酮胺化还原酶耐有机溶剂机制研究及其功能元件设计组装

Organic solvent tolerant reductive aminase (RedAm) are promising for preparation of high value-added chemicals and medical intermediates, such as chiral amine, for non-aqueous biocatalysis in industrial biotechnology. However, the key enzymes such as amine dehydrogenase (AmDH) and RedAm for the reductive amination of ketones to chiral amine shown low activity and poor stability, and it is no clear about tolerance mechanism of the enzyme in organic solvent. So, the new report important catalysts (RedAm) with broad substrate spectrum was used as the research object, the functional characterization, binding energy detection, dynamics simulation and docking and so on were selected as the technical support for this project, and mechanism resolution was used as the core. Then, the activity and catalytic properties of enzymes in organic solvents were first analyzed. After that, the interaction mechanisms between RedAm and organic solvents were revealed on the molecular level, and key action sites and regions were parsed and excavated. Finally, the functional element which plan an important role in improve the tolerance performance of enzyme was designed, modified and assembled to form new stress resistance RedAm with high activity. The strategies and methodologies arising from this project will provide theoretical basis and technical reference for analyzing the extremophilic mechanism of the RedAm and other enzyme on the molecular level, improving the performance of the RedAm and increasing the yield of key drug intermediates such as chiral amine.

耐有机溶剂的酮胺化还原酶可用于非水相体系催化，在工业催化制备手性胺等高附加值化学品和药物中间体中具有重要的应用前景。本课题针对催化酮还原胺化制备手性胺的关键酶如胺脱氢酶和酮胺化还原酶存在活性低、稳定性差、耐受有机溶剂机制不清楚等问题，以新报道的底物谱较宽的重要催化剂酮胺化还原酶为研究对象，以功能表征、结合能检测、动力学模拟与对接等为技术支撑，以机制解析为核心，分析酮胺化还原酶在有机溶剂中的活性及催化性能，分子层面揭示酮胺化还原酶与有机溶剂的相互作用机制，解析和挖掘关键的作用位点和区域，设计和改造对提高酶的耐受性能具有重要作用的功能元件并组装构建高活性的新型抗逆酮胺化还原酶。本项目研究工作的开展为从分子水平解析酮胺化还原酶和其它酶的嗜极机制、改善酮胺化还原酶的性能及提高手性胺等关键药物中间体的产量等提供了理论基础和技术参考。

酮胺化还原酶是工业催化制备手性胺等高附加值化学品和药物中间体的关键催化剂之一，其研究具有重要的意义。针对手性胺的不对称合成，从米曲霉中挖掘了酮胺化还原酶基因，分析了该酶的酶学性质和其对各种酮和胺的催化能力，结果表明该酶具有较宽的底物谱，且具有催化合成手性胺类药物的应用潜力。通过同源建模、分子对接、理性设计等构建该酶的突变体库，主要包括为了提高酶活、改善立体选择性进行的活性口袋改造，为了降低最适pH、提高有机溶剂耐受性的表面电荷改造等。通过设计突变等提高了该酶的温度稳定性、有机溶剂耐受性等并对其活性变化的机制进行了解析。使用3D打印技术结合超声波处理技术固定化酮胺化还原酶，通过单因素实验结合响应面分析，确定了最适打印条件。固定化的酶具有良好的重复利用性。本项目的研究对于该酶催化合成手性药物雷沙吉兰和普拉克索等具有重要的意义。