酶催化常用于获取中药活性成分，高效固定化游离酶则是其规模化应用的难点与关键。已完成的基金项目研究表明，由于固定化过程的随机性以及普通载体产生较大扩散限制，常规固定化酶在循环利用于水解产生薯蓣皂苷元和宝藿苷I时，催化性能和稳定性远低于预期。在此基础上，我们提出了“遗传修饰—共价结合—磁性纳米粒”固定化新策略。本项研究旨在解决“难以高效而规模化地应用酶水解获取中药活性成分”这一关键科学问题，拟采用定点突变技术对水解酶表面远离活性中心且富含Lys残基的区域进行遗传修饰，继而以共价结合法将酶突变体定向固定化于核壳型磁性纳米粒，用于构建新型高效酶水解体系，并揭示突变区域与位点、固定化条件、纳米载体性质等影响固定化酶性能的基本规律及内在原因。本项目可为酶催化获取中药活性成分及具有重要医药用途化合物的研究开辟一条新的途径；建立的关键共性技术可为最终应用于医药工业实践奠定基础，发展前景广阔，应用价值重大。

Enzymatic catalysis is often used to obtain active constituents of Chinese Medicines, and efficient immobilization of free enzymes is a challenging and key task for the large-scale application of enzymatic hydrolysis. In our completed projects, it has shown that the catalytic performance and stability of conventional covalently immobilized enzymes were much lower than expected level when they were repeatedly recycled in enzymatic hydrolysis for the production of diosgenin and Baohuoside I, due to the randomness of the immobilization process and the large diffusion limitation of the common carrier. On the basis of these, we proposed a new strategy of immobilization, namely "Genetic Modification - Covalent Binding - Magnetic Nanoparticles". This study aims to resolve one of key scientific problems “It is difficult to efficiently apply enzymatic catalysis for obtaining active constituents of Chinese Medicines on a large scale”, in which site-directed mutagenesis technique will be employed for genetic modification of non-active region with abundant Lys residues on the surface of wild-type hydrolase, with the incorporation of covalent bonding method to immobilize hydrolase mutants on core-shell type magnetic nanoparticles in a site-directed way. Afterwards, a new efficient system of enzymatic hydrolysis will be constructed using these immobilized enzymes. In addition, the basic rules and essential causes of how the region and site of mutation, conditions for covalent immobilization, and properties of magnetic nanoparticles effect on the efficiency of immobilized enzymes will be revealed. The aim of this proposed project is to provide a new approach for obtaining active constituents of Chinese Medicines and other important medicinal compounds by enzymatic hydrolysis. Besides, the key common technology to be established can lay a firm foundation for its final application in pharmaceutical industries, indicating its broad prospects and great application values.