分支葡聚蔗糖酶糖基化修饰类黄酮的催化识别机制及其分子改造研究

Flavonoids are natural plant secondary metabolites and exhibit various physicochemical and biological activities, including anti-oxidation, anti-bacterial, anticarcinogenic and cardioprotective et al. Thus, they represent very promising candidates to develop new active compounds of interest for the pharmaceutical, food and cosmetic industry. Enzymatic glycosylation is an effective way to generate flavonoid derivatives to enrich the structural diversity and improve the physicochemical properties of flavonoids, especially their solubility and stability. Branching sucrases of glycoside hydrolase family 70 (GH70)，a recently discovered subfamily of GH70，are very attractive transglucosylases, which use the cheap and renewable sucrose as a donor of glucosyl units. Notably, they are promiscuous on the acceptor substrate and are capable of glucosylating diverse hydroxylated acceptors, including flavonoids. However, the molecular mechanism of branching sucrase recognizing non-natural acceptor substrate flavonoids is unclear and their catalytic efficiency for flavonoid glucosylation remains limited. Our present study aims to elucidate the catalytic mechanism of flavonoids glucosylation by branching sucrase and improve catalytic efficiency of flavonoid glucosylation by enzyme evolution. Firstly, the glucosylation of different flavonoids by branch sucrases will be systematically investigated to analyze the effect of flavonoid structure features such as the number and position of hydroxyl groups, unsaturated double bonds and orientation of B ring on flavonoid glucosylation, especially on the regioselectivity. Secondly, the binding mode of flavonoids in the active site of branching sucrase will be analyzed using homology modeling and molecular docking to predict key amino acids for the binding of flavonoids. The functional roles of these key amino acids on the regioselectivity of the flavonoid glucosylation will be investigated by site-directed mutagenesis. Thirdly, iterative saturation mutagenesis will be performed to improve the flavonoid glucosylation efficiency of branching sucrase by directed evolution. Lastly, the glucosylation reaction parameters, including the concentration of acceptor substrate, the ratio of donor and acceptor substrate, the load of enzyme and reaction time, will be optimized. This project is expected to shed novel insight on the flavonoid glucosylation by branching sucrase and provide valuable tools for the glucosylation of flavonoids.

植物类黄酮是一类重要的生物活性物质，具有极高的药用价值。糖基化修饰是拓展类黄酮结构和功能多样性并改善其理化性质的重要途径。分支葡聚蔗糖酶具有天然的转糖苷活性，可以以廉价易得的蔗糖为糖基供体对类黄酮进行糖基化修饰，但是分支葡聚蔗糖酶糖基化修饰非天然受体底物类黄酮的催化识别机制及其催化效率的影响因素并不清楚。本项目拟首先系统分析分支葡聚蔗糖酶对具有不同结构特征的类黄酮物质的糖基化修饰反应，明确类黄酮羟基数目和位置、不饱和双键以及B环连接位置等对其糖基化修饰的影响；其次综合运用同源模建和分子对接等方法分析类黄酮受体底物在分支葡聚蔗糖酶活性中心架构中的结合模式，研究受体底物结合位点氨基酸在类黄酮糖基化位置选择性中的功能；进一步利用迭代饱和突变等技术，对酶分子进行改造，提高类黄酮糖基化效率。本项目有望阐明分支葡聚蔗糖酶糖基化修饰类黄酮的催化识别机制，为实现类黄酮糖苷化合物的大规模生产提供新的工具。

类黄酮等多酚类化合物具有抗氧化、抗菌、抗肿瘤、抗衰老和有效预防动脉硬化等多种生理活性，在医药、食品和化妆品等领域具有广泛的应用前景。但类黄酮等多酚类化合物存在溶解度低、稳定性差、生物利用度低和口感苦涩等问题，严重制约了其实际应用。糖基化修饰可以极大的改进多酚化合物的上述理化性质，因此发展高效的酶法糖基化技术，是促进类黄酮等多酚化合物开发应用的关键。GH70家族分支葡聚蔗糖酶具有高效的转糖苷活力和显著的受体底物杂泛性，具有糖基化修饰多酚化合物的巨大潜力。本项目研究系统分析了分支葡聚蔗糖酶GtfZ-CD2糖基化具有不同结构特征的类黄酮化合物的效率和产物组成，结果表明具有邻羟基结构的类黄酮化合物糖基化的效率普遍较高。但是影响糖基化效率的因素不仅只有羟基的数量和相对位置，还包括C环的不饱和双键和B环的连接位置。值得注意的是类黄酮的糖基化反应生成多个单糖基化合物和多糖基化合物，说明分支葡聚蔗糖酶GtfZ-CD2对羟基位置的选择性并不高。利用具有不同羟基相对位置的苯二酚类化合物研究了羟基位置选择性的分子机制，表明分支葡聚蔗糖酶对邻苯二酚的糖基化效率显著高于间苯二酚和对苯二酚。GtfZ-CD2糖基化修饰苯二酚的反应动力学分析发现该酶对邻苯二酚的Km显著低于间苯二酚和对苯二酚，说明其对邻苯二酚的结合能力明显强于间苯二酚和对苯二酚，从而使邻苯二酚的糖基化效率高于间苯二酚和对苯二酚。通过分支葡聚蔗糖酶GtfZ-CD2的同源模建和分子对接分析表明非攻击性羟基与分支葡聚蔗糖酶GtfZ-CD2受体底物结合位点氨基酸残基的相互作用的区别可能是其羟基相对位置选择性的重要影响因素。进一步对受体底物结合位点氨基酸的半理性突变改造得到对苯二酚糖基化效率显著提高的突变体L1560Y，并通过反应条件的优化使α-熊果苷产量达到55.8 mM，转化率为27.9%。该项研究的成果对类黄酮等多酚类化合物的工业化应用中存在的问题提供了可行的解决方案，也为葡聚蔗糖酶的开发应用奠定了重要的理论基础。