糖苷酶定向转化人参皂苷的分子识别机制

Ginseng, China's traditional Chinese medicine, with potent pharmacological effects, is widely used in potential therapeutics for cancer. The sugar-based directional modification and transformation of ginsenosides for the production of rare ginsenoside with high bio-anticancer activity has broad application prospects in tumor therapy. However, due to complex preparation process and high cost, rare ginsenoside can not be appplied in clinical. Enzyme specific conversion is the most feasible way to solve the bottleneck problem. At present, the key problems of enzymatic synthesis include: enzyme specificity activity is not high, the enzymes involved and the according catalytic mechanism are still not clarified, and studies based on molecular structure and gene of the enzyme is not systematically, respectively. Research on improving the catalytic charactors for enzymatic hydrolysis of ginsenoside is still in its initial stage. This project intends to carry out theoretical and application research on design of β-glycosidase with high regiospecificity for triterpenoid tailoring. Recombinant expression of glycosidases with specific activity were reasonably selected from various microbial populations; HPLC and LC/MS are used to determine the catalytic efficiency and the transformation path of the ginsenosides; according to the bioinformatics analysis, the glycosides hydrolysis mechanism is studied by a large number of site-directed or saturation mutagenesis, enzyme kinetics experiment with the help of computer simulation. From the model, the substrate binding pocket of the enzyme can be redesigned in a manner that preferentially hydrolyzes glycans at specific glycosylation sites of triterpenoids. Combined with chemical conversion，functional rare ginsenoside Rk1，Rg5 was synthesized to extended enzyme application scale. This study would give a theoretical guidance and basis on anticancer drugs development, high efficiency and large scale preparation, and the cancer treatment clinical application with ginsenoside involved.

人参是我国传统中药，药效显著、应用广泛。通过定向修饰与转化人参皂苷糖基，可产生高生物抗癌活性稀有人参皂苷。然而由于其制备工艺极其复杂、成本过高，不能应用于临床，酶法定向转化成为解决其瓶颈问题最可行手段。目前皂苷单体酶法合成关键问题包括：特异性酶活性不高、涉及酶及其催化机制不明确、结构生物信息学研究不系统，改善酶水解皂苷糖基专一性的研究尚处于起步阶段。为实现酶法定向转化的理论和应用研究，本项目拟针对这些问题开展如下工作：合理筛选得到重组表达活性高、专一性强的糖苷水解酶群及其微生物；以HPLC和LC/MS检测催化效率、皂苷转化路径；基于酶三维结构和功能信息，通过大量点突变、酶动力学实验及计算机模拟，系统研究糖苷酶水解机理和分子识别机制；结合化学手段得到Rk1，Rg5等功能性稀有人参皂苷，拓展酶的增效应用。本研究可为单体抗癌药物的开发、规模化制备和癌症治疗临床应用提供有用的理论和实验依据。

人参皂苷是人参属植物药理活性的重要物质，尤以稀有人参皂苷及苷元的抗肿瘤, 保护神经系统，保肝护肝等药理活性最为显著，而人参皂苷、次级皂苷和皂苷元等成分在人参属植物中含量较少，体内转化量和生物利用度极低，须通过体外总皂苷降解获得。因此，研究稀有人参皂苷转化方法，尤其以微生物糖苷水解酶群转化成为解决化学法瓶颈问题的有效手段。主要原人参二醇型Rb1和真正发挥药理作用的次级苷和苷元（Rg5，CK等）结构差异，主要在于人参皂苷达玛烷骨架C-3，C-20处支链所连接糖基种类和数量的不同。本研究从人参根土壤中经七叶苷-R2A固体培养基筛选获得高效转化人参皂苷的野生真菌，经Rb1→Rd→F2→CK途径获得人参皂苷CK，并通过发酵产酶工艺优化、反应体系构建及催化分子识别机制等方面进行了探究。黑曲霉（Aspergillus niger）菌丝体转化1mM Rb1，6天CK转化率为50.4%；A. niger发酵产酶，水解6 mM Rb1，60h CK转化率为90%。新型绿色溶剂低共熔溶剂（氯化胆碱：乙二醇）显著提高土曲霉（Aspergillus terreus）产β-糖苷酶酶对底物的亲和力和催化效率，A. terreus发酵产β-葡萄糖苷酶转化12 mM Rb1，60h CK转化率91%。以低共熔溶剂为基础的双水相体系能原位转化提取人参皂苷CK，在萃取生物转化最适工艺条件下，上、下相种产物和β-葡萄糖苷酶的收率分别为75.79%和61.14%。且低共熔溶剂和β-葡萄糖苷酶可回收用于CK的下一次提取转化。针对嗜热硫磺矿硫化叶菌Sulfolobus solfataricus糖苷酶（SS-bgly）产CK限速步骤Rd→F2，通过人参皂苷Rd和SS-bgly活性中心催化残基处的分子对接，确定了与Rd相互作用的候选突变残基，通过实验筛选催化效率提高的变异SS-bgly。最后，对人参皂苷Rg5对胃癌和乳腺癌的抗肿瘤功效和分子机制进行了体内外探究。本研究可为单体抗癌药物的开发、规模化制备和癌症治疗临床应用提供有用的理论和实验依据。