可设计生物膜材料固定化酶及在微流控体系中催化性能强化的研究

Gene engineering and protein engineering technologies develop fast recently, and they represent both opportunities and challenges for biocatalysis. Because of its high product efficiency, safety and extensibility, microfluidic technology provides a new idea for biocatalysis. Immobilized enzyme has been used in microreactor. However, immobilization faces several challenges that have not yet been fully addressed. The immobilization method can’t provide a high protein loading on the supports. Moreover, the immobilization method is not versatile in a sense that it can be readily adapted to different enzymes. There is a great need to develop new immobilization methods to solve the above problems. This topic is aiming to explore a new immobilization method—Biofilm Integrated Nanofiber Display which exploits the Curli system of E.coli to create a functional nanofiber network. We site-specifically immobilize a recombinant enzyme, fused to the attachment domain, onto the E.coli Curli fibers displaying complementary capture domains. The immobilization efficiency is improved by tuning the length and structure of Curli fibers. The recombinant enzymes are reprogrammed for enzyme immobilization by using genetic fusions of enzymes to attachment domain. Using enzymes from glycoside hydrolase family 13 as an example, we study the properties of the immobilized enzyme onto Biofilm Integrated Nanofiber Display. The mechanism of the immobilized enzyme in microreactor is discussed by their biocatalytic properties in microreactor. The optimizations of Curli fiber component and the immobilization conditions are studied based on the properties of immobilized enzyme in microreactor. We want to develop a versatile immobilization method and provide a new thought for immobilized enzyme in microreacter.

随着基因工程与蛋白质工程的发展，酶催化技术的应用面临新的挑战与机遇。微流控技术以高效、安全、可扩展的特性为酶催化技术提供了新的研究思路，但固定化酶在微流控中的研究仍然存在固定化效率低、固定化方法不通用等不足。本项目拟利用Curli纤维的自组装形成生物膜-自组装纳米纤维展示系统，通过载体蛋白将酶定向锚定在展示系统上。利用纳米纤维的长度及空间结构的可控性提高固定化酶的负载量，设计不同酶蛋白与载体蛋白的融合基因实现不同酶的固定化，获得一种高效、可设计的模块式固定化方法。以glycoside hydrolase 13家族的酶为示例，研究固定化酶在微反应器中的催化活性及稳定性等催化性能，阐释生物膜-自组装纳米纤维定向锚定的固定化酶在微流控体系中的反应机理，优化生物膜材料的结构设计及定向锚定的最佳条件，为固定化酶在微流控技术中的应用提供新的技术思路和参考。

为解决固定化酶在微流控应用中存在的固定化效率低、固定化方法不通用等问题，本项目通过基因工程技术构建了表达融合了SpyTag的curli纤维的重组菌株，并成功制备了生物膜-自组装纳米纤维展示系统。同时制备了融合SpyCatcher的海藻糖合成酶、β-淀粉酶等融合酶，利用SpyCatcher与SpyTag之间的相互作用将酶定向锚定至生物膜-自组装纳米纤维展示系统以制备固定化酶，建立了固定化酶在微反应器中的催化体系，研究固定化酶在微反应器中的催化性能，发展了一种高效、可设计的模块式的固定化方法以应用于微流控技术。结果表明：1.利用生物膜-自组装纳米纤维展示系统固定化海藻糖合成酶，固定化酶的酶活回收率较高，稳定性提高；2.利用生物膜-自组装纳米纤维展示系统组装构建一种胞内含海藻糖合酶而胞外curli纤维展示β-淀粉酶的双酶系统，其催化效果上与不固定的游离BA和TreS全细胞的共同作用的催化效率差异不显著，但双酶系统的温度、pH、有机溶剂稳定性更好，且实现了双酶系统的重复使用。3.建立了固定化海藻糖合成酶酶催化麦芽糖制备海藻糖的微反应器催化体系，并研究了反应条件对反应体系的影响，结果表明催化反应速率比传统反应器中加快了2.33倍。