尺寸与空间取向可控智能纳米酶的设计、性能及组装机制

The xylanase, which has simple structure but great potentials in catalyzing the hydrolysis of hemicellulose, will be chosen as the target enzyme. The overall objective of the project is to control the size of the nano-carriers and the orientation of the immobilized enzyme accurately and gently. We will rationally design the intelligent peptides based on the molecular simulation, and then construct the gene elements of the building blocks and splice them. After being transformed into the host strain and expressed, the intelligent peptide can covalently couple to the N-terminal (or C-terminal) of the target enzyme via the linker during the translation process. By regulating the environmental conditions, the intelligent peptides can self-assemble into nano-particles with uniform and proper size, and thus the target enzyme will be orientated during immobilization. We will use the machine learning algorithms to study the quantitative structure-property relationship (QSPR) in order to develop the regulation and control strategies for the global optimum in the process of immobilization, purification and catalysis. Combining the theoretical computation and experimental verification, we will seek the molecular mechanism of the peptide self-assemble and the influence law of the nano-carrier on the activity and stability of the xylanase. The proposed work will facilitate rational design of the active nano-carrier platforms, which has the potential to scientifically impact on the multibillion-dollar catalytic enzyme industry. With its successful development of new thinking and technology would lead to the transformation of the biocatalysis industry by providing a rapid, reliable, and covalent optimization method for oriented immobilization. Intellectual merit: The structure and thermodynamic interactions of the nano-carriers with the xylanase will be investigated using experimental nanoscale tools and computational methods to obtain insights into the mechanisms of how the intelligent peptides response to the environment. These insights will lead to design the optimal nano-carriers specific to the properties of the target enzyme. The project will lay the theoretical foundations of developing the intelligent and integrated nanobiocatalyst and expanding its application fields.

以应用广泛、底物空间位阻较大的木聚糖酶为对象，以温和手段精确控制纳米载体尺寸及酶空间取向为目标。基于分子模拟技术设计智能多肽，构建标准化基因元件并拼接，通过宿主菌基因表达，将智能多肽经连接臂与酶N-或C-末端共价结合；调控环境条件，使智能多肽自组装成粒径均一、尺寸合适的纳米颗粒，实现酶定向固定；籍助于机器学习算法研究纳米酶尺寸和性能与环境条件阈值的定量关系，研发实现固定化、纯化及催化过程工艺参数全局最优的调控策略；结合理论计算和实验验证，探寻智能多肽自组装的分子机制及纳米载体对酶活性和稳定性的影响规律。 本项目将开辟一条可控制备新型智能纳米载体材料的新途径，为酶在纳米载体上的定向固定提供新思路。其研究成果有助于揭示微观结构与表观性能之间的本质关系，为从分子水平设计最佳的纳米结构及其与酶定点组装奠定基础；将促进纳米酶的智能化和集成化，可望对纳米酶技术从实验室研究迈向工业应用产生推动作用。

本项目针对纳米酶在游离酶制备、纳米载体开发及酶与载体共价结合方法的研究和应用中亟待解决的问题，提出了尺寸和空间取向可控的智能纳米酶新思路。经过4年的研究，本项目完成了可控尺寸纳米酶的分子设计、性能及影响因素考察，结果发现在稳定性和循环使用次数上较游离酶有显著提升。系统探讨了不同拓扑结构ELPs相变特性和自组装条件对其粒径的影响，结果发现目标酶表面电荷状况及诱发ELPs发生自组装的盐类型是形成纳米酶的关键内外因素。构建了间歇酶反应器，考察了纳米酶催化特性并对该过程进行了优化，结果表明：酶在反应器中的水解反应不存在底物抑制现象，而存在产物抑制现象，产物抑制常数Kp=0.09 g∙L-1。通过超强分子粘合剂SpyTag/SpyCatcher环化目标蛋白及用于双酶的分离纯化和固定化的集成，结果表明：环化地衣多糖酶在100℃煮沸后仍保有80%的酶活力，而线性地衣多糖酶煮沸后基本失活；利用ELPs和超强分子粘合剂可以纯化双酶，其纯度可达95%，但自组装所形成的纳米颗粒重复使用性有待提高。此外，通过foldon和ELPs构建了寡聚化及线性地衣多糖酶/木聚糖酶，结果发现在在 ITC 纯化过程中，酶活回收率平均提高了9.3%，纯化倍数平均提高了2.2倍，更重要的是寡聚态酶催化效率（Kcat/Km）较单体酶提高了4-5倍，有望成为一种新型的酶分子改造以提其催化效率的方法。最后，本项目首次报道了连接有Spycather的ELPs40在碳酸钠和硫酸钠溶液中存在的不符合Hofmeister离子序的现象并解释了其机理。研究成果已申请发明专利2项，在Biotechnology for Biofuels、中国科学-化学 等期刊发表论文12篇，培养硕士研究生9名。.总之，本项目开辟了一条可控制备新型智能纳米载体材料的新途径，为酶在纳米载体上的定向固定提供了新思路，具有较重要的理论和应用价值。