基于右旋糖酐合成与降解制备低聚异麦芽糖的酶学基础

低聚异麦芽糖（IMO）是一种重要的功能性低聚糖，以异麦芽三糖为主的高聚合度组分是主要益生因子。随着新型4,6-α-葡聚糖基转移酶（4,6-α-GT酶）的发现，我们提出富含异麦芽三糖的IMO高效制备新思路：首先4,6-α-GT酶以淀粉为原料合成富含α-1,6键的右旋糖酐；然后该中间产物再经右旋糖酐酶降解成IMO。前期研究中我们发现，该思路得以顺利实施的关键因素为：提高4,6-α-GT酶合成产物的α-1,6键比率；降低右旋糖酐酶水解底物的非特异性。本项目拟阐明4,6-α-GT酶活性口袋作用模式及成键机制，通过分子改造提升酶合成α-1,6键特异性；解析异麦芽三糖右旋糖酐酶晶体结构，揭示酶产物链长特异性机制，分子改造提高产物异麦芽三糖比率；在此基础上，研究多酶复配反应机制及调控策略，构建产率高且富含异麦芽三糖的IMO制备体系。本研究将为IMO产业技术升级及品质改良提供重要的理论基础。

Isomaltooligosaccharide (IMO) is an important functional oligosaccharide, and the high molecular weight component of IMO mainly composed of isomaltotriose is the major probiotic factor. With the discovery of new 4,6-α-glucanyltransferase (4,6-α-GTase), we propose a new research approach for efficient preparation of IMO rich in isomaltotriose: firstly, starch is used as a raw material to synthesize dextran rich in α-1,6 bond; secondly, the intermediate product is degraded into IMO by dextranase. In the previous research, we found that the key factors for the successful implementation of this approach are increasing the α-1,6 bond ratio of the 4,6-α-GTase synthesis product and reducing the non-specificity of the substrate hydrolyzed by dextranase. This project intends to clarify the action mode of 4,6-α-GTase active pocket and the mechanism of bonding, and to improve the specificity of α-1,6 bond synthesis by molecular modification; the crystal structure of isomaltotriose dextranase and the specific mechanism of product chain length will be revealed, and the ratio of isomaltotriose produced by isomaltotriose dextranase will be increased by molecular modification; on this basis, the multi-enzyme complex reaction mechanism and regulation strategy will be clarified, and an IMO preparation system with high yield and rich in isomaltotriose will be constructed. This research will provide an important theoretical basis for IMO industrial technology upgrade and quality improvement.