酯类是决定白酒风味和品质的关键化合物，其合成深受脂肪酶催化的影响。目前脂肪酶催化酯类合成的研究主要集中于有机相体系，而在白酒酿造这个水相为主的过程中，基于水相体系的脂肪酶催化酯类合成机制尚待揭示。本项目拟以前期获得的一株产高性能酯类合成脂肪酶的细菌为基础，通过基因敲除/过表达，原位定位酯合成主要脂肪酶；通过脂肪酶的异源表达、性质分析和结构解析，确定酶的关键α-螺旋“盖子”结构并构建α-螺旋缺失脂肪酶；通过分子对接和量子计算，比较缺失前后脂肪酶催化活性中心、关键氨基酸位点、底物结合模式和微观催化行为等的差别；利用单点、双点和多点定点突变、反应动力学测定和三维结构预测，解析关键氨基酸残基对脂肪酶水相体系催化酯合成的影响；利用理性设计实现同家族中脂肪酶关键位点的催化特性对比，最终阐明白酒酿造水相体系中脂肪酶催化酯类合成的分子机制。项目的成功实施对推进传统酿造白酒产业的现代化进程具有重要意义。

Esters are one kind of important compounds which decide the flavor and quality of Chinese liquor. Lipases contribute greatly to the synthesis of esters. Pre-existing studies mainly focus on the synthesis of esters by lipases in the organic media, while in Chinese liquor, which is aqueous system, there is no report concern to the mechanism of ester synthesis by lipase. In previous studies, one strain was isolated from the sample of brewing Chinese liquor, which could efficiently catalyze the synthesis of esters. The present project aims to find the main lipase responsible for ester synthesis by gene mutation/overexpression. The lipase will be heterologous expressed, and the properties and structure will be analyzed, the α-helix "lid" structure will be determined and deleted. The differences in the catalytic activity center, key amino acids, binding mode with the substrate and microscopic catalytic process of the lipases with and without α-helix will be explored through molecular docking and quantum computing. The divergences of the key amino acid residues that affect the catalytic efficiencies of esters will be analyzed by single point, double point and multipoint mutation, reaction kinetics analysis and three-dimensional structure prediction, helping to design the representative lipases with different catalytic characteristics in the same families, finally, to modify and elucidate the molecular mechanism of the lipases for ester synthesis in aqueous system. The successful implementation of this project plays an important role to promote the traditional liquor brewing industry in the process of modernization.