Thermomyces dupontii 脂肪酶TDL2的分子识别机理及理性设计研究

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.

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。

脂肪酶（EC3.1.1.3）是生物催化中使用最多的一类酶，但由于细胞环境和工业化应用环境间的显著差异，天然酶的催化性能往往不能满足工业生产的需求。更加深入地认识酶的结构-功能关系，有助于酶的理性设计。本项目以Thermomyces dupontii脂肪酶TDL2（GenBank no. MN782511.1）为研究对象，进行酶基因表达、“结构-功能”关系及理性设计研究。项目通过基因克隆与表达，分别构建了表达脂肪酶TDL2的大肠杆菌和毕赤酵母工程菌；通过分离纯化和酶促反应，表征了TDL2的酶学性质；建立了罗丹明B平板的脂肪酶筛选模型，用于后续突变库中突变体的快速筛选；通过同源建模方法构建了脂肪酶TDL2的结构模型，并对其结构进行分析，揭示了脂肪酶TDL2的活性中心、底物结合位点、氧负离子洞、盖子结构等关键结构域和氨基酸残基；通过分子对接和计算机模拟方法，分析了脂肪酶TDL2的催化水解反应机理和底物识别机制；通过理性设计策略，采用定点突变/定点饱和突变方法，构建了分子突变文库，筛选到脂肪酶活力明显的提高或底物特异性发生变化的突变体Q4V、S58M、L60K、S83T、W89L、I90A、L206F、P207F等；综合利用包括同源建模、分子对接、分子动力学模拟、酶学性质表征等手段在内的“干实验”和“湿实验”方法，分析了各突变体酶学性质变化的分子机理，阐明脂肪酶TDL2生物催化特性与蛋白结构之间的关系；基于“结构-功能”关系研究基础上，对单突变进行基于组合和迭代的理性设计，获得了较野生型TDL2活力提高162倍的五突变体S83T/W89L/I90A/L206F/P207F；此外，本项目还以选择性水解CNDE为模式反应，研究了突变体脂肪酶的工业应用前景。研究成果进一步丰富了脂肪酶的基础理论研究，同时也为将为其他生物催化剂的理性设计提供指导。