新一代健康甜味剂甜菊糖苷D和M生物合成关键酶催化机理及酶活性优化的研究

This project aims to establish the method to prepare a new-generation of natural healthy stevia sweeteners rebaudioside D and M with low-calorie but high sweetness intensity from Stevia rebaudiana massively cultivated in China. Using the features of the ordered dual-site reaction of two glucosyltransferases found in the preliminary experiments, it is expected to convert the abundant stevia glucosides with bitter aftertaste to the premium rebaudioside D and M with much clean sweet taste by enzymatic catalysis, which helps to realize the impossible purification and application of rebaudioside D and M from leaves of S. rebaudiana in large scale due to their trace natural contents of about 0.6% of stevia glucosides. In this project, the crystal structures of two glucosyltransferases will be solved to elucidate the mechanism and reflect the whole procedure of the ordered dual-site reactions, examine the relevance between the structures and activities of the particular dual-site reactions by site-directed mutagenesis, discover the structural basis to control the activities of dual-site reactions. Based on the results in the project, it is desired to optimize the structures of two glucosyltransferases and increase the reaction velocities in the rate-limiting step during the catalysis. As the result, a high efficient in vitro catalysis system and an advanced, innovated technology will be established by using the collaboration of two optimized enzyme reactions to efficiently produce the premium stevia sweeteners of high quality, which provides the healthy sweeteners for food additives industry, promotes the development of sweetener crop stevia cultivation in China with high profits, and prevents obesity, diabetes and other diseases due to the excessive high-calorie sugar intake.

本项目拟从我国大量种植的甜料作物甜叶菊制备天然、低热量、高甜度的新一代健康甜味剂甜菊糖苷D和M。利用申请人最近发现的两个葡萄糖基转移酶所具有的双位点有序反应特性，通过酶促反应实现从有后苦味的甜菊糖主要成分到甜味纯正的甜菊糖苷D和M的生物转化，解决甜菊糖最佳成份（甜菊糖苷D和M）天然含量低（约0.6%），难以规模化制备和应用的难题；解析两个葡萄糖基转移酶及其复合物的晶体结构，深入研究其关键的双位点有序反应过程，揭示其催化机理；利用定点突变验证结构与独特酶学性质的对应关系，发现控制酶双位点反应活性的关键结构因素；优化酶结构，提高双位点有序反应限速步骤的速率；利用两个酶的协同作用，建立高效体外催化体系，形成自主创新的先进甜菊糖苷D和M高质、高效生物合成关键技术，为食品添加剂行业提供急需的健康甜味剂，对提高我国甜料作物甜叶菊种植业的经济效益，预防因高热量糖过量摄入引起的肥胖、糖尿病等具有重要意义。

产自甜叶菊的甜菊糖苷D和M甜度高、热量低，是新一代健康甜味剂，但其天然含量严重不足，无法通过直接提炼纯化的方法实现量产。前期工作发现两个糖基转移酶负责催化形成甜菊糖苷的必需糖苷键，是制约甜菊糖苷D和M生物转化和天然含量的关键因素。本项目具有“需求牵引，突破瓶颈”的科学问题属性，以解析两个关键酶与不同底物组合的9种复合物晶体结构为起点，发现罕见的酶和底物结合关系；系统分析酶、底物、产物的对应关系，揭示酶识别底物的具体部位和识别机制，摒弃了氢键在糖基识别中的常规认识，提出并验证“非方向性的疏水作用可主导底物结合方向”的新概念；与此同时，展现酶在催化过程中，底物可自由翻滚，发生分子的横向旋转及糖基基团的纵向翻转，从而将不同位置的反应基团递送到催化位点，阐明多样化糖基转移反应的结构基础以及酶解决催化反应的方向灵活性和成键特异性这对矛盾的策略；揭示酶匹配不同方向，不同形式糖基化反应的底物识别模式及其相互转换关系，剖析酶和底物多样化结合的新模式，发现提升酶特异性和催化效率的新途径；通过结构指导下的精准改造，升级甜菊糖苷D和M生物合成过程的必需催化成员，获得理想催化性质的新酶源，建立高效体外催化体系，形成甜菊糖苷D、M高效生物合成关键技术，解决规模化生产的瓶颈问题；为食品添加剂行业提供健康甜味剂，对提升甜菊糖苷深加工的经济效益，预防因高热量糖过量摄入引起的健康问题都具有重要意义。

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