Sn-2黑曲霉CCTCC No. M2012538脂肪酶的结构与功能研究

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。

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.