Thermomyces dupontii脂肪酶TDL2，是申请人前期研究中利用基因挖掘策略获得的、对2-羧乙基-3-氰基-5-甲基己酸乙酯和3-氰基-5-甲基己酸乙酯具有良好立体选择性的新酶。但由于TDL2活力偏低，且对其底物特异性和对映选择性规律认识的匮乏，其开发、改造和应用受到严重制约。本研究以认识TDL2的分子识别机理、探究酶结构与功能对应关系以及提高TDL2工业适用性为目标，综合利用同源建模、分子对接、分子动力学模拟、定点突变、定点饱和突变等手段在内的"湿实验"和"干实验"方法，准确定位影响脂肪酶底物识别、立体选择性识别、反应过渡态形成的氨基酸残基和结构功能域，揭示TDL2的底物分子识别机制，阐明酶结构与功能之间的关系。并在此基础上对TDL2进行理性设计，强化其催化活力。本项目不仅能丰富脂肪酶的基础理论研究，而且为相关酶的理性设计提供借鉴，具有重要的科学意义和实用价值。

The lipase TDL2 from Thermomyces dupontii, a new potential biocatalyst with excellent enantioselectivity in kinetic resolution of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester and 3-cyano-5-methylhexanoic acid ethyl ester, was discovered by gene mining in our previous study. Due to the low activity, the lack of deep understanding of substrate specificity and enantioselectivity law, the development, reconstruction and application of the lipase are severely restricted. This project aims to reveal the molecular recognition law, to investigate the relationship between structure-function, and to develop a novel biocatalyst with high process efficiency compatible with the industrial prerequisites. The "wet test" and "dry test" techniques, including homology modeling, molecular docking, molecular dynamics simulation, site-directed mutagenesis and site saturation mutagenesis, will be further applied to study the effects of key amino aicd residuces and functional domains that affect the lipase’s substrate recognition, stereoselective recognition, and reaction transition state formation, which can finally reveal the molecular recognition laws and structure-function relationships of TDL2. Based on the knowledge on structure-function relationships, the rational design will used to improve the catalytic efficiency of TDL2. The whole research has important scientific significance and practical value. It not only can enrich the basic theory research of lipase, but also provides reference for related enzymes' rational design.