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

(1)嗜碱芽孢杆菌的环糊精葡糖基转移酶。结晶，并用X-射线衍射仪(1.8)对其三维结构进行了分析。通过与酶活性直接相关的四个氨基酸残基的交换，对酶进行了修饰。(2)从克隆的cDNA中推导出嗜热杆菌α-葡萄糖苷酶的一级结构。(3)对大豆β-淀粉酶及其底物类似物进行了X射线结晶学分析。通过X-射线分析揭示了底物确证的机理。(4)动力学鉴定了格氏节杆菌异麦芽糖-葡聚糖酶的催化电离基团。(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(CD)上，合成了多相环糊精(CD)。(6)用亚位理论研究了环直链淀粉葡糖基转移酶的亚基结构，建立了以麦芽糖为原料合成异戊糖的方法。(8)用邻苯二甲醛化学修饰米曲霉α-淀粉酶。(9)化学修饰鉴定了活性部位的氨基酸序列和甜菜α-葡萄糖苷酶的催化氨基酸残基。(10)阐明了绿色木霉纤维素酶II-b的糖基化作用机理。(11)从油菜黄单胞菌中分离纯化了一种新的α-淀粉酶。阐明了在大肠杆菌中克隆的两种新的α-淀粉酶(TVA I和TVA II)的底物特异性，并对它们进行了比较。

(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.