-grown cells than in galactose-grown cells. No alpha-galactosidase activity was detected in glucose-grown cells. The expression of the aga-galKT operon was modulated by a regulator encoded by the upstream gene galR. The product of galR belongs to the LacI/GalR family of transcriptional regulators. In L. lactis, L. raffinolactis GalR acted as a repressor of aga and lowered the enzyme activity by more than 20-fold. We suggest that the expression of the aga operon in lactococci is negatively controlled by GalR and induced by a metabolite derived from the metabolism of galactosides.
44 citations
TL;DR: A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl-α-d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI.
Abstract: A DNA fragment, which complemented the growth of E. coli both on M9 medium containing raffinose and on LB medium containing ampicillin, IPTG and 5-bromo-4-chloro-3-indoxyl-α-d-galactoside, was isolated from the genomic library of Bifidobacterium longum SJ32, which had been digested with EcoRI. In the cloned DNA fragment, a gene encoding a sucrose phosphorylase (splP) and a partially cloned putative sucrose regulator gene (splR) were identified using the deletion analysis and sequence analysis. A 56 kDa protein was synthesized in E. coli and partially purified by DEAE-ion exchange chromatography. The partially purified enzyme did not react with melibiose, melezitoze and raffinose but did with sucrose. It had transglucosylation activity in addition to hydrolytic activity.
42 citations
TL;DR: The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra was studied and it was suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase.
Abstract: The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra (L.) was studied. The sugars used included monosaccharides (fructose, glucose, galactose, mannose, rhamnose), disaccharides (sucrose, maltose, trehalose, melibiose, lactose) and trisaccharides (melizitose, raffinose, erlose). Single-sugar solutions were tested on ant workers in a long-term laboratory bioassay in which acceptance of the solutions and ant survival were recorded. The acceptance of the sugars was confirmed in a second bioassay in which feeding time was established. Enzymatic hydrolysis of sucrose, maltose and melibiose was investigated through HPLC analyses of workers fed these disaccharides. Sugar acceptance and feeding time were related to ant survival. Considering the monosaccharide units of which the sugars are composed, fructose seems especially suitable as a short-term energy source, while glucose appears to be used both directly and for storage. The presence of a galactose unit appears to reduce sugar suitability. It is suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase. The gustatory perception is correlated with the profitability of sugars in further metabolic processes.
40 citations
TL;DR: α-Galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose and the product was confirmed by various analyses.
Abstract: Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.
38 citations
Journal Article•
TL;DR: The production of thermostable extracellular protease by Bacillus stearothermophilus Fl was influenced by the composition of culture medium and the presence of the metal ion at 4.5 mM enhanced the yield by two folds.
Abstract: The production of thermostable extracellular protease by Bacillus stearothermophilus Fl was influenced by the composition of culture medium. Peptone iv derived from soybean was the best organic compound for enzyme production. Sodium nitrate, ammonium salts and amino acids as sole nitrogen sources interfered with protease formation. The addition of most carbon sources to the peptone medium failed to improve the enzyme production. However, protease production was enhanced in the presence of melibiose and trehalose in sodium nitrate medium. Although the protease production was calcium independent, the presence of the metal ion at 4.5 mM enhanced the yield by two folds.
35 citations
TL;DR: An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli and it was concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.
Abstract: An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first α-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other α-galactosidases. Highest homology was found with α-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant α-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-α-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable α-galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by...
33 citations
TL;DR: Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements, consistent with xynT functioning as a symport.
Abstract: Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose. Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis. Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E. coli. The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43. The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na+/melibiose symporters and related proteins. These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K. oxytoca and recombinant E. coli. Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E. coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons. Based on similarities in protein sequence, the xynTB genes in K. oxytoca appear to have originated from a gram-positive ancestor related to L. lactis. Functional expression of xynB allowed ethanologenic E. coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements. 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase. The rate of xylodextrin utilization by recombinant E. coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport. Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate. Xylodextrins were utilized more rapidly by recombinant E. coli than K. oxytoca M5A1 (the source of xynT and xynB). XynB exhibited weak arabinosidase activity, 3% that of xylosidase.
27 citations
TL;DR: If verified in vivo, inhibition of this antibody by high serum glucose may possibly be among reasons for the increased susceptibility of diabetics to infection.
TL;DR: The results reported in this study indicate that Aspergillus flavipes is indeed an active source of extracellular α‐galactosidase.
Abstract: An extracellular α-galactosidase from the culture filtrate of Aspergillus flavipes grown on melibiose as a carbon source was partially purified by hydroxylapatite and diethylaminoethylcellulose chromatographies. Electrophoretic analysis showed protein bands corresponding to α-galactosidase and invertase activities. The optimum pH and temperature were determined as 4.5–5.0 and 45°C, respectively. The Km value for p-nitrophenyl-α-d-galactopyranoside was found to be 1.89 mm. The results reported in this study indicate that Aspergillus flavipes is indeed an active source of extracellular α-galactosidase. Copyright © 2003 John Wiley & Sons, Ltd.
TL;DR: The reported products are invaluable tools for studies on their possible biochemical function as donor substrates or inhibitors of Leloir glycosidase-catalyzed transglycosyltransferases.
Abstract: The enzymatic synthesis of nucleotide-activated disaccharides by glycosidase-catalyzed transglycosylation reactions was investigated. High product yields were obtained when the kinetically driven syntheses were performed in frozen aqueous solutions at −5 °C. β-Galactosidase from Bacillus circulans catalyzed the transfer of d -galactose from lactose onto the nucleotide sugar UDP-Gal stereo- and regioselectively, forming the nucleotide disaccharide Gal(β1–4)Gal(α1-UDP. α-Galactosidase from Bifidobacterium adolescentis accepted UDP-Gal and UDP-Glc only when the reaction was performed at −5 °C with melibiose as glycoside donor. Enzyme and donor concentrations were optimized for the preparative synthesis yielding Gal(α1–3)Glc(α1-UDP and Gal(α1–3)Gal(α1-UDP as analyzed by MALDI-TOF mass spectrometry and 1D and 2D 1 H and 13 C NMR spectroscopy. The reported products are invaluable tools for studies on their possible biochemical function as donor substrates or inhibitors of Leloir glycosyltransferases.
TL;DR: In this article, the authors isolated mutants of Escherichia coli HS4006 containing the melibiose-H+ symporter (MelY) from Enterobacter cloacae that had enhanced fermentation on 1% maltose MacConkey plates.
Abstract: We isolated mutants of Escherichia coli HS4006 containing the melibiose-H+ symporter (MelY) from Enterobacter cloacae that had enhanced fermentation on 1% maltose MacConkey plates. DNA sequencing revealed three site classes of mutations: L-88-P, L-91-P, and A-182-P. The mutants L-88-P and L-91-P had 3.6- and 5.1-fold greater maltose uptake than the wild type and enhanced apparent affinities for maltose. Energy-coupled transport was defective for melibiose accumulation, but detectable maltose accumulation for the mutants indicated that active transport is dependent upon the substrate transported through the carrier. We conclude that the residues Leu-88, Leu-91 (transmembrane segment 3 [TMS-3]), and Ala-182 (TMS-6) of MelY mediate sugar selection. These data represent the first MelY mutations that confer changes in sugar selection.
TL;DR: The mutant Saccharomyces cerevisiae ATCC 36858 was used in the production of ethanol and/or fructose from synthetic media in batch processes with raffinose, melibiose or sucrose.
Abstract: The mutant Saccharomyces cerevisiae ATCC 36858 was used in the production of ethanol and/or fructose from synthetic media in batch processes with raffinose, melibiose or sucrose. The mutant was able to hydrolyze all the sugars used and to selectively ferment glucose and galactose to ethanol while fructose accumulated in the fermentation medium. The fructose yield was above 89% of the theoretical value in the media with either raffinose or sucrose, when initial concentrations were between 131.5 g dm−3 and 242.0 g dm−3. The ethanol yields were 82% and 77% of the theoretical values in the media with melibiose and sucrose, respectively, and about 72% of the theoretical value when raffinose was used. The fructose fraction in the carbohydrate content of the produced syrups was more than 96% when raffinose concentration was below 189.1 g dm−3. However, even at a sucrose concentration of 187.9 g dm−3, the produced syrup contained 100% fructose. Some oligosaccharides were also produced in all tested media. The produced oligosaccharides were consumed by the end of the fermentation process. These findings can be useful in the production of ethanol and high fructose syrups using raw materials based on sucrose and raffinose such as molasses. Copyright © 2003 Society of Chemical Industry
TL;DR: Results indicated that this lectin belongs to the family of soluble -galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.
Abstract: A lectin from cat liver has been identified and purified by affinity chromatography on asialofetuin-Sepharose. One hundred micrograms of lectin was obtained from one cat liver with a purification factor of 1561. The lectin agglutinates trypsin-treated rabbit and cow erythrocytes. Hemagglutination was inhibited only by saccharides containing s-galactosyl residues, of which the 1-amine-1-deoxy-s-D-galactose was the most potent one by inhibiting hemagglutination at a concentration of 12.5 mM, followed by melibiose, trehalose and galactose. The lectin has a subunit molecular mass of 14.4 kDa determined by SDS-PAGE under reducing conditions and a pI of 4.85. Compared with the composition of lectins from calf heart and porcine heart, cat liver lectin contains approximately the same amount of cysteine, half the amount of glycine, twice as much arginine and threonine, and three times the amounts of tyrosine and methionine. Cat liver lectin contains four cysteine residues per subunit, all of them in the reduced form. Their lack of reactivity towards thiol-reactive supports suggests they are not exposed on the lectin surface. The protein apparently has a blocked N-terminus. The purified lectin was stable for up to 20 months stored at +4oC in buffer supplemented with 4 mM s-mercaptoethanol. Results indicated that this lectin belongs to the family of soluble s-galactoside-binding lectins, also known as galectins, which are expressed in a wide range of vertebrate tissues.