Journal of Applied Glycoscience
Online ISSN : 1880-7291
Print ISSN : 1344-7882
ISSN-L : 1344-7882
Volume 51 , Issue 2
Showing 1-16 articles out of 16 articles from the selected issue
Regular Papers
  • Kanefumi Kitahara, Takayuki Uchino, Ichiyo Okizono, Toshihiko Suganuma
    2004 Volume 51 Issue 2 Pages 81-85
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Sweetpotato leaves cultivated for three months were harvested at different times for two days, and starch structures in the leaves were examined. The starch content changed in a circadian rhythm, with the minimum at 05:00 and the maximum at 17:00. The starches in the leaves were extracted by maceration of the leaf debris in hot dimethyl sulfoxide and prepared by selective precipitation by iodine. Gel-permeation chromatography of the leaf starches and amylopectins on Toyopearl HW-75F revealed that the leaf starches had a higher amount of material eluted at a low molecular weight fraction compared to that of its root starch, and the low molecular weight fraction contained both amylose molecules and a large amount of small amylopectin molecules. It was also found that both molecular weight distributions of the leaf starch and amylopectin were shifted to high molecular weight during the daytime and to low molecular weight during the period from sunset to sunrise. After debranching the starches and amylopectins, the chain-length distributions and short-chain distributions were analyzed by high-performance gel-permeation chromatography and high-performance anion-exchange chromatography, respectively. The chain-length distributions of the leaf starches were clearly distinct from those of the root starch. The amylose content of the leaf starches, which was evaluated as the difference in long-chain fraction on chain-length distribution between starch and amylopectin, was low during the daytime, whereas the branch aspect of the leaf amylopectin was relatively constant throughout the whole day. Thus, the structural characteristics of the starch in sweetpotato leaf and their diurnal-nocturnal changes have been revealed.
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  • Kouichi Nozaki, Akemi Kano, Yoshihiko Amano, Takeomi Murata, Taichi Us ...
    2004 Volume 51 Issue 2 Pages 87-92
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    The possibility of transglycosylation and acceptor specificity of cellulases, CBH I, CBH II, EG II (Trichoderma reesei), Ex-1 (Irpex lacteus) and Exo-A (Aspergillus niger) was investigated by using cellotriose (G3) and p-nitrophenyl-β-D-glucoside (pNPG1) as a donor and an acceptor, respectively. Among exo-type cellulases, CBH I effectively accumulated two transfer products, pNP-β-D-cellotrioside and 3-O-β-D-cellobiosyl-pNP-β-D-glucoside. They were produced by the transfer of a cellobiosyl unit resulting in G3 hydrolysis to pNPG1 with β-1,4 or β-1,3 glycosidic bonds, respectively. On the other hand, endo-type cellulase EG II, produced pNP-β-D-cellobioside, pNP-β-D-gentiobioside and pNP-β-D-laminaribioside. These might be synthesized by the glucosyl transfer produced from G3 or pNPG1 to pNPG1 with β-1,4, β-1,6 or β-1,3 glycosidic bonds. Furthermore, CBH I and EG II catalyzed transglycosylation to various pNP-glycosides. From the study of acceptor specificity for CBH I and EG II, pNP-α-D-galactoside in addition to pNP-glycosides with β-configuration was used as the acceptor.
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  • Yoji Kato, Teruko Konishi, Yutaka Hidano, Yasushi Mitsuishi
    2004 Volume 51 Issue 2 Pages 93-99
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    A xyloglucan was isolated from the cell-walls of eggplant. This xyloglucan was hydrolyzed with a xyloglucan specific endo-1,4-β-D-glucanase (xyloglucanase) from Penicillium sp. M451. The resulting hydrolyzate was fractionated by Bio-Gel P-2 chromatography. Hexa-, hepta- and octa-saccharide fractions were obtained in a ratio of 35:43:22 as major oligosaccharide fractions. The xyloglucan oligosaccharide fractions were separately subjected to sugar composition and methylation analyses, high-performance anion exchange chromatography with pulsed amperometric detection, and matrix-assisted laser-desorption ionization time of the flight mass spectrometry before and after treatment with an isoprimeverose-producing oligoxyloglucan hydrolase purified from Eupenicillium sp. M9 or with β-D-galactosidase from Bacillus circulans. Hexa-, hepta- and octa-saccharide fractions might consist mainly of XXGG, of XLGG, LXGG and XSGG, and of LSGG and LLGG, respectively. In addition, the oligosaccharide units of xyloglucans from some solanaceous vegetables were comparatively analyzed. Xyloglucans from the cell-walls of sweet pepper, red pepper and tomato belonging to solanaceous vegetables were shown to be composed of hexa-, hepta- and octa-saccharide fractions in a ratio of 40:37:23, 41:34:25, and 45:37:18, respectively.
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  • Naoto Isono, Takeshi Senoura, Motoyo Yoshikawa, Yasutaka Sakurai, Kenj ...
    2004 Volume 51 Issue 2 Pages 101-107
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Starch synthases contribute to the elongation of glucan chains during starch synthesis, and therefore their characteristics are one of the dominant factors influencing the fine structure of starch. To understand the detailed properties of starch synthases in kidney bean (Phaseolus vulgaris L.) seeds, a cDNA clone (pvss22) for starch synthase II was isolated from developing seeds at the late stage by the combinations of reverse transcriptase-mediated PCR (RT-PCR), 5´-RACE (rapid amplification of cDNA end), and 3´-RACE. The pvss22 cDNA is 2486 bp in length and contains an open reading frame of 738 amino acids. The predicted amino acid sequence of the protein (designated PvSSII-2) encoded by pvss22 cDNA displayed substantial identity (more than 58%) to other dicot starch synthase II members. RNA gel blot analysis revealed that the pvss22 transcripts predominantly accumulate in developing seeds at the middle to late stages. The recombinant PvSSII-2 protein was produced as a major polypeptide in inclusion bodies of Escherichia coli cells. The antiserum raised against proteins extracted from the inclusion bodies recognized at least seven polypeptides in starch-granule fractions from seeds. Analyses of N-terminal sequences of starch-granule-bound proteins showed that three of the seven polypeptides reacting with the antiserum are encoded by the pvss22 gene.
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Note
  • Tomoko Maeda, Maryanto, Naofumi Morita
    2004 Volume 51 Issue 2 Pages 109-113
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Characteristics of taro corm starches of four kinds were studied as follows: Java and Celebes were harvested in Indonesia; Uhan in China; Satoimo in Japan. In addition, effects of acid- and heat-treatments on physical properties of native Java starch were examined. The particle size of Java starch was in the range of 3-17 μm with about 50% distribution of size of 9-13 μm. From SEM observation, the size of starch granules for Java, Celebes, Uhan and Satoimo was 3.0-13.0, 1.0-4.8, 1.5-5.8 and 0.5-3.4 μm, respectively. The native Java starch was mostly spherical, but some portions of the surface were square with a larger size than for other starch samples. The amylose content of the Java sample determined by the blue value method was 20.5% and number-average degree of polymerization was 880 and 2920 for amylose and amylopectin, respectively. The average chain length of amylose or amylopectin was 17 or 19, respectively. From the result of X-ray diffraction, the crystalline pattern of Java starch was A type. As for DSC results, Satoimo starch was gelatinized at a higher temperature, while the enthalpy was lower than those of other samples. In contrast, starch of Celebes or Uhan was gelatinized at lower temperature, as compared with native Java starch. The gelatinization enthalpy of heat-treated Java starch at 50°C showed the highest value among all samples. But, physical properties (DSC and SEM) of acid-treated Java starch were not distinctly different from those of native Java starch.
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Regular Papers (in Japanese with English Abstract)
  • Akiko Yamashita, Hiroyuki Hashimoto, Sumio Kitahata, Eriko Kikuchi, Ko ...
    2004 Volume 51 Issue 2 Pages 115-121
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    α-Linked galactooligosaccharide (α-GOS, 7.5 kg) was produced from galactose (18 kg; prepared from lactose hydrolyzate) by the reverse reaction of α-galactosidase from Aspergillus niger APC-9319. α-GOS contained 58% disaccharide, 28% trisaccharide and 14% oligosaccharides larger than trisaccharide The disaccharide in α-GOS consisted largely of α-1,6-galactobiose (73%). α-GOS was stable at neutral and acidic pHs, at temperatures up to 180°C (pH 4.5, 10 min). The water activity of α-GOS was 0.84 (25°C, 70% (w/v)). The powder preparation was highly hygroscopic, and its solution showed higher activity in retaining moisture than sucrose. The sweetness of α-GOS was 25% of that of sucrose. The taste of α-GOS was mild and mellow. α-GOS reduced acid taste, bitterness, astringency, and harsh taste without spoiling the original taste in various fruit juices, and emphasized the original flavor of food materials in various cooked foods. α-GOS was not hydrolyzed in an in vitro digestion method. In an oral α-GOS tolerance test, the blood glucose level did not change at all. α-GOS had a strong selective growth activity for Bifidobacterium sp. and Clostridium butyricum. An α-GOS intake in rats made the content of lactate and n-butyrate in the cecum higher, and pH in the cecum lower than for a control intake. The viable count of Candida albicans in the cecum and colon of mouse was decreased by an α-GOS intake more than by a raffinose intake.
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  • Izuru Kaneko, Kohsuke Hayamizu, Kyoko Tomita, Hiroto Kikuchi, Taizo Na ...
    2004 Volume 51 Issue 2 Pages 123-128
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Melibiose (6-O-α-D-galactopyranosyl-D-glucose) is a disaccharide, a small amount of which is contained in honey and soybeans. It was reported that raffinose, which is a trisaccharide built from melibiose and D-fructose, improved symptoms of atopic dermatitis. A 3-month open clinical study was performed using 31 patients with adolescent or adult-type atopic dermatitis to investigate whether melibiose is also useful for atopic dermatitis or not. In the study, 24 (77%) out of 31 patients showed improvement in subjective symptoms. Nineteen (61%) patients showed improvement in objective skin lesions after the treatment. Moreover, the extent of lesions and SCORAD Index were significantly decreased. In the clinical test, the number of eosinophil was significantly decreased after the treatment. No adverse event was observed. These results indicate that melibiose may be useful for atopic dermatitis.
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Proceedings of the Symposium on Amylases and Related Enzymes, 2003
  • Hiroshi Kamasaka, Daisuke Inaba, Kentaro Minami, To-o Kenji, Takahisa ...
    2004 Volume 51 Issue 2 Pages 129-134
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Phosphate ester groups are known to link to some glucosyl residues in starch molecules. We have prepared phosphoryl oligosaccharides (POs) from potato starch hydrolysates. The POs were composed of two fractions, PO-1 and PO-2. Fraction PO-1 was the main fraction, and it was composed of maltotriose, maltotetraose, and maltopentaose to which one phosphoryl group was attached. Fraction PO-2 was predominantly composed of maltopentaose and maltohexaose to which at least two phosphoryl groups were attached. POs had the ability to form a soluble complex with calcium and had an inhibitory effect on the formation of a calcium-phosphate precipitate. Based on the function of the POs, described above, we applied the POs of calcium (POs-Ca) as a food ingredient. POs-Ca was an advantageous food ingredient as a soluble calcium source. In relation to prevention of dental caries, POs cannot be fermented by cariogenic microorganisms or mutans streptococci, and they reduce the fall in plaque pH in vitro. Moreover, POs-Ca effectively enhanced the remineralization of enamel lesions. The aim of this study was to develop the application of POs-Ca for dental health to the enamel remineralization through the chewing of a sugar-free gum.
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  • Tomoyuki Nishimoto, Hajime Aga, Michio Kubota, Shigeharu Fukuda, Masas ...
    2004 Volume 51 Issue 2 Pages 135-140
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Cyclic tetrasaccharide (CTS; cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→}) is a cyclic oligosaccharide four D-glucosyl residues linked in an alternating α-1,3- and α-1,6-fashion. CTS has been reported for enzymatic synthesis from alternan, an α-(1→3)-α-(1→6)-D-glucan synthesized from sucrose by Leuconostoc mesenteroides NRRL B-1355 alternansucrase, by endo-glucanase (alternanase) from Bacillus sp. NRRL B-21195. While searching for nonreducing oligosaccharides that can be produced from α-1,4-glucan as the substrate, we screened bacteria isolated from soil in our own way, and obtained Bacillus globisporus C11, which produces CTS from starch. Two kinds of glycosyltransferase, 6-α-glucosyltransferase (6GT) and α-isomaltosyltransferase (IMT), were purified from the culture supernatant of this strain. It was found that CTS is produced by the sequential action of both enzymes. The genes for IMT (CtsY) and 6GT (CtsZ) were linked together on the chromosome, forming ctsYZ. Both of the gene products showed similarities to α-glucosidases belonging to glycoside hydrolase family 31 and conserved two aspartic acids corresponding to the putative catalytic residues of these enzymes. CtsYZ and four open reading frames upstream of ctsYZ form a gene cluster, ctsUVWXYZ. The reaction conditions for CTS synthesis were examined using 6GT and IMT from B. globisporus C11. The optimum reaction conditions to obtain CTS from Pinedex #100 (partial hydrolyzate of starch, 1.3%-hydrolysis) were the following: substrate concentration, 3%; pH, 6-7; temperature, 30°C; enzyme dosages, 1 U/g-dry solid 6GT, 10 U/g-dry solid IMT. In these optimum conditions the CTS yields reached 62% at the reaction time of 48 h. A mass-production method of highly purified CTS crystals at a reasonable cost was established, and the functions and characteristics of CTS were studied.
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  • Nozomu Yasutake, Kazuhide Totani, Yoichiro Harada, Shinobu Haraguchi, ...
    2004 Volume 51 Issue 2 Pages 141-147
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Alkyl β-lactosides were directly synthesized by β-lactosyl transfer reaction from p-nitrophenyl β-lactoside (Lacβ-pNP) to various 1-alkanols (n=2-12), utilizing a commercially available cellulase preparation of Trichoderma reesei C1. When 1-octanol and dodecanol were acceptors, octyl β-lactoside and dodecyl β-lactoside were obtained as transfer products, respectively. Furthermore, the enzyme catalyzed N-acetyllactosaminyl transfer reaction from p-nitrophenyl β-N-acetyllactosaminide (LacNAcβ-pNP) not only to 1-alkanol, but also to the OH-4 position of Man and Glc to produce the trisaccharides. As an alternative method, alkyl β-lactosides were synthesized by condensation reaction of lactose with alkanols. When various 1-alkanols (n: 2-8) were used, the corresponding alkyl β-lactosides were obtained in the yields of 1-4% based on lactose added. Further, condensation reaction between lactose and glycerol was effectively catalyzed to produce 1-O-β-lactosyl-(R,S)-glycerols and 2-O-β-lactosyl glycerol in a molar ratio of 7:3 and in a 20% yield based on lactose added. The enzyme also catalyzed the condensation with 2-propanol and allyl alcohol. In a similar manner, when LacNAc was used as a glycon substrate, the enzyme catalyzed the condensation with various alkanols and glycerol. The yields were lower than those obtained from reactions with lactose. Finally, purification of an enzyme possessing condensation activity was carried out by chromatographies using DEAE-Sepharose and Mono P columns, in order to reveal whether the same enzyme molecule would catalyze in both the lactosyl- and N-acetyllactosaminyl-condensing reactions. It was specified that a single enzyme works both condensation reactions, based on the substrate competition assay on hydrolysis.
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  • Hiromi Murakami, Akiko Seko, Masumi Azumi, Hirofumi Nakano, Sumio Kita ...
    2004 Volume 51 Issue 2 Pages 149-154
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    We aimed to develop microbial production systems for lactobionic acid (LA) for utilization of surplus lactose (Lac) from whey. Screened and selected strain Burkholderia cepacia No. 24 grew even in highly concentrated Lac syrup and had high oxidizing activity against Lac. We examined the conditions for fermentation and microbial conversion of Lac to LA. 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. Lac (150 g/L) was completely converted to LA by bioconversion with Burkholderia cepacia cells in 24 h.
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  • Yuji Honda, Motomitsu Kitaoka, Ken Tokuyasu, Kiyoshi Hayashi
    2004 Volume 51 Issue 2 Pages 155-160
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    To determine the reaction mechanism of ChiA from Serratia marcescens, we synthesized the partially and fully N-deacetylated 4-methylumberlliferyl chitobioside (1) derivatives, GlcN-GlcNAc-UMB (2), GlcNAc-GlcN-UMB (3), and (GlcN)2-UMB (4) as fluorogenic ChiA substrates and performed kinetic analyses of their hydrolysis by ChiA. With the exception of compound 4, ChiA released UMB from all compounds tested. The S-v curves of ChiA hydrolysis of 1 exhibited atypical kinetic patterns, which may have been due to the formation of enzyme complexes with multiple molecules of the substrate. Conversely, the S-v curves generated during ChiA cleavage of 2 and 3 exhibited typical Michaelis-Menten profiles. Compared with enzyme hydrolysis of 1, hydrolysis of 2 resulted in a 14-fold higher Km value, indicating that the N-acetyl group was recognized at the -2 subsite. The kcat value obtained with ChiA was identical to the kcat value observed for 1. Compared with the ChiA hydrolysis of 1, ChiA hydrolyzed 3 with a 5-fold greater Km and a 60-fold smaller kcat. The reaction mechanism of family 18 chitinases is discussed based on the results obtained from the hydrolysis of these compounds.
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  • Tetsuya Shimizu, Toru Nakatsu, Kazuo Miyairi, Toshikatsu Okuno, Hiroak ...
    2004 Volume 51 Issue 2 Pages 161-167
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Three crystal structures of endopolygalacturonase I (EndoPG I) from Stereum purpureum have been determined in this study: the unliganded EndoPG I, the binary and ternary complexes of EndoPG I with galacturonate. Consequently, the structural basis for substrate binding and the catalytic mechanism of EndoPG I have been elucidated by X-ray crystallography. Crystals of deglycosylated EndoPG Ia have been obtained using PEG4000 as precipitate with the hanging-drop vapor diffusion method. The crystal belongs to space group P1, with unit-cell parameters a=37.26 Å, b=46.34 Å , c=52.05 Å, α=67.17°, β=72.44°, γ=68.90°. The crystal diffracts to ultra-high (0.96 Å) resolution using synchrotron radiation of SPring-8. Crystal structures of EndoPG I were determined by the multiple wavelength anomalous dispersion (MAD) method. For MAD phasing, three wavelength data sets of K2PtCl4 derivative crystal were collected at SPring-8. The structure model was refined anisotropically with SHELXL-97, with an R factor of 11.4% and an Rfree factor of 14.0% at 0.96 Å resolution. The enzyme folds into a right-handed parallel β-helix with 10 complete turns. The crystal structures of its binary complex with one D-galacturonate and its ternary complex with two D-galacturonates were also determined to identify the substrate binding site at 1.0 and 1.15 Å resolutions, respectively. In the binary complex, one β-D-galactopyranuronate, GalpA, was found in the reducing end side of Asp153, Asp173 and Asp174, which are considered as candidates of catalytic residues. This reveals that the position of GalpA is the +1 subsite, thus proving the strong affinity of the +1 subsite expected from the bond cleavage frequency on oligo-galacturonates. In the ternary complex, an additional β-D-galactofuranuronate was found in the -1 subsite. In both subsites, the recognition of the galacturonate carboxy group is important in galacturonate binding. In the +1 subsite, the carboxy group interacts with three basic residues, His195, Arg226 and Lys228, which were conserved in all endopolygalacturonases. In the -1 subsite, the unique non-prolyl cis-peptide bond is believed to be involved in binding the carboxy group of the substrate. Based on the structures of GalfA and GalpA bound in the ternary complex, a structural model of the di-galacturonic acid part of the substrate molecule bound in both the -1 and +1 subsites across from the catalytic residues was constructed. The di-galacturonate model structure sheds light on the catalytic mechanism. Asp173 is at the appropriate position to be a proton donor to the fissile glycosidic bond. Asp153 or Asp174 seems to act as a general base to abstract a proton from the nucleophilic water.
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  • Hiroyuki Hashimoto, Akiko Yamashita, Koki Fujita, Masamichi Okada, Shi ...
    2004 Volume 51 Issue 2 Pages 169-176
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Oligosaccharides with α-D-galactosidic linkages, especially the positional isomers of α-galactobiose (α-Gal 2), participate in such biological processes as immunity and pathogen-to-cell adhesion on the basis of molecular mechanisms for ligand-receptor interaction. α-Linked galactooligosaccharide (α-GOS) containing positional isomers of α-Gal 2 produced from galactose by the reverse reaction of α-galactosidase, is a potential food for the prevention of infections by pathogens, neutralization of toxins, and regulation of immune system. α-GOS (7.5 kg) was produced from galactose (18 kg; prepared from lactose hydrolyzate) by the reverse reaction of α-galactosidase from Aspergillus niger APC-9319. α-GOS contained 58% disaccharide, 28% trisaccharide and 14% oligosaccharides larger than trisaccharide. The disaccharide in α-GOS consisted largely of α-1,6-galactobiose (73%). α-GOS was stable at neutral and acidic pHs, and high temperatures. The sweetness of α-GOS was 25% of that of sucrose. The taste of α-GOS was mild and mellow. α-GOS reduced acid taste, bitterness, astringency, and harsh taste without spoiling the original taste in various fruit juices, and emphasized the original flavor of food materials in various cooked foods. α-GOS was not hydrolyzed in an in vitro digestion method. In oral tolerance tests of α-GOS, the blood glucose level did not change at all. α-GOS had a strong selective growth activity for Bifidobacterium sp. and Clostridium butyricum. An α-GOS intake in rats made the contents of lactate and n-butyrate in the cecum higher, and pH in the cecum lower than for a control intake. The viable count of Candida albicans in cecum and colon of mouse was decreased by an α-GOS intake more than by a raffinose intake. Therapeutic oral treatment with α-GOS had a potent inhibitory effect on adjuvant arthritis in Wister rats and on type II collagen-induced arthritis in DBA/1 J mice. Dietary α-GOS ameliorated allergic airway eosinophilia at least partly via post-absorptive mechanisms in Brown Norway rats.
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  • Norio Shiomi, Keiji Ueno
    2004 Volume 51 Issue 2 Pages 177-183
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    Recently, we have investigated enzymic production of non-digestible oligosaccharides which have several functional activities as “tertiary functional ingredients” of foods. Fructo-oligosaccharides synthesized from sucrose by Eurotium repense fructosyltransferase had the effect of no elevation of blood glucose or insulin concentrations in rats. Fructosylxyloside formed from sucrose and xylose with fructosyltransferase action of Scopulariopsis brevicaulis cells suppressed serum glucose and insulin responses and / or promoted the absorption of calcium and magnesium ions in rats administered with sucrose. On the other hand, we studied purification and characterization of several fructosyltransferases; sucrose: sucrose 1-fructosyltransferase (1-SST), fructan: fructan 1-fructosyltransferase (1-FFT) and a new enzyme, fructan: fructan 6G-fructosyltransferase (6G-FFT) from asparagus roots. Previously, we reported that the 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 the saccharides selectively stimulated growth for Bifidobacteria. In this study, we have tried the isolation and expression of cDNAs encoding 6G-FFT, 1-FFT and 1-SST from asparagus for industrial use. The cDNAs encoding 6G-FFT, 1-FFT and 1-SST were isolated from a cDNA library of asparagus leaves or roots. The isolated cDNAs were named AoFT 1, AoFT 2 and AoFT 3, respectively. The deduced amino acid sequences of these cDNAs showed a high homology with those of plant fructosyltransferases. Expression of these cDNAs was done using Pichia pastoris. The recombinant protein from Pichia transformed with AoFT 1 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 show that 6G-FFT is expressed in P. pastoris. In the same way, recombinant protein from Pichia transformed with AoFT 2 produced nystose from 1-kestose and AoFT 3 recombinant protein produced 1-kestose from sucrose. These results also show that 1-FFT and 1-SST are expressed in P. pastoris. Three recombinant proteins had enzymatic properties similar to those of 6G-FFT, 1-FFT and 1-SST from asparagus roots, respectively.
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Award Address
  • Yoshiki Matsuura
    2004 Volume 51 Issue 2 Pages 185-192
    Published: 2004
    Released: March 24, 2008
    JOURNALS FREE ACCESS
    The crystal structure study of Taka-amylase (TAA) was started in early 1970th in the Institute for Protein Research of Osaka University. TAA has been extensively studied centered on this Institute by the group of Prof. Akabori. The elucidation of the three-dimensional structure of this enzyme has been the final goal of these studies. The first structure of TAA was published in 1979 at low resolution. The backbone model was published in 1980, and the complete molecular model at 3 Å resolution in 1984, with reference to the amino acid sequence determined by Prof. Narita’s group in 1982. Since then, the author and his group have determined the structures of six different enzymes of the α-amylase family: Taka-amylase, cyclodextrin glucosyltransferase, maltotetraose-forming amylase, isoamylase, trehalose synthase and neopullulanase. The three-dimensional structures of these α-amylase family enzymes are briefly reviewed focusing 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. The role of Asp 297 (in TAA), particularly, 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 the cleavage of 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 (α/β)8-barrel in the family enzymes is addressed.
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