ω-转氨酶不对称催化伯胺与酮之间的氨基转移反应，是合成手性胺的关键酶。但由于目前缺乏ω-转氨酶结构与其有机溶剂耐受性之间的构效关系研究，对现有酶进行分子改造难度较大，是制约其应用的重要因素。前期工作中，通过随机突变，我们获得了一个位于双吊桥形“门”结构上的有益突变（酶在50% DMSO中的半衰期增加了约2小时）。本项目拟分析该酶的晶体结构，饱和突变“门”结构上的氨基酸位点、高通量筛选，研究在不同有机介质中有义突变的位点/类型、“门”结构的柔/极性变化、酶的热/动力学；在分析“序列-结构-功能”关系的基础上，提出“门”结构调控ω-转氨酶有机溶剂耐受性的机制假说；进而利用该假说理性改造、快速构建高稳定性的ω-转氨酶，并结合分子模拟分析，验证或修正该假说。本项目的研究将有助于深入理解“门”结构与ω-转氨酶有机溶剂耐受性之间的构效关系，为高效改进ω-转氨酶在有机溶剂中的稳定性奠定基础。

ω-transaminase catalyses the asymmetric aminotransfer reaction between the primary amine and ketone. It is a key enzyme for the synthesis of chiral amines. However, its applications are hampered by its poor organic solvents tolerance. Therefore, it is necessary to study the structure-functional relationship between ω-transaminase’s structure and its organic solvent tolerance for increasing its stability in organic media by protein engineering. In the preliminary work, through random mutagenesis, we obtained a beneficial mutation located in the structure of the double-bridged "gate" (the half-life of it in 50% DMSO was increased by ~2 h). In this project, the sites/types of the beneficial mutations in different organic media will be firstly obtained by crystal structure analysis, site-satruation mutagenesis gene library generation and high-throughput screening. Importantly, the mechanism hypothesis of "gate" structure regulating the organic solvent tolerance will be summarized on the basis of analyzing the relationship of "sequence-structure-function". Furthermore, a highly stable ω-transaminase will be rapid constructed based on the verified or correct the hypothesis. The study of this project will help us to understand the structure-functional relationship between the "gate" structure and the organic solvent tolerance, thus laying the foundation for the efficient improvement of ω-transaminase’s stability in organic solvents.