Structural behaviour of adsorbed/immobilised enzymes on planar interfaces

Characterizing the structural deformation of enzymes upon immobilization is of paramount importance as it directly impacts the efficiency and effectiveness of heterogeneous biocatalysts. Gaining a deeper understanding of these structural changes will guide the immobilization protocols to obtain more robust and active immobilized enzymes with a wide range of application in biotechnology, pharmaceutical, cosmetic and the food industry. Here we will investigate the structural rearrangements undergone by a model lipase from Candida Antarctica (CALB) immobilized on silicon functionalized with different functional groups (hydrophobic and hydrophilic ones). These structural rearrangements at the interface between the solutions of enzymes and the solid surface are hard to characterize by conventional techniques (circular dichroism, fluorescence spectroscopy, etc), however neutron reflectometry (NR) can give us unprecedent information with spatiotemporal information about the molecular process that occur when an enzyme is bound to a solid surface, and how these bound enzymes behaves under temperature stress. We expect that NR will help us to create a bridge between structure and function of immobilized enzymes to better understand the computational and kinetic results. We select CALB because this is one of the most widely used materials for industrial applications, fundamental to design and fabricate more active and robust heterogeneous biocatalysts for industrial exploitation.

表征固定化过程中酶的结构形变，对非均相生物催化剂（heterogeneous biocatalysts）的催化效率与综合性能具有举足轻重的意义，因为此类结构变化直接影响非均相生物催化剂的整体效能。深入解析此类结构变化，可指导固定化工艺规程的优化，从而获得稳定性更强、催化活性更高的固定化酶，使其在生物技术、制药、化妆品及食品工业中拥有广阔的应用前景。
本研究将针对经不同官能团（疏水与亲水官能团）功能化的硅基载体所固定的模式脂肪酶——南极假丝酵母脂肪酶B（Candida Antarctica Lipase B, CALB）所经历的结构重排展开探究。
酶溶液与固体表面界面处的此类结构重排，难以通过常规表征技术（如圆二色谱（circular dichroism）、荧光光谱法（fluorescence spectroscopy）等）进行解析；而中子反射法（Neutron Reflectometry, NR）则可提供前所未有的时空尺度信息，揭示酶结合至固体表面时发生的分子过程，以及结合态酶在温度胁迫下的行为特性。
我们期望中子反射法能够搭建起固定化酶结构与功能之间的关联桥梁，从而助力我们更好地理解相关计算与动力学研究结果。
我们选择CALB作为研究对象，是因为其是工业应用中最为广泛使用的酶制剂之一，对于设计与制备活性更高、稳定性更强的非均相生物催化剂以实现工业化应用具有核心意义。