限域双亲性酶固定化材料设计合成与催化机理研究

Enzyme plays a vital role in the green development of the bio-industry, but the high cost of its "one-off" use limits its application to high-end industries. Enzyme immobilization enables efficient recovery and maintains activity in long-term use, reducing application costs. How to solve the contradiction between immobilized enzyme activity and stability is the bottleneck of the design and synthesis of immobilized carriers. This is because the dynamic changes of the enzyme conformation make its activity and stability highly susceptible to the carrier micro-environment and the reaction environment. Currently, however, the lack of in-depth study on the influence of the multi-environment on the catalytic performance of the enzyme limits the orientation design of the carrier. In this project, by taking advantage of the hydrophobic nature of the resin surface and the rich substitution position of the benzene ring, amphiphilic (hydrophilic/lipophilic) enzyme carriers with restricted domain structure (only for enzyme immobilization) are prepared by the innovative use of silica hard template-mediated multi-modifications of styrene resin. Through the immobilization of B. cepacia lipase, the conformational change mechanism of enzyme molecule during the immobilization process in immobilization multi-environment will be explored. Based on catalytic hydrolysis, esterification, and chiral enantiomer resolution reaction, the stabilization mechanism of the active center of immobilized enzyme molecules in the multi-environment of enzymatic reaction will also be investigated. The accomplish of this project will provide a basis for the rational design of stable and efficient immobilized enzyme.

酶在生物工业绿色化发展中具有重要作用，但其“一次性”使用带来的昂贵成本将其应用限于高端行业，酶固定化能够实现高效回收并在长期使用中保持活性，降低应用成本。如何解决固定化酶活性和稳定性之间的矛盾是固定化载体设计合成的瓶颈，这是因为酶构象的动态变化，使得其活性和稳定性极易受到多相环境即载体环境和反应环境的联合影响，但目前关于该多相环境对酶催化性能的影响规律缺乏深入研究，难以给固定化载体的定向设计提供支撑。本项目拟以苯乙烯树脂微球为基础，利用树脂表面疏水性质和苯环丰富取代位，创新采用二氧化硅硬模板介导的多重改性方式合成具有限域结构（限制性酶固定化区域）的双亲性（亲水/亲油）载体，通过对BCL脂肪酶的固定化，探究多相固定化环境中酶分子固载过程构象变化机制，同时基于催化水解、酯化和手性对映体拆分反应，解析多相酶促反应环境中固载酶分子活性中心稳定机制，为稳定高效固定化酶的理性设计提供依据。

酶在生物工业绿色化发展中具有重要作用，但其“一次性”使用带来的昂贵成本将其应用限于高端行业，酶固定化能够实现高效回收并在长期使用中保持活性。本项目创新采用二氧化硅硬模板介导的多重改性方式成功合成了具有限域结构（限制性酶固定化区域）的双亲性（亲水/亲油）酶固定化载体，并通过对BCL脂肪酶的固定化，明晰了多相固定化环境中酶分子固载过程构象变化机制，还基于脂肪酶分子N-糖基化的半理性设计、分子模拟和基因工程菌的筛选构建，获得了产高活性耐甲醇脂肪酶的基因工程菌株，为固定化酶材料的合成和高性能脂肪酶的筛选提供了新的思路。