淀粉分子链重构制备低热量糊精的酶学基础研究

Low caloric dextrin is a special form of soluble dextrin rich in α-1,2、α-1,3、α-1,6 glycosidic bonds or cyclic sugar chain structure. Due to its digestive enzyme-resisting properties, low calorie dextrin has been supplemented in diets as an effective intervention measure for the control of sub-health problems such as obesity. Low caloric dextrin is mainly obtained through molecular reconstruction using starch as the raw material. Current process for its preparation is low in efficiency and high in cost, and is thus unable to meet the demands of the consumer society. The key problems in this process are the lack of specific transglycosidases and the lack of an effective measure for controlling the enzymatic reaction. In the preliminary results, we have identified more than ten different enzymes for the preparation of low calorie dextrin, including branching enzyme, α-glucosidase, and α-cyclodextrin glycosyltransferase. The proposed project will further explore the identification of new enzymes; and investigate enzyme properties, structure and function relationship, as well as the mechanism of the transglycosylation reaction. Based on these studies, the function of enzymes will be optimized through molecular modification to improve their catalytic efficiency and applicability. The mechanism for the directional and quantitative control of the complex enzyme conversion system will also be elucidated. This project provides the indispensible theoretical basis not only for the mechanistic investigation of transglycosidases but also for the more efficient preparation of low calorie dextrin, and has significance to the development of starch deep processing industry.

低热量糊精是一类富含α-1,2、α-1,3、α-1,6糖苷键或环状糖链结构的可溶性糊精的总称。低热量糊精具有抗人体消化酶特性，增加其在摄入组分中的含量是防控肥胖等亚健康问题的有效膳食干预手段。低热量糊精主要是以淀粉为原料，通过分子链重构获得。目前制备效率低、生产成本高、难以满足大众消费者的需求。导致此问题的关键是缺乏适用于低热量糊精制备体系的特异性转糖苷酶和酶反应过程调控手段。前期工作中，本课题组已获得10余种可用于低热量糊精制备的转糖苷酶，包括分支酶、α-葡萄糖苷酶以及α-环糊精葡萄糖基转移酶。项目拟进一步挖掘新酶，研究各类酶特性、结构及转苷作用机制，并在此基础上，通过分子改造优化酶功能以提高其效率和应用适应性，阐明定向有序及定量可控的复合酶转化机制。本项目将为转糖苷酶作用机制研究以及低热量糊精高效制备提供重要的理论基础，对淀粉深加工行业的发展具有积极意义。

淀粉是食用碳水化合物的主要来源，在加工过程中可通过分子重构引入抗/慢消化性糖苷键或将糖链环化获得低热量糊精。低热量糊精具有抗人体消化酶特性，增加其在摄入组分中的含量是防控肥胖等亚健康问题的有效膳食干预手段。目前，低热量糊精制备中存在高性能转糖苷酶缺乏、作用机制不够明确、以及复合酶反应体系调控机制不清晰等关键科学问题，导致制备过程效率低、生产成本高，难以满足大众消费需求。针对该现状，项目围绕淀粉分子重构制备低热量糊精用转糖苷酶的基因挖掘、催化机制、分子改造和复配应用开展了多维、多角度、全链条式研究。在酶的基因挖掘方面，通过序列遗传进化和优势特征基序分析，挖掘并鉴定获得了应用性能优良的α-葡萄糖苷酶、分支酶、4,6-α-葡聚糖转移酶、分支蔗糖酶和环糊精葡萄糖基转移酶等转糖苷酶18个；在酶机制解析及分子改造方面，解析了新型转糖苷酶的晶体结构，利用前期建立的转糖苷酶多尺度模拟分子计算方法解析了该类酶催化转苷反应和键型特异性的分子机制，并进一步通过分子改造提升了转苷活性和产物中抗消化键型的比例，其中抗消化键比例最高可达92%；此外，项目还深入研究了制备低热量糊精的多酶协同反应路径及复合酶协同关系，成功构建了不同类型转糖苷酶的协同反应体系，实现了系列低热量糊精的高效酶法制备。项目共发表SCI论文21篇（第一标注17篇）；申请发明专利19件（美国专利3件），其中3件中国发明专利及2件美国专利获授权；培养研究生24名，其中博士研究生7名；此外，转糖苷酶基因挖掘相关成果是国家技术发明二等奖“淀粉加工关键酶制剂的创制及工业化应用技术”的重要组成部分。本项目研究不仅获得了新型转糖苷酶，丰富了该酶类家族结构与功能关系的酶学理论知识，还创立了高效低热量糊精复合酶转化体系，为低成本制造低热量糊精产品以满足大众人群需求提供了重要的理论基础和关键技术。