Microbial α-mannosidases

Microbial α-mannosidases are used in the analysis of glycopeptides and the developmental regulation of lysosomal enzymes. This survey presents comparison of properties of this high molecular weight, oligomeric protein from a number of microbial sources.     

Gluco-oligosaccharide synthesis by free and immobilized beta-glucosidase

Gluco-oligosaccharides were synthesized through the enzymatic condensation of D-glucose at high concentration using a commercial almond beta-glucosidase. The synthesis reactions were carried out with both free and immobilized enzyme, with or without sorbitol, an efficient depressor of water activity (a(w)) in the presence of different glucose concentrations. The yield and the composition of the gluco-oligosaccharides produced changed with the reaction mixture and the form of the enzyme used (free or immobilized). The use of 5 M glucose solution permitted only disaccharides to be obtained, whereas with a glucose concentration of 7.5 M glucose, di-, tri-, and tetrasaccharides were produced. A 7.5 M glucose solution used with 4.4 M sorbitol gave three times more disaccharides than the same solution without sorbitol. Moreover, the immobilized enzyme was much more active in synthesis. The synthesis yield (oligomers mg/mL . mg of enzyme) after immobilization was 573% compared to that of the free enzyme, when a 7.5 M glucose solution was tested. The effects of substrate concentration, sorbitol addition and enzyme immobilization were investigated. (c) 1993 John Wiley & Sons, Inc.     

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar‐proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.     

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.     

Synthesis of 2,5-anhydro-(beta-d-glucopyranosyluronate)- and (alpha-l-idopyranosyluronate)-d-mannitol hexa-O-sulfonate hepta sodium salt

Glycosidation of 2,5-anhydro-1,6-di-O-benzoyl-D-mannitol with methyl(2,3,4-tri-O-acetyl-alpha-d-glucopyranosyl-1-O-trichloroacetimidate)uronate in the presence of trimethylsilyl triflate afforded the corresponding 3-O-beta-glycoside, which after deprotection was converted into its hexa-O-sulfate with DMF x SO3 to give after treatment with sodium acetate and subsequent saponification of the methyl ester with sodium hydroxide the hepta sodium salt of 2,5-anhydro-3-O-(beta-d-glucopyranosyl uronate)-D-mannitol hexa-O-sulfate. Glycosidation of the same acceptor with the alpha-thiophenylglycoside of methyl 2,4-di-O-acetyl-3-O-benzyl-L-idopyranosyl uronate in the presence of NIS/TfOH afforded the corresponding 3-O-alpha-glycoside in very low yield, therefore the alpha-thiophenylglycoside of 2-O-acetyl-2,4-O-benzylidene-3-O-benzyl-L-idopyranose was used as donor. The terminal hydroxymethyl group of the obtained disaccharide was subsequently oxidised with NaOCl/TEMPO and the obtained iduronic acid derivative was converted into the hepta sodium salt of 2,5-anhydro-3-O-(-alpha-L-idopyranosyluronate)-D-mannitol hexa-O-sulfonate with DMF x SO3 and subsequent treatment with sodium acetate.     

Synthesis of galactofuranose-containing disaccharides using thioimidoyl-type donors

Four galactofuranose-containing disaccharides have been prepared utilising various thioimidates [Galf-SC(NR)XR'] and suitably protected acceptors as key precursors. We observed that the efficiency of the coupling reactions was particularly dependent on the aglycon present on the furanosyl donor when copper(II) ions were used as the promoter, and that activation could be correlated with the nature of the third heteroatom, X.     

The use of levoglucosenone and isolevoglucosenone as templates for the construction of C-linked disaccharides

Because of their functionalities (enone, ketone, and acetal) and their bicyclic structure (steric factors), levoglucosenone (1,6-anhydro-3,4-dideoxy-beta-D-glycero-hex-3-enopyran-2-ulose) and isolevoglucosenone (1,6-anhydro-2,3-dideoxy-beta-D-glycero-hex-3-enopyran-4-ulose) are useful templates for the convergent and combinatorial synthesis of (1-->2), (1-->3), and (1-->4)-linked C-disaccharides in reactions combining them with sugar-derived carbaldehydes. Synthetic methods relying on conjugate nucleophilic additions of these enones, their combination with aluminum reagents and aldehydes (Baylis-Hillman reaction) and modified Takai-Hiyama-Nozaki-Kishi couplings of enol triflates derived from them with sugar-derived aldehydes are reviewed. Highly stereoselective methods have thus been developed. These allow the generation of disaccharide mimetics with a high molecular diversity.     

A radioisotope method for assays of amylomaltase and D-enzyme

A method has been developed for the assay of amylomaltase based on the incorporation of a [14C]glucose moiety of uniformly 14C-labeled maltose into a maltodextrin fraction insoluble in aqueous ethanol. The presence of dextrin at a high concentration greatly enhances [14C]glucose incorporation and serves to minimize interference with the assay by contaminating enzymes that hydrolyze substrates and products in the assay mixture. Since a number of other enzymes are capable of forming glucose in the assay mixture, the 14C incorporation is a more specific method of enzyme assay than are previously reported assays based on glucose release.     

Influence of saccharides and glycine betaine on freezing of photosystem 2-enriched particles: a chlorophyll fluorescence study

Freeze-thaw treatment of photosystem 2 (PS2) enriched subchloroplast membranes induced changes in the relative proportion and antenna sizes of PS2α and PS2β. The addition of disaccharides (trehalose or saccharose) and glycine betaine before subjecting to freezing prevented these changes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Computer modelling studies on the modes of binding of some of theα-glycosidically linked disaccharides to concanavalin A

The possible modes of binding of kojibiose, nigerose, maltose and ManPα(1 → 2)Man to concanavalin A have been investigated using computer modelling studies. While α12 linked disaccharides bind to concanavalin A in two modes,i.e. by placing the reducing as well as non-reducing sugar units in the sugar binding site, nigerose or maltose can bind only in one mode,i.e. by placing the non-reducing sugar unit in the binding site. Though, both the sugar residues in α 12 linked disaccharides can reach the binding site, the preference is high for the non-reducing unit. When the non-reducing residue, in any of these disaccharides, enters the binding site, the allowed orientations and the possible hydrogen bonds with the protein seem to be independent of the glycosidic linkage. However, the number of hydrogen bonds the outward sugar residue forms with the protein are dependent on the type of linkage. Atleast one of the hydroxyl groups adjacent to the glycosidic linkage on the outward sugar residue is involved in the formation of a hydrogen bond with the protein suggesting the presence of an extended binding site. The orientation of the reducing sugar residue in the extended binding site is dependent on the linkage. Its orientation in nigerose is flipped when compared to that found in kojibiose or maltose leading to different non-covalent interactions with the protein which affect their binding affinities.