酶的改造：可见光/酶催化的不对称C-N键精准构建

在前期的仿生合成研究中，我们发现了能够不对称催化有机反应的可见光/酶双催化体系，针对其中存在的对映选择性不理想的问题，本项目拟首次以小麦胚脂肪酶为先导酶，采用易错PCR和定点饱和突变等分子进化生物技术，改造酶的分子结构；以亚胺与羰基化合物的烷基化反应和亚胺与烯酮的[2+2]环化两类反应为模型反应，通过对模型反应的测试和高通量筛选，寻找能够与可见光兼容并且能高效、高对映选择性地催化模型反应的进化小麦胚脂肪酶；进而研究反应机理。本项目创新性地将酶的定向进化和定点突变引入光合作用仿生研究中，以不需要任何辅因子的水解酶（小麦胚脂肪酶）的非天然催化功能为起点进行酶的定向进化，探索通过改造酶分子结构使其能与光催化相兼容并具有理想对映选择性的方法，发展光/酶联合不对称催化仿生合成新方法。该方向的研究将拓展有机合成的新领域，丰富绿色有机合成方法，对基础研究和工业生产都具有重要的意义。

In the previous study, we found a visible light/enzyme dual-catalyst system capable of asymmetrically catalyzing organic reactions. In view of the problem of unsatisfactory enantioselectivity, the project intends to use wheat germ lipase as the lead enzyme for the first time, using molecular evolutionary biotechnology such as error-prone PCR and site-directed saturation mutation to modify the molecular structure of the enzyme. The alkylation reaction of imines with carbonyl compounds and the [2+2] cyclization reaction of imines with enones will be selected as the model reactions. Through the test of the model reactions and high-throughput screening, we aim to find the evolutionary wheat germ lipase which is compatible with visible light and can efficiently and highly enantioselectively catalyze the model reactions, and then the reaction mechanisms will be studied. This project innovatively introduces directed evolution and site-directed mutagenesis of enzymes into bionic studies of photosynthesis. The directed evolution of the enzyme will be carried out starting from the non-natural catalytic function of the hydrolase (wheat germ lipase), which does not require any cofactors. By changing the molecular structure of the enzyme to make it compatible with photocatalysis and having the ideal enantioselectivity, we hope to develop new photo/enzyme asymmetric synthesis methods. This research will expand the field of organic synthesis, enrich the method of green organic synthesis, and have important significance for basic research and industrial production.