Janus结构磁性纳米复合载体材料的可控制备及其双酶特异共固定化研究

Janus structure nanocomposites have asymmetric physicochemical properties at the two sides, and have great potential for the use as carrier material for multi-enzyme co-immobilization and accordingly the application in the field of biocatalysis. However, the studies on Janus structure nanocomposites were mostly focused on the development of material synthesis and assembly at present. In this project, a technical route is developed for Janus structure magnetic nanocomposites based specific co-immobilization of dual enzymes. Janus structure Au-Fe3O4 nanocomposites will be prepared by use of controlled synthesis and then adopted with specific surface modification at the two sides. The Janus Au-Fe3O4 nanocomposites is intended to be introduced to two model dual-enzyme systems for site-specific co-immobilization of glucose oxidase/horseradish peroxidase and lactase/glucose isomerase for the construction of dual-enzyme catalyst. Based on the adjustable feature of the structure and properties in the two sides of Janus structure nanocomposites, influence of morphology, size, modification methods of the nanocomposites and experiment parameters of enzyme immobilization on the dual-enzyme systems will be investigated in term of the dual-enzyme loading amount, conformation, stability and catalytic performance. The mechanism of the interaction between Janus structure nanocomposites and dual-enzyme systems and the improvement of catalytic activity of dual-enzyme systems after co- immobilization will be elucidated after comprehensive analysis. The data gathered here would provide more theoretical evidence and experimental techniques for the nanocomposites based multi-enzyme co-immobilization system and is supposed to have practical significance for the industrial application of co-immobilized multi-enzyme biocatalyst.

Janus结构纳米复合材料由于具有两端物化性质不对称的特性，其作为多酶共固定化载体材料在生物催化领域有潜在的应用优势，不过目前对其研究还主要集中于材料合成和组装技术的开发。本项目拟发展一种基于Janus结构磁性纳米复合载体材料的双酶特异共固定化技术路线。选取Janus结构Au-Fe3O4材料进行可控合成，对其进行两端特异表面修饰，并将其作为模型载体材料引入葡萄糖氧化酶/辣根过氧化物酶及乳糖酶/葡萄糖异构酶两种模型双酶共固定化体系，构筑双酶催化剂；基于Janus结构载体材料结构及性质可调的特点，通过研究载体材料的结构、性质及双酶固定化实验参数对酶分子的负载量、构象、稳定性及催化性能的影响，揭示Janus结构载体材料与双酶的相互作用机制及酶活提高机理。本研究结果将为双酶体系高效共固定化提供更多的理论依据和实用技术，同时也对共固定化多酶生物催化剂的工业应用具有十分重要的现实意义。

Janus结构磁性纳米复合材料Au-Fe3O4具有两端物化性质不对称的特性，同时可兼具纳米Au的等离子体共振效应和纳米Fe3O4的超顺磁效应，因而作为酶固定化载体材料在生物催化领域有潜在的应用优势，不过对其研究目前还主要集中于材料合成和组装技术的开发。单酶固定化体系和多酶共固定化体系虽然相对于游离酶更能满足实际应用的需要，但目前设计合成合适的载体材料以提高酶活、稳定性以及可回收再利用的性质仍是单酶固定化和多酶共固定化领域急需解决的主要问题。本项目选取Janus结构磁性纳米复合材料Au-Fe3O4进行可控合成，并对形貌进行调控，随后对其进行特异表面修饰，并将其作为模型载体材料引入单酶固定化体系和双酶共固定化体系，构筑高效生物催化剂。通过两相界面法对Au-Fe3O4的Au端形貌进行调控，得到了多枝状的Au-Fe3O4，并在975 nm附近出现了很强的等离子共振吸收峰。多枝状Au-Fe3O4在980 nm激光辐照下展现了优越的光致变热性质，光热转换效率达到69.9%。以多枝状Au-Fe3O4为载体材料分别实现对β-半乳糖苷酶、葡萄糖氧化酶的固定化，发现在激光辐照下两种固定化酶的酶活相对于游离酶分别提高到196.6%和142.2%。揭示多枝状Au-Fe3O4结构载体材料的光热转换性质对于酶活提高起到了重要作用。以多枝状Au-Fe3O4为载体材料实现了对葡萄糖氧化酶（GOx）/辣根过氧化物酶（HRP）双酶进行共固定化，通过调控两种酶的加入量、温度和pH值，发现当GOx/HRP的质量比为15:2（质量比为2:1）、温度为40oC、pH为5.0-5.8时，固定化双酶体系的酶活最高，并可实现方便回收再利用。本研究结果将为单酶和双酶体系高效固定化提供更多的理论依据和实用技术，同时也对固定化酶生物催化剂的工业应用具有十分重要的现实意义。