离子液体双液相甾体生物催化体系构建研究

Low solubility of steroids in aqueous media considerably limits the productivity of the biotransformation system, which has been a bottle-neck in steroid bitransformation. Biphasic processes are usually applied in the whole-cell biotransformation to overcome low water solubility of substrate and/or product and their inhibitory effect of substrate and/or product on the biocatalyst. Furthermore, commonly used conventional organic solvents often damage the microbial cells. Ionic liquids (ILs) were recommended as promising alternatives to organic solvents in two-phase bioreactive systems, as they are environment friendly and can dissolve a wide range of chemical compounds. In this research project, we will construct new quantitative screening model, based on 15α hydroxylation of 13-ethyl-gon-4-en-3,17-dione by P. raistrickii, to evaluate the applicability of ILs for biphasic system according to the quantitative structure-toxicity relationship (QSAR), in which the distribution coefficient of substrate and product, as well as metabolic activity of P. raistrickii, is considered. The selected ILs, with higher distribution coefficients and lower toxicity to microorganism, are used to construct two-phase system for the production of 15α hydroxylation of 13-ethyl-gon-4-en-3,17-dione by IL-tolerant P. raistrickii. Through this project, the screening model of ILs for biphasic system will be constructed, the adaptive mechanism of IL-tolerant P. raistrickii and the the effect of ILs' physicochemical parameters on the microbial transformation of steroid are revealed. These studies will be very important for the potential industrial application of ILs-based biphasic systems for steroid biotransformation.

甾体化合物在水中溶解度极低，严重影响甾体转化反应的速度和产率，成为甾体生物催化工业中亟待解决的瓶颈问题；传统双液相体系设计用于全细胞生物催化，可解决底物和产物在水相中溶解度低的问题，但对细胞具有毒性且污染严重。非水溶性离子液体具有较高分配系数及绿色环保等优点，成为替代有机溶剂构建双液相体系最有前途的分配相。本项目以雷斯青霉生物催化左旋乙基甾烯双酮15α羟基化为研究模型，根据定量构效筛选方法（QSAR），结合代谢活力及分配系数等，构建离子液体定量筛选模型，高效筛选具有高分配系数、低毒性的离子液体，建立两液相生物催化体系，同时利用雷斯青霉离子液体耐受菌株，高效生产15α羟基化左旋乙基甾烯双酮。本项目通过对离子液体定量筛选模型构建，离子液体双液相生物催化反应规律及菌体对离子液体耐受机理研究，为构建离子液体双液相体系应用于甾体生物催化提供理论基础，对实现甾体生物催化产业的技术升级具有重要意义。

双液相体系用于甾体药物的生物催化，目的是解决底物溶解度低而造成转化效率低的问题。而传统双液相中的有机溶剂对菌体毒性较大，且对环境污染严重。因此“绿色溶剂”离子 液体因其独特的物理化学性质成为当前代替有机溶剂构建两相系统最有前途的分配相。本项目以以雷斯青霉生物催化左旋乙基甾烯双酮15α羟基化为研究模型，结合离子液体的生物相容性及分配系数等，提出绿色反应介质离子液体-水双液相发酵体系，旨在为工业化甾体羟基化反应存在难题提供一条新的研究思路。..本项目选用不同结构阳离子组合而成的不同离子液体，结合离子液体物理化学性质，研究离子液体对雷斯青霉、赭曲霉及简单节杆菌葡萄糖代谢能力及孢子存活能力的影响，揭示不同结构离子液体对雷斯青霉菌体细胞的生物相容性、生理生化特性之间的构效关系；系统考察离子液体对该反应体系中阳离子不同侧链基团以及阴离子对底物的溶解与传递、反应速率及转化效率等参数的影响；揭示离子液体双液相体系中甾体药物生物催化反应规律，为高效筛选离子液体构建甾体药物双液相生物催化体系提供理论依据。通过进一步考察离子液体双液相体系中水相组成、离子液体相与水相比例、反应温度和搅拌形式等条件对甾体化合物传质及生物催化效率的影响。构建高效离子液体双液相甾体药物生物催化体系，有助于实现实现甾体生物催化关键技术和应用研究的新突破。最后，本课题组探索了基于新型离子液体低熔点共溶溶剂进行简单节杆菌催化甾体化合物脱氢反应的可行性。