Showing papers on "Melibiose published in 1976"

Genetic co-regulation of galactose and melibiose utilization in Saccharomyces.

Abstract: The gal3 mutation of Saccharomyces, which is associated with an impairment in the utilization of galactose, has been shown to be pleiotropic, causing similar impairments in the utilization of melibiose and maltose. Milibiose utilization and alpha-galactosidase production are directly controlled by the galactose regulatory elements i, c, and GAL4. The fermentation of maltose and the induction of alpha-glucosidase are regulated independently of the i, c, GAL4 system. The production of alpha-galactosidase and galactose-1-phosphate uridyl transferase is coordinate in galactokinaseless strains. Galactose serves as a nonmetabolized, gratuitous inducer of alpha-galactosidase in strains lacking the genes for one or more of the Leloir pathway enzymes.

Raffinose metabolism in Escherichia coli K12. Purification and properties of a new alpha-galactosidase specified by a transmissible plasmid.

Abstract: The utilization by Escherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an alpha-galactosidase specified by the Raf-plasmid D1021. The alpha-galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme. alpha-Galactosidase hydrolyzes p-nitrophenyl-alpha-D-galactoside (Km 0.14 mM), methyl-alpha-D-galactoside (Km 30mM), melibiose (Km 3.2 mM) and raffinose (Km 60 mM). The enzymatic activity is strongly inhibited by Ag+, p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of one form of alpha-galactosidase with an isoelectric point of 5.1. The purified enzyme has an sw,20 value of 11.7 +/- 0.3S and a molecular weight of 329000 +/- 4000; this value is not reduced at high dilutions. When examined by dodecylsulphate gel electrophoresis, purified alpha-galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits. Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half-cystine residues. No carbohydrate moiety has been detected. High resolution electron micrographs and Markham rotation of alpha-galactosidase show enzyme molecules of approximately 11 x 11 nm containing four globular subunits in a tetragonal arrangement. The plasmid-coded alpha-galactosidase differs from the homologous E. coli enzyme by substrate affinities, cofactor requirements, stability and toluene resistance. It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf-plasmids.

Characterization of the neoagarotetra-ase and neoagarobiase of Cytophaga flevensis.

Abstract: The degradation of neoagarotetraose and neoagarobiose by Cytophaga flevensis was investigated The organism possesses an enzyme that hydrolyzes the tetramer by cleavage of its central β-galactosidic linkage The product of this reaction, neoagarobiose, is further hydrolyzed enzymatically to d-galactose and 3,6-anhydro-l-galactose Both enzyme activities were localized in the cytoplasm Attempts were made to partially purify the respective enzymes and although at 30–40-fold purification was achieved, the final preparation contained both neoagarotetra-ase and neoagarobiase activities Evidence was obtained that these activities were due to different enzymes Neoagarotetra-ase is highly specific for oligosaccharides containing neoagarobiose units; the rate of hydrolysis is greatest with neoagarotetraose It cannot hydrolyze pyruvated neoagarotetraose Optimal conditions for its activity were pH 70 and 25 C Neoagarobiase hydrolyzes only neoagarobiose and neoagarobiitol and optimal conditions for activity were pH 675 and 25 C Both enzymes were inhibited by Ag+, Hg2+ and Zn2+ ions and by p-CMB, which indicates that thiol groups are present in their active centres Both enzymes were induced by neoagaro-oligosaccharides and melibiose and were repressed when glucose was added to the medium Neoagarobiase was also induced by d-galacturonic acid In continuous culture, the rate of enzyme production was maximal at a dilution rate of 01 h-1

Temperature-dependent cultural and biochemical characteristics of rhamnose-positive Yersinia enterocolitica.

Abstract: Clinical isolates of rhamnose-positive Yersinia enterocolitica (Y.e.rh+) were compared with typical rhamnose-negative Y. enterocolitica (Y.e.rh-) and with Yersinia pseudotuberculosis. The Y.e.rh+ differed from the Y.e.rh- and Y. pseudotuberculosis in their ability to ferment raffinose and lactose, utilize citrate and in their inability to grow on Hektoen enteric agar at 22 or 37 C, on Salmonella-Shigella agar at 37 C, and scant on xylose-lysine-deoxycholate agar at 37 C. An extensive temperature-dependent profile of characteristics was established for the Y.e.rh+: motility, acetoin production, citrate utilization, growth on Salmonella-Shigella agar, and ampicillin resistance occurred at 22 C but not 37 C; fermentation of melibiose, raffinose, and cellobiose occurred within 24 h at 22 C, but not before 5 days at 37 C; fermentation of rhamnose and production of beta-galactosidase occurred within 24 h at 22 C, but not before 48 h at 37 C; greater resistance to ampicillin, chloramphenicol, streptomycin, kanamycin, carbenicillin, and gentamicin was observed at 22 than 37 C; and good growth on xylose-lysine-deoxycholate agar occurred at 22 but not 37 C. For optimal recovery of Y.e.rh+ from mixed culture, e.g., stools, two MacConkey plates should be inoculated and incubated, one at 37 C, and one at 22 C. Lactose-negative colonies appearing after 48 h on the 22 C MacConkey agar but not the 37 C MacConkey agar should be considered possible Y.e.rh+. Biochemicals should be tested in duplicate, one set incubated at 22 C, one set at 37 C. Antibiotic susceptibility tests of Y.e.rh+ isolates should be incubated at both 37 C and at a lower temperature to allow the greatest expression of resistance of these organisms to the various antibiotics.

The Conformations of Oligosaccharides. III. The Crystal and Molecular Structure of Melibiose Monohydrate

Abstract: The crystal structure of α-melibiose monohydrate (C12H22O11·H2O) was determined by direct methods using two-dimensional cosine invariants. The space group is P212121, with Z=4 and unit cell dimensions of a=15.814(5), b=10.924(5), c=8.903(4) A. The structure was refined to R=0.044 for 1439 reflections measured with MoKα radiation. Difference synthesis indicated the partial (15%) random substitution of α-melibiose molecules by the β-anomer. The molecular conformation of α-melibiose is almost the same as that previously found in the melibiose moiety of raffinose, although some of the bond lengths and angles are significantly different. The molecules are linked by complex hydrogen-bond systems which consist of six-link finite chains and five-membered closed loops.

Purification and Properties of α-Galactosidase from Escherichia coli subsp. communior IAM 1272

Abstract: Galactosylsucroses contained in soybeans are not digestible. Thus we wished to detect α-galactosidase (EC 3. 2. 1. 22) in intestinal bacteria. The strain of E. coli in the title was found to produce considerably this enzyme adaptively. We could prepare rather pure solution of the enzyme from the sonicate of the strain. It was purified about 142-fold. It showed optimum pH and temperature at 6.8 and 37°C, respectively, with the substrate p-nitrophenyl-α-D-galactoside (PNPG). Dilute enzyme solutions were very unstable even at 0_??_5°C. However, concentrated solutions were considerably stable. The Michaelis constant (M) was 1.07×10-4, 2.33×10-3, and 3.65×10-1 for PNPG, melibiose, and raffinose, respectively, The maximum velocity (mole/min/mg protein) was 2.72×10-5, 2.67×10-5, and 2.04×10-5, respectively for the same three substrates. This enzyme had a weak transferase action.

.ALPHA.-Galactosidase of alkalophilic microorganisms. I. Identification and growth characteristics of .ALPHA.-galactosidase-producing microorganisms.

Abstract: Two kinds of α-galactosidase-producing microorganisms, strain No. 31–2 and strain No. 7–5, have been isolated from soil and subjected to a determinative study. On the basis of the morphological and physiological characters, the strain No. 31–2 was identified to be belonged to genus Micrococcus and the strain No. 7–5 to genus Bacillus. The former strain, Micrococcus sp. No. 31–2, produced exclusively an intracellular α-galactosidase, and the latter one, Bacillus sp. No. 7–5, secreted the enzyme into culture medium. The cell growth and enzyme production of both strains were observed to reach the maximum under an alkaline culture condition. The intracellular α-galactosidase of Micrococcus sp. No. 31–2 was inducible by galactose, melibiose, and raffinose, while the α-galactosidase of Bacillus sp. No. 7–5 was produced constitutively.

Properties of α-Galactosidases of Alkalophilic Bacteria

Abstract: The properties of α-galactosidases of alkalophilic strains of Micrococcus sp. No. 31-2 and Bacillus sp. No. 7-5 were examined. The enzymes were partially purified by ion exchange and gel filtration chromatography. The enzymes had pH optima between 6.5 and 7.5. The enzyme of the Micrococcus strain was stable at pH 7.5_??_8.0; that of the Bacillus strain was stable at rather broad pH ranging 6.0 to 8.5. The enzymes were both unstable at temperature higher than 35°C. Metal ions such as Ag+, Hg2+, etc. caused complete loss of activity. The enzymes were also sensitive to sulfhydryl reagents. Among the substrates tested, the order of the hydrolysis rate by these enzymes was o-nitrophenyl-α-D-galactoside>melibiose>raffinose.

Some kinetic properties of .ALPHA.-D-galactosidase from Streptomyces 9917S2.

Abstract: Some kinetic properties of the Streptomyces 9917S2 α-d-galactosidase were investigated. This enzyme could use α-d-galactoside and α-d-fucoside as substrates, but not β-l-arabinoside, nor the anhydro and uronic acid derivatives of α-d-galactoside. Both Km and V values for phenyl α-d-fucoside were much higher than those for p- and o-nitrophenyl and phenyl α-d-galactosides, melibiose, and raffinose. So far as examined, substrate inhibition was observed only in the hydrolysis of p-nitrophenyl α-d-galactoside. β-d-Galactosides and d-glucose inhibited the hydrolysis of α-d-fucoside but did not the hydrolysis of α-d-galactoside. The types of inhibition by various sugars and their derivatives were competitive. The Ki values of inhibitors in the hydrolysis of α-d-galactoside were always ten times higher than those in the hydrolysis of α-d-fucoside.

Regulation of agarase production by resting cells of cytophaga-flevensis

Abstract: The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3mm calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3mm calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme.

Uptake and binding of disaccharides in human erythrocytes

Abstract: Disaccharides (sucrose, lactose, melibiose, cellobiose, trehalose, maltose, and isomaltose) are not transported across the human erythrocyte membrane. Maltose alone is bound in appreciable amounts to the intact cell as well as ghost membranes and competes mutually for uptake with D-glucose. In (NH4)2SO4-precipitated membrane preparations, maltose binds more strongly than other disaccharides (KD = 1.3 × 10−5 M; maximum binding capacity, 71 pmol/mg protein) and again competes mutually with D-glucose. Phloretin inhibits the binding of glucose much more than that of maltose.

Raffinose metabolism in Gluconobacter oxydans

Abstract: Metabolism of raffinose has been examined in experiments with the growing culture and washed cells of Gluconobacter oxydans L-1. Degradtion of the trisaccharide was found to be catalyzed by levansucrase, levan being synthesized, and melibiose and small quantitites of fructose being liberated in the reaction. Melibiose is not hydrolyzed and is not used by the bacterium as a source of carbon, but is oxidized to melibionic acid. Fructose is assimilated by the bacterium in constructive metabolism, being oxidized to gluconic, 2-ketogluconic acids and 5-ketofructose.

The conformations of oligosaccharides. iii. the crystal and molecular structure of melibiose monohydrate

Abstract: The crystal structure of α-melibiose monohydrate (C12H22O11·H2O) was determined by direct methods using two-dimensional cosine invariants. The space group is P212121, with Z=4 and unit cell dimensions of a=15.814(5), b=10.924(5), c=8.903(4) A. The structure was refined to R=0.044 for 1439 reflections measured with MoKα radiation. Difference synthesis indicated the partial (15%) random substitution of α-melibiose molecules by the β-anomer. The molecular conformation of α-melibiose is almost the same as that previously found in the melibiose moiety of raffinose, although some of the bond lengths and angles are significantly different. The molecules are linked by complex hydrogen-bond systems which consist of six-link finite chains and five-membered closed loops.