Multi-disciplinary Studies with Protein Engineering Approaches on Proteinaceous Proteinase Inhibitors of Microbial Origin

SSI在此表示链霉菌枯草杆菌蛋白酶抑制剂，SMPI表示链霉菌金属蛋白酶抑制剂。SSI对链霉菌属金属蛋白酶SGMPA和SGMPB也有抑制作用，用一种新的荧光底物测定了Ki值，克隆了SSI基因，对其进行了定点突变，并对突变体SSI的特性和SSI表达系统的特性进行了研究，建立了产生抗SSI单克隆抗体IgG 1和IgM的克隆，并对单克隆抗体IgG 1和IgM的表位进行了NMR分析; SSI晶体的X射线衍射数据用同步辐射技术获得了（野生型和突变型）枯草杆菌蛋白酶BPN'复合物，并对其进行了结构修饰，测定了嗜热蛋白酶Aqualysin I基因的碱基序列，研究了该酶的结构和功能;利用已克隆的SMPI基因，探索了SMPI细胞外生产的最佳条件;本文建立了稳定同位素标记乳清蛋白的方法学，并研究了位置-研究了SSI分子结构的变化规律，分析了SSI高级结构上的特异性氨基酸取代，用氘核磁共振分析了SSI分子结构的涨落，建立了灵敏的SH/SS化合物微量分析的电化学方法，并对SSI的结构进行了表征。对SSI突变体与枯草杆菌蛋白酶BPN'和SMPI与嗜热菌蛋白酶的相互作用进行了统计学和动力学分析，确定了从海洋微生物中分离的丝氨酸蛋白酶抑制剂Marinostatin D的一级结构;测定了新型胃蛋白酶抑制剂不敏感羧基蛋白酶抑制剂酪蛋白抑制剂的一级结构，揭示了新型嗜热羧基蛋白酶的独特性质及其严格的底物特异性。

SSI denotes here Streptomyces subtilisin inhibitor and SMPI, Streptomyces metallo-proteinase inhibitor. SSI was found to inhibit also Streptomyces metallo-proteinases, SGMPA & SGMPB, and Ki was determined with a novel fluorogenic substrate; SSI gene was cloned, site-directed mutagenesis was applied on it, and characters of the mutant SSI's and of SSI-expression system were elucidated; Clones producing mondclonal antibodies, IgG1 & IgM, against SSI were established, and the epitopes were analyzed by NMR; X-ray diffraction data on the crystals of SSI (wild and mutant)-subtilisin BPN' complexes were obtained with syncrotron radiation, and structural refinement was made; Base sequence of the gene of a thermophilic proteinase, Aqualysine I, was determined, and the structure and function of the enzyme was studied; Optimal conditions were searched for extra-cellular production of SMPI by making use of a cloned gene; Methodology of Orotein NMR by using stable isotope labelling was established and the effects of site-specific amino acid replacement on the higher structure of SSI was analyzed; Fluctuation in SSI molecular structure was analyzed by deuterium NMR; A sensitive electro-chemical method for micro-analysis of SH/SS compounds was established; Interactions of SSI mutants-subtilisin BPN' and of SMPI-thermolysin were analyzed statistically and kinetically; The primary structure of Marinostatin D, a novel serine-proteinase inhibitor isolated from a marine micro-organism, was determined; The primary structure of Tyrostatin, a novel inhibitor of Pepstatin-insensitive carboxyl-proteinases, was determined; Unique properties of a novel, thermophilic carboxyl-proteinase and its strict substrate specificity were shown.