基于分子模型的糖类化合物与脂肪酶超分子构象构效关系研究

Enzymatic engineering is one of the core technics of biological engineering. Currently, most researches were focused on the structure of the enzyme itself and the immobilization method. Nevertheless, the enzymatic engineering is a systematic engineering; it includes not only the transformation of the enzyme molecule and immobilization, but also the additive in the enzyme preparation process. However, few studies were focused on the investigation of mechanism of additive function, which caused applications of the additives were mostly from practical experience. This project will focus on the seldom researched saccharide additives of lipase, basing on the computer simulation technic and modern instrumental analysis, and with the instruction of the theory of structural chemistry, interfacial chemistry, molecular dynamics, the investigation of the changes of hydrogen bonds, ionic bonds, electrostatic interactions, hydrophobic interactions and the structure changes of supramolecule would be carried out. The project will establish the model based on the structure-function relationship of the saccharides and lipase supermolecular, and lucubrate the mechanism of the saccharide additives influenced the function of enzyme molecules. The mechanism and the model could be helpful for the selection of specific saccharide additive for different reaction systems, and providing a viable theory to guide the selection of additives for industrial enzymatic catalysis.

酶工程是生物工程的核心技术之一，目前关于酶工程的基础研究偏重于酶本身的结构及固定化。然而酶工程是系统工程，不仅包含酶分子改造、固定化等研究工作，制剂过程中的小分子添加剂亦具有关键作用。但相关的机理研究基础研究较少，多为经验摸索。本项目从目前关注较少的制剂添加剂出发，深入研究其与酶分子相互作用规律。借助先进的计算机模拟技术结合现代分析仪器表征手段，及结构化学、界面化学、分子动力学等理论，从酶蛋白分子内和分子间氢键、离子键、静电相互作用、疏水作用及超分子空间结构变化等方面，构建糖类化合物与对脂肪酶相互作用机理模型。并从蛋白超分子空间结构变化及其所带来的构型构效关系变化等多方面揭示分子层面上糖类化合物促进脂肪酶催化效果作用机理。该模型的建立及机理探明可用于预判酶制剂过程中添加剂选择，特别是针对于不同反应类型选择特定糖类化合物作为添加剂，为酶制剂工业添加剂的选择提供可行的理论指导。

脂肪酶是一种重要的工业催化合成酶，其可在有机相中催化合成多种有机化学品。为提高酶制剂的稳定性及催化活性，降低生产成本，添加剂是酶制剂中的关键成分。目前酶制剂添加剂技术多为国外公司技术垄断，而其作用机理尚无统一解释，导致酶制剂用添加剂研发工作进展缓慢。因而从机理层面研究添加剂对脂肪酶的作用机理，从而开发相应高效低成本的脂肪酶制剂添加剂具有重要的科学意义和实用价值。本研究以一种脂肪酶（Yarrowia lipolytica Lipase 2- YLLIP2）作为研究对象，基于脂肪酶催化机理（bi-bi-ping-pong）阐述了该酶的热失活和极性有机溶剂失活机理，以分子动力学模拟手段结合蛋白质结构及超分子构效关系，进一步解释了β-环糊精通过外环分子羟基基团作用于该酶分子表面极性氨基酸残基的温度和溶剂耐受性改善机理。同时，研究以羟基官能团为线索，重点研究了葡萄糖分子对脂肪酶（YLLIP2）的表面修饰作用，得出葡萄糖通过形成糖-酶超分子构象阻止甲醇分子对酶结构扰动的机理结论。最后，开发了可用于生物柴油生产用的以葡萄糖为主的脂肪酶催化添加剂，生物柴油（脂肪酸甲酯）转化率提高了15.9%；将该类添加剂用于其他油脂产品生产，同样可取的良好效果。本研究的成果，对于设计针对不同反应体系的酶制剂添加剂具有较重要理论意义和应用价值。

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