源于Burkholderia pyrrocinia脂肪酶影响白酒酯类合成的分子机制

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.

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。

白酒酿造过程是以水相为主的环境，决定白酒风味和品质的关键化合物酯类的合成需在水相体系中具有酯合成能力脂肪酶的参与。本项目以一株高产酯类合成脂肪酶性能的细菌Burkholderia pyrrocinia为研究对象，通过双水相体系纯化获得电泳纯脂肪酶，详细分析其性质，该酶最适pH 8.0，最适反应温度50℃，且具有较好的酯合成特性。分子克隆表达Burkholderia pyrrocinia脂肪酶Lip1739，对其发酵条件进行了优化；基因分析发现该菌存在与脂肪酶具有类似催化三联体和盖子结构的阿魏酸酯酶，通过克隆表达成功获得此酶，经研究发现该酶具有较好的耐酸性和酯类化合物的水解效果。本项目着重探讨了脂肪酶盖子结构等关键位点催化酯类合成的分子机制。首先，考察了模拟白酒发酵条件下具有高己酸乙酯合成能力的Monascus purpureus来源脂肪酶LIP05，蛋白质同源建模获得LIP05的三维结构，构建盖子结构缺失蛋白LIP05-50，证实脂肪酶盖子结构的存在，对于维持酶的稳定性具有重要作用，而盖子结构的去除却对酶活力提升有帮助； LIP05-50在最优酯化条件下催化酸与乙醇生成对应乙酯时，其最适底物由LIP05最适的癸酸变为辛酸，表明盖子结构的去除直接影响了酶的底物特异性。超声耦合条件下考察LIP05-50催化效率，3倍加酶量耦合超声催化酯化时，1 h辛酸乙酯的产量达1614.65±73.77 mg/L，高达理论转化率的93.73%。项目研究了脂肪酶盖子螺旋数量对催化速率的影响。来源于Candida rugosa的具有多个螺旋作为盖子的脂肪酶催化速率较快，且乙酸乙酯合成量较高；基于Ping Pang bi bi双底物抑制酶反应动力学模型考察乙醇和乙酸摩尔比例对脂肪酶的催化效率影响，发现提高乙醇的摩尔比例可以显著提升脂肪酶催化酯类物质的合成效率；而当乙醇的摩尔浓度不变时，提高乙酸的摩尔比例则显著抑制脂肪酶催化酯类的合成效率。本项目针对脂肪酶分子关键位点对其催化特性影响的研究，对于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提供了大量的基础数据和实验结果，为进一步解析白酒酿造过程微生物酶的作用机理，进而推动生产工艺过程的科学阐释具有重要的实际意义。