共价固定化酶循环利用于高效催化产生中药活性成分的研究

After the reaction for the production of active ingredients from Chinese medicine by enzymatic catalysis, the free enzymes haven't been reused as they are destroyed or it is hard to recycle the enzyme from the reaction system, also a low efficiency is undesirable if using fermentation to produce enzymes, and both of which cause the difficulties while continuous, efficient and large-scale application of the enzymes. Immobilization, is not only the most effective way for recycling enzymes, especially those enzymes reacting with the chemical constituents from Chinese medicine, but also the key process for large-scale application. Hydrolases of Dioscorea saponins (HDSs), as model enzymes in this study, will be covalently immobilized based on the structure analysis data. Enzyme immobilization will overcome the free enzymes' disadvantages of bad stability and recovery difficulty, as well as other immobilization methods' shortages. Covalently immobilized HDSs having the best catalytic activity and recycling performance will be obtained and their stability, kinetic property and molecular structure, etc, will be detected and analyzed. The results will reveal the effective factors on their catalysis and recycling performance. The recyclable covalently immobilized HDSs will be used on producing active ingredient diosgenin highly effectively. The aim of this study is to build up a new research approach to obtain active ingredients from Chinese medicine more effectively, and to develop key techniques as a base for producing target compounds. So this work has a promising perspective and a significant value for industrial application. In addition, this study is benefit for science of Chinese medicine cooperated with other research fields such as bioinformatics, material technology, and enzyme technology, therefore it has a great academic significance.

在酶催化产生中药活性成分的反应结束之后，游离酶因被破坏或难以被回收而未能再利用，而发酵产酶亦存在效率低下等不足，均不利于酶的连续、高效和规模化使用。固定化是酶得以高效循环利用的最有效途径及其规模化应用的关键，本研究拟以薯蓣皂苷水解酶为模型，根据其结构特征，采用共价结合法进行固定化以克服游离酶稳定性差、回收困难以及其它固定化方法的不足，并获得具有最佳催化和重复利用性能的共价固定化酶；同时，分析与比较稳定性、动力学性质、立体构象等，揭示影响固定化薯蓣皂苷水解酶的催化及重复利用性能的内在原因，并最终实现其循环利用于高效催化水解薯蓣皂苷产生活性成分薯蓣皂苷元。本项目旨在为高效获取中药活性成分开辟一条新的研究途径，所建立的关键技术可为最终应用于生产实践奠定一定的基础，因此有着广阔的发展前景和重大的应用价值；并且本研究可促进生物信息学、材料学、酶工程等与中药学的有机结合，具有重要的学术意义。

在酶催化产生中药活性成分的反应结束之后，游离酶因被破坏或难以被回收而未能再利用，而发酵产酶亦存在效率低下等不足，均不利于酶的连续、高效和规模化使用，固定化则是酶得以高效循环利用的最有效途径及其规模化应用的关键。.本研究选择了具有一定代表性的中药苷类成分为对象，包括甾体皂苷（薯蓣总皂苷）、黄酮苷（淫羊藿苷、香椿叶黄酮苷、柚皮苷），首先建立了薯蓣根茎综合利用新途径以及各个类别中药活性成分的分析方法，继而运用双相催化水解法为核心技术，从而产生多个类别的中药活性成分（薯蓣皂苷元、宝藿苷I、山柰酚和柚皮素等）。.课题研究中采用了共价结合法将薯蓣皂苷水解酶固定化于Ni0.5Zn0.5Fe2O4@SiO2磁性纳米颗粒。结果表明，beta-葡萄糖苷酶和薯蓣皂苷糖苷酶的最佳固定化时间分别为4 hrs和8 hrs，最适酶浓度均为0.10 mg/mL，pH 8.0时固定化效率最高，固定化酶与游离酶水解反应的最适温度相一致，均为45℃，经过固定后薯蓣皂苷水解酶的热稳定性得以增加。固定化薯蓣皂苷水解酶催化水解薯蓣皂苷产生薯蓣皂苷元的得率和固定化效率达到4.13%和113%；固定化酶经连续11次间歇操作使用，催化活性有所下降，经第5次重复使用后，活性下降幅度减小且趋于稳定；在第11次使用后，表观活性仍然保持了初始活性的66.1%。共价固定化酶与游离水解酶以及吸附法制备的固定化酶相比，具有明显的优势。此外，采用共价结合法将beta-葡萄糖苷酶固定于Ni0.5Zn0.5Fe2O4@SiO2并用于催化水解淫羊藿苷制备宝藿苷I，水解率达到98.5%，明显高于包埋法固定化酶和吸附固定化酶。.本项目的研究工作为高效获取中药活性成分开辟一条新的途径，有助于解决“酶循环利用于高效催化产生中药活性成分”这一共性关键科学问题；同时所建立的关键技术也可为最终应用于生产实践奠定一定的基础，有利于提升我国薯蓣皂苷元、淫羊藿黄酮产业的核心竞争力。