基于底物结构特征的黄原胶内切酶的催化性能解析及分子改造

黄原胶降解产生的低聚糖具有良好的生理活性，是一种有价值的新型功能性寡糖。然而，黄原胶的复杂结构造成其主链酶解效率低且切割位点随机，获得的寡糖产物为聚合度分散的混合物，不利于后续构效分析和工业化制备。因此，基于黄原胶结构特征的内切酶XCD的催化性能解析及分子改造成为亟待解决的问题。本项目拟在梳理黄原胶多级结构与酶解效率关系的基础上，建立低剂量离子液体-黄原胶内切酶水解体系，实现黄原胶主链的高效水解；同时，阐明糖结合模块SBM识别黄原胶侧链的分子机理，并通过分子改造提高SBM离子液体耐受性；最后，构建多组件XCD-SBM嵌合体文库，利用SBM“引导”黄原胶内切酶特异性识别黄原胶五糖单元，实现低剂量离子液体中无序化黄原胶的高效、精准切割。本项目将进一步阐明黄原胶的结构特征影响其内切酶水解效率和底物识别切割方式的分子机理，获得的新信息和新材料对精准化研制功能性寡糖具有重要理论意义和经济价值。

The xantho-oligosaccharide produced by xanthan degradation has excellent physiological activities. Therefore, xantho-oligosaccharide has proved to be a valuable and novel functional oligosaccharide. However, due to the complex structure of xanthan, the enzymatic cleavage of xanthan backbone is hard and random, leading to mixed oligosaccharides with different degree of polymerization, which obstacles the study of structure-activity relationship. Thus, it is important to reveal and rationally improve the catalytic performance of endo-xanthanase based on xanthan structural properties. In this project, to achieve high-efficient hydrolysis of xanthan backbone, we aim to establish a low-dose ionic liquid-enzymatic hydrolysis system, based on clarifying the correlation between the tertiary structure of xanthan and enzymatic hydrolysis efficiency. Meanwhile, the recognition mechanism of xanthan side chain-binding module SBM is going to be revealed, and ionic liquid-tolerant SBM will be constructed by saturation mutagenesis. Finally, to achieve high-efficiency and precise cleaving of disordered xanthan in low-dose ionic liquid, the multimodular XCD-SBM chimera library will be constructed, and with the "guidance" of SBM, XCD will specifically recognize and cleave the pentasaccharide unit of xanthan. Our project will further elucidate the effect of xanthan structural characteristics on the hydrolysis efficiency and behavior of xanthanase, and is significant for precise production of functional oligosaccharides at food grade.