新型海洋脂肪酶MAS1高效催化甘油三酯型DHA/EPA合成的机制研究

Enzymatic modification of lipids offers an effective way to produce high-purity DHA/EPA-rich triacylglycerols (TAG). However, DHA/EPA-rich TAG are difficult to be synthesized efficiently by most of lipases. Our previous studies have found that highly efficient synthesis of high-purity DHA/EPA-rich TAG can be achieved by new lipase MAS1 from marine Streptomyces sp. strain W007. Thus, it is conferred that the catalytic mechanism of lipase MAS1 may be different from those of other lipases. In this project, lipase MAS1 will be employed to synthesize DHA/EPA-rich triacylglycerols and its characteristics in the reaction process will be investigated. Computational biology approaches, such as molecular docking, will be utilized to analyze the relationship between the possible key amino acid moieties and the catalytic characteristics. The mutants of lipase MAS1 will be produced by site-directed mutagenesis and their catalytic characteristics will be investigated in the production of triacylglycerols. The catalytic mechanism of lipase MAS1 will be revealed by analyzing catalytic parameters and three-dimensional structure. The research is of significant for the basic research of enzyme engineering, and will push the innovative proceeding of the synthesis of DHA/EPA-rich triacylglycerols. The research also provides a theoretical basis for the engineering application of lipase MAS1.

酶法催化脂质改性是制备高纯度的甘油三酯型DHA/EPA产品的有效途径，但现有的绝大多数脂肪酶难以实现高效快速地催化合成高纯度产品。我们的前期研究发现，来源于海洋放线菌的新型脂肪酶MAS1可以高效催化合成高纯度的甘油三酯型DHA/EPA，由此推测脂肪酶MAS1的催化机制可能不同于其他脂肪酶。本课题拟重点研究脂肪酶MAS1在甘油三酯型DHA/EPA合成过程中的催化特性，再结合脂肪酶MAS1的结构，采用分子对接等生物计算手段分析催化特性与酶分子活性氨基酸变化的相关性；对其活性氨基酸位点进行突变，并研究突变体在甘油三酯合成反应中的催化特性，揭示脂肪酶MAS1分子高效催化的反应机制。期望通过此研究，可推动酶工程领域的基础学科研究和甘油三酯型DHA/EPA生产工艺研究的源头创新，同时为脂肪酶MAS1的工程化应用提供坚实的理论基础。

本课题在前期研究的基础上，对脂肪酶MAS1的结构与功能关系及其在甘油三酯型DHA/EPA合成中的应用进行了系统研究。首先，进行了脂肪酶MAS1的重组表达与性质研究，利用毕赤酵母表达脂肪酶MAS1，进行了蛋白纯化和不同甘油酯底物水解特性研究；然后开展了脂肪酶MAS1的晶体学研究，初步探索了其甘油酯底物特异性与酶蛋白结构的关系；考察了脂肪酶MAS1的酶学性质及其在甘油三酯型DHA/EPA合成中的应用，探讨了甘油/n-3 PUFA摩尔比、加酶量和反应温度对酯化反应的影响，结果表明无位置选择性脂肪酶MAS1能够高效催化合成高纯度的TAG产物；通过对脂肪酶MAS1晶体结构的分析，特异性地对H108、V202、V233三位点进行定点突变，分析并比较野生型及突变体的比酶活和三油酸甘油酯水解特性，结果显示脂肪酶MAS1-H108A和MAS1-H108W的比酶活得到了较大地提高，分别为野生型的1.6倍和2倍，并且脂肪酶MAS1-H108W变成了一个具有强1,3位选择性的脂肪酶，初步阐明了脂肪酶MAS1的位置选择性机制；并对脂肪酶MAS1突变体在甘油三酯型DHA/EPA合成中的应用进行了考察，初步揭示了脂肪酶MAS1高效催化合成甘油三酯型DHA/EPA的反应机制。