麦芽四糖生成酶的催化机制及其产物定向合成研究

Maltotetraose is a new sugar source having nutrition and function. With growing potential applications of maltotetraose in the food industry, it is particularly important to achieve high-performance in the production of maltotetraose. In order to overcome technical bottlenecks, the project focuses on the applied basic researches concerning catalytic mechanism of maltotetraose-forming amylase (G4-amylase) and regioselective synthesis of maltotetraose. First of all, G4-amylase gene will be gained by the combination of direct screening and directional mining, and the food-grade expression system will be constructed for secretory expression of G4-amylase gene in Bacillus subtilis. Using structural alignment, computer simulation, and site-directed mutagenesis, the underlying reasons mainly producing maltotetraose by G4-amylase will be clarified, and the catalytic mechanism of G4-amylase be revealed. The correlation between product specificity and structure of G4-amylase will be analyzed, and the mutants with ideal product specificity be constructed, which can significantly improve maltotetraose synthesis by G4-amylase. Effects of substrate structure on maltotetraose synthesis influence of enzymatic synthesis will be elucidated, and the methods for screening and rationally designing the suitable substrates be established. Finally, highly efficient and regioselective enzymatic synthesis of maltotetraose will be achieved. These researches will lay a solid theoretical foundation for the industrial production of maltotetraose, promote the rapid development of maltotetraose-related industries, and provide theoretical supports for developing other maltooligosaccharide-forming enzymes and their products.

麦芽四糖是一种集营养与功能于一体的新糖源。随着麦芽四糖在食品工业中的应用潜力越来越大，着力于实现其高效生产显得尤为重要。为了突破技术瓶颈，本项目主要在麦芽四糖生成酶（G4-淀粉酶）的催化机制及其产物定向合成方面进行一定的应用基础研究。首先，开展基于直接筛选和定向“钓取”相结合的G4-淀粉酶基因挖掘，并构建酶基因的食品级枯草芽孢杆菌分泌表达系统；利用结构比对、计算机模拟以及定点突变等手段，阐明G4-淀粉酶主产麦芽四糖的内在原因，揭示酶的催化机制；深入解析G4-淀粉酶产物专一性与其结构的相关性，构建具有理想产物专一性的酶突变体，显著提高酶合成麦芽四糖的能力；明确淀粉分子结构对麦芽四糖酶法合成的影响规律，建立底物筛选及理性设计方法，最终实现麦芽四糖的酶法高效、定向合成，为麦芽四糖工业化生产打下坚实的理论基础，促进麦芽四糖相关产业的快速发展，并为其它麦芽低聚糖生成酶及其产物的开发提供理论参考。

麦芽四糖是一种集功能和营养为一体的新糖源，随着麦芽四糖在食品工业中的应用潜力越来越大，着力于实现其高效生产显得尤为重要。为了突破技术瓶颈，本项目主要在麦芽四糖生成酶的催化机制及其产物定向合成方面进行了一定的应用基础研究。本项目挖掘并高效分泌表达了具有突出产物特异性的麦芽四糖生成酶基因，发现其水解淀粉生产麦芽四糖的比例达75%以上；在此基础上，利用结构比对、计算机模拟以及定点突变等手段，揭示了酶的催化机制，并深入研究了结构特征对产物特异性的影响，发现糖苷配基位点与底物非还原端之间分子间接触的强弱对底物的定位起主导作用，进而决定了产物类型；同时，结合具有不同产物特异性的麦芽低聚糖生成酶，研究并验证其构效关系，并且基于理性设计构建具有理想产物专一性的酶突变体W139Y和G109D，显著增强了对产物类型的定向调控。此外，本项目采用麦芽低聚糖生成酶和普鲁兰酶协同催化技术，得到了单一聚合度麦芽低聚糖高效、定向合成的技术路线，底物转化率达93%以上，麦芽低聚糖产率超过83%，为麦芽四糖工业化生产打下坚实的理论基础，促进麦芽四糖相关产业的快速发展，并为其它麦芽低聚糖生成酶及其产物的开发提供理论参考。