寡聚化介导的糖基转移酶热稳定性分子改造机制探究

Glycosyltransferase is capable of transforming carbohydrate into functional sugar. Due to the high temperature when glycosyltransferase used, how to improve thermostability is crucial for its efficient application. Enlightened by the novel theory of “oligomerization is a general evolution pattern for obtaining stable protein”, which presented by Eugene Shakhnovich, a famous professor in the field of protein folding and evolution, the candidate developed an innovative thought to improve the thermostability of glycosyltransferase by oligomerization. The thought was proved to be feasible by the practical oligomerization experiments of several glycosyltransferases. Based on the above, the current project firstly intends to use glycosyltransferases with different structure features as models, aims to obtain enzyme mutants which can form oligomer resulting in higher thermostability by site-specific mutagenesis and directed evolution. Subsequently, to reveal the molecular, thermodynamics and dynamics mechanism of the oligomerization of glycosyltransferases. Finally, according to the studies above, to obtain the universal strategy of enhancing the thermostability of glycosyltransferase by oligomerization and further apply it on the modification of other glycosyltransferase. This project will enable us to expand the methodology of thermerstability reconstruction for enzymes significantly, and also ultimately promote the further application of food enzymes in industry.

糖基转移酶可催化转化糖质资源生成功能糖，鉴于糖基转移酶的高温应用条件，提高其热稳定性成为亟待解决的问题。申请人受蛋白质折叠进化领域著名教授Eugene Shakhnovich提出的“寡聚化是一种普遍的获得稳定蛋白的热适应性进化方式”新理论的启发，形成了基于寡聚化的糖基转移酶热稳定性改造新思路，并通过部分糖基转移酶的改造初步验证了该思路的可行性。在此基础上，本项目拟以具有不同结构特征的糖基转移酶作为模型蛋白，通过定点突变和定向进化方法构建热稳定性提高的糖基转移酶寡聚突变库；对糖基转移酶寡聚体形成的分子机制及热力学动力学机制进行剖析；基于上述研究获得糖基转移酶寡聚化热稳定性改造的普遍方法，并应用于其他糖基转移酶的改造。本项目的实施不仅对拓展酶分子稳定性改造的方法论具有重要价值，而且对推动食品酶乃至其他工业酶的产业化应用具有深远意义。

项目针对目前糖酶应用过程中热稳定性普遍较差，不能满足工业需求的现状，以淀粉加工用关键糖酶蔗糖异构酶、β-淀粉酶及α-淀粉酶等为模型，分别应用氨基酸 B 因子热稳定性改造、易错PCR结合高通量定向进化、结构域保守区域及loop环理性设计改造、构建寡肽融合蛋白、引入二硫键形成寡聚体等手段，获得了热稳定性显著提高的糖酶突变体，并在此基础上对热稳定性提高的机理进行探究，获得了适用于糖酶热稳定性改造的重要方法。相关研究成果发表论文9篇（其中SCI论文5篇），申请发明专利6件，获国家技术发明二等奖1项。培养博士研究生3名，硕士研究生4名。相关产品在企业实施了成果转化及应用。本项目的研究不仅对拓展酶分子热稳定性改造的方法论具有重要价值，而且对推动食品酶乃至其他工业酶的产业化应用具有深远意义。