微环境可控的脂肪酶介孔微阵列构筑及其定向酯化机制研究

酯化修饰是拓展油脂应用，提升其功能和效益的重要途径。脂肪酶固定化是实现定向酯化的关键技术，但仍缺乏载体理性设计，存在酶易受损和流失、活性中心易被掩盖、活性和稳定性差等瓶颈难题。本项目拟在多年研究基础上，以安全稳定、孔径适中可调和表面易改性的介孔氧化硅为载体，运用多重吸附等温和方法将脂肪酶引入其孔穴，构筑脂肪酶介孔微阵列。通过对介孔尺寸、形状、内部亲/疏水性、电荷、拥挤程度等微环境精确调控，诱导脂肪酶的“界面激活”和“分子匹配”效应，使微阵列展现出高活力与稳定性。针对该微阵列对脂肪酶构象和综合性能影响不明且定向酯化机制欠缺的现状，拟在构建出微环境可控脂肪酶介孔微阵列的基础上，以底物专一性和区域选择性酯化为模型，阐明介孔微环境对酯化效率、稳定性和选择性的调控规律；采用分子对接模拟结合脂肪酶构象及底物结构解析，揭示脂肪酶微阵列的定向酯化作用机制与关键控制点，为其在脂质修饰领域的应用提供理论依据。

Esterification modification is an important solution to expand the application of oil, and improve its function and efficiency. Lipase immobilization is the key technology to realize targeted esterification. However, the reliability designs of carriers still quite lack. There are several bottleneck problems, for instance, easy damage and loss of enzyme, easily be covered of enzyme active center, poor stability and activity of enzyme. Base on the recent research, this project is proposed to choose the mesoporous silica as the carrier because of its safety and stability, moderate and adjustable pore size, and easy modified surface. The lipase is introduced into the mesoporous by mild methods (e.g. multiple adsorptions) for constructing the lipase mesoporous microarray. By precise controlling the microenvironment (e.g. mesoporous size, morphology, hydrophilic/hydrophobic, charge, crowding), the "Activate Interface Effect" and "Molecular Match Effect" of lipase were induced, which lead lipase microarray to exhibit high activity and stability. The effect of microarray on lipase conformation and comprehensive performance is unclear and the targeted esterification mechanism is deficient. On the basis of building lipase mesoporous microarray with microenvironment controllable, the substrate specificity and regional selective esterification were selected as the model reactions, which are used to clarify the effect of mesoporous microenvironment on the esterification efficiency, stability and selectivity. By using the molecular docking combined with lipase conformation and the structure analysis of substrates, this project will reveal the targeted esterification mechanism of lipase microarray and the key control points of the reaction. This project will further provide the theoretical foundation for the application of lipase mesoporous microarray in the field of lipid modification.