基于底物选择性机制解析的脂肪酶理性设计及ω-3多不饱和脂肪酸甘油酯的定向合成

ω-3 polyunsaturated fatty glyceride is one of the best lipids. Each configuration of ω-3 polyunsaturated fatty glyceride has specific physiological activity and distinct bioavailability. However, its main source, natural fish oil, has complex ingredients, which are difficult to separate. Therefore, the research and application of monocomponent are hindered severely. In our previous study, several lipases with ω-3 polyunsaturated fatty acid esterification activity were screened out and the research condition was well established. In this study, three main research contents will be conducted. First, the catalytic characteristics of the lipases to saturated and unsaturated fatty acids will be researched thoroughly. Second, the structure analysis and computational simulation will be applied to analyze the differences of binding mode and binding free energy between saturated and unsaturated fatty acids. Subsequently, the influence of the differences of hydrogen bond, electrostatic interaction and conjugated affinity, which is caused by alkane and alkene on the substrate binding, will be investigated to elucidate the molecular mechanism of lipase substrate selectivity to saturated and unsaturated fatty acids. And the rational design of lipase with high specificity will be realized. Third, an efficient controllable and assembly Aspergillus oryzae expression system will be established to explore the matching characteristics of lipase and to optimize its expression. Ultimately, the efficient and oriented synthesis of ω-3 polyunsaturated fatty glyceride with high purity will be achieved. This project will provide theoretical and technical support for the production of more high-performance esters with specific configuration, and will be valuable for the enrichment of unsaturated glycerides library and the improvement of the quality of lipids.

ω-3多不饱和脂肪酸甘油酯被誉为最优质的食用油脂之一，其各单一构型均具有独特的生理活性和不同的生物利用度。但其主要来源鱼油成分复杂，单组份分离纯化困难，严重制约其研究与应用。项目以前期筛选的具有ω-3多不饱和脂肪酸酯化性能且建立良好研究基础的脂肪酶为对象，首先研究其对饱和／不饱和脂肪酸的催化特性；其次采用结构认知和计算模拟等手段分析其饱和／不饱和脂肪酸结合模式及结合自由能等差别，并研究由烯烃／烷烃键不同导致的氢键、静电和共轭亲和等作用力的差异对底物结合的影响，从而深入揭示饱和/不饱和脂肪酸底物选择性的分子机制，并以此为依据实现高专一性脂肪酶的理性设计；进而通过构建高效可控组装的米曲霉表达系统，探索脂肪酶的适配特性并优化表达，最终实现高纯度特定构型ω-3多不饱和脂肪酸甘油酯的高效定向合成。项目为更多特定构型功能酯的生物合成提供了理论和技术支持，对丰富不饱和甘油酯库、提升油脂品质具有广泛意义。

食用油脂中营养价值最高的为多不饱和脂肪酸，其各单一构型均具有独特的生理活性和不同的生物利用度。项目以具有ω-3多不饱和脂肪酸酯化性能且建立良好研究基础的脂肪酶LipK107和MAS1为对象，首先基于脂肪酶的结构确定了其催化中心、解析了其催化机制；其次，开发了一套能够充分挖掘酶的催化潜力的计算模拟算法Crius，并通过脂肪酶等进行了实验验证，为开发能够有效预测酶底物的计算机算法开辟了新的思路；进而课题组提出了“对于酶的底物选择性，除了酶与底物的结合力以外，合理的构象也同样非常重要”的理论，并设计了一套新颖、高效的基于电荷作用力的in silico的虚拟改造方案并获得验证。上述理论与方法，在理论上也同样适用于不同底物的选择性机理的认知与研究，具有广阔应用前景；接下来，项目设计搭建了针对脂肪酸和甘油酯的反应体系和检测平台，研究了脂肪酶对饱和／不饱和脂肪酸的催化特性；针对底物小分子进行核磁解析与高斯模拟，证明多不饱和脂肪酸因其双键结构存在及相互作用比饱和脂肪酸更加弯曲。随后通过动力学模拟验证得知“盖子”的存在很大程度上影响着酶的活性和稳定性，同时确定了脂肪酶活性受到关键残基原子间的距离——S79 中的 OG 原子到脂肪酸中羧基氧 O2 原子的距离的影响；进而基于MM/GBSA结合自由能计算，深入分析了饱和／不饱和脂肪酸结合模式和作用力等差别，并以此为依据理性设计高专一性、高活性的脂肪酶。项目最后对理性设计的系列突变体并进行了性能测定。筛选获得水解酶活提升233% 的突变体，以及对多不饱和脂肪酸酯化选择性提升537%的突变体，从而实现了多不饱和脂肪酸甘油酯的定向合成。项目开发了底物预测算法，解析了脂肪酶选择性催化多不饱和脂肪酸的分子机制，并基于理论基础设计并获得了高选择性脂肪酶，该研究为更多特定构型功能酯的生物合成提供了理论和技术支持，对丰富不饱和甘油酯库、提升油脂品质具有广泛意义。