基于定量构效关系的铜绿假单胞杆菌脂肪酶及其折叠酶的分子改造

脂肪酶在有机相中能催化酯化、酯的醇解、酯的氨解和酯的酸解等各种化学反应。然而脂肪酶在有机相中的催化活性比水相中低数个数量级，因此需用提高脂肪酶在有机相中的活性。栗精胺-6-O-丁酯具有抗多种病毒活性。在栗精胺-6-O-丁酯的合成中，利用脂肪酶催化酯化反应具有高度区域选择性的特点，就可以起到简化有机合成反应步骤和提高产率的效果。本研究以自主筛选的Pseudomonas aeruginosa CS-2为材料，构建其脂肪酶及折叠酶基因突变库，并测得脂肪酶突变体在有机溶剂中催化合成栗精胺-6-O-丁酯的产率。在此基础上，建立脂肪酶及其折叠酶的定量构效关系模型，并以此模型为基础进行脂肪酶及折叠酶的分子改造。同时通过分析脂肪酶及折叠酶的分子改造过程中出现的各种阳性和阴性结果，采用抗蛋白酶作用实验和表面等离子共振方法研究脂肪酶和折叠酶的相互作用，为铜绿假单胞杆菌脂肪酶的分子改造提供更完善的理论基础。

Lipases catalyze various chemical reactions such as esterification,alcoholysis,aminolysis and acidlysis in organic solvents . However, lipase in organic solvents often exhibit a few orders of magnitude lower activity than in aqueous solutions.Therefore, it is necessary to improve lipase performance in organic solvents by all kinds of ways. Castanospermine-6-O-butyrate has antiviral activity. In order to simplify reaction steps and increase reaction yield, lipase is used for the synthsis of castanospermine-6-O-butyrate owing to its high regiospecificity. Genes mutant libraries of lipase and its cogante foldase from Pseudomonas aeruginosa CS-2 are built in the project, and the yields of castanospermine-6-O-butyrate are assayed in the lipase mutants-catalyzed reactions. Based on these results, the quantatitive activity-structure realtionship(QASR) models are built, and the molecular engineering of lipase and foldase is carried out, aided by the QASR models. According to the positive and negative results obtained from molecular engineering of lipases and foldases, the interactions between lipases and foldase are studied by anti-protease metohod and surface plasmon resonance method in order to support theory basis for the molecular engineering of lipase from P.aeruginosa.