1994 年度実績報告書

糖質分解・合成酵素の糖認識機構解析、機能改変および高度利用

研究課題

研究課題/領域番号	05303010
研究機関	北海道大学
研究代表者	千葉誠哉北海道大学, 農学部, 教授 (30001449)
研究分担者	檜作進鹿児島大学, 農学部, 教授 (90041590) 坂野好幸東京農工大学, 農学部, 教授 (70014959) 南浦能至大阪市立大学, 理学部, 教授 (20047129) 鈴木讓京都府立大学, 農学部, 教授 (50046471) 山根國男筑波大学, 生物科学系, 教授 (20013336)
キーワード	糖質分解酵素 / 糖質合成酵素 / 糖認識機構 / 糖質分解酵素の高度利用 / アミラーゼ / グリコシダーゼ / サイクロデキストリン合成酵素
研究概要	1.好アルカリBacillus sp.のサイクロデキストリン合成酵素を結晶化し、1.8Aでの解析をした。本酵素の活性に直接関係する4個のアミノ酸残基の改変酵素を作成した。 2.Bacillus srearothermophilus α-glucosidaseの一次構造を遺伝子より演繹した。Bacillus cereus α-glucosidaseの活性残基と基質結合残基を部位特異的変異により決定した。 3.ダイズβ-アミラーゼと基質アナログとの複合体のX線結晶構造解析を行った。α-シクロデキストリンおよびマルトースを用いた複合体の解析結果より、本酵素の基質認識機構を明らかにすることができた。 4.Arthrobacter globiformis イソマルトデキストラナーゼの活性解離基を同定し、その遺伝子クローニングにより全一次構造を決定し、酵素の大腸菌での発現に成功した。 5.α-ガラクトシダーゼおよびα-マンノシダーゼの糖転移作用を利用して、シクロデキストリン(CD)あるいは分岐CDにガラクトシル基やマンノシル基を転移結合させたヘテロ分岐CDを合成し、その構造を決定した。 6.マルトースを切り難く、マルトシル-のCDの枝をよく切るグルコアミラーゼを見い出し、分岐CDの生成率を90%以上に高める方法を確立した。 7.Cycloamylose glucanotransferase反応による生成糖を定量して本酵素のサブサイト構造を推定し、マルトースを基質とするCGTase反応によるisopanoseの効率よい製産法を確立した。 8.株化した甘藷塊根細胞の培養細胞から4量体β-アミラーゼの活性を有するモノマーを収量を落とさずに常法に2-3倍の速度で培養時間を短縮することに成功した。 9.Asp.oryzae α-アミラーゼをオルトタルアルデヒドにより化学修飾してその反応特異性を検討し、親和標識剤としての特性を明らかにした。 10.化学修飾によりテンサイα-グルコシダーゼの活性中心附近のアミノ酸配列と活性に直接関与しているアミノ酸残基(Asp)を同定した。 11.Trichoderma virideのセルラーゼで、天然セルロースの酵素糖化に主役を演ずるセルラーゼII-bの特異なグリコシレーション(縮合・転移・水解)の作用機作を明らかにした。 12.Xanthomanos campestrisから新規α-アミラーゼを精製し、その酵素化学的性質を明らかにした。 13.大腸菌にクローン化したTVA IとTVA IIを比較した結果、TVA IはTVA IIより高分子の基質を容易に分解するが、低分子基質は逆にTVA IIにより良く分解された。

研究成果
(39件)

すべてその他

すべて文献書誌 (39件)

[文献書誌] A.Nakamura: "The transglycosylation reaction of cyclodextrin glucanotransferase is poerated by a Ping-Pong mechanism" FEBS Lett.337. 66-70 (1994)
[文献書誌] K.Haga: "Crystallization and preliminary X-ray studies of cyclodexterin glucanotransferase from alklophilic Bacilus sp.1011" J.Mol.Biol.237. 163-164 (1994)
[文献書誌] A.Nakamura: "Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp.1011:effects of replacements on substrate binding and cyclization characteristics" Biochemistry. 33. 9929-9936 (1994)
[文献書誌] K.Nakamura: "The Bacillus subtilis SRP54 homologue,Ffh,has an instrinsic GTPase activity and forms a ribonucleoprotein complex with small cytoplasmic RNA in vivo" Biochem.Biopohy.Res.Commun.199. 1394-1399 (1994)
[文献書誌] M.Nishiguchi: "Structural requirements of Bacillus subtilis small cytoplansmic RNA for cell qrowth,sporulation,and extracellular enzyme production" J.Bacteriol.176. 157-165 (1994)
[文献書誌] K.Watanabe: "Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase.Irrefragable proof supporting the proline rule" Eur.J.Biochem.226. 277-283 (1994)
[文献書誌] B.Mikami: "Crystal Structures of Soybean β-Amylase Reacted with β-Maltose and Maltal:Active Site Components and Their Apparent Roles in Catalysis" Biochemistry. 33. 7779-7787 (1994)
[文献書誌] B.Mikami: "Two Sulfhydryl Groups Near the Active Site of Soybean β-Amylase" Biosci.Biotech.Biochem.58. 126-132 (1994)
[文献書誌] B.Mikami: "Crystallization and Preliminary X-ray Diffraction Studies of Bacterial Alginate Lyase" J.Ferment.Bioengineer.77. 691-692 (1994)
[文献書誌] J.H.Roh: "Crystallization and Preliminary X-ray Analysis of Copper Amine oxidase from Escherichia coli K-12" J.Mol.Biol.238. 635-637 (1994)
[文献書誌] A.B.Gidamis: "Crystallization and Preliminary X-ray Analuysis of Soybean Proglysinins Modified by Protein Engineering" Biosci.Biotech.Biochem.58. 703-706 (1994)
[文献書誌] T.Takayanagi: "Identification of Ionizable Groups Essential to the Actiivity of Isomalto-dextranase from Arthrobacter globiformis" Biosci.Biotech.Biochem.58. 1995-1999 (1994)
[文献書誌] S.Onodera: "Substrate Specificity and Subsite Affinities of Rabbit Liver Acid α-Glucosidase" J.Biochem.116. 7-11 (1994)
[文献書誌] K.Hara: "Acceptor Speciticities of α-Mannosidases from Jack Beans and Almond,and Transmannosylation of Branched Cyclodextrins" Biosci.Biotech.Biochem.58. 60-63 (1994)
[文献書誌] K.Fujita: "Chemical Srtuctures of Hetero-oligosaccharides Produced by Arthrobacter sp.K-1 β-Fructofuranosidase" Biosci.Biotech.Biochem.58. 239-243 (1994)
[文献書誌] Y.Okada: "Separation and characterization of five positional isomers of trimaltosyl-cyclomaltoheptaose(trimaltosyl-β-cyclodextrin)" Carbohydr.Res.254. 1-13 (1994)
[文献書誌] K.hara: "Galactosylation of Cyclodextrins and Branched Cyclodextrins by α-Galactosidases" Biosci.Biotech.Biochem.58. 652-659 (1994)
[文献書誌] S.Mori: "Purification and Properties of Cyclodextrin Glucanotransferase from Brevibacterium sp.No.9605" Biosci.Biotech.Biochem.58. 1968-1972 (1994)
[文献書誌] W.Tsuzuki: "Esterase Activity and Free Fatty Acid Accumulation in the Brain of Selected Rice Cultivars" Cereal Chem.71. 162-165 (1994)
[文献書誌] M.Ohnishi: "Tryptophan residues of Bacillus cycloamylose glucanotransferase:Effect of modification with N-bromosuccinimide on the enzymecatalysed synthesis of cyclomaltoheptaose from maltotriose" Starch. 46. 272-275 (1994)
[文献書誌] M.Ohnishi: "Cycloamylose glucaonotransferase-catalyzed cyclization for a substrate maltose. Modification with N-bromosuccinimide on the tryptophan residues" Starch. 46. 276-279 (1994)
[文献書誌] M.Ohnishi: "Transition in the subunit of Strptomyces xylose isomerase:Its relation with the catalytic activity and substrate binding" J.Glycoscience. 41. 419-424 (1994)
[文献書誌] K.Ito: "Purification and Characterization of Fungal Nuclease Composed of Heterogeneous Subuits" Arch.Bichem.Biophys.308. 160-167 (1994)
[文献書誌] T.Suzuki: "A Novel Generation of Optical Active 1,2-Diols from the Racemates by Using Haloydrin Dehydro-Dehalogenase" Tetrahedron Asymmetry. 5. 239-246 (1994)
[文献書誌] T.Suzuki: "A Novel Enzymatic Dehalogenation of (R)-3-Chloro-1,2-Propanediol in an Alcaligenes sp.DS-S-7G" Appl.Microbiol.Biotechnol.42. 270-279 (1994)
[文献書誌] T.Suzuki: "Microbial Production of Opticaly Active 1,2-diols Using Resting Cells of Alcaligenes sp.DS-S-7G" J.Ferment.Bioeng.78. 194-196 (1994)
[文献書誌] K.Ito: "Evidence for an Essential Histidine Residue on Active Site of Human Urinary DNase I:Carboxymethylation and Carbethoxylation" Arch.Biochem.Biophys.313. 126-130 (1994)
[文献書誌] M.Kobayashi: "Cross-linked stabilization of trypsin with dextran-dialdehyde" Biosci.Biotech.Biochem.58. 275-278 (1994)
[文献書誌] Y.Yamagata: "Functional changes of dextran-modified alkaline proteinase from alkalopohilic Bacillus sp." Enzyme Microb.Technol.16. 99-193 (1994)
[文献書誌] T.Oguma: "Purification and properties of a novel enzyme from Bacillus sp.T-3040 which catalyzed the conversion of dextran to dextran to cyclic isomaltooligosaccharides" FEBS.Lett.345. 135-138 (1994)
[文献書誌] M.Kobayashi: "Water-soluble calbodiimide for the fluorescent measurement of the carboxyl group produced by enzyme reactions" Anal.Biochem. 219. 189-194 (1994)
[文献書誌] T.Yoshida: "Chemical modification of Penicillum 1,2-α-D-mannosidase:indentication of a catalytically important aspartic acid residue" Biochem.J.303. 97-103 (1994)
[文献書誌] A.Iwai: "Molecular Cloning and Expression of an isomalto-Dextranase Gene from Arthrobacter globiformis T6" J.Bacteriol.176. 7730-7734 (1994)
[文献書誌] T.Tonozaka: "A convenient enzymatic synthesis of 4^2-α-isomaltosylisomaltose using Thermoactinomyces vulgaris R-47 alpha-amylase(TVA II)" Carbohydr.Res.261. 157-162 (1994)
[文献書誌] J.Abe: "Purification and Characterization of Periplasmic α-Amylase from xanthomonas campesrtris K-11151" J.Bacteriol.176. 3584-3588 (1994)
[文献書誌] 志村進: "カワラツメイのタンニン含有画分のリパーセ阻害効果" 日食工誌. 41. 561-564 (1994)
[文献書誌] 深井洋一: "酵素処理による3種起源澱粉粒の特性改変" 日農化誌. 68. 793-800 (1994)
[文献書誌] 海野剛裕: "β-グリコオリゴ糖含有シラップの生産とその諸性質" 応用糖質科学. 41. 317-324 (1994)
[文献書誌] Amylase Res.Soc.Japan: "Enzyme Chemistry and Molecular Biology of Amylases and Related Enzymes" CRC Press,Inc., 206 (1994)

1994 年度 実績報告書

糖質分解・合成酵素の糖認識機構解析、機能改変および高度利用

研究代表者

千葉 誠哉 北海道大学, 農学部, 教授 (30001449)

研究成果

[文献書誌] A.Nakamura: "The transglycosylation reaction of cyclodextrin glucanotransferase is poerated by a Ping-Pong mechanism" FEBS Lett.337. 66-70 (1994)

[文献書誌] K.Haga: "Crystallization and preliminary X-ray studies of cyclodexterin glucanotransferase from alklophilic Bacilus sp.1011" J.Mol.Biol.237. 163-164 (1994)

[文献書誌] A.Nakamura: "Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp.1011:effects of replacements on substrate binding and cyclization characteristics" Biochemistry. 33. 9929-9936 (1994)

[文献書誌] K.Nakamura: "The Bacillus subtilis SRP54 homologue,Ffh,has an instrinsic GTPase activity and forms a ribonucleoprotein complex with small cytoplasmic RNA in vivo" Biochem.Biopohy.Res.Commun.199. 1394-1399 (1994)

[文献書誌] M.Nishiguchi: "Structural requirements of Bacillus subtilis small cytoplansmic RNA for cell qrowth,sporulation,and extracellular enzyme production" J.Bacteriol.176. 157-165 (1994)

[文献書誌] K.Watanabe: "Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase.Irrefragable proof supporting the proline rule" Eur.J.Biochem.226. 277-283 (1994)

[文献書誌] B.Mikami: "Crystal Structures of Soybean β-Amylase Reacted with β-Maltose and Maltal:Active Site Components and Their Apparent Roles in Catalysis" Biochemistry. 33. 7779-7787 (1994)

[文献書誌] B.Mikami: "Two Sulfhydryl Groups Near the Active Site of Soybean β-Amylase" Biosci.Biotech.Biochem.58. 126-132 (1994)

[文献書誌] B.Mikami: "Crystallization and Preliminary X-ray Diffraction Studies of Bacterial Alginate Lyase" J.Ferment.Bioengineer.77. 691-692 (1994)

[文献書誌] J.H.Roh: "Crystallization and Preliminary X-ray Analysis of Copper Amine oxidase from Escherichia coli K-12" J.Mol.Biol.238. 635-637 (1994)

[文献書誌] A.B.Gidamis: "Crystallization and Preliminary X-ray Analuysis of Soybean Proglysinins Modified by Protein Engineering" Biosci.Biotech.Biochem.58. 703-706 (1994)

[文献書誌] T.Takayanagi: "Identification of Ionizable Groups Essential to the Actiivity of Isomalto-dextranase from Arthrobacter globiformis" Biosci.Biotech.Biochem.58. 1995-1999 (1994)

[文献書誌] S.Onodera: "Substrate Specificity and Subsite Affinities of Rabbit Liver Acid α-Glucosidase" J.Biochem.116. 7-11 (1994)

[文献書誌] K.Hara: "Acceptor Speciticities of α-Mannosidases from Jack Beans and Almond,and Transmannosylation of Branched Cyclodextrins" Biosci.Biotech.Biochem.58. 60-63 (1994)

[文献書誌] K.Fujita: "Chemical Srtuctures of Hetero-oligosaccharides Produced by Arthrobacter sp.K-1 β-Fructofuranosidase" Biosci.Biotech.Biochem.58. 239-243 (1994)

[文献書誌] Y.Okada: "Separation and characterization of five positional isomers of trimaltosyl-cyclomaltoheptaose(trimaltosyl-β-cyclodextrin)" Carbohydr.Res.254. 1-13 (1994)

[文献書誌] K.hara: "Galactosylation of Cyclodextrins and Branched Cyclodextrins by α-Galactosidases" Biosci.Biotech.Biochem.58. 652-659 (1994)

[文献書誌] S.Mori: "Purification and Properties of Cyclodextrin Glucanotransferase from Brevibacterium sp.No.9605" Biosci.Biotech.Biochem.58. 1968-1972 (1994)

[文献書誌] W.Tsuzuki: "Esterase Activity and Free Fatty Acid Accumulation in the Brain of Selected Rice Cultivars" Cereal Chem.71. 162-165 (1994)

[文献書誌] M.Ohnishi: "Tryptophan residues of Bacillus cycloamylose glucanotransferase:Effect of modification with N-bromosuccinimide on the enzymecatalysed synthesis of cyclomaltoheptaose from maltotriose" Starch. 46. 272-275 (1994)

[文献書誌] M.Ohnishi: "Cycloamylose glucaonotransferase-catalyzed cyclization for a substrate maltose. Modification with N-bromosuccinimide on the tryptophan residues" Starch. 46. 276-279 (1994)

[文献書誌] M.Ohnishi: "Transition in the subunit of Strptomyces xylose isomerase:Its relation with the catalytic activity and substrate binding" J.Glycoscience. 41. 419-424 (1994)

[文献書誌] K.Ito: "Purification and Characterization of Fungal Nuclease Composed of Heterogeneous Subuits" Arch.Bichem.Biophys.308. 160-167 (1994)

[文献書誌] T.Suzuki: "A Novel Generation of Optical Active 1,2-Diols from the Racemates by Using Haloydrin Dehydro-Dehalogenase" Tetrahedron Asymmetry. 5. 239-246 (1994)

[文献書誌] T.Suzuki: "A Novel Enzymatic Dehalogenation of (R)-3-Chloro-1,2-Propanediol in an Alcaligenes sp.DS-S-7G" Appl.Microbiol.Biotechnol.42. 270-279 (1994)

[文献書誌] T.Suzuki: "Microbial Production of Opticaly Active 1,2-diols Using Resting Cells of Alcaligenes sp.DS-S-7G" J.Ferment.Bioeng.78. 194-196 (1994)

[文献書誌] K.Ito: "Evidence for an Essential Histidine Residue on Active Site of Human Urinary DNase I:Carboxymethylation and Carbethoxylation" Arch.Biochem.Biophys.313. 126-130 (1994)

[文献書誌] M.Kobayashi: "Cross-linked stabilization of trypsin with dextran-dialdehyde" Biosci.Biotech.Biochem.58. 275-278 (1994)

[文献書誌] Y.Yamagata: "Functional changes of dextran-modified alkaline proteinase from alkalopohilic Bacillus sp." Enzyme Microb.Technol.16. 99-193 (1994)

[文献書誌] T.Oguma: "Purification and properties of a novel enzyme from Bacillus sp.T-3040 which catalyzed the conversion of dextran to dextran to cyclic isomaltooligosaccharides" FEBS.Lett.345. 135-138 (1994)

[文献書誌] M.Kobayashi: "Water-soluble calbodiimide for the fluorescent measurement of the carboxyl group produced by enzyme reactions" Anal.Biochem. 219. 189-194 (1994)

[文献書誌] T.Yoshida: "Chemical modification of Penicillum 1,2-α-D-mannosidase:indentication of a catalytically important aspartic acid residue" Biochem.J.303. 97-103 (1994)

[文献書誌] A.Iwai: "Molecular Cloning and Expression of an isomalto-Dextranase Gene from Arthrobacter globiformis T6" J.Bacteriol.176. 7730-7734 (1994)

[文献書誌] T.Tonozaka: "A convenient enzymatic synthesis of 4^2-α-isomaltosylisomaltose using Thermoactinomyces vulgaris R-47 alpha-amylase(TVA II)" Carbohydr.Res.261. 157-162 (1994)

[文献書誌] J.Abe: "Purification and Characterization of Periplasmic α-Amylase from xanthomonas campesrtris K-11151" J.Bacteriol.176. 3584-3588 (1994)

[文献書誌] 志村 進: "カワラツメイのタンニン含有画分のリパーセ阻害効果" 日食工誌. 41. 561-564 (1994)

[文献書誌] 深井 洋一: "酵素処理による3種起源澱粉粒の特性改変" 日農化誌. 68. 793-800 (1994)

[文献書誌] 海野 剛裕: "β-グリコオリゴ糖含有シラップの生産とその諸性質" 応用糖質科学. 41. 317-324 (1994)

[文献書誌] Amylase Res.Soc.Japan: "Enzyme Chemistry and Molecular Biology of Amylases and Related Enzymes" CRC Press,Inc., 206 (1994)

1994 年度実績報告書

千葉誠哉北海道大学, 農学部, 教授 (30001449)

[文献書誌] 志村進: "カワラツメイのタンニン含有画分のリパーセ阻害効果" 日食工誌. 41. 561-564 (1994)

[文献書誌] 深井洋一: "酵素処理による3種起源澱粉粒の特性改変" 日農化誌. 68. 793-800 (1994)

[文献書誌] 海野剛裕: "β-グリコオリゴ糖含有シラップの生産とその諸性質" 応用糖質科学. 41. 317-324 (1994)