Sn-2黑曲霉CCTCC No. M2012538脂肪酶的结构与功能研究

Sn-2 lipase is of great value in the preparation of functional oils. However, Sn-2 lipase is exiguous. We previously screened a new strain of Aspergillus niger, CCTCC No.M2012538 (GZUF36), which produced a lipase with a high selectivity for hydrolyzing triglycerides at the Sn-2 position. At present, the catalytic mechanism of lipase has been clarified, but the position-selective catalytic mechanism is still unclear. In this project, we wolud first clone and express Sn-2 lipase holoenzyme using E.coli, and then use SAXS and bioinformatics to characterize the three-dimensional structure and domain of the enzyme. Subsequently, we would use E.coli to clone and express the catalytic domain and non-catalytic domain, respectively. Next, the catalytic domain and non-catalytic domain would be co-resolved using NMR and SAXS. On this basis, a high-resolution structure of Sn-2 lipase would be constructed, and the spatial conformation and position selection and enzyme activity of the enzyme would be changed by further changing conditions, and the conformational change of the enzyme in the solution was analyzed by SAXS, and the position selectivity of the enzyme as well as the enzymatic would be analyzed at the same time. Thus, the structure-activity relationship of Sn-2 lipase, ie, relation between the conformation of the lipase and the Sn-2 position selectivity mechanism and enzyme activity would be revealed. The implementation of this project would help to in-depth analyze the three-dimensional structure of Sn-2 lipase from Aspergillus GZUF36, reveal the relationship between its position selectivity and spatial conformation, and further reveal its position selective catalytic mechanism. It would be of great significance for the subsequent rational molecular modification and potential industrial applications. It would also help to enrich the theoretical and practical content of lipase catalysis.

Sn-2脂肪酶在制备功能油脂方面有重要应用价值，不过Sn-2脂肪酶很少。前期课题组筛选到一株产高Sn-2位选择性水解甘油三酯的脂肪酶的黑曲霉新菌株CCTCC No.M2012538。目前脂肪酶的催化机制已阐明，但位置选择性催化机制研究还不清楚。本课题拟首先利用大肠杆菌克隆表达Sn-2脂肪酶，利用SAXS并结合生物信息学初步分析酶的三维结构和结构域；然后再利用大肠杆菌分别克隆表达催化结构域和非催化结构域；接着利用NMR和SAXS共同解析催化结构域和非催化结构域。在此基础上构建Sn-2脂肪酶全酶的高分辨度结构，并进一步变化条件改变酶的空间构象和位置选择、酶活，通过SAXS分析溶液中酶构象变化，以揭示Sn-2脂肪酶的构效关系即构象与其Sn-2位选择性机制和酶活间的关系，进而揭示其位置选择性催化机制。这对于其随后理性改造和潜在的工业化应用都有重要意义，同时有助于丰富脂肪酶催化的理论和实践内容。

黑曲霉脂肪酶具有高的位置选择性，但对黑曲霉脂肪酶的结构和位置选择性机制方面研究偏少，缺少关键的结构方面的数据支撑。本项目对黑曲霉GZUF36的胞外脂肪酶EXANL1粗酶液进行了交联酶聚集体（CLEAs）制备，获得了酶活力高、稳定性强且可多次回收利用的固定化脂肪酶。接下来通过丙酮沉淀与反胶束萃可对黑曲霉CZUF36胞外脂肪酶进行了纯化，获得了电泳纯的脂肪酶，并获得了其氨基酸组成基本序列。将此纯化的脂肪酶用于制备CLEAs-ANL（即CLEAs-EXANL1）并研究其酶学性质、二级结构、形态外貌以及选择性进行研究。.根据获得的EXANL1氨基酸组成基本序列，对其在大肠杆菌、毕赤酵母分别进行了表达、酶学性质、酶结构和固定化研究，获得了几种性质获得大幅提升的固定化脂肪酶。.物特异性试验表明EXANL1最适底物为对硝基棕榈酸酯，却几乎不水解短链脂肪酸（C2-C8）。并进行了底物特异性机制研究。基于同源建模和小角X射线散射（SAXS）对毕赤酵母表达重组酶的结构进行了表征，并初步分析了其位置选择性机制。.进行结晶条件初筛，出现了疑似为PEXANL1的晶体，但遗憾没有获得高纯度EXANL1晶体。进行了m-PEG修饰和离子液体改性纳米磁性Fe3O4固定化对重组的黑曲霉GZUF36胞外脂肪酶的结构和性质影响；同时进行了Sn-1,3 黑曲霉 GZUF36 胞外脂肪酶的高级结构分析与提升其催化性能研究，获得了较好结果。.对黑曲霉的GDSL型酯酶INANE1首次进行了克隆表达、纯化、酶学性质、结晶与结构初步解析研究。结果表明INANE1是其冷活性和高乙酰酯选择性，其也是sn-1,3位置选择性。更重要的是我们获得了其晶体结构，并进行了初步的结构解析。.而且通过项目实施在发表SCI论文27篇；申请国家发明专利5项；出版学术专著一部；做会议报告4篇；培养和造就相关领域的中青年专家2名，培养毕业研究生8名，形成一脂肪酶/酯酶科技创新团队。