液芯微囊化Penicillium purpurogenum Li-3细胞的制备及其催化性能研究

In order to improve hydrolysis of glycyrrhizin (GL) into glycyrrhetinic acid monoglucuronide (GAMG) possessing best biological function, and make full use of the advantageous glycyrrhizae resources of Xinjiang, and aim at actual condition of glycyrrhizin and glycyrrhetinic acid not giving of its best as medicine or food sweetener, and shortage of glycyrrhizae products requiring high-precision techniques in Xinjiang, penicillium purpurogenum Li-3 isolated by our research team will be chosen as research object in this project, which can produce β-glucuronidase（β-GUS）. The key techniques and technological conditions used for preparation of immobilized penicillium purpurogenum Li-3 by alginate-chitosan liquid core microcapsule will be studied. The correlation existing between technological conditions for preparation of microencapsulated penicillium purpurogenum Li-3 strain cell and structure characteristics of immobilized cell, and the relevance of structure characteristics of immobilized cell to its usable performance will be investigated. The effect of compositions, properties and content of the ions liquid in bio-catalytic medium on the mass transfer efficiency of microcapsule, the laws of the growth and metabolism of microencapsulated cell will be observed and studied so as to explore the possible mechanism and set up effective bio-catalytic system. Study on kinetics regularity of bio-catalytic process with immobilized cells will be carried out, leading to set up of corresponding kinetics models to calculate kinetics parameter so as to clarify the variation of cell concentration, substrate concentration and product concentration with time and establish the adjustment strategy for bio-catalytic process. This project will lay down a foundation for design and application of new clean immobilized cells reactor and technology to improve hydrolysis of GL into GAMG with self-owned intellectual property rights .

本项目利用新疆优势甘草资源，针对甘草酸（GL）和甘草次酸(GA)均不能发挥最佳生物功效，以及新疆甘草精深加工技术匮乏的现状，以生物转化GL定向合成最佳生物功效的单葡萄糖醛酸基甘草次酸(GAMG)为目的，选择课题组筛选的、能高效表达β-葡萄糖醛酸苷酶（β-GUS）的产紫青霉（Penicillium purpurogenum Li-3）细胞为研究对象，拟研究制备液芯微囊化产紫青霉细胞的关键技术和条件，探讨工艺参数与结构特性之间、结构特性与细胞使用性能之间的相关性；考察催化介质中离子液体的组成、性质和浓度对液芯微囊化细胞的传质和细胞的生长代谢及催化效率的影响，探讨作用机理，建立高效催化介质体系；对液芯微囊化细胞的催化过程进行动力学分析，考察细胞浓度、GL及GAMG浓度随时间的变化规律，确定对催化过程的高效调控策略。为建立具有自主知识产权的新型清洁化细胞反应器和反应工艺设计奠定基础。

本项目以生物转化甘草酸（Glycyrrhizin, GL）定向合成最佳功效的单葡萄糖醛酸基甘草次酸(Glycyrrhetinic acid monoglucuronide, GAMG)为目的，选择课题组筛选的、能高效表达β-葡萄糖醛酸苷酶的产紫青霉（Penicillium purpurogenum Li-3）细胞为研究对象，首先研究了海藻酸钠-壳聚糖液芯微囊化产紫青霉细胞制备的关键技术与条件；然后考察了离子液体（Ionic liquids, ILs）对产紫青霉细胞的生长代谢、膜通透性及催化效率的影响，验证了液芯微囊化细胞在ILs介质提高催化效率的可行性；针对ILs作为催化介质，存在使用量大、难回收、价格贵、使用成本高的问题，本研究进一步创新，尝试将ILs和菌株细胞一起负载于微胶囊中来实现GL向GAMG的高效转化。分别研究和建立了两种负载ILs液芯微囊化细胞的制备技术。一种以海藻酸钠和壳聚糖为囊材制得负载[BMIM]PF6液芯微囊化细胞，研究显示，GAMG的产率增加了16.8%，这与负载ILs后，囊膜表面孔隙增多、传质性能增强及ILs能够促进细胞的催化活性有关。负载ILs微囊化细胞重复利用第9次，GAMG的产率仍达65.0%。而另一种以海藻酸钠和羧甲基纤维素钠为载体材料制得负载[MBPy]Tf2N液芯微囊化细胞，结果显示，GAMG产率增加了16.1%，重复使用第9次，微囊化细胞相对活性仍达70.8%。通过对催化过程影响因素的考察与动力学分析，研究还确定了实现高效催化过程的最优条件。由此说明，负载ILs微胶囊技术应用于固定化细胞生物催化体系，可以充分发挥固定化细胞技术和ILs在生物催化中的优势，而且更利于ILs的循环利用，大大减少ILs的使用量，降低使用成本。本项目为建立具有自主知识产权的新型清洁化细胞反应器和反应工艺设计奠定了基础。