基于手性特点的D型苯丙氨酸脱氨酶的从头设计与创制

项目名称： 基于手性特点的D型苯丙氨酸脱氨酶的从头设计与创制

项目编号： No.21506172

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 有机化学

项目作者： 刘义

作者单位： 西华大学

项目金额： 21万元

中文摘要： D型苯丙氨酸脱氨酶（D-PAL）因可催化合成重要的医药中间体-D型苯丙氨酸而受到重视。目前通过分子改造相应L型酶（L-PAL）得到D-PAL的效果很不理想，关键在于D-PAL与L-PAL立体结构差异较大。分子改造一般只适合结构差异不大的酶改造，因而迫切需要开拓新的方法解决此类问题。近年来基于蛋白骨架选择理论的酶完全从头设计方法已成功应用，但因其设计难度相对较大而不易实现。本项目在预研基础上，创造性的采用酶手性特点结合从头设计方法的新策略，参考已有L型设计D型PAL，降低设计难度因而较易成功。经量子化学计算获得可靠性较高的D-PAL活性中心，装配到稳定性较好的TIM蛋白骨架中，能量优化和结构评估获得一系列D-PAL基因，利用E. coli pET高效表达D-PAL进行实验验证，比较实验数据与设计方案进行分子模拟验证。本项目旨在为部分结构差异较大的手性酶设计策略的制定提供科学依据和方法学参考。

中文关键词： 酶促反应；对映体选择性；构效关系

英文摘要： D-phenylalanine ammonia-lyase (D-PAL) is an important enzyme, which could catalyze and synthesize D-phe, an important pharmaceutical intermediate. It is hard to be obtained through molecular modification based on L-phenylalanine ammonia-lyase (L-PAL) up to now, due to the much structural difference between the D-PAL and L-PAL. New strategies are pursued since the molecular modification is only fit for the modification of limited structural difference of enzymes. Enzymatic de novo design based on the theory of selection of protein scaffold is a good strategy to solve this problem. However, it is a little difficult to achieve as a result of the difficulty of design. Based on pre-research foundation, this project creatively employs a new strategy of enzymatic chirality and the method of de novo design, to decrease the difficulty of design based on the L-PAL. The model of active center of D-PAL is obtained by the quantum calculation. It is then to install in the TIM scaffold with more advanced properties such as the stability. The modeled and matched structures are assayed through energetic minimum. A series of designed D-PAL gene sequences are obtained, transferred to the E. coli, expressed for verification, and further verified by the molecular simulation through the data of D-PAL activity. The aim of this project is to provide scientific basis and methodical reference for the strategy of certain chiral enzymatic design that need much structural modification.

英文关键词： enzyme catalysis;enantioselectivity;structure-activity relationship