脂肪酶的定向修饰对油脂醇解反应的调控及机理研究

Lipase-catalyzed alcoholysis of oils is one kind of main reactions for the preparation of functional structured lipids (for examples, symmetrically structured triacylglycerols). However, during the alcoholysis of oils, the presence of short-chain alcohols resulted in the deactivation of lipases. In the project, in order to overcome the deactivation of lipases in polar alcohol system and mass transfer limitation of the immobilized lipases, and according to our previous work, Candida antarctica lipase A (CALA) will be selected and used as the model lipase. MALDI-TOF, CD and DSC will be used to analyze the characteristics of the modified lipases; and different polymers will be used to directly modify the amino group of the protein on the surface of CALA to design and prepare the novel lipase-fluids. The catalytic activity and stability of CALA in polar alcohol system will be enhanced by controlling and adjusting the structures of the polymers. And the structure-activity relationships between directional modification methods, the molecule structures of polymers with the fluid characteristics (mass transfer limitation of reaction system and reuse performance) and reaction selectivity of CALA will be established. Moreover, other lipases (Candida antarctica lipase B and Lipase from Thermomyces lanuginosus) will also be used to prepare the lipase-fluids and compare with CALA-fluids. The regulation mechanism of the directional modification of lipases for the alcoholysis of oils will be achieved.

脂肪酶催化油脂醇解是制备功能性脂质（如对称性结构甘三酯）的一个主要反应，但短链醇的存在会导致脂肪酶活性的丧失。为解决极性醇体系中脂肪酶失活和固定化酶反应传质限制问题，本课题在前期工作基础上，拟采用脂肪酶Candida antarctica lipase A （CALA）为研究对象，运用MALDI-TOF、CD、DSC等分析手段，通过聚合物对CALA分子表面的氨基酸基团进行定向修饰，设计和构造一种新型脂肪酶CALA流体。通过控制和调变聚合物结构，提高脂肪酶CALA在极性醇体系中的催化活性和稳定性，研究定向修饰、聚合物结构与脂肪酶的流体学特性（反应体系传质和重复利用性能）和反应选择性的构效关系。同时与其它脂肪酶（Candida antarctica lipase B，Lipase from Thermomyces lanuginosus等）进行对比，建立脂肪酶定向修饰对油脂醇解反应的调控机理。

油脂醇解是一类用于生产生物柴油、甘一酯、甘二酯等的主要反应，但存在脂肪酶在醇解反应体系易失活等问题。本项目团队针对此问题，通过四年的研究工作，已圆满完成项目计划内容。先研究几种游离的液体脂肪酶（CALA，Lipozyme TL100L和CALB等）在不同醇解（甘油解、甲醇解和乙醇解）反应体系的特点，在此基础上，采用具有流体性质的两亲性物质Pluronic F-127等对几种液体脂肪酶进行定向修饰，通过扫描电镜图谱、SDS-PAGE凝胶电泳、傅利叶红外光谱等手段对改性前后的脂肪酶进行表征分析，表明修饰成功。修饰后的脂肪酶的活性、重复利用性能等均显著提升，克服其在醇解反应体系失活问题，实现了酶的定向修饰对酶活性和醇解反应的调控。同时也建立了反应的动力学和热力学，提出了醇解反应机制。相关结果已发表SCI论文14篇，其中JCR一区10篇。申请专利2件（含1件国际专利），授权专利1件。获得全国粮食行业青年拔尖人才和河南省教育厅学术技术带头人称号，同时获得中国粮油学会青年科技奖。培养研究生5人，其中3人获河南工业大学优秀硕士学位论文一等奖和二等奖，1人论文被推荐省级优秀硕士学位论文。参加国际会议2次并作报告2次，同时承办国际会议1次；参加国内会议3次，作报告1次。