基于中药活性成分生物转化的逆流色谱反应器研究

Bioreactor research is one of the front and hot directions in today's scientific community. Research and development of reactors with high biotransformation ability has important significance. Countercurrent chromatography plays an important role in the field of separation and purification, for its unique advantages such as non-solid phase adsorption, samples activity retention and good separation ability. Our previous study found that extracellular β-glucosidase NG-1 produced by Lactobacillus brevis BL1 has the ability to efficiently biotransforming the active ingredients of chinese herbs, such as biotransforming ginsenoside Rb1 to rare ginsenoside C-K. This project intends to develop a countercurrent chromatography reactor with efficient biotransformation capacity, by the ginsenoside biotransformation mentioned above as a model, based on liquid-liquid distribution model of the countercurrent chromatography, and the latest research results of countercurrent chromatography solvent system. In detail, the two-phase solvent system of the countercurrent chromatography will be screened, the enzymatic properties of the solvent system will be investigated, the parameters of the countercurrent chromatography reactor will be optimized, the kinetic law of the countercurrent chromatography catalytic reaction and the properties of the transfer process within the reaction phase will be explored, and an empirical kinetic mathematical model will be established. The countercurrent chromatography reactor constructed by this project will be widely used in the biotransformation of the active ingredients in chinese herbs, and also has potential application in the biotransformation activity with microbial or cell as carrier, so there is significant scientific value to study the countercurrent chromatography reactor.

生物反应器的研究是当今科学界的前沿和热点之一，研究和开发具有高效生物转化能力的反应器具有重要意义。逆流色谱在分离纯化领域以其无固相吸附、样品活性保持、良好的分离能力等独特的优点，发挥着重要的作用。课题组前期研究发现短乳杆菌BL1产胞外β-葡萄糖苷酶NG-1具有高效生物转化中药活性成分人参皂苷Rb1为稀有人参皂苷C-K的能力。本项目拟以上述人参皂苷的生物转化为模型，基于逆流色谱的液-液分配模式，结合最新的逆流色谱溶剂系统研究成果，研制具有高效生物转化能力的逆流色谱反应器。具体为：筛选合适的逆流色谱两相溶剂系统，考察溶剂系统中的酶学性质，对逆流色谱反应器的参数进行优化，探索逆流色谱催化反应动力学规律和反应相内传递过程的特性，建立经验的动力学数学模型。本项目构建的逆流色谱反应器可广泛应用于中药活性成分的生物转化，同时以微生物、细胞等为载体生物转化中也有潜在的应用前景，具有重要科学价值。

生物反应器的研究是当今科学界的前沿和热点之一，研究和开发具有高效生物转化能力的反应器具有重要意义。逆流色谱在分离纯化领域以其无固相吸附、样品活性保持、良好的分离能力等独特的优点，发挥着重要的作用。本项目以天然产物活性成分尤其是糖苷类成分的生物转化为模型，基于逆流色谱的液-液分配模式，结合最新的逆流色谱溶剂系统研究成果，筛选合适的逆流色谱两相溶剂系统，考察溶剂系统中的酶学性质，对逆流色谱反应器的参数进行优化，探索逆流色谱催化反应动力学规律和反应相内传递过程的特性，建立经验的动力学数学模型，研制具有高效生物转化能力的逆流色谱反应器。. 结果显示，正己烷、氯仿溶剂体系的酶活力下降显著，而乙酸乙酯溶剂体系48 h后酶活力高于99%，可作为逆流色谱生物反应器的基础溶剂体系。通过加入曲拉通X-100作为增溶剂，酶活力无明显变化，可作为逆流色谱反应器的助溶剂。反转正接和正转反接两种运行模式下固定相保留率均高于83%，且随流速的增大呈下降趋势，当流速增至10 mL/min时，两种模式下固定相保留率仍高于60%。固定相保留率随转速的增大而增大，最终优选出800 rpm为最适转速。采用构建的新型逆流色谱反应器实现了虎杖苷、大豆异黄酮苷、京尼平苷和染料木苷的高效转化，转化率高于90%。以逆流色谱作为生物转化反应器高效、可行，适合于中药中糖苷类成分的高效生物转化，为中药中活性成分的生物转化提供了新的工具，丰富了酶生物反应器的类型，同时在以微生物、细胞等为载体的生物转化中也具有潜在的应用前景，具有深入研究的价值。