Studies on Mechanism of Substrate Confirmation, Modification of Function, and Utilization for Carbohydrate-Hydrolase and Synthase

碳水化合物水解酶和合酶的底物确认、功能修饰和利用机制研究

• 批准号: 05303010
• 负责人: CHIBA Seiya
• 金额: $ 11.84万
• 依托单位: HOKKAIDO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Co-operative Research (A)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05303010/
• 关键词: Carbohydrate-hydrolase; Carbohydrate-Synthase; Mechanism of substrate confirmation; Utilization of carbohydrase; Amylase; Glucosidase; Cyclodextrin synthase; グリコシダーゼ

(1) A cyclodextrin glucanotransferase from alkaliphilic Bacillus sp. was crystallized, and its three dimensional structure was analyzed by x-ray (1.8*) . The enzyme was modified by exchanging the four amino acid residues directly involved in catalytic activity.(2) Primary structure of B.stearothermophilus alpha-glucosidase was deduced from cloned cDNA.(3) Soybean beta-amylase and a substrate analog complex was analyzed by x-ray crystallography. The mechanism in the substrate confirmation was revealed by the x-ray analysis.(4) Catalytic ionizable groups of Arthrobacter grobiformis isomalto-dextranase were kinetically identified. The complete primary structure of the enzyme was deduced from the analysis of cDNA.(5) Heterogeneous cyclodextrins (CD) were synthesized through transfer reaction of alpha-galactosyl or alpha-mannosyl residue to branched CD by alpha-galactosidase and alpha-mannosidase.(6) Subsite structure of cycloamylose glucanotransferase was evaluated by the subsite theory, and the production method of isopanose from maltose was established by the enzyme.(7) From the cell suspension culture of sweet potato tuber, a monomeric beta-amylase having the same level activity as tetrameric enzyme was successfully obtained in a high yield.(8) Reaction specificity of Aspergillus oryzae alpha-amylase was elucidated by a chemical modiification with omicron-phthalaldehyde.(9) Amino acid sequence in active site and a catalytic amino acid residue of sugar beet alpha-glucosidase were identified by chemical modification.(10) Action mechanism of novel glycosylation (condecsation, transfer reaction and hydrolysis) was elucidated for Trichoderma viride cellulase II-b.(11) A novel alpha-amylase was purified from Xanthomonas campestris, and the properties were examined.(12) Substrate specificties of two novel alpha-amylases (TVA I and II) cloned in E.coli were clarified and compared with each other.

(1)从嗜碱芽孢杆菌（Bacillussp.）中分离出一种环糊精葡聚糖转移酶，并对其进行了结晶，用X射线衍射（1.8*）分析了其三维结构。通过交换直接参与催化活性的四个氨基酸残基来修饰酶。(2)从克隆的cDNA推导出嗜热脂肪B. (3)通过X射线晶体学分析大豆β-淀粉酶和底物类似物复合物。通过X射线衍射分析揭示了基质确认的机理。(4)对球形节杆菌异麦芽糖葡聚糖酶的催化电离基团进行了动力学鉴定。通过对cDNA的分析，推测出该酶的完整一级结构。(5)通过α-半乳糖苷酶和α-甘露糖苷酶将α-半乳糖基或α-甘露糖基转移到支链环糊精上，合成了多相环糊精。(6)利用亚位点理论对环直链淀粉葡聚糖转移酶的亚位点结构进行了分析，并建立了利用该酶从麦芽糖生产异潘糖的方法。(7)从甘薯块茎细胞悬浮培养中，成功地获得了一种具有与四聚体酶相同活性的单体β-淀粉酶。(8)用邻苯二甲醛对曲霉α-淀粉酶进行化学修饰，以阐明其反应特异性。(9)用化学修饰法鉴定了甜菜α-葡萄糖苷酶活性中心的氨基酸序列和催化氨基酸残基。(10)阐明了绿色木霉纤维素酶II-b的新型糖基化作用机理（缩合、转移反应和水解）。(11)从野油菜黄单胞菌（Xanthomonascampestris）中分离纯化了一种新型α-淀粉酶，并对其性质进行了研究。(12)阐明了两种新的α-淀粉酶（TVA I和II）在大肠杆菌中克隆的底物特异性，并相互比较。

项目成果

K. Nomura: "The role of SH and S-S groups in Bacillus cereus β-amylase." J. Biochem.118. 1124-1130 (1995)

K. Nomura：“SH 和 S-S 基团在蜡状芽孢杆菌 β-淀粉酶中的作用。J. Biochem.1124-1130 (1995)。

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)

B.Mikami：“大豆 β-淀粉酶与 β-麦芽糖和麦芽糖反应的晶体结构：活性位点成分及其在生物化学中的明显作用。”33. 7779-7787 (1994)

深井 洋一: "酵素処理による澱粉粒の特性改変" 澱粉科学. 40. 263-269 (1993)

Yoichi Fukai：“通过酶处理改变淀粉颗粒的性质”《淀粉科学》40. 263-269 (1993)。

K.Hara: "Acceptor Specificities of α-Mannosidases from Jack Bean and Almond and Transmannosylation of Brancbod Cyclodextrins" Biosci.Biotech.Biochem.,. 58. 60-63 (1994)

K.Hara：“来自杰克豆和杏仁的 α-甘露糖苷酶的受体特异性以及 Brancbod 环糊精的转甘露糖基化”Biosci.Biotech.Biochem., 58. 60-63 (1994)

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)

Yoichi Fukai：“酶处理对三种类型淀粉颗粒的特性改性”Nippon Noka Kasai 68. 793-800 (1994)。