甲基天冬氨酸氨裂解酶EcMAL的半理性改造及其不对称催化D-氨基酸的合成

D-氨基酸是手性药物和精细化学品合成的重要结构砌块，具有重要的应用价值，开发高效、绿色的合成方法是当前的研究热点。当前，酶催化法是合成D-氨基酸的重要途径，但仍存在反应过程复杂、产物光学纯度较低、酶稳定性较差等瓶颈问题。本项目拟以甲基天冬氨酸氨裂解酶EcMAL为研究对象，对EcMAL进行半理性改造，重塑其底物结合口袋，翻转其立体选择性，并基于偶联D-天冬氨酸氧化酶的高通量筛选方法，获得可高选择性一步转化富马酸为D-天冬氨酸的突变酶。揭示该酶立体选择性的分子机制，阐明其构效关系，实现烯酸类底物一步法转化为D-氨基酸，为非天然D-氨基酸的合成提供新途径。同时，辅以溶剂工程技术，提高酶的稳定性，以此建立高效、绿色合成D-氨基酸的生物催化体系。项目的开展将为氨裂解酶的催化功能改造奠定理论基础，推动氨裂解酶在非天然氨基酸绿色制造中的应用，为我国医药化工行业的可持续发展做出积极贡献。

D-amino acids are the important building blocks of chiral drugs and fine chemicals and have significant value in lots of applications. The development of efficient and green synthesis pathways has attracted much attention. At present, enzymatic catalysis is an important method to synthesize D-amino acids. However, the complex reaction process, the relatively low optical purity of the products, and the poor enzyme stability limit the enzymatic synthesis. This project intends to use the methylaspartic acid ammonia lyase EcMAL as the research object and engineer EcMAL by semi-rational design to reshape the substrate-binding pocket and switch its stereoselectivity. Based on the high-throughput screening method coupled with D-aspartic acid oxidase, we expect to afford the EcMAL mutant which can stereoselectively one-step transform fumaric acid to D-aspartic acid. At the same time, the molecular mechanism of the stereoselectivity of EcMAL will be revealed and the structure-activity relationship will be clarified. The one-step conversion of olefine acid substrates to D-amino acids will be achieved, thus opening a new way for the biocatalytic synthesis of unnatural D-amino acids. Besides, in the favor of solvent engineering strategy to improve the stability of EcMAL, an efficient and green biocatalytic system for the synthesis of D-amino acids will be established. The project will provide a theoretical basis for the engineering of ammonia lyase and promote the application of ammonia lyase in the green manufacturing of unnatural amino acids. It will also contribute greatly to the sustainable development of the medicine and chemical industry in our country.