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

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.

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。

酯化修饰是实现油脂高值化与多元化利用，提高油料加工综合效益的重要途径。高活性的固定化脂肪酶是实现定向酯化的关键，针对当前酶活性不高、使用中易受损、流失，传质效率低等产业瓶颈难题。本项目采用模板法、相分离法等制备具有不同比表面积、孔径大小、孔结构的二氧化硅、碳球和纤维素整体型的介孔微阵列载体材料，探明了不同模板剂和制备条件对微阵列比表面积、孔径大小、形状等结构的影响；筛选出适合酯化修饰且微环境可控的脂肪酶介孔微阵列8种。基于纤维素基整体柱，制备出连续流动酶反应器。与传统的间歇搅拌反应模式相比，催化效率增加了6.1倍。持续不间断反应200小时未发现催化活性下降，创造了酶法制备甾醇酯（258 g/g整体柱）的最高产量记录。评价了微阵列的介孔大小、结构和分布及拥挤程度等空间微环境和孔道亲/疏水性、电荷分布等化学微环境变化对脂肪酶微阵列酯化活性、稳定性和选择性的影响；揭示了水相、非水和乳液体系及介孔微环境对脂肪酶“界面激活”和“诱导匹配”效应的影响。选择水相、非水相和乳液等不同的酶促反应体系，采用圆二色光谱对酯化反应前后的脂肪酶构象变化进行表征，结合模型反应转化率和选择性的变化，探明了脂肪酶微阵列在超声和乳液双重激活条件下的活力提升机制：脂肪酶在油水界面上可以发生二级构象的变化，α螺旋减少，β折叠增加，暴露出更多的活性位点，发挥显著的界面激活效应，使脂肪酶处于高活性状态。基于脂肪酶微阵列稳定的皮克林乳液酶反应体系，创建了绿色、高效无溶剂的“乳液酶反应工厂”，效率是两相反应体系的20倍。解决了效率低和有机溶剂污染等油脂酶法修饰的共性难题，为食品级功能脂质的酶法制备提供了新思路。该反应体系创造了反应速度和单位催化剂产量等多项纪录，还具有无溶剂、超高效、酶可回收和多次重复使用，易于规模化放大等优点，可以广泛应用于不同结构和功能的重构脂质绿色制备，为脂质的多元化、高值化利用提供了新途径。