Journal of Applied Glycoscience
Online ISSN : 1880-7291
Print ISSN : 1344-7882
ISSN-L : 1344-7882
Volume 50 , Issue 2
Showing 1-39 articles out of 39 articles from the selected issue
  • Katsuo Takahashi, Mutsumi Shimada, Masatake Ohnishi
    2003 Volume 50 Issue 2 Pages 111-115
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    In the presence or absence of urea, binding of a substrate maltose (G2) and a transition-state analogue glucono-1, 5-lactone (GLN) to glucoamylase (GA) from Rhizopus niveus was studied by means of thetransient-phase kinetics using the stopped-flow method. The steady-state kinetics of the GA-catalyzed reactionfor G2 was carried out to evaluate kinetic parameters, Km and k0. Urea is known as a specific reagent for de naturation of proteins, but under the specified conditions of the experiments, we did not find a large denaturation shown by blue-shift in the difference absorption-spectra, indicating that urea-induced change in the conformation of GA was little. The binding kinetics of GLN and G2 agreed well with a two-step mechanism andit was found that urea affects k+2 and k-2 of the two-step mechanism, and Km of the steady-state. These values are closely related with a productive binding of G2 and GLN to subsite 1. Urea is known to effect a weakening of the hydrophobic interaction, so it was suggested that the effect of urea was weakening of the interaction of a saccharide with tryptophan residue(s) located at subsite 1 and in the consequence preventing the formation of a productive complex during the slow unimolecular process.
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  • Hiromi Murakami, Akiko Seko, Masumi Azumi, Natsuko Ueshima, Hajime Yos ...
    2003 Volume 50 Issue 2 Pages 117-120
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
    Lactobionic acid (LA) is derived from lactose (Lac) and has been reported to have several useful functions. LA has been supplied only as a reagent for study because it has been produced chemically in a small scale in Japan. We aimed to develop microbial production systems of LA and examined LA fermentation conditions. A three-day batch culture produced 200 g/L of LA. A ten-day fed-batch culture with periodic additions of Lac and CaCO3 accumulated 400 g/L of LA. There was no by-product and no loss of Lac, and the yield of LA-conversion was 100%. The addition of ethanol (75 % (v/v), final concentration) to culture supernatant resulted in recovery of 98 % of LA as precipitate.
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  • Sayuri Akuzawa, Akiko Kawabata
    2003 Volume 50 Issue 2 Pages 121-126
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    The physicochemical properties of starch from arrowroot, cassava, corn, edible canna, indian lotus root, katakuri, kuzu, potato, sago and sweet potato were measured. Potato exhibited a remarkably high value for swelling power of 88.0 at 95°C, next being cassava with 45.3. The swelling power of indian lotus root was 30.5, while the values for the other starches were in the range of 16.2-28.0. Cassava was the most soluble of the starch samples, corn being the lowest in solubility. Amylography represented the features of starch samples well, the temperature for increasing viscosity being in the 52.2-81.0°C range. The 6-12 chain length distribution of amylopectin was characteristic to each sample, the proportion of 6-9 chain length in particular being different. Data for the swelling power, solubility, amylograph and some physicochemical properties that had previously been measured in our laboratory were studied by principal component analysis. The component scores for the first three principal components, which related to characteristics under large deformation, those of negligible deformation, and those of starch granules, were plotted in a three-dimensional space. It was showed that the starches examined were classified into four groups by the plotted position.
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  • Jiemei Chen, Yoshio Nishida, Makoto Hisamatsu
    2003 Volume 50 Issue 2 Pages 127-131
    Published: April 20, 2003
    Released: February 23, 2011
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    Starches from a variety of Sake-rice (Yamadanishiki, Gohyakumangoku, Miyamanishiki, Kitanishiki and Wakamizu) which are suitable for Sake brewing were investigated by comparing them with Koshihikari starch to recognize their structural properties of amylopectins. The partial hydrolysate (about 15% hy drolysis) of starch with isoamylase was fractionated into three fractions (fr.1, fr.2 and fr.3) by gel permeation chromatography (GPC) with a Toyopearl HW-50S, and then the short-amylose chains derived mainly from the outer portion of the amylopectin collected in the fr.3 were analyzed by a HPAEC-system equipped with a pulsed amperometric detector (HPAEC-PAD). From comparison of the chain-length distributions between Sake-rice starches and that of Koshihikari, it was found that Sake-rice amylopectins were plentiful in longish chains around DP 13-23 and scarce in shortish chains around DP 6-12, suggesting a structural property susceptible to retrogradation. The pasting properties of Sake-rice starches analyzed by Rapid Visco Analyzer (RVA) showed higher pasting temperature (PT), higher setback (SB), and lower breakdown (BD) than Koshi hikari starch, suggesting the outer portions of amylopectin had abundant longish debranched short side chains. These results suggested that rice starches with amylopectins susceptible to retrogradation are adequate for Sake-rice.
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  • Jiemei Chen, Masako Watanabe, Tomoko Nakamori, Makoto Hisamatsu
    2003 Volume 50 Issue 2 Pages 133-137
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    Starches from three waxy rice cultivars (Hakuchoumochi, Hiyokumochi and Koganemochi) showing different physical properties were studied in order to identify the relationship between physical properties and their amylopectin structures. Pastes of Hakuchoumochi, Hiyokumochi and Koganemochi are known as soft, intermediate and hard, respectively. Pasting properties of the soft type starch showed the lowest setback (SB) and the highest breakdown (BD) among the three. The hard type showed the reverse relationship and the middle type showed an intermediate property. To follow these physical properties, partial hydrolysates of starches with debranching enzymes were prepared and the distributions of debranched short side-chains in amylopectin existing mainly in the outer layer of the amylopectin were analyzed by HPAEC-PAD. Hakuchou mochi was composed of plentiful shortish chains and scarce longish chains and suggested to be resistant toretrogradation. Koganemochi had the reverse properties and was suggested to be sensitive to retrogradation. Hiyokumochi was intermediate. From these results, it could be presumed that a feature of chain-length distribution on the outer layer of the amylopectin molecule influenced the physical properties relating to retrogra dation.
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  • Tomoko Nakamori, Tetsuji Yanagihara, Jun Kato
    2003 Volume 50 Issue 2 Pages 139-142
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
    Glutinous rice exhibits the phenomenon of turning opaque during the drying process after harvest. This phenomenon is called "Haze." "Mihaze" grains are translucent even after artificial drying. To make clear the factors causing the appearance of Mihaze grains of glutinous rice in Hokkaido, moisture content of grains and cultivation conditions were investigated. As the moisture content of grains of glutinous rice was decreased by experimental drying, most of the grains turned opaque at a moisture content of less than 15.0%. However a few of them remained translucent at a moisture content below 15.0%, suggesting that there were Mihaze grains even at the low moisture content as commercial products. Therefore it was concluded that there are factors causing the appearance of Mihaze grains besides moisture content. The frequency of Mihaze grains was not affected directly by contents of protein and starch, and starch granule size. It was observed that there was no difference in contents of moisture, protein and starch, or starch granule size and iodine absorption spectrum of each prepared starch between Haze and Mihaze grains.
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  • Birte Svensson, Haruhide Mori, Kristian Sass Bak-Jensen, Morten Tovbor ...
    2003 Volume 50 Issue 2 Pages 143-145
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    The mutational analysis of the roles of specific side chains at individual subsites have been conducted for barley α-amylase 1(AMY1) across the ten subsites long substrate binding cleft. The present study specifically focuses on such mutants in which the AMY2 structure has been mimicked. Generally the kinetics parameters for mutants at subsites accommodating the substrate glycone part showed decreased affinity for oligosaccharide and amylose DP 17 whereas an aglycon binding subsite +4 AMY2 mimic had increased affinity but reduced activity. Among barley α-amylase/subtilisin inhibitor (BASI) recognition mimics, further more mutation at one site provided weak inhibition of an otherwise insensitive AMYL type hybrid, whereas a mimic in another area of the protein-protein interface was not affected by BASI.
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  • John F. Robyt
    2003 Volume 50 Issue 2 Pages 147-155
    Published: April 20, 2003
    Released: February 23, 2011
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    One hundred years ago, at the beginning of the 20th Century, enzymes were just beginning to be recognized as powerful catalysts that had a high degree of specificity. Michaelis-Menten kinetics indicated that catalysis occurred after an enzyme-substrate complex was formed. Studies on several carbohydrate enzymes, invertase, phosphorylase, dextransucrase, α-amylases, β-amylases, lysozyme, sucrose phosphorylase, cellulose synthase, and starch synthase have contributed a large body of information about subsite binding specificity, the formation of enzyme-substrate complexes, the formation of a covalent enzyme-product-intermediate from an SN2 reaction that undergoes a second reaction to give the final product(s). Mechanisms for several carbohydrate enzymes are presented.
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  • Y. Sakano, Y. Nakamura, T. Kuriki, H. Matsui, M. Tsuzuki, G. Okada
    2003 Volume 50 Issue 2 Pages 157
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
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  • Tamao Hatta, Seiko Nemoto, Keiji Kainuma
    2003 Volume 50 Issue 2 Pages 159-162
    Published: April 20, 2003
    Released: February 23, 2011
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    The outermost surface structure and internal structure of starch granules were linked using AFMand SEM. Within a potato starch granule, there are radial linear structures that extend from the hilum and grow wider with increasing distance from the center. The many convexoconcave structures and small raised nodules on the outermost surface of starch granules consist of aggregates of molecular chain terminals, or the minimum structural units of starches. By analyzing the outermost surface of starch granules using XPS, we obtained spectra from the Cls orbital which show lower binding energies typical of starches.
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  • Yasuhito Takeda, Isao Hanashiro
    2003 Volume 50 Issue 2 Pages 163-166
    Published: April 20, 2003
    Released: February 23, 2011
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    The fluorescent labeling/HPSEC method was developed and the molar-based distribution of amylopectin unit-chains, and molecules of amylose and amylopectin from various sources was examined. The molar-based distribution of A and B chains enabled the estimation of the number of chains per cluster. Cereal amylopectins had a larger number of chains in a cluster than root and tuber amylopectins. A-Crystalline type starches contained amylopectin with a larger number of chains per cluster than B-type crystalline. The molar distribution of C chain suggested the presence of long and short C-chains, which appeared to connect two clusters and were in single cluster, respectively. Amylopectin comprised three molecular species. The large species was major in amount by mole and weight, but the medium and small species were relatively large in amount by mole but very small by weight. The three species appeared to be built up with a similar cluster in structure and differed in the number of clusters. The number of clusters suggested that the large and medium species might be blocklets in granule while the small species immature and/or degraded products of the large species. Amylose comprised several molecular species with different size, and their proportions differed by plant sources. The small amylose species was predominant in cereal amyloses while the large amylose species in root and tuber amyloses. The number-average degree of polymerization of amyloses and the number average chain length of amylopectins determined by the labeling/HPSEC method were in good agreement with those determined by conventional colorimetric methods.
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  • Jay-lin Jane, Zihau Ao, Susan A. Duvick, Maria Wiklund, Sang-Ho Yoo, K ...
    2003 Volume 50 Issue 2 Pages 167-172
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
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  • Alison M. Smith, Samuel Zeeman, Totte Niittylä, Heike Kofler, Dav ...
    2003 Volume 50 Issue 2 Pages 173-176
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    Study of mutants of the model plant Arabidopsis is providing new information about the nature and regulation of starch degradation in leaves. The onlyoc-amylase currently predicted to be in the chloroplast is not necessary for the initial attack on the starch granule, and the enzyme(s) responsible for this attack remain unknown. A starch-water dikinase that phosphorylates glucose residues in amylopectin is necessary for starch degradation, and it seems likely that the enzyme(s) responsible for the initial attack require either the dikinase itself or phosphorylated regions of amylopectin for their activity. At least four chloroplastic enzymes could potentially debranch glucans released by the initial attack on the granule: the relative importance of these enzymes is not yet known. The degradation of linear glucans to monomers is catalysed by β-amylasesrather than starch phosphorylase. Plants lacking chloroplastic starch phosphorylase have normal rates of starch degradation. The fate of the maltose produced by β-amylolysis of linear glucans is being investigated through study of maltose-accumulating mutants. Other malto-oligosaccharides too short to be attacked by β-amylase are metabolised by disproportionating enzyme to produce longer chains susceptible to further attack. The process of starch degradation is subject to strong diurnal regulation. The nature of this regulation is not understood, but new approaches to the problem are suggested.
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  • Jennifer M. Imparl-Radosevich, Jamin R. Gameon, Angela McKean, Deborah ...
    2003 Volume 50 Issue 2 Pages 177-182
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    We have previously reported the kinetic properties of maize starch synthase I (SSI), SSIIa and SSIIb using glycogen and amylopectin as primer and have determined that these SS enzymes can be distinguished based on their kinetic properties. However, it is not clear how starch synthesis is initiated and what the native primer for SS in vivo is. Therefore, in this study, we determined whether maize SS can catalyze the de novo synthesis of α-glucan and studied how maize SS utilizes potential native primers such as maltooligosaccharides (MOS) and developing native starch granules. Using purified recombinant maize SSI, we provide evidence that maize SSI cannot catalyze the de novo synthesis of α-glucan and the “unprimed activity” may be due to the presence of primers in ADPG. However, maize SSI, SSIIa and SSIIb, can use maltose as a primer, although much less efficiently than MOS with a DP of 3 or greater. All MOSs tested can be elongated by SSI, SSIIa and SSIIb with more than one glucose residues. For the first time we describe here that purified maize SS can further elongate the chains of native starch granule isolated from developing maize waxy endosperm (18 days after pollination). Compared with their kinetic properties in the presence of soluble amylopectin and glycogen, differences in the Vmax and optimum temperature of these three enzymes were ob served in the presence of native starch granules. The differences between SSI, SSIIa and SSIIb in their primer preferences using MOS and native starch granule may provide some clues as to the functions of different SS in starch synthesis.
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  • Hirokazu Matsui, Hiroyuki Ito
    2003 Volume 50 Issue 2 Pages 183-186
    Published: April 20, 2003
    Released: February 23, 2011
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    We isolated two SBE cDNA species (pvsbel and pvsbe2) from immature seeds of kidney bean (Phaseolus vulgaris L.) and characterized the enzymatic properties of the coded recombinant enzymes (rPvSBE1 and rPvSBE2). The primary sequences of pvsbel and pvsbe2 displayed significant similarity to other class B and A SBEs, respectively. Northern blot analysis revealed that pvsbe2 showed maximum steady state transcript levels at the mid-stage of seed maturation, whereas pvsbel reached peak levels at a later stage. Immunoblot analysis showed that PvSBE1 was associated with the starch granule fraction, whereas PvSBE2 was present in the soluble fraction. In addition, rPvSBE1 and rPvSBE2 showed different kinetic properties and substrate preferences. Native PvSBE2 from immature seeds has a molecular mass (82 kDa) significant smaller than those reported for the other class A SBEs. Based on the observations of smaller molecular mass of PvSBE2 and its partial localization, we suspected the occurrence of a larger form of PvSBE2 (LF-PvSBE2) containing an extended N-terminal region. Indeed, LF-PvSBE2 was observed in both the soluble and starch granule fractions of developing seeds. Immunoblot and molecular analyses suggest that the two isoforms, LF-PvSBE2 and PvSBE2 are products of the same gene. Analysis of the products by 5'-RACE (rapid amplification of cDNA ends) indicated that the two transcripts for pre-LF-PvSBE2 and pre-PvSBE2 are generated by alternative splicing of the first two exons. Recombinant LF-PvSBE2 (rLF-PvSBE2) showed much higher affinity for amylopectin than rPvSBE2. These results suggest that the N-terminal extension of LF-PvSBE2 plays a critical role for subcellular localization in starch-granules by altering its enzymatic properties.
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  • Steven Ball, Luc Liénard, Fabrice Wattebled, Martin Steup, Glen ...
    2003 Volume 50 Issue 2 Pages 187-189
    Published: April 20, 2003
    Released: February 23, 2011
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    Bacterial glycogen and plant starch metabolism both require the presence of malto-oligosaccharide assimilation enzymes. In Escherichia coil maltotetraose is generated through debranching of the glycogen limit dextrin produced by glycogen phosphorylase. This maltotetraose if further metabolised through the combined action of amylomaltase (an α-1, 4 glucanotransferase) and maltodextrin phosphorylase. In the starch accumulating alga Chlamydomonas reinhardtii we show that a deficiency in D-enzyme (the plant α-1, 4 glucanotransferase) leads to a severe decrease in starch content and a modification in amylopectin structure as well as a modification in amylose content. We further show that there are 2 distinct plastidial phosphorylases in Chlamydomonas. Kinetic and genetic studies suggest these forms may be related to the maltodextrin and glycogen-type of phosphorylases from bacteria.
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  • Jason R. Dinges, Martha G. James, Alan M. Myers
    2003 Volume 50 Issue 2 Pages 191-195
    Published: April 20, 2003
    Released: June 28, 2010
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    The functions of the two known plant classes of α(1→6) glucan hydrolase were investigated by genetic analysis in maize. A null mutation in the zpul gene, coding for the pullulanase-type starch debranching enzyme (DBE), was coupled with three different alleles of the sul gene, coding for the isoamylase-type DBE . In two instances the starch biosynthesis abnormality conditioned by the isoamylase-type DBE mutation, namely a decrease in starch content and an increase in phytoglycogen content, was exacerbated by the additional loss of pullulanase-type DBE activity. These data confirm previous findings that pullulanase-type DBE contributes to starch biosynthesis in developing endosperm. An appreciable level of starch was still formed, however, in mutant endosperm bearing null mutations for both classes of DBE, and in this instance the pullulanase-type DBE had no effect on the amount of starch produced. The data suggest that a critical level of isoamylase-type DBE protein or activity may be required in order for pullulanase-type DBE to be able to function productively in starch biosynthesis. Furthermore, the data show clearly that a significant degree ofstarch biosynthesis, approximately 40% of normal, occurs in the absence of any detectable DBE activity in the endosperm of maize kernels.
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  • Yasunori Nakamura, Naoko Fujita, Akiko Kubo, Sadequr Rahman, Matthew M ...
    2003 Volume 50 Issue 2 Pages 197-200
    Published: April 20, 2003
    Released: February 23, 2011
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    Metabolic engineering of the structure of amylopectin in rice endosperm has been performed by manipulating the activities of the major isoforms of soluble starch synthase, starch branching enzyme, and starch debranching enzymes. Our results show that each isoform distinctly affects amylopectin cluster structure and that the change leads to the synthesis of novel starches with varied physicochemical properties . This paper describes these experimental results and discusses the advantage of the molecular approach for elucidation of the starch biosynthesis system and for production of novel starches.
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  • Kelly M. Gibson, Seon-Kap Hwang, Gerald E. Edwards, Thomas W. Okita, C ...
    2003 Volume 50 Issue 2 Pages 201-206
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
    Plant productivity and yield depend on the capacity of leaves to fix CO2 during photosynthesis and on the capacity of sink tissues such as developing seeds or fruits to convert the fixed carbon into dry matter. The capacity to convert triose-phosphate to sucrose or starch in the leaf can limit the maximal rate of photo synthesis, and we have demonstrated that photosynthetic capacity and growth correlate with the capacity to make leaf starch. The findings presented here reveal that in Arabidopsis thaliana with increased activity of the major starch regulatory enzyme, ADPglucose pyrophosphorylase (AGPase), there is a greater capacity for starch biosynthesis compared to wildtype and a starch-deficient mutant. This resulted in increased CO2 as similation due to suppression of photosynthetic feedback and in higher starch turnover rates in these plants. Collectively these effects translated to increased growth and seed biomass. Previous studies from our laboratory showed that rice is limited by the capacity to convert triose-P to carbohydrates under ordinary environ mental conditions. Therefore, productivity may benefit by increasing the conversion of triose-P into leaf starch through increased AGPase activity.
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  • Christophe Colleoni, Alan M. Myers, Martha G. James
    2003 Volume 50 Issue 2 Pages 207-212
    Published: April 20, 2003
    Released: June 28, 2010
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    Starch metabolizing enzyme isoforms are known to exhibit functional interactions. This research employed one and twodimensional starch zymograms to investigate pleiotropic enzymatic effects in starch branching enzyme (BE) and debranching enzyme (DBE) mutants of maize. Activity bands are shown to correspond with specific gene products by immunoblot analysis or biochemical assay. Multiple migratory forms are observed for BEI and BEIIa, and a single form for BEIIb. SU1 isoamylase-type DBE migrates as three distinct forms and ZPU1 pullulanase-type DBE as a single form. For each BE and DBE, a genetic null mutation conditions complete loss of all forms of the respective enzyme. The BE mutation amylose-extender also results in loss of two BEI migration forms, and the sul-starchy and zpul-204 DBE mutations result in a loss of BEIIa activity. In both DBE mutants, BEIIa is of normal abundance and size, indicating the effect is posttranslational and directly impacts enzyme function. Zymogram analysis of the uncharacterized opaque5 (o5) mutant shows o5 endosperm is deficient in β-amylase activity, providing insight into a biochemical defect resulting from the mutation. Finally, a novel enzyme activity identified in starch zymograms was characterized and found to be a glucan hydrolase of the β-amylase type. This research demonstrates that activity gel analysis is an effective approach for uncovering pleiotropic enzymatic effects that might not be discovered using RNA microarray or proteomics technologies, and indicates that some mutational effects on starch metabolizing enzymes are likely due to altered protein complex associations.
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  • Takayuki Umemoto, Noriaki Aoki, Takeshi Ebitani
    2003 Volume 50 Issue 2 Pages 213-216
    Published: April 20, 2003
    Released: July 01, 2011
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    We genetically analyzed cooked rice quality differences between japonica and indica rice varieties . By using backcross inbred lines, substitution, and near isogenic lines, we confirmed that at least 3 chromosomal regions affect cooked rice quality as measured by a Mido Meter. Each of these regions contains a locus for the starch synthase isoform, granule-bound starch synthase I (GBSSI), starch synthase I, and starch synthase ha (SSIIa). We found naturally occurring variations in GBSSI (Wx protein) amount and SSIIa activity that greatly affect cooked rice quality by altering amylose content for GBSSI or amylopectin structure for SSIIa. In sensory tests, effects of GBSSI variation were clear even just after cooking, but those of SSIIa became clear only after cooling.
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  • Matthew K. Morell, Ahmed Regina, Zhongyi Li, Behjat Kosar-Hashemi, Sad ...
    2003 Volume 50 Issue 2 Pages 217-224
    Published: April 20, 2003
    Released: February 23, 2011
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    The synthesis of starch in wheat and barley is an important topic for research because of the exten sive utility of starch from these crop species in human and animal foods, and in industrial processes. Wheat and barley starches are highly characteristic due to their granular architecture and multi-modal granule size distribution. This granule architecture is important because it defines the ways in which wheat and barley starches behave during food processing. The core starch biosynthetic genes of wheat have been cloned and shown to exist as homeologous sets of genes represented on each of the three wheat genomes. While hexaploidyrepresents a major impediment to the selection of altered starch phenotypes by phenotypic screening, the availability of methods for identifying the products of homeologus genes from each of the wheat genes has provided methods for the selection of triple null lines from waxy, starch synthase ha, and branching enzyme I genes. In barley, direct phenotypic selection has resulted in the identification of waxy, amol and SSIIa mutations. In this paper, we review the state of knowledge of starch synthesis in wheat and barley and discuss the relationships between individual genes and their roles in starch biosynthesis.
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  • Hikaru Satoh, Aiko Nishi, Naoko Fujita, Akiko Kubo, Yasunori Nakamura, ...
    2003 Volume 50 Issue 2 Pages 225-230
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    We isolated various kinds of mutants participating in the biosynthesis of endosperm starch by the MNU treatment of fertilized egg cells in japonica rice cultivars. There were at least 6 mutant loci lowering amylose content including the wx locus. These low amylose mutations altered the amylose content without affecting the amylopectin structure. The amylose content did not affect the initiation of gelatinization, but influenced greatly the swelling of gel, indicating that amylose content affects the swelling power of gel and the termination of gelatinization. BEIIb mutation decreased specifically the short chains with DP less than 17 in amylopectin whereas BEI mutation was characterized by a significant decrease in long chains with DP longer than 38 and short chains with DP 12 to 23, suggesting that BEIIb and BEI contributes to the synthesis of A chains, and B, and B3 chains of amylopectin, respectively. Notable alteration on the chain length profile was not observed in BEIIa mutation. Each BE mutation showed distinct effect on the thermal properties of endosperm starch. The sug2 mutation lowering the expression level of PUL increased shorter chains of amylopectin and accumulated a significant amount of the water-soluble polysaccharides as well as ISA (sugl ) mutation. The fl o2 mutation decreased simultaneously the expression levels of some of the enzymes including BEI, PUL and GBSS, suggesting that the wild type Flo2 gene probably encodes a regulatory protein that modulates the expression of the genes involved in starch biosynthesis. Some mutants involved in the biosyn thesis of ADP-glucose were also isolated.
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  • Ian J. Tetlow, Caroline G. Bowsher, Edward F.A.L. Scrase-Field, Emma J ...
    2003 Volume 50 Issue 2 Pages 231-236
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
    Recent studies of the subcellular compartmentation of starch synthesis have revealed differences between events in monocotyledenous endosperm compared with other starch storing tissues and species . ADPglucose pyrophosphorylase (AGPase) has been found to be located in both amyloplasts and cytosol of barley, rice and maize. We have found a similar distribution in developing endosperm of wheat, although as much as 30% of total cellular AGPase is localized in amyloplasts, a far higher proportion than for other species. Intriguingly the wheat amyloplast enzyme is activated only 2-fold by 3-phosphoglyceric acid (3-PGA) when assayed in the direction of ADPglucose (ADPG) synthesis, and 3-PGA does not reactivate the enzyme when it is inhibited by high concentrations of inorganic orthophosphate . In view of the cytosolic localisation of the majority of AGPase activity, we have studied the transport of ADPG into amyloplasts . The transporter has been reconstituted into artificial lipid bilayers and shown to counter-exchange ADPG with ATP, ADP or AMP, but the protein does not transport UDPglucose (UDPG). The import of ADPG from the cytosol into amyloplasts offers a means of regulating both the flux of carbon to the enzymes of starch synthesis, and the nature of the end product. The role of ADPG transport in controlling the partitioning of carbon between amylose and amylopectin will be discussed.
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  • Budsaraporn Ngampanya, Taito Takeda, Jarunya Narangajavana, Akira Iked ...
    2003 Volume 50 Issue 2 Pages 237-240
    Published: April 20, 2003
    Released: June 28, 2010
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    Sucrose and hexoses derived from sucrose breakdown are importance for early grain development in cereals. These assimilates were used to promote division and elongation, differentiation of the cells and deposition in storage organs. OsMST5 (Oryza sativa monosaccharide transporter 5), OsSUT1 (Oryza sativa sucrosetransporter 1) and OsSUT2 (Oryza saliva sucrose transporter 2) have been cloned from rice. All mRNAs of these functional sugar transporters were detected during flowering and grain filling stage. The OsMST5 and OsSUT2 mRNAs were exclusively detectable before the pollinating stage by Northern analysis and the OsSUT2 mRNA was localized in archespores and pollen mother cells by in situ mRNA detection, indicating that these gene products are involved in import of sucrose to promote pollen development at the early stage. Moreover, the OsSUT1 whiclh is known to be phloem companion cell-specific expression in source organs, was also expressed in the sink organs as well as modified aleurone cells of the developing endosperm. These results indicatethat the OsSUT1 plays a role in re-uptake of sucrose from endosperm cavity to accumulate starch in grain filling stage.
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  • Pedro M. Coutinho, Emeline Deleury, Bernard Henrissat
    2003 Volume 50 Issue 2 Pages 241-244
    Published: April 20, 2003
    Released: February 23, 2011
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    A novel classification scheme for glycoside hydrolases was introduced in 1991 to overcome some of the limitations of the classical EC number system. The new system, based on amino acid sequence similarities, was subsequently extended to other carbohydrate-active enzymes such as the glycosyltransferases and the polysaccharide lyases. Here we examine the current status of this classification scheme which provides a unified framework for the analysis of the structures and mechanisms of carbohydrate-active enzymes in the genomic era.
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  • Antoni Planas, Magda Faijes, Mireia Abel
    2003 Volume 50 Issue 2 Pages 245-251
    Published: April 20, 2003
    Released: July 01, 2011
    JOURNALS FREE ACCESS
    Bacillus 1, 3-1, 4-β-glucanases are retaining endo-glycosidases of family 16 GH. The hydrolyticmechanism of the Bacillus licheniformis enzyme follows a double-displacement reaction involving a catalytictriad with G1u134 as enzyme nucleophile, G1u138 as general acid-base, and a third residue, Asp136, that assistsin catalysis. It is shown that the carboxylate side chain of Asp136 participates in both glycosylation and deglycosylation steps contributing to the pKa modulation of the general acid-base during the enzyme cycle. Upon mutation of the catalytic nucleophile to alanine, the B. licheniformis 1, 3-1, 4-β-glucanase is a highly efficientendo - glycosynthase, with strict specificity for formation of 11-1, 4 glycosidic bonds. It is shown that Gin 138 acts as a general base in the transglycosylation mechanism. Strategies to control donor-acceptor condensation, donor self condensation, elongation and polymerization reactions have been devised, and examples are here shown for their application in oligosaccharide assembly or polysaccharide synthesis.
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  • Ulla Rudsander, Stuart Denman, Sami Raza, Tuula T. Teeril
    2003 Volume 50 Issue 2 Pages 253-256
    Published: April 20, 2003
    Released: July 01, 2011
    JOURNALS FREE ACCESS
    Most plant cellulases belong to the glycosyl hydrolase family 9, which is both enzymatically and structurally well characterized in microbes. The microbial enzymes include both randomly acting endoglucanases active on soluble and amorphous substrates and processive endoglucanases, which can also degrade crystalline cellulose. The corresponding plant enzymes have been difficult to purify and express in heterologoushosts and thus their modes of action have remained obscure. Here we have taken a molecular modelling approach and describe some structural features characteristic for soluble and membrane-bound family 9 cellulases in plants and filamentous fungi.
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  • Susumu Ito, Tohru Kobayashi, Katsuya Ozaki, Kazuaki Igarashi, Hiroshi ...
    2003 Volume 50 Issue 2 Pages 257-262
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    The market in industrial enzymes for detergents, starch, food, fabric, and so on has been estimated at more than 1600 million US dollars in 1999. The usage of detergent enzymes, such as protease, cellulase, aamylase, and lipase, forms 35% of the world market. The enzymes now are incorporated into more than 80% of heavy-duty laundry detergents in the world. Furthermore, alkaline protease and alkaline aamylase have been frequently used in automatic dishwashing detergents especially in the US and Europe. We have exploited large-scale production of alkaline cellulases, alkaline proteases, and an alkaline α-amylase, and the enzymes have been incorporated into heavy-duty compact detergents and/or bleaches. The problems are that traditional alkaline proteases and alkaline α-amylases are seriously inactivated by chemical oxidants and chelating reagents and are thermally unstable, especially when the enzymes are used in automatic dishwashers. Here, we describe the improvement of thermostability and oxidative stability of our alkaline liquefying α-amylase designated LAMY. Furthermore, we show a novel amylase that is extremely resistant to chemical oxidants and chelating reagents.
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  • Hans Leemhuis, Lubbert Dijkhuizen
    2003 Volume 50 Issue 2 Pages 263-271
    Published: April 20, 2003
    Released: July 01, 2011
    JOURNALS FREE ACCESS
    The α-amylase family is a large group of enzymes that act on α-glycosidic bonds. Within this family, about twenty different reaction and product specificities can be found, including exo/endo-specificity, preferencefor α-(1, 4)- or α-(1, 6)-glycosidic bonds, and hydrolysis/transglycosylation reaction specificity. The combinationof three-dimensional structure elucidation and the biochemical characterization of dozens of site directed mutants has yielded a detailed insight into the catalytic mechanism employed by α-amylase family enzymes. Nevertheless, the variation in reaction and product specificity is far from understood. The purpose of this review is to give an overview of recent developments in the understanding of the hydrolysis and transglycosylation reaction specificity within cyclodextrin glycosyltransferase (CGTase), which is one of the best studied α-amylase family enzymes.
  • Hiroshi Kamasaka, Kazuhisa Sugimoto, Hiroki Takata, Takahisa Nishimura ...
    2003 Volume 50 Issue 2 Pages 273-275
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    Neopullulanase was the key enzyme to open the door for the formulation of the concept of the aamylasefamily. The enzyme catalyzes both hydrolysis and transglycosylation at α-1, 4- and α-1, 6-glucosidic linkages through one active center. We found a unique macromolecule recognition by the enzyme. A mixture of amylose and amylopectin from various sources was used as the substrate. Neopullulanase completely hydrolyzedamylose to maltose and a small amount of glucose, but scarcely hydrolyzed amylopectin. Although the molecular mass of potato amylopectin (approximately 108 Da) decreased slightly, the degradation of amylopectincompletely halted at the molecular mass of approximately 107Da. This difference in action on two macromolecules was also found in cyclomaltodextrinase and maltogenic amylase from Bacillus licheniformis.
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  • Christine Finnie, Ole Østergaard, Kristian Sass Bak-Jensen, Pet ...
    2003 Volume 50 Issue 2 Pages 277-282
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    Proteomes of barley seeds were described by 2-D gel electrophoresis and spots selected for proteinidentification by mass spectrometry and database searches. Proteins were categorised according to temporalappearance during seed development and maturation. Fragments of β-amylases appeared transiently at midgrain filling and during germination. The α-amylase/trypsin inhibitors increased during grain filling andtypical housekeeping enzymes were present throughout the period. Germination altered the proteome and dissection of micromalted seeds enabled localization of selected proteins to specific tissues. The embryo was particularly rich in soluble proteins. Barley α-amylase/subtilisin inhibitor (BASI) and its target α-amylaseisozyme 2 occurred in endosperm and aleurone. Immunoblotting showed how the α-amylase changed duringgermination. Mutational analysis in a-amylase and BASI was guided by the three-dimensional structures.Mutants along the 10 binding subsites in barley a-amylase 1 addressed roles of individual residues in thepreference for starch over oligosaccharide substrates and vice versa, action patterns on oligosaccharides, andmultiple attack on amylose. Although the wild-type enzyme was proficient in transglycosylation, mutants ofthe catalytic nucleophile did not act as glycosynthase. C-terminal fusion of a starch binding domain from Aspergillus niger glucoamylase to barley α-amylase isozyme 1 enhanced degradation of starch granules. Recent heterologous expression of BASI allowed mutation of residues critical in enzyme binding as monitored by activity inhibition and surface plasmon resonance assays. A fully hydrated Ca2+ at the protein interface secured contact between BASI and the catalytic residues in the α-amylase.
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  • [in Japanese]
    2003 Volume 50 Issue 2 Pages 283-313
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
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  • Yoshiki Matsuura
    2003 Volume 50 Issue 2 Pages 315-319
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    The three-dimensional structures of various types of α-amylase family enzymes are briefly reviewed focused on their substrate specificities. Through these studies, it was shown that a conserved network structure composed of amino acids and a water exists at the active site of the enzymes of this family . The roles of the three essential catalytic residues are discussed based on the substrate-complexed structures of mutant enzymes. Particularly, the role of Asp297 (in TAA), which has not been clarified before, is discussed and shown to work crucially for evoking a distortion on the glucose ring at subsite -1, which leads to cleavage of the glucosidic bond. A reaction scheme involving the three essential catalytic residues is presented. Furthermore, the characteristic binding mode of the substrate amylose with respect to the (β/α)8-barrel in the family enzymes is addressed.
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  • Shinichi Kitamura
    2003 Volume 50 Issue 2 Pages 321-325
    Published: April 20, 2003
    Released: July 01, 2011
    JOURNALS FREE ACCESS
    This review mainly describes the progress of recent studies on the inclusion properties of large-ring cyclodextrins with guest molecules in aqueous solution. Complex formations of large-ring cyclodextrins (CDs) having degree of polymerization 11-40 with iodine and sodium dodecyl sulfate (SDS) were examined mainly by isothermal titration calorimetry. The term CDn is used to represent a generalized form for a CD, where represents the degree of polymerization. The curves obtained for the titration of the CD21 to CD33 with iodinecannot be analyzed by a model based on a single set of identical sites, but, rather, by a model assuming 1 : 2complex formations with identical interacting sites. These two binding constants, defined relative to the progressof saturation, and the corresponding enthalpies of binding were determined. These thermodynamic data indicate that the binding process is enthalpically driven. The titration curve for CD26 with SDS can be analyzedusing a model that assumes 1 : 2 complex formation with identical interacting sites as well, and the binding of a second SDS molecule to CD26 is rather strong. For other CDs, the titration curves are consistentwith a 1 : 1 complex formation with weak interaction in contrast to the case of CD26-SDS. The strong binding of the second SDS molecule to CD26 was simulated using molecular dynamics calculations based on the recently reported crystal structure of CD26.
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  • Haruko Ogawa, Maho Amano, Ako Hijikata, [in Japanese], Tomoko Kamidair ...
    2003 Volume 50 Issue 2 Pages 327-331
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    Plant N -linked oligosaccharides of complex type generally possess a characteristic core structure with xylose β-2 linked to β-mannose and fucose a 1-3 linked to N-acetylglucosamine at the reducing end, which rarely found in animals. Such glycans of plant glycoproteins have been found to induce immunogenic responses in animals. This brief report introduces the current knowledge on the structure, antigenicity and allergenicity of plant carbohydrate epitopes (plant glycotopes) obtained from our studies on the allergens of Japanese cedar pollinosis and baker's asthma. It also presents relationship between CCD (cross-reactive carbohydrate determinant) of allergens from common vegetables or fruits and that of the Japanese cedar pollen in this study.
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  • Norimasa Hosoya, M. D., Ph. D.
    2003 Volume 50 Issue 2 Pages 333-339
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
    A diet with a balanced amount of nutrients is only a standard of good health. One's health and nutritionalstatus must be checked by nutritional assessment, and any nutrient that is lacking must be supplemented by an adequate intake of dietary supplements. Here, I will explain the present regulatory system in Japan and express my opinion on the future outlook of the role of dietary supplements.
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  • Keiko Abe
    2003 Volume 50 Issue 2 Pages 341-344
    Published: April 20, 2003
    Released: February 23, 2011
    JOURNALS FREE ACCESS
    The taste molecular biology has suggested that, among the five basic taste, sweet, bitter, and umami tastes are received by G protein-coupled receptors on taste bud cells. Recent studies on the molecular entity of the receptors have disclosed the presence of T 1 R and T 2 R. Our group has investigated the intracellular signalingmechanism following the taste reception has elucidated the involvement of the phosphoinositol turnover passway with phospholipase Cβ2 playing a central role.
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  • Atsuhiko Shinmyo
    2003 Volume 50 Issue 2 Pages 345-349
    Published: April 20, 2003
    Released: June 28, 2010
    JOURNALS FREE ACCESS
    World population reached 6.1 billion in 2002 and it will become 9.5-10 billion in 2050. Programs for increasing crop yield are (1) Expansion of arable land, (2) Increase of yield per unit area, and (3) Plant breeding, but the former 2 programs can not be expected. The remaining possibility is plant breeding, i.e., development of high-yield and high-quality varieties and exploitation of stress-tolerant plants for extensive farming. Various physical and biological environmental stresses reduce the productivity of plants by 80% even in US. Traditional plant breeding by crossing requires at least a decade to establish improvement of one trait. Only one possible solution is a molecular breeding of plants. Transgenic crops with characters of herbicide-, insect-, and virus-resistance, long-life, high oleic and lauric acids content were produced. Safety assessment ofgenetically modified plants as food and feed is judged in various aspects. a) Effect of gene disruption on productionof toxic compounds. b) Transfer of maker gene, such as antibiotics-resistant gene, to intestinal microbes. c) Toxicity and allergen of foreign protein. These items were carefully studied by chemical analysis of transgenic plants, sequence of transgene, comparison of structure of transgene product (protein) with known toxic proteins and allergenic proteins, digestibility of proteins in stomach and intestine, and animal test. In conclusion, foods and feeds from transgenic plants so far certificated were substantially identical to usual foods and feeds.
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