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

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.

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。

本项目按照研究计划书实施研究计划，并达到预期研究目标。具体而言，首先构建高质量的宏基因组库，然后利用Rec A蛋白结合磁珠亲和纯化法专一的富集钓取菊糖果糖基转移酶基因，结合pH切换结合DNS还原糖测定法进行进一步筛选和功能验证。对于其中有潜力的菊糖果糖基转移酶，通过分子定向进化的方式进一步改造。在酶的筛选与定向改造过程中，综合运用基于intein的读码框选择法和pH切换结合DNS还原糖测定法提高酶活筛选效率。对于催化活性高而且对低聚果糖底物特异性强的酶，进行酶学性质分析，从而为实现酶的进一步改造奠定基础。在此基础上，选择对低聚果糖催化效率最高的菊糖果糖基转移酶与来自黑曲霉的β-蔗糖果糖基转移酶进行纯化，采用新的介孔CLEAs-可得然胶-二氧化硅复合微粒（CLEAs-Curdlan-SiO2）以及基于ELP标签和磷酸钙的纳米花方法进行固定化，并用固定化酶以蔗糖为底物进行偶联催化。同时本项目实施过程中建立的一系列新方法，包括基于宏基因组区段改组库的构建方法，基于ELP标签建立了新型蛋白表达和纯化系统，对于提高突变文库建库质量，提升重组蛋白的表达水平，提高不溶性蛋白纯化效率等方面，具有重要的参考价值。此外，针对分子克隆和突变库构建过程中存在的问题，本研究建立了一系列高效的质粒载体。这些质粒载体具备更加高效、容易、假阳性少等优点，在国内外多家实验室中已获得应用，并展示出比经典载体更出色的特征。