Transport and hydrolysis of disaccharides by Trichosporon cutaneum.

Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose, and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of all investigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 +/- 3 mM sucrose or lactose and 5 +/- 1 mM maltose, which is of the same order of magnitude as those reported for Saccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucrose and that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose. Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysis before or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such. The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast, there were no indications of the involvement of gradients of H+, K+, or Na+ in the uptake of maltose. The uptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases for cellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and the corresponding glycosidases is constitutive.     

A general survey of the proton spin-lattice relaxation-times of some oligo- and poly-saccharide derivatives

A Fourier-transform method has been used to measure the spin-lattice relaxation-times (T₁ values) of the anomeric protons of a selection of oligo- and poly-saccharide derivatives. Although systematic variations are found for the substances of lower molecular weight, these variations are essentially non-existent at higher molecular weights. Data for the disaccharides cellobiose, maltose, lactose, gentiobiose, and melibiose demonstrate that proton T₁-values may provide a powerful method for evaluating conformations of oligosaccharides.     

Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by β-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).     

Uptake and binding of dissaccharides in human erythrocytes.

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)2-SO4-precipitated membrane preparations, maltose binds more strongly than other disaccharides (KD = 1.3 X 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.     

Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α-N-1-(1-deoxyglucitol)-lysine and ?-N-1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ?-amino groups of lysine.     

Investigation of disaccharide recognition by molecularly imprinted polymers

The selectivity of carbohydrate-imprinted polymers for several disaccharides, namely cellobiose, maltose, lactose and gentiobiose, is investigated. An ternary ligand–Cu(II)–carbohydrate complex was formed in alkaline solution and captured afterwards in the polymer. The accessibility of the polymer matrix for disaccharides was investigated by HPLC analysis, refractometry and ¹H NMR spectroscopy applying excess of the original template during rebinding experiments under saturation conditions in unbuffered, aqueous solution at neutral pH and 20°C. The selective discrimination of the - and -glycosidic linkage of cellobiose and maltose is demonstrated. It is further shown, that the disaccharide-imprinted polymers slightly distinguish between the 1,4-- and the 1,6--glycosidic linkage of cellobiose and gentiobiose, while cellobiose and lactose are not selectively recognized. Due to the weak apparent binding constant of the functional Cu(II) monomers with the targeted disaccharides at physiological pH, the recognition process is dominated by the shape of the created imprinted cavity under the applied conditions.     

Vacuum-ultraviolet circular dichroism study of saccharides by synchrotron radiation spectrophotometry

Vacuum-ultraviolet circular dichroism (VUVCD) spectra of five monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, and D-lyxose) and five disaccharides (maltose, isomaltose, cellobiose, gentiobiose, and lactose) were measured to 160 nm using a synchrotron-radiation VUVCD spectrophotometer in aqueous solution under high vacuum at 25 degrees C. Most of the saccharides show a positive peak with some shoulders at around 170 nm, except for D-galactose and lactose, which show two distinct negative peaks at around 165 and 177 nm. These spectra are influenced by such structural factors as alpha and beta anomers at C-1, axial and equatorial hydroxyl groups at C-2 and C-4, trans (T) and gauche (G) conformations of the hydroxymethyl group at C-5, and the type of glycosidic linkage. Deconvolution of the VUVCD spectra of D-glucose, D-mannose, and D-galactose into six independent Gaussian components for alpha-GG, alpha-GT, alpha-TG, beta-GG, beta-GT, and beta-TG conformations suggests that the alpha anomer has red-shifted spectra relative to the beta anomer, and that GG and GT conformations have positive and negative circular dichroism signs, respectively, while the sign for TG conformation is anomer dependent. These speculations from the deconvolution analyses are also supported by the VUVCD spectra of disaccharides. These results give new insight into the equilibrium conformations of saccharides, demonstrating the usefulness of synchrotron-radiation VUVCD spectroscopy.     

A critical study of the taxonomic value of some tests of assimilation used for the classification of the sporogenous yeasts

Six texts of assimilation used in the taxonomy of yeasts, (lactose, maltose, cellobiose, trehalose, melibiose, sucrose) have been critically tested by the examination of intracellular enzymic systems. The results obtained among the sporogenous species ofSaccharomyces, Kluyveromyces, Pichia, Hansenula, Debaryomyces indicate that cellobiose, lactose, maltose and trehalose tests no longer supply an important value for the speciation, because the number of cryptical osidases is so high.     

Partial characterization of Pseudomonas phage 2 receptor

The lipopolysacharide from Pseudomonas aeruginosa strain BI contains the receptors for phage 2 and strongly inactivates this phage in vitro (95-98% within 15 min). Several mono- and di-saccharides tested reduced phage 2 inactivation to 50% when present at the following concentrations: D-glucosamine, 0.25 M; maltose, 0.3M; lactose and cellobiose, 0.5 M; D-glucose, L-rhamnose, D-mannose, 2-deoxy-D-glucose, and sucrose, 1.0 M; D-galactose, D-xylose, and N-acetyl-D-glucosamine, 1.4 M; and melibiose. greater than 1.6 M. These results suggest the possibility that phage 2 receptors in lipopolysaccharide contain L-rhamnose, D-glucosamine, and (or) D-glucose, or a structurally related molecule. Either one of the latter two could be located at a terminal position alpha-linked to the adjacent residue, or located internally in the polysaccharide chain linked through its C-4 position.     

Solvent effects in the reaction of 2,3,6-tri-O-methyl D-glucopyranose and phenyl isocyanate

The reaction of a 3:1 mixture of 2,3,6-tri-O-methyl-α- andβ-D-glucopyranose (1) with phenyl isocyanate, in acetone, benzene, dimethyl sulfoxide, 1,4-dioxane, pyridine, and tetrahydrofuran, showed the isomer ratio in the product mixture to be solvent-dependent. The ratio varied from 4.55β/α in benzene to 0.49 in Me₂SO. It is proposed that an activated complex formed between 1 and a 1-isocyanate complex provides for the simultaneous attack of a nucleophile on the anomeric hydroxyl proton, and of an electrophile on the ring-oxygen atom of 1, causing mutarotation. The rate of mutarotation of the activated complex is dependent on the degree of solvation of the anomeric hydroxyl group. Solvent association is highest in ME₂SO and lowest in benzene. The reaction rate is higher in benzene than 1,4-dioxane and is slowest in Me₂SO. The hydroxyl group at C-1 is ≈ 3 times as reactive as the one at C-4.     

Hydrolysis of disaccharides over solid acid catalysts under green conditions

The hydrolysis of the three most important disaccharides: sucrose, maltose and cellobiose, has been comparatively studied in mild conditions (50-80°C) in water over several solid acid catalysts. Strong acidic resins (Amberlite A120 and A200), mixed oxides (silica-alumina and silica-zirconia), and niobium-containing solids (niobic acid, silica-niobia, and niobium phosphate) have been chosen as acid catalysts. The hydrolysis activity was studied in a continuous reactor with fixed catalytic bed working in total recirculation mode. Rate constants and activation parameters of the hydrolysis reactions have been obtained and discussed comparing the reactivity of the α-1,β-2-, α-1,4-, and β-1,4-glycosidic bonds of the employed disaccharides. The following order of reactivity was found: sucrose > maltose > cellobiose. The sulfonic acidic resins, as expected, gave complete sucrose conversion at 80°C and good conversions for cellobiose and maltose. Among the other catalysts, niobium phosphate provided the most interesting results toward the disaccharide hydrolysis, which are here presented for the first time. Relations between activity and surface acid properties are discussed.     

Biosynthesis of Riboflavin Glycosides by Plant Grains

An enzyme preparation from glutinous millet grains has been found to synthesize various riboflavin glycosides from riboflavin and disaccharides other than maltose (such as cellobiose, melibiose and lactose). Each of these riboflavin glycosides has been isolated in crystalline form and shown to have the structure, 5′-D-riboflavin-β-d-glucopyranoside, 5′-d-riboflavin-α-d-galactopyranoside and 5′-d-riboflavin-β-D-galactopyranoside.     

Simple preparations of alkyl and cycloalkyl alpha-glycosides of maltose, cellobiose, and lactose

Alkyl, cycloalkyl, allyl, 4-pentenyl, and benzyl alpha-glycosides of maltose, cellobiose, and lactose were prepared (17-77% yield; alpha/beta=70/30-96/4) via a direct reaction of the free disaccharides with a binary AcBr-AcOH mixture, followed by glycosidation with alcohol using FeCl3 in MeNO2 or CH2Cl2, Zemplén deacetylation, and resolution of the anomeric mixture of glycosides by chromatography. Using MeCN as solvent for the glycosidation step, the corresponding beta-biosides were also prepared (16-61% yield; alpha/beta=25/75-5/95).     

Synthesis and reactions of 1′,2:4,6-di-o-isopropylidene-sucrose

The reaction of sucrose with a combination of 2,2-dimethoxypropane, N,N-dimethylformamide, and toluene-p-sulphonic acid (reagent A) gave, after acetylation followed by chromatography, 1′,2:4,6-di-O-isopropylidenesucrose tetra-acetate (1) in 15% yield. The structure of 1 was determined on the basis of p.m.r. and mass spectrometry, and by chemical transformations. Treatment of 1 with aqueous acetic acid afforded sucrose 3,3′,4′,6′-tetra-acetate 2. Reacetalation of 2 using reagent A gave 1 in 80% yield. The p.m.r. spectrum of 2 confirmed the presence of hydroxyl groups at C-2 and C-4. The following sequence of reactions showed that the remaining two hydroxyl groups were located at C-6 and C-1′. Selective tritylation of 2 gave 1′,6-di-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (3) as the minor, and 6-O-tritylsucrose 3,3′,4′,6′-tetra-acetate (4) as the major, product. When tritylation was carried out under forcing conditions, 2 gave 3 as the major product. Acetylation of 4 afforded 6-O-tritylsucrose hepta-acetate. Mesylation of 2 gave the tetramethanesulphonate 5, which afforded the 6-dcoxy-6-iodo derivative 6 on treatment with a refluxing solution of sodium iodide in butanone. Treatment of 3 with methanesulphonyl chloride in pyridine gave the disulphonate 7, which on detritylation followed by acetylation gave 2,4-di-O-methanesulphonylsucrose hexa-acetate (9). Treatment of 9 with sodium benzoate in hexamethylphosphoric triamide displaced the 4-sulphonate, with inversion of configuration, to give the galacto derivative 10.     

The reaction of ammonia with acylated disaccharides. X. Octa-O-benzoyl-lactose and other benzoyl derivatives of lactose

From the reaction of octa-O-benzoyl-lactose (1) with methanolic ammonia, lactose, 1,1-bis(benzamido)-1-deoxy-4-O-β-D-galactopyranosyl-D-glucitol (2), and N-benzoyl-4-O-β-D-galactopyranosyl-D-glucopyranosylamine (3) were obtained. The behavior of some other octabenzoylated disaccharides in the ammonolysis reaction is discussed.     

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10⁶ Da) with ～85.5% α-(1→6) linear and ～14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.     

Galactan sulfate from the test of tunicates

Methyl and benzyl 3-O-β-d-xylopyranosyl-α-d-mannopyranoside were prepared by way of d-xylosylation (Koenigs-Knorr) of methyl and benzyl 4,6-O-benzylidene-α-d-mannopyranoside (1 and 17). Analogous 2-O-β-d-xylopyranosyl-α-d-mannopyranosides could not be prepared efficiently by this procedure. However, methyl and benzyl 3-O-acetyl-4,6-O-benzylidene-α-d-mannopyranoside, prepared by limited acetylation of 1 and 17, respectively, could be d-xylosylated by the same method, and afforded, after removal of protective groups, methyl and benzyl 2-O-β-d-xylopyranosyl-α-d-mannopyranoside. Hydrogenolysis of benzyl 2-O- and 3-O-β-d-xylopyranosyl-α-d-mannopyranoside yielded the corresponding, reducing disaccharides. In addition to these disaccharides, disaccharides containing an α-d-xylopyranosyl group, and trisaccharides having d-xylopyranosyl groups at both O-2 and O-3 were obtained as minor products.     

Continuous Enzymatic Regeneration of Electron Acceptors Used by Flavoenzymes: Cellobiose Dehydrogenase-Catalyzed Production of Lactobionic Acid as an Example

An efficient enzymatic bioprocess is described in which lactose, an abundant renewable resource produced by the dairy industry, is completely and efficiently converted with a specific productivity of up to 32 g (kU h)^?1 into lactobionic acid, without the formation of any by-products. The key biocatalyst of this new process is the fungal enzyme cellobiose dehydrogenase which oxidizes several β-1,4-linked disaccharides including lactose specifically at position C-1 of the reducing sugar moiety to the corresponding lactones. The electron acceptor employed in this reaction is continuously regenerated with the help of laccase, a H₂O-producing, copper-containing oxidase, and therefore has to be added in low, catalytic amounts only. Redox mediators that were successfully employed in this novel process and hence are compatible with the laccase regeneration system include benzoquinone, ABTS, ferricyanide, or ferrocene, amongst others. Factors affecting operational stability of the biocatalysts employed in this process include the redox mediator used, the temperature, and importantly the volumetric gas flow necessary for maintaining the dissolved oxygen tension. Lactobionic acid is a mild and sweet tasting acid with excellent chelating properties. These useful characteristics have lead to a growing number of patents for diverse applications in the food, pharmaceutical and detergent industries.     

Equilibration of alditol anhydrides in acetic acid

Dehydration of pentitols in acetic acid containing an acidic catalyst parallels that in aqueous sulfuric acid; 1,4(2,5)-dehydration occurs with inversion of configuration at C-2 or C-4. Acetylated alditols undergo similar processes via intermediates having free hydroxyl groups. Configurational inversion of 1,4- or 1,5-anhydroalditols is attributed to intermediate acyloxonium ions that are also proposed as intermediates in the structural isomerisation. Drastic treatment of each alditol gives equilibrium mixtures. The equilibrium concentrations are used to calculate free-energy differences.     

Interaction of (1→4)- and (1→6)-linked disaccharides with the fenton reagent under physiological conditions

Reactions of (1→4)- and (1→6)-linked disaccharides, mainly of maltose and isomaltose, with the Fenton reagent under physiological conditions were studied. Chemical characterization of oxidation products was conducted by g.l.c. and g.l.c.-m.s. of their trimethylsilyl derivatives, and the results demonstrated that (1→6)-linked disaccharides are more reactive with the hydroxyl radical (·OH) generated by the Fenton reagent than (1→4)-linked disaccharides. About 35–40% of (1→6)-and 15–20% of (1→4)-linked disaccharides were oxidatively degraded to smaller molecules after incubation for 24 h. Of the four disaccharides examined, namely, maltose, isomaltose, cellobiose, and gentiobiose, the α-(1→6)-linked disaccharide isomaltose exhibited the highest reactivity, whereas the β-(1→4)-linked disaccharide cellobiose showed the lowest. These results suggest the existence of a relationship between the configuration of the glycosidic linkage and the reactivity with ·OH in aqueous solution.