The polysaccharide (AF2S-2 and BF2S-2) was isolated from two types (A and B) of fruit-body of Hericium erinaceum via hot water extraction, fractional precipitation by ammonium sulfate, and DEAE-sephadex A-50 column chromatography. It was concluded from the results of the methylation study and Smith degradation that the polysaccharide was composed of a backbone of β-(1→6) -linked D-glucopyranosyl residues, and had β-D-glucopyranosyl and β-laminarabiosyl groups joined through 0-3 of n-glucose of the backbone. The structure was supported by the results of 1H- and 13C-NMR analyses and of enzymic hydrolysis of the polysaccharide. The molecular weight was estimated by gelfiltration to be 13, 000 (AF2S-2) and 22, 000 (BF2S-2).
Cyclodextran glucanotransferase (CITase) synthesized cyclodextrans (CIs) from the substrate dextran. A new assay method of CITase by the convenient measurement of its cyclization reaction was devised by using ODS C18 cartridge (Sep-Pak). By this method, the kinetic parameters of CITase for the cyclization reaction were measured. The Km values for the dextrans T-40, T-110, and T-500 were 7.5, 1.3, and 0.5 mM glucose equivalent, and the Vmax values were 122.3, 72.2, and 48.5 dumol of glucose/min, respectively. Moreover, the hydrolysis reaction of CITase was measured with dextrans and CIs by the reducing sugar assay of the Neocuproine method, and the Km values for the hydrolysis of dextrans were almost comparable with the values for cyclization reaction.
Cyclodextrin glucanotransferase (CGTase) produced cyclodextrin from the nonreducing terminals of substrate starch molecule, and this would provide a clue for the classification of CGTase into an exotype of enzyme. However, random cyclization reaction was recently demonstrated by an analysis of the initial action of CGTase. Further evidence for the random attack of CGTase on soluble starch was obtained by the following experiments. In a comparison of the cleavage pattern of CGTase on the fluorescent-labeled soluble starch with Taka-amylase A (endo-type) and glucoamylase (exo-type), the characteristics of CGTase were much closer to an endo-type action than to an exo-type action; namely, a rapid decrease in the substrate molecular weight was observed. A very low production of reducing sugar, in spite of a large decrease in blue value, was another characteristic obtained for the CGTase reaction, which would suggest that the production of oligomer fragments having a reducing end group was much less than that of cyclized fragments. These results show that CGTase attacked the starch substrate not only at sites close to the nonreducing terminals in an exo-type of cleavage, but also at sites in the middle of the substrate molecule in cleavage much like an endo-type.
The non-Newtonian behavior and dynamic viscoelasticity in aqueous solution of potato amylopectin were measured with a rheogoniometer, and the molecular origin for the rheological characteristics were discussed in comparison with those of rice amylopectin. The flow curves, at 25t, of potato amylopectin showed shear-thinning behavior at 2.0 and 4.0% concentration, but plastic behavior at 6.0%, the yield value of which was estimated to be 0.8 Pa. Although the dynamic viscoelasticity of rice amylopectin showed a constant value, it decreased in potato amylopectin solution when the temperature was increased. The tan s value was 1.15 at a concentration of 6.0% even at 0°C. The dynamic modulus decreased with the addition of urea (4.0 M) and in alkaline solution (0.05 N and 0.10 N NaOH) during an increase in the temperature. An increase of the dynamic modulus, however, was observed in 85% DMSO. A slight intramolecular association in potato amylopectin molecules seems to beinvolved in aqueous solution. The phosphate groups attached at C-6 seem to prevent intramolecular associations of potato amylopectin molecules in aqueous solution.
The effect of waxy-barley flour substitution on the physical properties of wheat dough and bread was studied. The substitution of more than 20% waxy-barley flour in the presence of 0.3% calcium stearoyl-2-lactylate (CSL) decreased the loaf volume significantly, compared with the control, but no distinct differences between the loaf volume made with waxy and nonwaxy (draft) barley flours were observed. In farinograph mixing, 20% substitution of waxy-barley flour in wheat dough did not change the arrival time of the dough, but the substitution of the waxy-barley flour with lipase or CSL, or both, decreased the development and stability times from those of the control, whereas the water absorption ratio was significantly greater than in the control. Similarly, the compression stresses, modulus of elasticity, and viscosity coefficient of 20% substitution of waxy-barley flour in dough with lipase, CSL, or both were significantly lower than those of the control. The substitution of waxy-barley flour in dough tended to slightly increase the gelatinization temperature and enthalpy over that of the control. Under the scanning electron microscope, the substitution of waxy-barley flour in dough was somewhat smooth in appearance, and the starch granules were completely covered with gluten. The firmness of bread crumbs during storage was significantly increased only at 30% substitution of waxy-barley flour in wheat flour.
We performed a kinetic study to estimate the action mode of two α-amylases, TVA I and TVA II, on glucosyl cyclodextrins. The enzyme genes from Thermoactinomyces vulgaris R-47 were individually cloned and expressed in Escherichia coli, and the enzymes were prepared from the transformants. TVA I hydrolyzed glucosyl-γ-cyclodextrin well, whereas TVA II hydrolyzed glucosyl-α- and -β-cyclodextrins. TVA I and TVA II hydrolyzed glucosyl cyclodextrins to produce glucose, maltose, panose, and 62-α-glucosylmaltotriose.
Bacillus stearothermophilus designated strain SA0301 produces extracellular oligo-1, 6-glucosidases during the stationary phase of growth. The enzyme was purified to an electrophoretically homogeneous form. Its molecular mass was estimated to be 63 kDa by SDS-PAGE, and its N-terminal amino acid sequence was MERKWWKEAVVYQIYP-. The enzyme was shown to hydrolyze isomaltose, isomaltotriose, and panose, but not trehalose, sucrose, or maltose.
The oligosaccharide units of xyloglucans from some leaf and root vegetables were comparatively analyzed by enzymatic digestion followed by anion-exchange chromatography with pulsed amperometric detection. The enzymes used were a xyloglucan-specific endo-1, 4-β-D-glucanase (xyloglucanase) from Penicillium sp. M451 and an isoprimeverose-producing oligoxyloglucan hydrolase from Eupenicillium sp. M9. The oligosaccharide units of the polysaccharides were XXXG, XXLG, XLXG, XXFG, XLLG, and XLFG [where each (1→4) -β-linked D-glucosyl residue in the backbone is given a one-letter code according to its substituents: G, β-D-Glc; X, cr-D-XyI-(1→6)-R-D-GIc; L, R-D-GaI-(1→2) -α-D-XyI- (1→6) -β-D-GIc; F, α-L-Fuc- (1→2) -β-D-Gal- (1→2) -α-D-XyI- (1→6) -β-D-GIc] in an approximate molar ratio of 35 : 6 : 5 : 22 : 3 : 29 for cabbage, of 35 : 9 : 5 : 24 : 2 : 25 for Chinese cabbage, of 38: 6:5:22:5:24 for spinach, of 35:14:6:19:4:22 for chingentsuai, of 33:7:4:31:3:22 for lettuce, of 35: 3 : 8:17: 4 : 33 for turnip, of 34 : 2:10:13: 3 : 38 for Japanese radish, of 34 : 3 : 4 : 27: 4 : 28 for edible burdock, of 33 : 7 : 4 : 29 :1: 26 for carrot, and of 41: 2 : 5 : 37: 2 :13 for East Indian lotus.
Asn-linked oligosaccharides on glycoproteins are divided into four subgroups: mannan-, high-mannose-, hybrid- and complex-type. Complex-type oligosaccharides have SA-Gal-G1cNAc-branches on terminal mannosides of the core structure (Man3GlcNAc2), and they are identified as a bi-, tri-, or tetra antennary structure depending on how many branches they have. A bisect structure that has a GlcNAc linked to the β1-4 mannose of the core structure has also been observed in many species. The branched portions of oligosaccharides affect 1) the interaction of the outer parts of oligosaccharides with other molecules such as lectins by increasing the multivalency of ligands, and 2) the turnover of proteins in circulation mainly because of the bulky structure of branched oligosaccharides. Several factors determine oligosaccharide branchings. The enzymes that catalyze branchings are N-acetylglucosaminyltransferases, some of which require products of other enzymes or inhibit actions of other enzymes. Furthermore, the timing when a-mannosidase II and galactosyltransferase react on the substrate oligosaccharides may also influence the number of branches. The activities and distribution in the Golgi apparatus of these enzymes are enzymatic factors that determine the branchings. A higher structure of peptide backbones may also influence the size of sugar chains. All the mammalian enzymes regulating branched structures are now available because GnT-IV, the missing link of GnTs, has been purified and cloned by our group. Several attempts to alter sugar-chain structures by using some of those enzymes have already been reported. In the future, a method to freely control the structures of Asn-linked oligosaccharides will be developed by regulating the expressions of these enzymes.
Chitin and its N-deacetylated derivative, chitosan, are manufactured on a large scale from the outer shells of crustaceans (crabs and shrimps), which are attractive as functional natural materials. In the past, a major trend of applied research on chitin and chitosan has been directed toward the use of these polysaccharides. But it is important, by using their hydrolysates, chitin-oligosaccharides and chitosanoligosaccharides, which have unique characteristics that are different from polymers, to achieve highutilization. This review deals with the properties and uses of these oligosaccharides by focusing on recent developments.