Paracoccus kondratievae来源酶HylD1和HylD2水解邻苯二甲酸酯侧链酯键的分子识别机制

食品源邻苯二甲酸酯污染严重威胁人体健康，酶法水解其侧链酯键是降低毒性的有效方法之一。邻苯二甲酸酯酯键连接侧链结构多样，显著影响酶对底物的分子识别和酯键水解。由于食品常被多种邻苯二甲酸酯污染，以及酶的底物谱差异和分子识别机制匮乏，酶法水解的应用被极大限制。因此探究酶对邻苯二甲酸酯侧链酯键水解的分子识别机制十分必要。前期研究从自主知识产权的白酒来源微生物中获得分别具有短直链和长支链邻苯二甲酸酯水解能力的酶HylD1和HylD2。拟通过同源建模、分子对接和酶工程改造，对比分析影响酶底物谱差异的关键位点；利用理性设计和分子动力学计算，从催化关键节点和微观动态过程双角度对比原酶与突变酶底物分子识别的差别，解析关键位点影响酶对底物分子识别的催化过程的差异；对关键位点分别引入不同类型氨基酸突变，结合催化动力学和模拟计算分析，总结关键位点影响酶对底物分子识别的机制，为酶法有效水解邻苯二甲酸酯提供理论依据。

Phthalate esters pollution in food is a serious threat to human health. Enzymatic hydrolysis of the side-chain ester bond of phthalate esters is one of the effective methods to reduce the toxicity. The side-chain of phthalate ester linked by ester bond is varied in structure, and significantly affects the molecular recognition and hydrolysis of the substrate. As always polluted by kinds of phthalate esters, the specificity of enzyme and the lack of substrate recognition mechanism, the application of enzymatic hydrolysis is greatly limited. Therefore, it is quite necessary to explore the molecular recognition mechanism of the enzyme toward the hydrolysis of side-chain ester bond of phthalate esters. In our previous work, the enzymes HylD1 and HylD2 with respective hydrolysis capabilities toward short and straight side-chain, and long and branched side-chain ester bonds of phthalate esters were cloned from Baijiu microorganism with independent intellectual property rights. The key sites were compared affecting the divergence of substrate spectrum through homologous modeling, molecular docking and enzyme engineering. By rational design and molecular dynamic calculation, the differences of substrate recognition mechanisms between the original enzyme and the mutant ones were compared and summarized from the two perspectives of key catalytic nodes and micro-dynamic process. The divergence of the catalytic processes affected by the key sites of the enzymes for substrate molecular recognition were revealed. The key site was mutated by different types of amino acids to summarize the affections to the enzyme for substrate molecular recognition combined with catalytic kinetic and simulate computation analysis. This will provide theoretical basis for the efficient hydrolysis of phthalate esters by enzymes.