载体微环境调控强化AchE酶稳定性的构象匹配机制

The stability of extracellular enzyme is always a bottleneck in the industrialization and marketization of enzyme. Through lots of research and practice，people have realized that the microenvironment with the extracellular enzyme directly affect the conformation and catalytic processes of the enzyme. Among other technologies, constructing the optimal microenvironment of enzyme carrier has been considered to be the most feasible. However, little work has considered the mechanism and the interaction relationship between the enzyme and the surface properties of carrier microenvironment. In this proposal, the conformation matching mechanism will be studied by using Quantum chemical calculations and molecular simulation as well as experimental test with enzyme biosensor system. The correlation among the regulation of carrier microenvironment, the stability of enzyme and the conformation evolution of AChE enzyme during its immobilization process will be discussed at the molecular level in order to clarify the influence of the AChE conformation from the regulation of carrier microenvironment in immobilization process, to understand the relationship between the carrier microenvironment and the AChE enzymeconformation, and to establish the optimal matching mechanism which can effectively improve the stability of AChE. Finally, the regulation principles and methods of carrier microenvironment based on conformational matching mechanism are proposed. This study is important to better understand the conformational change, reactivity and mechanism of enzyme during the immobilization process. Hopefully it will also provide both methods and theories for further rational design to improve the stability of extracellular enzymes.

胞外酶的稳定性始终是酶的工业化与市场化的瓶颈问题。经过大量研究和实践，人们认识到胞外酶存在的微环境直接影响酶的构象及催化过程，其中构建固定化酶载体的最适微环境被认为是最可行的稳定化技术。但目前尚未考虑酶与载体微环境表面性质的相互作用关系与反应机制。为此，本项目将AChE酶稳定性及其固定化过程中AChE酶构象演变与载体微环境调控进行优化匹配。采用量子化学计算与分子模拟相结合方法，通过载体微环境调控，构建AChE酶生物传感体系，将酶的微观结构参数与固定化酶的宏观催化效能相关联，明确固定化过程中载体微环境调控对AChE酶构象的影响，建立载体微环境及其调控与有效提高AChE酶稳定性的优势酶构象的最优匹配机制，提出基于构象匹配机制的载体微环境调控原理和方法。为更好地理解酶在固定化过程中的构象变化、反应活性、作用机制提供理论依据，也为进一步合理设计改善胞外酶的稳定性提供科学依据和技术原理。

针对胞外酶稳定性差的问题，本项目以乙酰胆碱酯酶（AChE）稳定性及其固定化过程中AChE 酶构象演变与载体微环境调控进行优化匹配。采用计算化学与分子模拟等手段，通过载体微环境调控，构建AChE 酶固定化体系，将酶的微观结构参数与固定化酶的宏观催化效能相关联，明确固定化过程中载体微环境调控对AChE 酶构象的影响，建立载体微环境及其调控与有效提高AChE 酶稳定性的优势酶构象的最优匹配机制，提出基于构象匹配机制的载体微环境调控原理和方法。.研究结果表明：.通过综合性分析AChE酶蛋白表面性质信息及结构特点（疏水性、电性、氢键受体/供体、凹陷结构），设计确定基于PAMAM树枝状分子和功能性固定化小分子配基的两类具有不同载体微环境的AChE固定化方法。.选用氨基修饰磁性纳米颗粒为固定化酶的基础载体，构建基于PAMAM树枝状分子（PAMAM-NH2和PAMAM-OH）和功能性固定化小分子配基（NAR、CAT、GLL）的固定化AChE体系，同时采用多种手段加以调控表征。.固定化酶活性实验及多种稳定性（pH稳定性、温度稳定性、重复使用稳定性）实验结果表明，基于PAMAM树枝状分子和功能性固定化小分子配基的固定化AChE活性及稳定性均远优于游离态酶和传统固定化酶。.荧光光谱实验证明PAMAM树枝状分子和功能性固定化小分子配基均与AChE酶表面具有相互作用，且相互作用强度存在明显差异。其结合强为为PAMAM-OH > PAMAM-NH2和NAR > HES > QUE >> EPG, TAX > CAT, EPI, FLU >GLL>>ATR, HYO。.通过使用分子动力学模拟方法对多个固定化AChE体系进行研究，获得基于PAMAM树枝状分子和功能性固定化小分子配基的固定化AChE体系的载体微环境匹配性机制。PAMAM分子与酶蛋白表面多个区域可发生随机性的结构匹配，稳定分子构象。功能性固定化小分子配基只与AChE酶蛋白上酶活性区域特定位点发生特异性结合，以达到稳定酶分子结构的目的。