Sn-2位选择性黑曲霉CCTCCNo.M2012538脂肪酶水解法高效制备1,3-甘油二酯

1,3-甘油二酯是一种健康油脂，但天然含量低。本项目拟利用前期筛选的来自新菌株黑曲霉CCTCC M2012538 的胞外Sn-2位选择性的脂肪酶进行水解制备1,3-甘油二酯。拟通过单因素试验、PB试验、快速登高试验和中心组合试验分析发酵条件、反胶团萃取条件、交联酶聚体固定化条件、水解条件对酶活和稳定性的影响规律以及分析分子蒸馏法纯化条件对纯化产物的影响，在此基础上实现黑曲霉Sn-2位脂肪酶的高密度发酵、丙酮沉淀、反胶团萃取纯化及交联酶聚体固定化并构建其催化的水解体系和分子蒸馏纯化产物，实现1,3-甘油二酯的高效制备，而且初步分析此Sn-2位脂肪酶的选择性机制。所以本项目的实施有利于开发一种绿色简单经济制备1,3-甘油二酯的方法，有利于丰富脂肪酶学的理论和实践内容，项目集成的高密度发酵、反胶团萃取纯化、交联酶聚体固定化、水解体系优化技术和分子蒸馏纯化技术，亦可为其它酶的研究提供参考。

1,3-diglyceride (1,3-DG) is a kind of healthy fats and the natural content is low. This project aims to use extracellular lipase with Sn-2 positional selectivity from previously screening new strain of Aspergillus niger CCTCC M2012538 to prepare 1,3-diglyceride by high selective hydrolysis. Effects of fermentation conditions, reverse micellar extraction conditions, immobilization conditions by cross-linked enzyme aggregates on the lipase activity and selectivity as well as effects of conditions of molecular distillation on purification of DG were analyzed by means of single factor test, Plackett-Burman (PB) test, rapid high experiment and central composite design. Then, Sn-2 lipase from Aspergillus niger were produced by high density fermentation, purified by acetone precipitation and reverse micellar extraction, and immobilized by cross-linked enzyme aggregates. After that, 1,3-DG were efficiently produecd by construction of hydrolysis system through the immobilized lipase as well as 1,3-DG purified by molecular distillation. Moreover, the selective mechanism of the Sn-2 position lipase was preliminarily analyzed. Therefore, the implementation of the project is helpful to develop a simple, green and economic method to prepare 1,3-diglyceride, and to enrich the theory and practice of Enzymology. The integrated high density fermentation and reverse micellar extraction purification, cross-linked enzyme aggregates immobilization, hydrolysis system optimization and purification by molecular distillation technologies can also provide a reference for the study of other enzymes.