ω-转氨酶双吊桥形“门”结构与其有机溶剂耐受性的构效机理研究

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.

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。

手性胺是指小分子化合物手性中心含有官能团“-NH2’’的一类化合物，是治疗高血压、糖尿病和心血管疾病等众多药物的中间体或终产物，在日常生活和国民经济中占据重要地位。ω-转氨酶(EC 2.6.1.X)是工业上生产手性胺等重要药物中间体和精细化学品的常用生物催化剂之一。鉴于生物催化的高效率、高对映体选择性、高区域选择性、反应条件温和及环境友好等优点，采用ω-转氨酶制备手性胺类化合物已成为应用酶学的研究热点。. 本项目以ω-转氨酶为研究对象，利用ω-转氨酶直接不对称催化潜手性酮类化合物的策略合成光学纯(R)-手性胺西他列汀中间体PTfpB。首先从实验室已有的ω-转氨酶库筛选到一株来源于卡明斯杆菌(Arthrobacter cumminsii ZJUT212)活力较高的ω-转氨酶；将其命名为AcATA，并作为分子改造出发酶。接着，结合AcATA的三维结构、底物结合口袋以及丙氨酸扫描结果选定T134、M122、Q155为潜在有益位点，建立点突变基因文库，使用高效液相色谱进行分析。经过三轮突变，成功筛选到2个高活力酶突变体，分别是：AcATA M1+M122H和AcATA M1+T134G。它们不仅显示出改进的活性和底物亲和力，而且在包含20% DMSO的缓冲液中显示出更高的稳定性。随后的研究酶学性质研究，确定了AcATA M1、M1+T134G和M1+M122H最佳反应温度分别为45°C、50°C和50°C；最适反应pH均为9.0，表明AcATA催化反应需要碱性环境。通过对其稳定性的测定可知，AcATA M1、M1+T134G和M1 + M122H在50°C的半衰期分别为47 h、61 h和81 h；进一步，从比酶活、动力学参数比较可知，突变体AcATA M1+M122H酶活最高，催化效率（kcat/Km）达到0.048 mM-1 s-1。使用2 g/L含有AcATA M1+M122H基因的冻干大肠杆菌细胞作为生物催化剂，反应30 h后成功转化50 g/L PTfpB合成40g/L西他列汀中间体，转化率超过82%，产物ee> 99%。结构分析表明，“门“结构和底物通道氨基酸残基的柔性降低是AcATA稳定性增加的结构基础。