界面原位亲疏水可调的脂肪酶二维纳米反应器的构建及催化机理研究

Molecular modification is an important way to expand the function of lipids and immobilized lipase plays a key role in the industrialization of functional lipids. There are still several drawbacks in lipase immobilization, for instances, irrationality of carriers design, damage and loss of enzyme, insufficient active site exposure, poor enzyme activity and stability, and unknown catalytic mechanism. Based on the “interface activation” effect of lipase, we plan to prepare a series of lipase reactors by the advantages of the features of two-dimensional nanomaterials such as graphene/graphene oxide, graphite phase carbon nitride, etc. Their components of interfaces can be in situ modified by ultraviolet light. We can design some nano-biointerfaces with different composition by adjusting the hydrophobicity of the substrate material and light responsive layer. Systematical studies will be carried out to clarify the relationship between interface engineering and the state of “lid”, establishes the database of interface composition, immobilization method, enzyme activity, “lid” state, and finds the “the optimum interface” to maintain “lid open”. Through the application of reactors in the synthesis of structured lipids, according to the reaction mechanism of free lipase, revealing the key steps that affect enzymatic reaction, and using a variety of on-line monitoring methods to infer the possible reaction mechanism of the reactors in the structured lipids synthesis process. At last, we will provide a theoretical support and new technical approach for the design, synthesis, application and industrialization of novel structured lipids.

脂质修饰是拓展油脂功能的重要途径，固定化脂肪酶是功能脂质产业化技术中关键催化剂。目前固定化脂肪酶仍存在载体设计非理性化、酶易受损和流失、活性中心暴露不充分、酶活及稳定性较低、反应机制不明确等问题。本项目根据脂肪酶“界面活化”效应，利用石墨烯/氧化石墨烯、石墨相氮化碳等二维纳米材料在界面研究上的优势，制备光原位调节载体材料/脂肪酶界面亲疏水性的脂肪酶反应器，通过基底材料与光感层的亲疏水性调节，构建不同组成的纳米生物界面，系统研究界面工程与脂肪酶“盖子”结构开闭的关系，建立界面组成、固定方法、酶活、“盖子”状态四者之间的数据集，找出维持“盖子打开”的“适宜界面”，通过反应器在结构脂质合成中的实际应用，揭示影响酶催化反应的关键步骤，结合游离脂肪酶的反应机理，利用多种在线监测手段，推测反应器参与结构脂质合成可能的反应机制，为新型结构脂质的设计、合成、应用和产业化提供坚实的理论支撑与新的技术途径。

脂质修饰是拓展油脂功能，发掘新型功能脂质的重要途径，而固定化脂肪酶是功能脂质产业化技术中关键催化剂。目前固定化脂肪酶仍存在载体设计非理性化、酶易受损和流失、活性中心暴露不充分、酶活及稳定性较低、反应机制不明确、酶活测试方法复杂等问题。本项目从酶与纳米载体材料之间的界面出发，从界面设计、修饰的角度，基于一系列具有界面活性的纳米材料设计制备了多个脂肪酶界面反应器，包括：二维Janus纳米片脂肪酶两相界面反应器、具有持续磁分离回收性能的碳纳米管脂肪酶反应器、石墨相中大孔碳脂肪酶反应器、中空方形碳脂肪酶反应器等，系统研究材料微观结构、形貌、界面组分等参数对脂肪酶反应器的反应活性、稳定性、环境耐受性以及循环使用性能的影响，并成功应用于植物甾醇酯、叶黄素酯等新型功能脂质的高效制备，为脂肪酶反应器的理性设计及新型功能脂质的绿色高效制备提供研究思路和参考；设计合成了一系列小分子荧光探针，利用这些探针分子来构建具有高灵敏度、快速的脂肪酶、淀粉酶活性测试体系，系统研究了体系的灵敏度、测试时间及其在实际样品中的应用，并且实现了多个酶活性的同时分析，对新型生物催化剂的创制，新型结构脂质的合成具有重要意义。在项目的资助下，以项目负责人为第一作者或通讯作者发表论文12篇，申请国家发明专利3件，1件已获授权，获得省部级奖励1项。