多酶共固定化催化微环境的非均一性调控机制及设计

For the production of more complex products, multi-enzyme catalysis attracts more and more attention and leads the upsurge of biocatalysis. The core idea of it is the construction of multi-enzyme catalytic system in vitro. The construction involves two aspects of issues. On the one hand, the optimum microenvironment of multi-enzyme is quite different, so the reaction rate of different enzymes is difficult to control and match. On the other hand, the reaction process is complex that result in the transfer and reaction rate of substrate and intermediate product are difficult to match. In order to solving these problems, co-immobilization of multi-enzyme is developed as an effective method. It is a frontier research in the field of enzyme engineering, whose core technology is the selection of carriers. The structure and properties of the carriers largely create a suitable microenvironment for multi-enzyme system and regulate the relative positions of multi- enzyme. Baesd on this point, the realization of substrate channel and the maintenance of enzyme activity can be obtained. .In this project, mesoporous titanium oxide with adjustable surface structure was introduced as a carrier for multi-enzyme co-immobilization. The effect of microenvironment and confined environment that construct by pore structure on the catalytic performance and intermediates transfer properties of glucose oxidase and catalase were investigated. Futhermore, rotating ring disk electrode analysis of the redox reaction electron transfer method was applied to confirmed whether the reaction of enzymes or the transfer of intermediates is the control step affecting the construction of the catalytic system. This method lays the foundation for the application of electroanalytical chemistry in multi-enzyme catalytic system, for example, to provide theoretical support for the construction of multi-enzyme catalytic system and to offer the possibility of conducting biosensing, biocatalysis and biomedical applications.

多酶催化正引领生物催化研究热潮，但普遍存在反应效率低、速率低和成本高的“两低一高”难题。究其本质原因是多酶催化体系涉及到多种酶、多种中间产物的反应和传递协同难题：多酶需要的最佳pH和温度微环境差异较大；底物与中间产物的传递速率与反应速率的匹配难度大。解决这些难题的有效途径是通过共固定化调控多酶催化的微环境，并通过底物通道调控反应速率与物质、能量传递速率。本项目创新的引入表面结构性质可调的介孔氧化钛作为多酶共固定化介质，考察介孔氧化钛表面非均一与受限微环境的构筑对于葡萄糖氧化酶和过氧化氢酶催化性能的影响。将旋转环盘电极分析氧化还原反应过程中电子转移的方法应用于多酶催化体系，考察反应与传递孰是控制步骤，并实现调控。阐明酶与介质表面非均一性调控微环境的机制，为多酶催化体系的构建和调控提供理论支撑，也为电分析化学在多酶催化体系中的应用奠定基础，并开发在生物传感、生物催化和生物医药等领域的应用技术。

多酶反应在科学和工业应用方面经历了快速增长，特别是生物转化、生物传感器和生物医学工程，正引领生物催化研究热潮。此外，多酶过程被认为是生产许多药品、生物燃料和精细化学品的替代途径。多酶级联反应可在一个锅内发生至少两个连续的过程，表现出显著的优势，不需要分离或纯化中间产物，从而减少副产品的产生和加工成本。但普遍存在反应效率低、速率低和成本高的“两低一高”难题。究其本质原因是多酶催化体系涉及到多种酶、多种中间产物的反应和传递协同难题。本项目创新的引入表面结构性质可调的介孔氧化钛、氧化石墨烯、金属有机框架作为共固定化介质，考察介质表面非均一性与受限微环境的构筑对于多酶催化性能的影响。.我们取得了以下几点突破：.1）合成了具有可控孔径的异质性二氧化钛表面，使用650℃煅烧并经乙烯三乙氧基硅烷（ETS）改性的TiO2作为脂肪酶固定化载体的活性最高，达到428.04%，并表现出良好的稳定性；.2）生物催化系统中使用葡萄糖氧化酶（GOD）和过氧化氢酶（CAT）的级联反应实现高效的一步法生产葡萄糖酸，理性设计底物通道与介质孔径，提升了多酶催化效率及稳定性，在反复利用生产葡萄糖酸钠和葡萄糖酸的过程中保持了90%以上的活性。.3）构建了以功能化二氧化钛，氧化石墨烯及金属有机框架UIO-66等介质作为固定化载体的尿苷-胞苷激酶（UCK）和乙酸激酶（ACK）的共固定化双酶级联催化体系，基于电分析化学的定量检测手段，调控反应底物、辅因子和中间产物在酶表面的集聚状态，从而达到反应效率的最优化，实现胞苷酸（CMP）低成本、高效率生产。在亲和介质的基础上进一步设计聚赖氨酸（EPL）修饰可使共固定化酶的酶活回收率提高至101.6%，固定率接近100%，在重复使用 10 次之后残留活性依然高于 70.4%。