菊糖果糖基转移酶偶联催化制备双果糖酐III中的关键问题研究

双果糖酐III是一种新型天然甜味食品，而采用双酶偶联催化法比传统的单酶催化法在降低双果糖酐III生产成本方面具有明显的优势。但现有菊糖果糖基转移酶活力低，而且对低聚果糖底物的催化效率很低，严重影响了该偶联催化反应的效率。因此，本研究将首先通过构建宏基因组库，并利用基于RecA蛋白结合磁珠亲和纯化法对宏基因组库中的目的基因进行特异性钓取，进而筛选获得高效催化低聚果糖底物的菊糖果糖基转移酶。然后构建DNA shuffling库和随机突变库，再采用基于蛋白内含子intein的读码框选择和新设计的功能筛选策略，进一步提高该酶对低聚果糖底物的催化效率。然后采用新型的介孔CLEAs-可得然胶-二氧化硅复合微粒固定化酶，并通过偶联催化实现高产双果糖酐III的目标。本研究还将对新菊糖果糖基转移酶进行晶体结构解析，定点突变和生物信息学分析，深入研究酶的催化机理和底物特异性机制，并为酶的进一步改造奠定基础。

Difructose anhydride III is a novel natural sweet food. The cost of producing difructose anhydride III should be much lower if coupled enzyme reaction is performed instead of single enzyme reactions. However, the activities of the current inulin fructotransferases are all very low, and inefficient in catalyzing oligofructose substrates, which has seriously affected the efficiency of the coupled enzyme reaction. Therefore, in this study, metagenomic libraries should be constructed, and the target genes which can encode inulin fructotransferases with higher activity for catalyzing oligofructose substrates should be fished by Rec A protein and magnetic beads-based affinity purification strategies. Then libraries should be constructed by DNA shuffling and random mutagenesis, which should be screened by reading frame selection and a newly designed specific screening strategy to isolate genes encoding inulin fructotransferases with higher activities towards oligofructose substrates. Then the enzymes should be immobilized by novel mesoporous CLEAs-Curdlan-SiO2 microspheres, followed by coupled enzyme reaction to efficiently produce difructose anhydride III. In this study, the novel inulin fructotransferases should be solved spatial structures, followed by site-directed mutagenesis and bioinformatic analysis to reveal their catalytic mechanism and mechanism of substrate specificity. These studies will lay the foundation for further modification of the enzymes.