米曲霉脂肪酶AOL的底物识别机制及分子改造研究

Due to Aspergillus oryzae lipase (AOL) with excellent thermal stability, solvent resistance, region-selectivity and stereo-selectivity, which has a huge industrial application potential in the field of chiral synthesis and oil processing, it is an extremely important lipase. But the research on the mechanism of substrate recognition and the structure-function relationships of AOL is insufficient at present, which greatly limits its application range. In this study, we aim to construct the Pichia pastoris for efficient expression of A. oryzae lipase AOL, to investigate its enzymatic properties and catalytic characteristics (substrate specificity and stereo-selectivity), and combining with homology modeling and molecular docking to analysis the three-dimensional structure of the enzyme, to reveal the mechanism of enzymatic substrate recognition and the structure-function relationships of AOL. Based on the knowledge of the structure-function relationships of AOL, site directed mutagenesis is performed on the key amino acid sites and structural functional domains (activity center, substrate binding pocket, substrate entrance tunnel etc.), to improve the catalytic activity and substrate specificity (towards medium and long-chain fatty acid substrates). This project will further expand the application range of lipase, enrich the basic theory research of lipase, and also provide a basic guidance for the rational design of related enzymes.

米曲霉脂肪酶AOL具有良好的热稳定性、溶剂耐受性、立体选择性和区域选择性等特性，在手性合成和油脂加工领域中具有巨大的工业化应用潜力，是一种不可多得的脂肪酶。但是目前关于米曲霉脂肪酶AOL的底物识别机制和结构与功能关系的研究不足，大大限制了其应用范围。本研究拟利用毕赤酵母表达系统对米曲霉脂肪酶基因aol进行高效表达，考察其酶学性质和底物特异性、立体选择性等催化特性，结合同源建模和分子对接等方法分析酶的立体结构，揭示酶的底物识别机制及其结构-功能关系。在此基础上对关键氨基酸位点和结构功能域（活性中心、底物结合口袋、底物入口通道等）进行定点突变，增强其催化活性和底物特异性（中-长链脂肪酸特异性）。本项目能进一步拓展脂肪酶的应用范围，丰富脂肪酶的基础理论研究，同时也为相关酶的理性设计提供基础性指导。

脂肪酶（EC3.1.1.3）属于羧基酯水解酶类，广泛存在于自然界。本项目以米曲霉脂肪酶（GenBank no. KP975533）为研究对象，进行酶基因表达、底物识别机制及分子改造研究。首先根据毕赤酵母的密码子偏好性优化密码子，构建重组质粒pPICZɑA-aol，电转化法导入毕赤酵母成功构建了毕赤酵母工程菌X-33/αA-aol。利用共价结合法将脂肪酶AOL固定化，应用于(R, S)-苯氧丙酸酯拆分反应。通过将米曲霉脂肪酶的氨基酸序列在NCBI蛋白质库中进行对比，以蛋白4PSE和4PSC为模板，对米曲霉脂肪酶进行了同源建模。以苯氧丙酸乙酯为底物，利用Auto dock软件进行了分子对接，分析了米曲霉脂肪酶的催化水解反应机理和底物识别机制。实验通过易错PCR构建分子突变文库，通过两轮易错PCR和高通量筛选，获得了2个催化特性有较大提高的突变子。其中脂肪酶第38位的苯丙氨酸(F)突变为天冬酰胺(N)，脂肪酶第230位的缬氨酸(V)突变为精氨酸(R)，突变体命名为AOL-3F38N/V230R。AOL-3F38N/V230R和原始酶AOL-3比酶活性是分别是186.81 U/mg和46.64 U/mg。突变体AOL-3F38N/V230R对不同碳链长度脂肪酸乙酯的水解特异性发生了变化，优先水解的碳链长度从C4变为C6-C10，对中长链乙酯的水解活性有所提高。脂肪酶突变体AOL-3F38N/V230R的Km1.92 mM和Vmax12.22 mM/min。通过对原始酶AOL-3和脂肪酶突变体AOL-3F38N/V230R的同源建模以及分子对接，发现两个突变位点分别在盖子上以及氧负离子洞附近，影响了催化过程中活性中心与底物结合的状态，从而改变了酶活性以及底物特异性。本研究将为米曲霉脂肪酶分子改造提供了理论技术基础。