己酸乙酯含量偏低是传统酿造浓香型白酒品质差和优质酒出酒率低的主要原因之一。脂肪酶是催化合成己酸乙酯的关键酶，发掘底物特异性好的脂肪酶并探究催化机制，对保障酯的稳定合成尤为关键。脂肪酶的盖子是影响催化的关键结构域，对底物的特异性分子识别具有重要作用。前期研究发现，紫色红曲霉来源脂肪酶LIP05是特异性催化合成己酸乙酯的重要酶，且编码序列具有新颖性，但其盖子结构域和底物识别机制尚不清楚，具有重要应用基础研究价值。项目拟进行该酶的表达和纯化，利用晶体结构解析准确定位盖子结构域；通过盖子定点突变和酶学性质比较，确定关键位点，结合同源建模和分子对接，解析关键位点突变对酶的结构和催化过程作用力的影响；利用理性设计获得特异性识别功能提高的酶，并与野生型酶进行结构和酶与底物作用力差别分析，揭示酶的底物分子识别作用过程，阐明该酶特异性催化合成己酸乙酯的分子识别机制，为酿造过程己酸乙酯的稳定合成提供理论依据。

The low concentration of ethyl hexanoate is one of the main reasons for the poor quality and yield of traditionally brewed strong flavour Chinese Baijius. Lipases are the key enzymes for the synthesis of ethyl hexanoate. Exploring of the lipase with improved substrate specificity and investigating the catalytic mechanism is crucial to ensure the stable synthesis of the ester. The lid of the lipase is the key domain affecting catalysis, which is very important for the specific molecular recognition of the enzyme toward the substrate. In the previous study, a lipase LIP05 originated from strain Monascus purpureus was found to be an important enzyme for specifically catalyzing the synthesis of ethyl hexanoate. Its encoding gene showed sequence novelty, but the lid domain and mechanism of substrate recognization was still not clear for this lipase. Therefore, the enzyme had great value to the applied basic research. In this project, the lipase was expressed and purified. The accurate lid domain of the lipase was identified through crystal structure analysis. The key amino acids were determined by site-specific mutagenesis of the lid domain and comparison of enzymatic properties. Homologous modeling and molecular docking were used to investigate the influences of the key amino acids on enzyme structures and forces during the catalytic process. The lipases with improved specific recognization property were obtained through rational design. The divergence of structures and the forces of the enzymes toward the substrates were compared between the wild type and engineered ones, to reveal the specific recognization process of the enzyme toward the substrate. Finally, the molecular recognition mechanism of ethyl hexanoate synthesis was revealed of this lipase, which would provide theory basis for the stable synthesis of ethyl hexanoate during the brewing process.