1989 年度実績報告書

酵素蛋白質の計画的改変のための基礎技術の確立

研究課題

研究課題/領域番号	01616002
研究機関	東京大学
研究代表者	別府輝彦東京大学, 農学部, 教授 (80011873)
研究分担者	徳重正信京都大学, 理学部, 教授 (20025266) 稲田祐二桐蔭学園横浜大, 工学部, 教授 (40016035) 岡田弘輔大阪大学, 工学部, 教授 (20028947) 太田隆久東京大学, 農学部, 教授 (30011844) 大塚栄子北海道大学, 薬学部, 教授 (80028836)
キーワード	蛋白質工学 / 部位特異的変異 / 分泌シグナル配列 / アクアライシン / トリプトファナーゼ / キモシン / グルコース脱水素酵素 / ガングリオシド
研究概要	Mucorからクローン化したレンニン遺伝子より酵母で発現される蛋白の糖鎖付加部位を同定した。また大腸菌内で封入体として発現されるウシキモシンの再生過程を追跡し、N末の数アミノ酸の重要性を明らかにした(別府)。チミン2量体除去修複酵素T4エンドヌクレアーゼを化学合成し、大腸菌での大量発現に成功してin vitroと in vivoで活性の異なる変異体を多数取得した(大塚)。高度好熱菌のプロテアーゼアクアライシンの特異的な分泌機構を明らかにした(太田)。グルコース脱水素酵素の部位特異的変換で、アミノ酸置換と特異性の変化に関する情報を得た(岡田)。サイクロデキストリンを生成するCGTase遺伝子をクローン化し、全構造の決定および大腸菌での分泌生産に成功した(正木)。ポリエチレングリコール修飾酵素の有機溶媒中での反応特性を明らかにし、また磁性化酵素を磁力を用いて生体の特定の部位に誘導できた(稲田)。四量体アスパルターゼサブユニットのハイブリッド化による活性と調節機能の変換を達成し、またトリプトファナーゼの大量発現と結晶化に成功した(徳重)。大腸菌外膜蛋白(水野)と枯草菌アミラーゼ(山根)の分泌に関するシグナルおよび成熟体部分の構造と機能の相関を解析した。アデノウィルスE1A遺伝子による癌関連糖脂質抗原GD3の発現には、GD3合成酵素阻害物費の転写抑制が関与する(永井)。ズブチリシンとその蛋白性阻害剤の結合についてX線解析を行った(三井)。トリプトファン合成酵素のalphaサブユニット中のプロリン残基が安定性と構造形成に及ぼす影響を検討した(油谷)。ヒトプロスロンビンと1:1の分子複合体を形成しつつスロンビン活性を発現するスタフィロコアギュラーゼ遺伝子をクローン化し、全塩基配列の決定および大腸菌での大量発現に成功した(岩永)。

研究成果

(37件)

すべてその他

すべて文献書誌 (37件)

[文献書誌] J.suzuki: "Alteration of catalytic properties of chymosin by site-directed mutagenesis" Protein Eng.2. 563-569 (1989)
[文献書誌] R.Hiramatsu: "Secretion by yeast of the zymogen form of Mucor rennin,an aspartic proteinase of Mucor pusillus, and its conversion to the mature form" J.Biol.Chem.264. 16862-16866 (1989)
[文献書誌] I.Terada: "Unique precursor structure of an extracellular protease,aqualysin I,with N- and C-terminal prosequences and its processing in Escherichia coli" J.Biol.Chem.
[文献書誌] H.Takagi: "Enhancement of the thermostability of subtilisin E by the introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease" J.Biol.Chem.
[文献書誌] M.Yamaizumi: "Microinjection of T4 endonuclease V produced by a synthetic den V gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells" Mutation Res.337. 90-93 (1989)
[文献書誌] T.Inaoka: "Affinity of single-or doubleーstranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V" J.Biol.Chem.264. 2609-2614 (1989)
[文献書誌] M.Ishida: "In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region" Biochemistry.
[文献書誌] Y.Makino: "Stability-increasing mutants of glucose dehydrogenase from Bacillus megaterium IWG3" J.Biol.Chem.264. 6381-6385 (1989)
[文献書誌] T.Nagao: "Stability-increasing mutants of glucose dehydrogenase" FEBS Lett.253. 113-116 (1989)
[文献書誌] T.Mitamura: "Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium" Eur.J.Biochem.186. 389-393 (1989)
[文献書誌] M.Tokushige: "Role of cysteine residues in tryptophanase for monovalent cationinduced activation" Biochemie. 71. 711-720 (1989)
[文献書誌] P.Bjurling: "Synthesis of β-^<11>C-labelled L-tryptophan an 5-hydroxy-1-tryptophanusing multi-enzymatic reaction routes" J.Chem.Soc.Perkin Trans.1331-1334 (1989)
[文献書誌] H.Imaishi: "Preparation of active hybrid enzymes composed of the native and chemically inactivated aspartase subunits from Escherichia coli" Biotechnol.Appl.Biochem.
[文献書誌] H.Imaishi: "Characterization of intermediate species during the molecular assembly of aspartase" Physiol.Chem.Phys.Med.NMR.
[文献書誌] S.Tani: "Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli" Biotechnol.Appl.Biochem.
[文献書誌] H.Lee: "Substrate specificity of polyethylene glycol-modified papain catalyzed peptide bond synthesis in benzene" Kinki Chem.Soc.4. 253-256 (1989)
[文献書誌] T.Yoshimoto: "Targetting of urokinase by magnetic force" Drug Delivery System. 4. 121-122 (1989)
[文献書誌] K.Ohkawa: "Synthesis of N^α,N^δ-dicarbobenzoil-L-ornithine-β-alanine benzyl ester, a delivative of salty peptide,in 1,1,1-trichloroethane with polyethylene glycol-modified papain" Biotechnol.Lett.7. 499-502 (1989)
[文献書誌] K.Kimura: "Expression of the -cyclodextrin glucanotransferase gene of an alkalophilic Bacillus sp.#1011 in Escherichiacoli cells and characterization of the synthesized enzyme" Agric.Biol.Chem.
[文献書誌] T.Yamamoto: "Nucleotide sequence of the diaminopimerate-decarboxylase gene from Bacillus Subtilis" Nucleic Acids Res.17. 10105-10115 (1989)
[文献書誌] K.Nakamura: "Cloning and chracterization of a Bacillus subtilis gene homologous to E.coli secY" J.Biochem.
[文献書誌] S.Itonori: "Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody:the case of an" Glycoconjugate J.6. 551-560 (1989)
[文献書誌] Y.sanai: "Induction of GD3 ganglioside by adenovirus E1A gene 13S-and 12S-mRNA products in rat 3Y1 cells" J.Biochem.
[文献書誌] K.Ogasahara: "Comparison of CD spectra in the aromatic region on a resies of variant proteins substituted at a unique position of tryptophan synthtase α-subunit" Proteins. 5. 211-217 (1989)
[文献書誌] S.Kikkawa: "Esterification of chiral secondary alcohols with fatty acid in organic solvents by polyethylene glycol-modified lipase" Biochem.International. 19. 1125-1131 (1989)
[文献書誌] A.Mizutani: "Biosynthesis of gefarnate with reusable lipase modified with polyethylene glycol" J.Biotechnol.10. 121-126 (1989)
[文献書誌] C.Hikita: "Structural and functional characterization of OmpF and OmpC porins in Escherichia coli outer membrane:studies with chimeric proteins" Res.Microbiol.140. 177-190 (1989)
[文献書誌] A.Shinkai: "Insertion of a signal pepetide-derived hydrophobic segment into the mature domain of OmpC,an outer membrane protein,does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E.coli" J.Biochem.106. 323-330 (1989)
[文献書誌] K.Nakamura: "Modification of length,hydrophobic properties and electric charge of Bacillussubtilis α-amylase signal peptide and their different effects on the production of secretory proteins" Mol.Gen.Genet.216. 1-9 (1989)
[文献書誌] H.Sasamoto: "Signal peptide of Bacillussubtilis α-amylase" J.Biochem.106. 376-382 (1989)
[文献書誌] S.Sawada: "Assignment of tyrosine resonances in proton NMR spectrum of tryptophan syntase -subunit;monitering conformational changes due to substitutions at position 49" Eur.J.Biochem.
[文献書誌] Y.Sugisaki: "Scanning calorimetric study of tryptophan synthase -subunit from Escherichia cooi,Salmonellatyphimurium,and interspecific hybrid" Themochimica Acta.
[文献書誌] S.Kaida: "Nucleotide sequence and deduced amino acid sequence of staphylocoagulase gene from Staphylococcusaureus strain 213" Nucleic Acids res.1. 8871 (1989)
[文献書誌] Y.Sanai: "Gangliosides and Cancer(ed.H.F.Oettgen)" VCH,Weinhei,pp.70-77 (1989)
[文献書誌] K.Yamane: "Bacillus Subtilis:Molecular Biology and Industrial Application(ed.B.Maruo & H.Yoshikawa)" Kodansha and Elsevier Press,Inc., pp.162-190 (1989)
[文献書誌] Y.Mitsui: "GBF Monographs,Vo1.12 Advances in Protein Design" VCH Publishers, pp.177-183 (1989)
[文献書誌] Y.Mitsui: "新基礎生化学実験法" 丸善, pp.158-180 (1989)

1989 年度 実績報告書

酵素蛋白質の計画的改変のための基礎技術の確立

研究代表者

別府 輝彦 東京大学, 農学部, 教授 (80011873)

研究成果

[文献書誌] J.suzuki: "Alteration of catalytic properties of chymosin by site-directed mutagenesis" Protein Eng.2. 563-569 (1989)

[文献書誌] R.Hiramatsu: "Secretion by yeast of the zymogen form of Mucor rennin,an aspartic proteinase of Mucor pusillus, and its conversion to the mature form" J.Biol.Chem.264. 16862-16866 (1989)

[文献書誌] I.Terada: "Unique precursor structure of an extracellular protease,aqualysin I,with N- and C-terminal prosequences and its processing in Escherichia coli" J.Biol.Chem.

[文献書誌] H.Takagi: "Enhancement of the thermostability of subtilisin E by the introduction of a disulfide bond engineered on the basis of structural comparison with a thermophilic serine protease" J.Biol.Chem.

[文献書誌] M.Yamaizumi: "Microinjection of T4 endonuclease V produced by a synthetic den V gene stimulates unscheduled DNA synthesis in both xeroderma pigmentosum and normal cells" Mutation Res.337. 90-93 (1989)

[文献書誌] T.Inaoka: "Affinity of single-or doubleーstranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V" J.Biol.Chem.264. 2609-2614 (1989)

[文献書誌] M.Ishida: "In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region" Biochemistry.

[文献書誌] Y.Makino: "Stability-increasing mutants of glucose dehydrogenase from Bacillus megaterium IWG3" J.Biol.Chem.264. 6381-6385 (1989)

[文献書誌] T.Nagao: "Stability-increasing mutants of glucose dehydrogenase" FEBS Lett.253. 113-116 (1989)

[文献書誌] T.Mitamura: "Enzymatic properties of isozymes and variants of glucose dehydrogenase from Bacillus megaterium" Eur.J.Biochem.186. 389-393 (1989)

[文献書誌] M.Tokushige: "Role of cysteine residues in tryptophanase for monovalent cationinduced activation" Biochemie. 71. 711-720 (1989)

[文献書誌] P.Bjurling: "Synthesis of β-^<11>C-labelled L-tryptophan an 5-hydroxy-1-tryptophanusing multi-enzymatic reaction routes" J.Chem.Soc.Perkin Trans.1331-1334 (1989)

[文献書誌] H.Imaishi: "Preparation of active hybrid enzymes composed of the native and chemically inactivated aspartase subunits from Escherichia coli" Biotechnol.Appl.Biochem.

[文献書誌] H.Imaishi: "Characterization of intermediate species during the molecular assembly of aspartase" Physiol.Chem.Phys.Med.NMR.

[文献書誌] S.Tani: "Overproduction and crystallization of tryptophanase from recombinant cells of Escherichia coli" Biotechnol.Appl.Biochem.

[文献書誌] H.Lee: "Substrate specificity of polyethylene glycol-modified papain catalyzed peptide bond synthesis in benzene" Kinki Chem.Soc.4. 253-256 (1989)

[文献書誌] T.Yoshimoto: "Targetting of urokinase by magnetic force" Drug Delivery System. 4. 121-122 (1989)

[文献書誌] K.Ohkawa: "Synthesis of N^α,N^δ-dicarbobenzoil-L-ornithine-β-alanine benzyl ester, a delivative of salty peptide,in 1,1,1-trichloroethane with polyethylene glycol-modified papain" Biotechnol.Lett.7. 499-502 (1989)

[文献書誌] K.Kimura: "Expression of the -cyclodextrin glucanotransferase gene of an alkalophilic Bacillus sp.#1011 in Escherichiacoli cells and characterization of the synthesized enzyme" Agric.Biol.Chem.

[文献書誌] T.Yamamoto: "Nucleotide sequence of the diaminopimerate-decarboxylase gene from Bacillus Subtilis" Nucleic Acids Res.17. 10105-10115 (1989)

[文献書誌] K.Nakamura: "Cloning and chracterization of a Bacillus subtilis gene homologous to E.coli secY" J.Biochem.

[文献書誌] S.Itonori: "Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody:the case of an" Glycoconjugate J.6. 551-560 (1989)

[文献書誌] Y.sanai: "Induction of GD3 ganglioside by adenovirus E1A gene 13S-and 12S-mRNA products in rat 3Y1 cells" J.Biochem.

[文献書誌] K.Ogasahara: "Comparison of CD spectra in the aromatic region on a resies of variant proteins substituted at a unique position of tryptophan synthtase α-subunit" Proteins. 5. 211-217 (1989)

[文献書誌] S.Kikkawa: "Esterification of chiral secondary alcohols with fatty acid in organic solvents by polyethylene glycol-modified lipase" Biochem.International. 19. 1125-1131 (1989)

[文献書誌] A.Mizutani: "Biosynthesis of gefarnate with reusable lipase modified with polyethylene glycol" J.Biotechnol.10. 121-126 (1989)

[文献書誌] C.Hikita: "Structural and functional characterization of OmpF and OmpC porins in Escherichia coli outer membrane:studies with chimeric proteins" Res.Microbiol.140. 177-190 (1989)

[文献書誌] A.Shinkai: "Insertion of a signal pepetide-derived hydrophobic segment into the mature domain of OmpC,an outer membrane protein,does not interfere with the export of the following polypeptide chain across the cytoplasmic membrane of E.coli" J.Biochem.106. 323-330 (1989)

[文献書誌] K.Nakamura: "Modification of length,hydrophobic properties and electric charge of Bacillussubtilis α-amylase signal peptide and their different effects on the production of secretory proteins" Mol.Gen.Genet.216. 1-9 (1989)

[文献書誌] H.Sasamoto: "Signal peptide of Bacillussubtilis α-amylase" J.Biochem.106. 376-382 (1989)

[文献書誌] S.Sawada: "Assignment of tyrosine resonances in proton NMR spectrum of tryptophan syntase -subunit;monitering conformational changes due to substitutions at position 49" Eur.J.Biochem.

[文献書誌] Y.Sugisaki: "Scanning calorimetric study of tryptophan synthase -subunit from Escherichia cooi,Salmonellatyphimurium,and interspecific hybrid" Themochimica Acta.

[文献書誌] S.Kaida: "Nucleotide sequence and deduced amino acid sequence of staphylocoagulase gene from Staphylococcusaureus strain 213" Nucleic Acids res.1. 8871 (1989)

[文献書誌] Y.Sanai: "Gangliosides and Cancer(ed.H.F.Oettgen)" VCH,Weinhei,pp.70-77 (1989)

[文献書誌] K.Yamane: "Bacillus Subtilis:Molecular Biology and Industrial Application(ed.B.Maruo & H.Yoshikawa)" Kodansha and Elsevier Press,Inc., pp.162-190 (1989)

[文献書誌] Y.Mitsui: "GBF Monographs,Vo1.12 Advances in Protein Design" VCH Publishers, pp.177-183 (1989)

[文献書誌] Y.Mitsui: "新基礎生化学実験法" 丸善, pp.158-180 (1989)

1989 年度実績報告書

別府輝彦東京大学, 農学部, 教授 (80011873)