褶皱假丝酵母脂肪酶Lip1识别2-取代芳基丙酸对映体的分子基础及催化性能协同优化研究

In the preparation of chiral chemicals through biocatalysis, the enantioselectivity and coexistence of beneficial catalytic properties of a biocatalyst are crucial for its industrial application. 2-arylpropionic acid is one kind of important non-steroidal anti-inflammatory drug. The molecular basis of chiral discrimination mechanism and the coexistence of enzymatic activity and stability based on the biomacromolecular structure, and further cooperative enhancement of all catalytic properties is the key issue for the understanding of molecular mechanism for preparation of 2-arylpropionic acid, function modulation and industrial development of enzymes. Based on previous studies, this research is focused on the effects of structure factors on the 2-arylpropionic acid enantiomer discrimination, enzymatic activity and stability and the search for the interactions between structural factors and catalytic properties. Based on these, this project is also aimed to obtain the mutant forms of Candida rugosa lipase (Lip1) with cooperatively improved catalytic properties and further evaluate the mutant Lip1 by more effective and comprehensive indicator. This research will be significant for not only understanding the molecular mechanism of enantiomer discrimination of 2-arylpropionic acid, but also functional modulation and cooperative enhancement of catalytic properties from the viewpoint of industrial application potential.

在生物催化制备手性化学品过程中，生物催化剂的对映体选择性优劣以及其与其他催化指标的协同共存是决定一个拆分制备过程是否具有工业应用前景的关键前提。2-芳基丙酸类手性化合物是一类重要的非甾体类消炎药。如何从生物大分子结构因素入手，认识脂肪酶对2-取代芳基丙酸类物质的手性识别的分子基础，以及深入理解这种手性识别能力与其反应活性和稳定性之间的相互关系，并进一步协同优化脂肪酶的各项催化指标是利用脂肪酶不对称催化制备2-取代芳基丙酸的关键科学问题。本项目拟在前人研究的基础上，从蛋白质结构因素出发，研究褶皱假丝酵母脂肪酶对外消旋2-取代芳基丙酸的手性识别机理以及对反应活性与稳定性的影响，并总结出各结构因素与各催化指标之间的相互作用关系。在此基础上，通过蛋白质改造获得各催化性能指标协同优化的突变脂肪酶，并使用更加有效的评价指标进行快速综合评价，最终为不对称合成领域新型生物催化剂的开发提供理论和实践基础。

2-芳基丙酸类手性化合物是一类重要的非甾体类消炎药。从蛋白质结构因素入手，认识脂肪酶对2-取代芳基丙酸类物质的手性识别的分子基础以及这种手性识别能力与反应活性和稳定性之间的相互关系，进而协同优化脂肪酶的各项催化指标对进一步提高脂肪酶催化效率实现2-取代芳基丙酸的有效制备具有重要意义。本项目根据已报道的褶皱假丝酵母脂肪酶CRL（Lip1）的基因序列进行了基因全合成，并将目标基因片段插入酵母表达载体pPIC9K中，在Pichia pastoris GS115中进行了异源表达，通过表达融合标签实现了Lip1可溶性活性表达以及目标蛋白的高效亲和层析纯化，同时进行了重组脂肪酶Lip1的诱导表达条件优化。并从蛋白质结构因素出发，建立了布洛芬对映异构体手性分析方法，确定了R与S-布洛芬丙酸酯与脂肪酶CRL（Lip1）的结合模式及突变热点