Glucosylpsicose was synthesized by transglycosylation with α-cyclodextrin and D-psicose using cyclomaltodextrin glucanotransferase (EC 126.96.36.199; CGTase). The structure of glucosylpsicose was analyzed by HPLC to determine sugar composition and molecular mass, by methylation analysis using GC-MS and by α- and β-glucosidase treatments to determine linkage, and 1H- and 13C-NMR spectrometries to obtain the anomeric configuration of the glycosidic linkage. By chemical analysis, it was found that the structure of glucosylpsicose is 1-o-α-D-glucopyranosyl-D-psicose.
The molecular structures and some properties of starches of Hokkaido rice (Hoshinoyume, Kirara 397 and Aya) were examined and compared with Akita rice (Akitakomachi). The Hokkaido cultivars showed a Ca type of X-ray diffraction pattern, and Akitakomachi showed an A type. The actual amylose contents of Hoshinoyume and Kirara 397 were 18%. Pasting properties determined with RVA showed that Hoshinoyume and Kirara 397 had smaller maximum viscosity than Aya and Akitakomachi, and smaller breakdown and higher setback than Akitakomachi. Iodine affinity of amylose in Hoshinoyume and Kirara 397 was almost 19. The average degree of polymerization (DPn) showed that Hoshinoyume and Kirara 397 (-900) were similar to Akitakomachi, and smaller than Aya (-1000). The average number of chains was 2-3. The molar-based distribution of the three Hokkaido varieties was wider than for Akitakomachi. The iodine affinity of the amylopectin of Hoshinoyume and Kirara 397 was 2.5 times higher than for Aya and Akitakomachi. The DPn of Akitakomachi (9400) was the biggest, and of Aya (7600) was the smallest. The average chain lengths of the Hokkaido varieties are 1-2 residues shorter than Akitakomachi. The β-amylolysis limit was a little higher than that of Akitakomachi. The amylopectin super-long chain contents of Hoshinoyume and Kirara 397 were about 3.5 times higher than for Akitakomachi. From these results, it was concluded that in terms of eating qualities of cooked rice, Aya has better ones than the other Hokkaido cultivars because of its lower apparent amylose content and consequently, lower setback even though the setback of Aya was still slightly higher than that of Akitakomachi. Thus, besides amylose content, the amylose molecular weight distribution and the amylopectin chain-length distribution appeared to be factors affecting starch pasting properties, suggesting that the molecular structure, especially the super-long chain content of amylopectin, should be paid more attention in rice breeding for better eating quality.
Waxy rice is used for staple and processed foods, and for its effective breeding an evaluation/selection index has been required. Here, the chain-length distribution of amylopectin from japonica waxy rice varieties with different hardness of rice cake were analyzed by the fluorescent labeling/HPSEC method, and the relation of the unit chain-length distribution to the hardness was examined. The difference among varietals was found in the amylopectin structure. Waxy rice producing soft cake, such as Hakuchoumochi, has more short chains (A+B1), and fewer long chains (B2+B3), than that producing hard cake, such as Koganemochi. The molar ratio of short chains (A+B1)/long chains (B2+B3), which is considered to be the number of chains comprising a cluster of amylopectin, was 10.1-11.7, being different among the varieties tested. The molar ratio determined by fluorescent response was much more reliable than the weight ratio determined by IR response because of the stable base line during HPLC. A negative correlation was found between the molar ratio and the hardening of rice cake. Therefore, the molar ratio (A+B1)/(B2+B3) of amylopectin unit-chains seemed to be one of useful evaluation/selection indexes for breeding of waxy rice. An index, or molar ratio, lower than 11 appeared to be suitable for processing use, and one higher than 11.5 for principal food use.
Formation of δ-tocopherol (δ-Toc) and cyclodextrin (CDs) complexes were studied by two kinds of methods. The first was a method (the CD wrap method) newly developed by the author, in which the mixture of starch and δ-Toc was reacted with cyclodextrin glucanotransferase (CGTase) in a 20% ethanol solution. The other was an inclusion method, in which the mixture of δ-Toc and CDs was vigorously stirred to form complexes in a water solution. The products prepared by the two methods were analyzed by fast atom bombardment (FAB) mass spectrometry. The peaks corresponding to δ-Toc-α-CD and δ-Toc-β-CD complexes were found in mass spectra of samples prepared by the CD wrap method, whereas such peaks were not found in the mass spectra of the inclusion method samples.
There are more than 500 kinds of oligosaccharide occurring in nature, and all of these are synthesized by chemical and enzymatic reactions. Various studies have recently identified physiological and physical applications of such oligosaccharides in agricultural chemistry, nutrition and medicine. New technologies for the production of oligosaccharides from natural resources have been developed in the food industry and several oligosaccharides are now produced on a large scale as ingredients in animal feed. We have investigated the isolation of fructo-oligosaccharides, as well as the enzymatic synthesis of oligosaccharides that have functional activities as tertiary functional ingredients in food. We have studied the purification and characterization of several fructosyltransferases from asparagus roots and onion bulbs, including sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT), and the novel enzyme fructan:fructan 6G-fructosyltransferase (6G-FFT). We have also reported that asparagus 1-FFT synthesized new functional oligosaccharides elongated with one or two additional fructose units by fructosyltransfer from 1-kestose to 4G-β-D-galactosylsucrose, and this compound selectively stimulates the growth of Bifidobacteria. For industrial applications, we attempted the isolation and expression of cDNAs encoding 6G-FFT, 1-FFT and 1-SST from asparagus plants. The cDNAs encoding 6G-FFT, 1-FFT and 1-SST were isolated from a cDNA library of asparagus leaves or roots, and the isolated cDNAs were designated aoft1, aoft2 and aoft3, respectively. The deduced amino acid sequences of these cDNAs showed high homology with those of plant fructosyltransferases. Expression of these cDNAs was performed using Pichia pastoris. The recombinant protein from Pichia transformed with aoft1 produced 1F,6G-di-β-D-fructofuranosylsucrose, neokestose and sucrose from 1-kestose, while the transformant with an empty vector produced no saccharides. These results confirmed that 6G-FFT was expressed in P. pastoris. Similarly, the recombinant protein from Pichia transformed with aoft2 produced nystose from 1-kestose and the recombinant protein with aoft3 produced 1-kestose from sucrose. These results confirmed that 1-FFT and 1-SST were expressed in P. pastoris, and that the recombinant proteins had enzymatic properties similar to those of 6G-FFT, 1-FFT and 1-SST from asparagus roots. We then examined the conversion of substrate specificity from 6G-FFT to 1-FFT by point mutations in the β-fructosidase motif. The asparagine in this motif in aoft1 was changed to serine, and the mutant recombinant protein was characterized. We found that this amino acid substitution in wild-type aoft1 changed the substrate specificity from 6G-FFT to 1-FFT. Finally, we studied the activities of the main enzymes involved in the synthesis and hydrolysis of fructo-oligosaccharides during the post-harvest life of onion bulbs and asparagus spears, and discuss the mechanisms triggering these enzyme activities, as well as the mechanisms by which fructo-oligosaccharides contribute to the quality and perishability of the vegetables.
This paper describes our studies on the fine structures and functions of the important hemicellulose, xyloglucan and glucuronoarabinoxylan, of the primary cell-walls of all higher plants. Xyloglucan: 1) A method for rapidly identifying six of the most commonly found xyloglucan oligosaccharide units, XXXG, XLXG, XXLG, XLLG, XXFG and XLFG was developed by high-performance anion exchange chromatography with pulsed amperometric detection before and after digestion with purified isoprimeverose-producing oligoxyloglucan hydrolase (IPase). The usability of this method was confirmed strongly by the studies of the compositional analysis of oligosaccharide units of xyloglucans isolated from the cell-walls of thirteen vegetables and eleven fruits. In our sequential experiments, most fucose-containing xyloglucans were shown to have essentially a small quantity of XLLG, XLXG and XXLG besides XXXG, XXFG and XLFG as major structural oligosaccharide units. 2) The structures of xyloglucans from Solanaceae (eggplant, sweet pepper, red pepper and tomato) and Gramineae (barley and bamboo shoot) were investigated using xyloglucan-specific enzymes (xyloglucanase, IPase, oligoxyloglucan reducing endo-specific cellobiohydrolase), β-D-glucosidase and β-D-galactosidase. The former was built from hexa-(XXGG), hepta-(XLGG, LXGG, and XSGG) and octa-saccharide units (LSGG), and the latter was built from tetra-(XX), penta-(XXG), hexa-(XXGG), hepta-(XXGGG and LXGG), octa-(XLGGG, LXGGG, XXGGGG, and GXXGGG) and nona-saccharide units (XLGGGG, LXGGGG, GXLGGG, GLXGGG, and XXGGGGG). 3) A comparative study of the structures of xyloglucans prepared from the cell-walls of apple fruit obtained at various development stages showed that the structural modifications of cell-wall xyloglucan occur during development of apple fruit. Glucuronoarabinoxylan: 1) Feruloylated glucuronoarabinoxylan liberated from Zea shoot cell-walls by treatment with a purified endo-(1→4)-β-D-xylanase had a xylan backbone which contained (1→4)-β-D-xylopyranosyl residues, with 60 to 70% substitution at the C-2 or C-3 position with arabinose, glucuronic acid, and other substituents. Ferulic acid occurred at 3-O-arabinosyl branch-points of linear backbones of (1→4)-linked β-D-xylopyranosyl residues in the glucuronoarabinoxylan. 2) Feruloyl and diferuloyl esters between polysaccharides were found to be involved in the aggregation of cultured rice cells. 3) The rootlets from the kilned malt were hydrolyzed with acid followed by treatment with an enzyme to give carbohydrate fragments containing ferulic acid. Feruloylated mono- and tri-saccharide fractions were prepared from the mixture of fragments. These fractions were tested for effects on the growth of COLO 201 human tumor cells before and after treatment with esterase. The cell growth was reduced by feruloylated mono- and tri-saccharides, especially feruloylated tri-saccharide, and was not reduced by ferulic acid, monosaccharide or trisaccharide.
Carbohydrates are the most abundant material produced by plants and processes for their efficient conversion and utilization are of major importance in biotechnology. Their enzymatic conversion can be systematically optimized if suitable enzymes of known degradative activity are available. However, the process is complicated for pectins, in which the carbohydrate structures and sugar composition are varied. Pectins consist of homogalacturonan regions and rhamnogalacturonan regions carrying neutral sugar side-chains. Galacturonic acid residues in the homogalacturonan may be replaced by different compounds such as methanol, acetic acid and xylose. Arabinans and galactans are most frequently found in rhamnogalacturonan regions of pectins. Furthermore, ferulic acid is associated almost exclusively with the neutral sugar side-chains. In order to achieve effective or selective degradation of pectins, I have studied various pectin-degrading enzymes with different catalytic properties. This paper deals with characterizations of arabinanases, ferulic acid esterases, a rhamnogalacturonase and an exo-polygalacturonase.
The mechanism of the suppression of lipid peroxidation by saccharides was investigated. Trehalose effectively inhibited the heat- or radical-induced peroxidation of unsaturated fatty acid (UFA). Several other saccharides, such as sucrose, maltose and neotrehalose, showed negligible effect on the peroxidation, but maltitol's effectiveness was second to that of trehalose. Maltitol inhibited the peroxidation of UFA by its radical-scavenging effect. Thus, among the sugars studied so far, trehalose is a unique antioxidant whose reaction mechanism has not been clarified. We started this study with the hypothesis that trehalose interacts directly with oxidation-sensitive parts of UFA and consequently protects it from the autoxidation. NMR experiments performed, including 1H-1H NOESY measurements, indicated that trehalose selectively interacts with the cis-olefine proton pair in the above UFA with 1 : 1 stoichiometry, and the C-3 (C-3´) and C-6´ (C-6) sites of the sugar are responsible for the interaction. Similar interactions were not observed for the mixtures of the UFA and other saccharides. Quantum chemical study indicates that the OH-3 and OH-6 groups of trehalose bind to the olefin double bonds through OH…π and CH…O types of hydrogen bonds, respectively. Furthermore, the activation energies were calculated for the hydrogen abstraction reactions from the activated methylene group of heptadiene by a methyl radical. The activation energy drastically increased to ca. 30 kcal mol-1 on the complexation with trehalose. These results strongly support the antioxidant mechanism deduced in the previous study and indicate that the formation of unique multiple hydrogen bonds between trehalose and cis-olefin bonds is a rather general event, not confined to the case of UFA. This is the first study to elucidate the antioxidant function of trehalose and maltitol.