ω-转氨酶对映体识别的分子基础及其改造

Biocatalysis technology has become one of the hot research fields due to its advantages of high efficiency and green characteristic. The core of biocatalysis is the biocatalyst-enzyme. But the poor performance and low efficiency of the existing enzymes limited their applications in industrial process with non-natural substrate and environment. The key problem is that the molecular recognition mechanism of the enzyme catalysis process have not been resolved. It is very difficult to reform the performance of enzyme, thus the industrial enzyme source is limited. S-aminotransferase from C. violaceum DSM3019 was selected as the target biocatalyst and the transamination between alanine and acetophenone was selected as the model reaction. Based on the crystal structure, the interaction between the ω-aminotransferase and the substrate and their dynamic structure changes will be studied by using the methods of molecular dynamics/quantum mechanics. The molecular recognition mechanism and regulation principles of the enantioselectivity of ω-aminotransferase will be resolved at the molecular level. Employing the enantiomer recognition promiscuity of ω-aminotransferase, the construction of highly selective R-aminotransferase will be realized via rational transformation of S-aminotransferase. Based on the thermodynamics/kinetics studies, the substrate engineering, medium engineering and online removing of by-products via multiple-enzyme coupling process will be comprehensively used and systematically evaluated to intensify the process and improve the reaction efficiency. This project is not only a very important supplement to the existing theory of aminotransferase with significant theoretical value, but also has very clear and broad application prospects.

生物催化过程高效绿色，已成为当前热点研究领域之一。生物催化剂-酶是生物催化的核心。但是现有催化用酶在工业应用过程中，面对非天分子然底物和非天然环境，存在性能差、效率低、应用难等问题。问题的关键在于缺乏对酶催化过程分子识别机制的深入研究和解析，酶分子性能改造困难，来源有限。本项目拟选择来源于C. violaceum DSM3019的S-转氨酶作为研究对象，以丙氨酸和苯乙酮的转氨反应作为模型，基于晶体结构，采用分子动力学/量子力学方法研究ω-转氨酶和底物间相互作用和动态结构变化，在分子水平上解析转氨酶对映体识别机制和调控原理；利用ω-转氨酶对映体识别的混乱性，理性改造S-转氨酶，构建高选择性R-转氨酶；深入研究催化过程热/动力学，综合运用和系统评价底物工程、介质工程和多酶偶联在线去除副产物等技术手段，实现过程强化，改善反应效率。本项目研究不仅具有重要的理论价值，同时其应用前景也非常明确和广阔。

转氨酶是一种具有重要工业价值的生物催化剂，其对映体选择性是其得以广泛应用的最为重要的特性之一，在用于制备具有重要应用价值的手性化学品上显得尤为关键。此外，转氨酶催化的反应存在反应平衡，需要对过程进行强化提高制备手性胺类化学品的效率。通过项目研究，成功克隆、表达了23种不同来源、具有不同底物特异性的S-转氨酶，并根据其空间结构、底物结合位点和反应机理解析其对映体识别的分子机制。基于其机制，结合计算机模拟计算对S-转氨酶进行分子改造获得多个位点的大量突变株，但是大部分突变株的催化活力严重下降，部分菌株选择性下降，仍不能满足高R-选择性的需求。因而克隆、表达了11个R-转氨酶并考察其在非天然氨基酸和手性胺的制备中的应用。对高活力、高选择性的转氨酶进行表达优化，包括培养基、培养条件、表达宿主、质粒拷贝数、融合标签等，将活力提高5倍。通过对表达宿主中辅酶PLP合成途径的强化或重构，使得重组转氨酶的生产能实现辅酶PLP的自给自足，不需额外添加。通过补料分批发酵实现转氨酶重组菌株的高密度发酵，大大提高酶的生产效率。分别构建转氨酶/乙烯合成酶双酶偶联体系和转氨酶/氨基酸脱氢酶/辅酶循环三酶偶联催化体系强化转氨反应过程，解决转氨反应固有的反应平衡问题，提高底物转化率。最后，实现精草铵膦的高效制备，经12 h反应，底物完全转化获得90 g/L的精草铵膦，得率为98.3%，并使用抗溶剂结晶法获得纯品，收率为75%。本项目有望在5年内实现精草铵膦的产业化。