以淀粉为底物高效制备海藻糖的酶学基础研究

Trehalose is a non-reducing disaccharide that comprises two glucose molecules connected by an α-1,1 linkage, which is used as high quality sweetener and natural food preservative. It is an urgent problem to be solved that how to prepare trehalose with high efficiency and low cost to meet its growing demand. Maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) are the key enzymes in the preparation of trehalose using starch as substrate. It is indicated from literature and the previous results of applicants that the poor maltotetraose/maltotriose affinity of MTSase, and the production of by-products maltose and glucose catalyzed by MTHase act as the major limitations for increasing the trehalose yield. According to these factors, this project aims to explore the mechanisms of the substrate affinity of MTSase and the by-products production by MTHase, and subsequently obtain new MTSase and MTHase by gene mining and function reconstruction. Furthermore, the project intends to reveal the enzyme conversion mechanism and regulation strategy, based on the complexity of the starch substrate and versatility of products in the reaction process, and then construct the high-efficient enzyme catalysis system for preparation of trehalose. This project will provide the significant theoretical basis not only for the structure and function of MTSase and MTHase, but also for the more efficient preparation of trehalose.

海藻糖是由两个葡萄糖通过α-1,1-糖苷键连接而成的非还原性二糖，是一种优质食用甜味糖和天然食品保鲜剂。如何高效低成本制备海藻糖以满足其日益增长的需求为目前亟需解决的问题。麦芽寡糖基海藻糖合成酶(MTSase)和麦芽寡糖基海藻糖水解酶(MTHase)是以淀粉为底物制备海藻糖的关键酶。综合文献资料及申请人前期工作分析发现，MTSase对麦芽四糖和麦芽三糖亲和性差，以及MTHase水解麦芽寡糖产生麦芽糖和葡萄糖副产物为制约海藻糖产率提升的关键因素。针对这些问题，本项目将在研究MTSase底物亲和性和MTHase副产物产生机制基础上，开展酶基因挖掘和功能优化，获得新型MTSase和MTHase；针对淀粉底物复杂性和中间产物多样性，探索酶反应机制和调控策略，构建高效制备海藻糖的酶反应体系。本项目将为MTSase和MTHase结构功能研究以及海藻糖高效制备奠定重要的理论基础。

海藻糖由两个葡萄糖基通过α-1,1糖苷键连接形成，是一种安全、稳定的天然二糖，素有“生命之糖”的美誉，作为优质食用甜味糖和天然食品保鲜剂，广泛用于饮料、糖果、烘焙和速冻食品等领域。为了解决海藻糖制备用酶应用性能低成本高的问题，本研究结合晶体结构解析和分子改造，获得了催化性能和热稳定性提升的麦芽寡糖基海藻糖合成酶突变体，并通过定向进化获得了热稳定性提升的麦芽寡糖基海藻糖水解酶突变体，提高了海藻糖产量，同时有助于降低微生物污染机率，并进一步探究了相关分子机制；针对海藻糖制备过程中寡糖中间体难以利用的瓶颈问题，提出了利用高特异性歧化反应将寡糖分子延长再利用的思路，并通过基因挖掘和分子改造获得了对麦芽寡糖亲和力强的环糊精葡萄糖基转移酶；将以上三种海藻糖制备用酶在枯草芽孢杆菌中进行重组表达和发酵制备，并构建了以淀粉为底物制备海藻糖的多酶偶联催化体系，获得了海藻糖高效酶法制备。相关研究成果发表论文13篇（其中SCI论文8篇），授权发明专利9件（国际专利2件），申请发明专利2件，获国家技术发明二等奖和中国商业联合会科技进步奖特等奖。培养博士研究生2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海藻糖制备用酶的功能优化具有重要指导意义，而且为推动海藻糖产业革新发展以及其他食品酶生产和应用提供了重要借鉴。