基于生物组合催化技术的微量活性成分三七皂苷ST4的定向转化

The directional improvement of rare active ingredients based on biocatalysis technology, with the advantages of high efficiency, specificity, environmentally friendly and easy to industrialization, is regarded as a new and important method to conquer the shortage of resources and to develop innovation medicines..Panax notoginseng, a kind of unique traditional Chinese medicinal materials in China, has a long tradition in clinical application. Notoginsenoside ST4 belonging to tetracyclic triterpenoid of protopanaxadiol type, has shown tempting development prospects in anastalsis and antiviral. But its content is incredibly low in Panax notoginseng, which seriously results in difficult to carry out systemic pharmacology researches and further evaluate the development of new drugs. Notoginsenoside Fc is a very ideal candidate substrate because it is rich in the stems and leaves of P. notoginseng and makes a similar structure to ST4..This project intends to apply technologies on protein engineering, structural biology and biocatalysis to develop biocatalysts possessing highly effective and directional catalytic hydrolysis effects on the xylose and glucose of notoginsenoside Fc at C-20 sugar chain, to achieve batch preparation of active ingredient ST4 by the combination catalysis method of “Two-step in one pot” and further to reveal the molecular mechanisms which influence the catalytic activity of triterpenoid saponins glycoside hydrolase on substrates. The research will not only help broaden the application of glycoside hydrolases in triterpenoid saponins of dammarane type, but also provide reference for other types of triterpenoid saponins on biotransformation.

基于生物催化技术的中药稀有活性成分的定向修饰，具有高效、专一、环境友好和易于工业化等优点，为解决中药药用资源短缺和创新药物开发提供了一条新的重要途径。.三七是我国特有的名贵中药材，具有悠久的临床应用历史。研究表明，原人参二醇型三七皂苷ST4，在止血和抗病毒等方面展现出诱人的开发前景；但其在三七中的含量极低，难以开展系统深入的药理学及进一步的新药开发评价研究。富含于三七茎叶中的三七皂苷Fc与其结构相似，是一种非常理想的“候选底物”。.本项目拟运用蛋白质工程、结构生物学和生物催化等技术，开发出高效、定向催化水解三七皂苷Fc C-20位木糖和葡萄糖的生物催化剂，“二步一锅煮法”组合催化实现稀有活性成分三七皂苷ST4的批量制备，并从蛋白空间结构上揭示影响三萜皂苷糖苷水解酶催化活力的分子机制。该研究不仅有助于拓宽达玛烷型三萜皂苷糖苷水解酶的应用，还为其它类型三萜皂苷的生物转化研究提供借鉴。

三七皂苷ST4在三七中的含量极低，这严重限制了相关药理研究及新药开发。三七皂苷Fc是三七茎叶皂苷中的主要化学成分，与三七皂苷ST4结构相似，是转化生产ST4的理想底物。本研究运用中药学、化学和生物学等多学科交叉技术，围绕三七皂苷ST4的组合生物催化，开展了一系列相关研究。采用基因挖库的方法，挑选出30个糖苷水解酶；完成了对应基因的分子克隆和重组蛋白的异源表达、分离纯化与功能检测，成功筛选出一种具有定向催化水解三七皂苷 Fc C-20 位外侧木糖基作用生成越南人参皂苷R7的生物催化剂KfGH01；通过定向进化和半理性设计等蛋白质结构改造技术，获得一株活力是母本KfGH01 50倍的突变体KfGH01I248F/Y410R；利用该突变体和HaGH03两种酶组合催化，“二步一锅煮法”实现了稀有三七皂苷ST4的定向制备；并解析出KfGH01及突变体KfGH01I248F/Y410R两种蛋白三维结构；并深入开展了三七皂苷糖苷水解KfGH01及其突变体KfGH01I248F/Y410R的分子动力学模拟实验，探索与阐明了突变体催化活力提高机制。目前本项目发表SCI论文2篇，申请专利1项，获得晶体PDB号2个（6M6M和6M6L）。本项目顺利完成了预期目标，实现了含有五糖基三七皂苷Fc的C-20位木糖基和葡萄糖基的定向催化水解，有利于拓宽三萜皂苷糖苷水解酶的应用，为其它类型三萜皂苷的生物转化研究提供了研究借鉴。