紫色红曲霉来源脂肪酶LIP05催化合成己酸乙酯的分子识别机制

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.

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。

以己酸乙酯为代表的脂肪酸乙酯含量偏低是浓香型白酒品质差和优质酒出酒率低的主要原因之一。脂肪酶是催化合成乙酯的关键酶，探究其在白酒发酵水相体系的催化机制，对保障白酒风味乙酯的稳定合成尤为关键。项目聚焦白酒源紫色红曲霉来源水相体系酯合成酶LIP05，系统解析了酶催化脂肪酸乙酯合成的分子机制。研究建立基因5′-端密码子优化技术，成功实现酶LIP05的异源高效表达。利用蛋白结构分析软件，解析LIP05高精度三维结构，准确定位盖子结构域为N-端50 bp的两个α-螺旋和螺旋之间的loop区域。通过盖子结构域和催化活性区域关键位点定点突变和酶学性质比较，确定酶催化酯合成相关的10个关键位点并分别引入5种不同的氨基酸残基突变，包括非极性氨基酸Ala和Val、极性氨基酸Ser/Thr、碱性氨基酸Lys和酸性氨基酸Asp。结果表明，Ile30和Leu37的空间位阻作用，Gly73和Thr74的氢键作用，Leu83的空间位阻和疏水相互作用的叠加，Tyr116和Tyr149氨基酸残基的苯环与底物的互作，以及Ile204、Ile211和Leu216的疏水相互作用，均影响酶的催化效率。其中，Ile30和Leu37位于盖子结构域。Gly73和Thr74与催化活性中心Ser150空间相对距离3–5 Å，相关位点与底物产生氢键相互作用，引入任何性质氨基酸残基均导致酶活力显著降低，表明两个位点应是酶的氧阴离子洞，对于维持酶催化过程的四面体过渡态具有重要作用。利用理性设计，获得底物特异性识别明显改变的突变酶LIP05-I30D，并与野生型酶进行结构和酶与底物作用力和结合能量差别分析，发现LIP05-I30D与产物己酸乙酯作用的氢键距离变长，结合能增高，利于产物己酸乙酯的释放，表明LIP05盖子结构域30位点氨基酸极性的改变影响酶对底物的分子识别和催化酯化的效率。在上述研究基础上，项目进行了系统的拓展性研究，以“风味与健康”为导向，聚焦酯合成的底物小分子脂肪酸的微生物代谢，以及白酒酯类物质中潜在有害酯的微生物降解研究，对于确保发酵白酒风味乙酯的稳定合成和产品的饮用安全性，提供了重要的基础数据依据。项目发表SCI论文9篇，EI论文1篇，授权国家发明专利6件，另申请3件。