基于分子模拟的功能性离子液体化学修饰脂肪酶分子改造研究

Chemical modification based on molecular simulation is an important strategy in the field of enzyme molecular alteration at present. However, due to the structure diversity of enzymes and the species limitation of traditional modifiers, the activity, selectivity, thermal stability and organic solvent tolerance of enzymes are difficult to be improved simultaneously. Functional ionic liquids have been used as novel and efficient modifiers to enhance the catalytic performances of lipases in the previous studies, while this method exists inevitable blindness and the molecular mechanisms of modification haven't been elucidated clearly yet. In view of typical lipases with specific structure, the structure and function of novel ionic-liquid modifiers are rationally designed by molecular simulation technology in this project. Then, the screened novel ionic liquids are used to improve the enzymatic properties by covalent bonding with specific amino acid residues of enzymes. Based on the simulation analysis of the interaction between modifiers and enzymes, combined with modern spectroscopic characterization techniques and enzymatic experiments verification, the mechanisms of catalytic properties change of lipases owing to modification with functional ionic liquids are to be revealed reasonably from molecular level. Thus, the selection of modifiers, modification sites and modification degrees could be conducted rationally to realize the efficient regulation of catalytic performances of lipases. Finally, a novel and efficient method for lipases modification with functional ionic liquids to enhance their catalytic properties based on molecular simulation is intensely expected to be developed. The research results can provide theoretical guidance and technical support for the industrial application transformation of lipases.

基于分子模拟的化学修饰是当前酶分子改造的重要手段，然而由于酶分子结构多样性和传统修饰剂种类的局限，修饰酶的活性、选择性、热稳定性、有机溶剂耐受性等各种催化性能难以得到同时提升。在前期研究中功能性离子液体作为一种新型修饰剂可成功应用于脂肪酶的催化性能强化改造，但修饰存在盲目性，且修饰的分子机制尚未明确。在此基础上，本课题拟通过分子模拟技术对新型离子液体修饰剂的结构和功能进行理性设计，再以模拟优筛得到的修饰剂对特定氨基酸残基进行共价修饰以实现酶催化性能强化改造；在分子模拟计算解析影响酶与修饰剂分子相互作用关键因素的基础上，结合现代光谱学表征技术和酶学实验合理解析脂肪酶的分子改造机制，进而指导修饰剂、修饰位点和修饰度的理性组合，实现脂肪酶催化性能强化高效调控。本课题预期发展一种基于分子模拟研究的功能性离子液体化学修饰脂肪酶的高效分子改造新技术，为脂肪酶工业应用改造提供理论指导和技术支撑。

本项目设计了多种不同结构的功能性离子液体修饰剂，并拟合出符合本研究体系的离子液体及其修饰氨基酸的分子力场。采用分子动力学模拟等手段预测了离子液体在洋葱伯克霍尔德菌脂肪酶（BCL）和猪胰脂肪酶（PPL）上的修饰位点，并对修饰前后脂肪酶的三维结构进行分子动力学模拟，预测了修饰脂肪酶空间构象体系的变化和酶学性能变化。在此基础上，优选了可能具有较好修饰效果的咪唑和/胆碱型、脯氨酸型离子液体，将其分别共价接枝到BCL和PPL上，实验证实修饰酶的活性和稳定性都得到了显著提升。. 分子动力学模拟预测脯氨酸型离子液体对南极假丝酵母脂肪酶B (CALB)也具有较好的修饰效果，并且构型对其修饰效果有较大影响。实验证实四种不同结构的脯氨酸型离子液体均能有效提高CALB的活性和稳定性，而且L-脯氨酸型离子液体的修饰效果优于D-脯氨酸型离子液体。分子动力学模拟显示修饰后CALB的RMSD和总能量均小于未修饰CALB，RMSF计算表明修饰后CALB的刚性得到了提高，溶剂可及面积(SASA)计算表明修饰后酶蛋白的亲水性和疏水性都得到了改善，水径向分布函数（RDF）计算证实活性位点附近的水分子也增加了，这些模拟结果都证实了酶修饰后活性和稳定性可以得到改善。分子模拟计算结果结合荧光光谱、圆二色谱等光谱学结构表征合理解析了修饰后CALB 酶学性能变化的分子机制。. 在此基础上进一步完善了离子液体及其修饰的非天然氨基酸的力场参数，针对CALB和PPL，基于分子模拟计算分别设计了甜菜碱型离子液体与侧链带长链疏水烷基的L-脯氨酸型离子液体，实验结果证实CALB和PPL的酶学性能得到了更大程度的提升。本项目发展了一种基于分子模拟研究的功能性离子液体化学修饰脂肪酶的分子改造新技术，解决了当前化学修饰改造方法中存在的盲目性、机理不清晰等问题，拓宽了分子模拟技术和离子液体在酶分子改造领域的应用。