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

(1)从嗜碱性芽孢杆菌中分离出一种环糊精葡聚糖转移酶，通过x射线（1.8*）对其三维结构进行了分析。通过交换直接参与催化活性的四个氨基酸残基对酶进行了修饰。(2)从克隆的cDNA中推导出嗜热嗜脂杆菌α -葡萄糖苷酶的初级结构。(3)用x射线晶体学分析了大豆β -淀粉酶及其底物类似物。x射线分析揭示了衬底确认的机理。(4)对grobiformis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，推断出该酶的完整一级结构。(5)利用α -半乳糖苷酶和α -甘露糖苷酶将α -半乳糖基或α -甘露糖基残基转化为支链环糊精，合成了非均相环糊精。(6)利用亚位理论对环直链糖葡聚糖转移酶的亚位结构进行了评价，建立了该酶由麦芽糖生产异二糖的方法。(7)通过红薯块茎细胞悬浮培养，成功高产地获得了与四聚体酶活性相当的单体β -淀粉酶。(8)用邻苯二醛对米曲霉α -淀粉酶进行化学修饰，确定了米曲霉α -淀粉酶的反应特异性。(9)用化学修饰法鉴定了甜菜α -葡萄糖苷酶活性位点的氨基酸序列和催化残基。（10）阐明了绿色木霉纤维素酶II-b的新型糖基化（冷凝、转移和水解）作用机理。（11）从油菜黄单胞菌中纯化了一种新的α -淀粉酶，并对其性质进行了研究。（12）澄清了在大肠杆菌中克隆的两种新型α -淀粉酶（TVA I和TVA II）的底物特异性，并对其进行了比较。

(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.