β－葡萄糖苷酶水解萜烯糖苷的底物特异性关键氨基酸研究

In food, the aromatic precursors (flavor precursor) are glycoside compounds, which can be hydrolyzed by beta-glucosidase and release volatile aromatic compounds. Thus, beta-glucosidase play an very important role in food itself and food processing for flavor release. However, lack of knowledge about molecular foundation of the substrate specificity, it is hard to provide a basis for food aromatic precursors hydrolysis to select and mutate the appropriate beta-glucosidase. This study is based on our research on relationships between protein structure and activity and research on molecular design and modification of enzyme. It will be performed in the way that analysis of the key amino acids participated in binding and catalysis of substrate in the process of beta-glucosidase hydrolysis of glycosidic perpenses compounds. With the aid of molecular docking technique, protein point modification techniques and isothermal titration calorimethy techniques, the molecular mechanism of beta-glucosidase hydrolysis of glycosidic terpenses compounds is analyzed. Then, the relationships of amino acid with glycosidic perpenses substrate would be revealed. This study would provide a strong basis for the directed mutation of beta-glucosidase and liberation of volatile aromatic compound in food.

食品中的芳香类前体物质是糖苷类化合物，β-葡萄糖苷酶具有水解芳香前体物质的能力，是食品自身以及食品加工中进行前体物质水解释放香味不可缺少的酶。但目前缺乏β-葡萄糖苷酶水解芳香前体物质的底物特异性分子基础的研究资料，无法为分子改造和选择适合的β-葡萄糖苷酶用于食品芳香前体物质水解提供基础。本研究在我们所拥有的蛋白质大分子结构和活性关系研究以及酶蛋白质分子改造研究的基础上，分析β-葡萄糖苷酶在水解萜烯醇糖苷芳香前体物质过程中，β-葡萄糖苷酶参与底物结合、底物催化等反应的关键氨基酸。通过分子对接模拟技术、蛋白质定点改造技术以及等温滴定量热技术等的结合，分析和揭示β-葡萄糖苷酶蛋白质分子水解萜烯醇糖苷的作用机理，以及各种氨基酸和底物的相互关系。为定向突变改造β-葡萄糖苷酶、为食品工业中的芳香前体物质的水解释放提供研究基础。

本课题研究了不同微生物来源的β-葡萄糖苷酶对芳香前体物萜烯糖苷类黄酮底物和甜菊糖苷底物的水解活性，获得了目前报道对黄酮底物具有最高的亲和力以及水解催化效率的葡萄糖苷酶，获得了能够作用甜菊糖苷不同位点的糖苷键的葡萄糖苷酶。通过蛋白质结构建模比对分析这些不同来源的β-葡萄糖苷酶，利用易错PCR和定点突变改造技术等手段对这些β-葡萄糖苷酶基因进行突变研究，获得二十几个具有活性的突变体，从中找到了2个对黄酮底物中的黄豆苷和染料木苷水解活性有变化的突变体。然后对这2个突变体进行相关的结构分析和序列分析，一一排除部分氨基酸残基，发现147位点位于活性中心位点附近、557位点的氨基酸位于蛋白表面，推测该位点氨基酸的电荷性与酶活有一定的关联、推测208、218位点的氨基酸跟CBGL与底物对接有一定的关联。