基于纤维素内切酶活性中心的印迹模拟酶构建及降解纤维素机制研究

Endoglucanase is a key enzyme of cellulose degradation, but it is difficult to extract and purify. Considering natural enzyme’s high production cost, poor tolerance, low recycling and reusing rate, it is urgent to develop mimic enzymes with biomimetic catalytic activity. Aiming to solve the problems of natural enzyme, this project plans to develop the bio-derived crosslinking monomers based on the endoglucanase active center. By using molecular simulation, spectroscopic characterization, high-throughput screening methods and etc., the highly specific imprinting of the transition state analogues of the endoglucanase will be gained, from which the catalytic molecularly imprinted nanospheres can be prepared for efficient degradation of cellulose. The structure-activity relationship of the mimetic enzyme will be analyzed after characterizing its recognition sites, catalytic performance and substrate structure and etc. With the various influencing factors of mimetic enzyme considered, it will be further compared with the active domain of the natural endoglucanase to explore the catalytic mechanism of cellulose degradation and the law of bionic design of high-performance imprinted catalyst. In this study, the active center of the natural enzyme is simulated by molecular imprinting technology, which is an innovative way of using the chemical method to deduce and verify the biological principle. The designed crosslinking monomers improve the imprinting specificity and simplify the preparation process. The implementation of this project will provide theoretical basis for construction of highly specific imprinted catalyst and technical support for rational utilization of cellulosic biomass.

纤维素内切酶是纤维素降解中的关键酶之一，但天然酶提纯困难、生产成本高、耐受性差且难以回收与重复利用，亟需研发具有仿生催化活性的模拟酶。本项目针对天然酶存在的问题，拟制备基于纤维素内切酶活性中心的生物源性交联功能单体，采用分子模拟、光谱学验证和高通量筛选等方法，进行纤维素内切酶过渡态类似物的高特异性印迹，以获得印迹模拟酶的纳米微球，实现纤维素的高效降解。通过对模拟酶的识别位点、催化活性和底物结构等方面进行表征，分析其构效关系，并与天然纤维素内切酶的活性结构域比对，结合影响模拟酶性能的各种因素，揭示模拟酶催化纤维素降解的机制，探索高性能印迹模拟酶的仿生设计规律。本研究采用分子印迹技术模拟天然酶的活性中心，应用化学方法推演和验证生物学原理。所设计的交联功能单体提高了印迹聚合物的特异性，简化了制备工艺。项目的开展将为高特异性印迹模拟酶的构建提供理论依据，为纤维素生物质的合理利用提供技术支撑。

纤维素是自然界储量最丰富的可再生生物质资源，充分利用天然纤维素，将其高效和洁净地转化为人类所需能源，是解决当前能源短缺与环境污染的可行途径。酶解是纤维素有效利用的关键环节，但天然酶提纯困难、生产成本高、耐受性差，难以回收和重复利用。人工模拟酶具有稳定性好、催化和再生效率高、便于制备和纯化等优势。依据天然酶活性位点和识别方式，结合分子印迹技术，仿生设计和创制分子印迹模拟酶已成为高效催化的可行途径。本项目以结构信息较为明确的里氏木霉纤维素内切酶Ⅰ为模拟对象，通过分析天然酶催化中心的活性位点，制备相应的生物源性交联功能单体，经虚拟筛选和实验验证，进行纤维素内切酶过渡态类似物的高特异性印迹，获得分子印迹模拟酶纳米微球，实现纤维素的高效降解。催化性能的表征结果显示，在模拟酶添加浓度为10%时，酶活力为15.34 U mL-1，最佳的pH值为5.2，最适的反应温度为45 ℃，米氏常数为5.7 mg mL-1，能够在0.5 h内完全降解底物，催化性能为空白印迹微球的10倍。本研究还分析了影响催化和识别性能的各种因素，探索了高性能印迹模拟酶的仿生设计规律，提出并发展了仿生印迹的新策略。项目的开展，有助于进一步丰富模拟酶设计的理论内涵，推进不同学科方法和手段的融合，促进纤维素生物质的生物转化与合理利用。在本项目的资助下，发表SCI论文4篇，EI论文1篇，授权发明专利1项，获省级和市厅级科技奖励各1项，荣获省级优秀学术论文奖励1项，市级青年科技奖1项。培养硕士研究生3名，本科毕业生16名，指导本科生和研究生分别获得相关奖励各1项。