基于酶的纯化、固定化和快速分离的多功能磁性三维多孔复合材料的设计与应用研究

Enzyme immobilization techniques make the enzyme can be reused and effectively improve its structure stability, but it also suffers from the problems of lower activity, poor cycling stability and difficult in purification etc. Therefore, this project will build a magnetic three-dimensional porous Ni@NiO/PMG/DTA-M/MAO5 composite material based on the multi-function of enzyme purification, immobilization and separation, in which, the DTA-M2+ component can realize the enzyme purification and immobilization with on-step; the magnetic Ni@NiO component can separate the enzyme fastly; the PMG/DTA component can as a flexible support of the enzyme to enhance its catalysis reactivity; the three-dimensional porous structure can enhance effectively the laoding amount and structure stability of the immobilized enzyme. The relationship among the immobilizing conditions, the structure of the Ni@NiO/PMG/DTA-M2+ composite material and the catalytic performance of the MAO5 will be investigated systematically. Moreover, the struacture transformation of the MAO5 and its mechanism during cycling will be further studied. More important, this study tries to develop a new technology and approach of enzyme immobilization, which can effectively resolve the key fundamental problems of the lower activity, poor cycling stability and lower reactant substrate concentration of the MAO5 and provide an effectively approach and technique of the preparation of chiral amine compounds by biocatalysis with MAO5.

酶固定化技术虽然可以实现酶的循环使用和提高酶的结构稳定性，但固定化酶仍然面临活力低、循环稳定性差和纯化难等关键科学问题。本项目拟构建一种集酶的纯化、固定化和快速分离多功能于一体的磁性三维多孔Ni@NiO/PMG/DTA-M2+复合材料。其中，DTA-M2+实现对酶一步纯化和固定化；磁性Ni@NiO实现固定化酶的快速分离；PMG/DTA作为酶连接的柔性支架提高酶催化反应活性；多孔结构提高酶的固载量和结构稳定性。将这种具有特殊结构和功能复合材料的用于一步纯化和固定化单胺氧化酶，有效解决其活力低、循环稳定性差和反应底物浓度低等关键科学问题，实现对喹啉类药物的高效手性拆分。重点研究Ni@NiO/PMG/DTA-M2+/单胺氧化酶与其催化性能之间的构效关系；探索固定化条件对其催化性能的影响及其规律；研究固定化酶循环过程中结构变化与其活力的关系，揭示其关键变构因素及机理。

生物催化技术在手性药物合成中具有广泛的应用，然而生物催化酶存在稳定性差、活性低等关键科学问题，酶固定化技术虽然可以实现酶的循环使用和提高酶的结构稳定性。本项目基于基因克隆技术制备了单胺氧化酶和P450等氧化酶，系统地研究了氧化酶热稳定性和催化活性影响的关键因素，同时制备各种磁性三维多孔复合材料及生物质多孔复合材料用于氧化酶的纯化和固定化。研究结果发现，基因克隆表达获得的酶稳定性及活性非常差，但是通过稳定剂修饰、辅因子改性和固定化后，其热稳定性、催化活性和循环使用性得到提升。固定化酶的温度耐受性、有机溶剂耐受性和金属离子耐受性也得到改善。此外，在本项目研究过程中，我们也有很多的新发现：首先我们发现了NADH辅酶因子在氧化酶生物催化中的关键作用，同时构建了基于CdS光催化NADH再生系统，有效地推动NADH的循环再生；其次，我们还发现制备的三维多孔复合材料可以极为温和条件下催化喹啉类化合物的氧化脱氢。本项目的研究极大地推动了氧化酶在生物催化氧化合成手性胺和手性喹啉类药物的发展与应用。