嗜热鼠李糖苷酶底物特异性机制及其定向改造

Compared with rutin and quercetin, isoquercitrin showed better bioactivity and bioavailability. Alpha-L-rhamnosidase is the key enzyme for the biotransformation from rutin to isoquercitrin.. Through the preparation of protein crystal and analysis of the structure, α-L-rhamnosidase from Thermotoga petrophila was firstly used as the research object in this project to clarify the relationship between the structure and function of the enzyme, the catalytic specificity of the thermophilic α-L-rhamnosidase and the molecular mechanism for the inefficiency in the catalysis of flavonoids containing rhamnoside (rutin et. al). Based on experimental data, the α-L-rhamnosidase from T. petrophila is site-directed modified by using genetic engineering technology. To construct a novel α-L-rhamnosidase with thermostability and high rutin-hydrolytic efficiency, α-L-rhamnosidase from T. petrophila and Aspergillus niger were used as the parent gene, and the catalytic domain will be recombined. . This study filled the blank in the crystal structure of thermophilic α-L-rhamnosidase. The theory about the catalytic properties and structure-activity relationship of the enzyme will be build. It’s expected that novel α-L-rhamnosidase mutants and related recombinant stains will be obtained. The results will provide theoretical basis and guidance for the deeply research, modification and application of α-L-rhamnosidases. In addition, the results will lay the foundation for bio-preparation of isoquercitrin and accelerate its application in food and medicine.

与芦丁、槲皮素相比，异槲皮素在多个方面具有更好的生物活性和生物利用度。α-L-鼠李糖苷酶是转化芦丁制备异槲皮素的关键酶。课题首次以极端嗜热菌T. petrophila 的α-L-鼠李糖苷酶基因为研究对象，通过结晶条件优化，制备蛋白晶体及酶蛋白质与底物共结晶的复合物；通过结构解析，阐明结构与功能关系，阐明酶催化特异性及不能有效水解芦丁等含鼠李糖苷的黄酮化合物的分子机理；利用基因工程技术定点突变酶基因，并以该基因和黑曲霉α-L-究鼠李糖苷酶基因为亲本，重组催化结构域，构建同时具有嗜热和高效催化芦丁的鼠李糖苷酶。研究将填补极端嗜热鼠李糖苷酶晶体结构的空白，将首次建立该酶的催化特性、结构与功能的关系等理论，有望能获得新型嗜热α-L-鼠李糖苷酶突变基因及其基因工程菌。结果将为进一步改造该酶的催化性能和挖掘酶的新用途提供理论和方法的指导，将为异槲皮素的生物制备及其在食品、医药等领域中的应用奠定基础。

与芦丁、槲皮素相比，异槲皮素在多个方面具有更好的生物活性和生物利用度。α-L-鼠李糖苷酶是转化芦丁制备异槲皮素的关键酶，且可用于定向转化其它含鼠李糖基的黄酮类化合物，以获取相应稀有天然活性成分。课题克隆表达和分离纯化了来源于T. petrophila DSM 13995、A. terreus CCF 3059等微生物的α-L-鼠李糖苷酶基因；系统研究了它们的酶学特性，获得了耐高温、底物特异性和键型特异性强、具有很大应用潜力的优异鼠李糖苷酶基因；探讨了鼠李糖苷酶TpeRha的结晶条件；利用同源建模分别获得了TpeRha、AtRha和BtRha等鼠李糖苷酶三维结构；基于 B-factor 饱和突变等策略对 AtRha 等3种鼠李糖苷酶进行了定向分子改造，阐明了其热稳定性提高的机制，解析了酶的结构与功能的关系，获得了优良突变体 AtRha-D594Q、AtRha-G827K-D594Q、BtRha-E39W；通过密码子优化和高拷贝基因工程菌构建，获得了4种同时具有嗜热和高效催化芦丁等底物的鼠李糖苷酶突变基因及其基因工程菌；通过表达条件优化和高密度发酵调控策略的建立，显著提升了目的酶的活力。在此基础上，优化了新型鼠李糖苷酶定向降解芦丁制备异槲皮素、定向降解淫羊藿苷制备淫羊藿苷元的主要影响因子；同时，利用鼠李糖苷酶的转糖基功能，构建了合成含鼠李糖基的系列黄酮糖苷，其中，槲皮素3-O-鼠李糖苷、3,7-O-双鼠李糖苷的产量可分别达到1106 mg/L和390 mg/L。研究结果为进一步改造α-L-鼠李糖苷酶的催化性能和挖掘酶的新用途提供了理论和方法的指导，为异槲皮素等植物中稀有黄酮的制备及其在大健康产品、医药等领域中的应用奠定了基础。