新型壳聚糖酶结构-功能解析及其理性改造研究

Chitooligosaccharide, the hydrolyzed product of chitosan, is a beneficial functional oligosaccharide, and exhibits numerous biological functions such as modulating intestinal flora, immune activation and antifungal. However, as the endo-type catalytic mode of natural chitosanases, the preparation of purified chitooligosaccharides with defined degree of polymerization is still difficult. The bioactivities of chitooligosaccharides products are often determined using heterogeneous and/or relatively poorly characterized oligosaccharides mixtures, and it is difficult to determine which molecules are responsible for the observed biological effects. So it is clearly essential to explore novel chitosanases for the preparation of chitooligosaccharide with defined degree of polymerization for both scientific and industrial application value. Here, on the basis of the structure and function of chitosanases, the proposal will solve the crystal structures of glycoside hydrolase (GH) family 46 and GH family 75 chitosanases and their complexes with substrates and investigate the catalytic mechanisms, enzymatic properties and molecular mechanisms of their substrate recognition. Furthermore, these chitosanases will be modified by structure-based rational design with the aim of increasing the ratio of chitooligosaccharides with specific degree of polymerization in the enzymatic hydrolysate. This study will confer structural insights on molecular mechanism of products of various chitosanases with different degree of polymerization. It will not only provide theoretical basis for the catalytic mechanism of different GH family chitosanases, but also have important application potential in oligosaccharides industries.

壳寡糖作为一种重要的功能性低聚糖，在调节肠道菌群、提高机体免疫力和抗真菌等方面具有广阔的应用前景。然而由于天然壳聚糖酶的内切型反应机制，目前特定聚合度区间壳寡糖的制备还存在诸多难题，造成壳寡糖单体构效关系不明确，限制了壳寡糖的进一步研究和应用。本课题基于壳聚糖酶的结构与功能关系，通过X-射线晶体法解析GH46和GH75家族新型壳聚糖酶的蛋白质结构，探索壳聚糖酶的关键氨基酸残基位点及底物识别机制。结合结构与功能研究阐明不同家族壳聚糖酶的催化特性及催化机理。进而基于结构信息对壳聚糖酶进行理性设计改造，优化酶解产物聚合度分布，阐明影响壳聚糖酶产物聚合度差异的分子机制，获得适宜于特定聚合度区间壳寡糖制备的特异性壳聚糖酶。本研究从结构、功能及应用方面对壳聚糖酶展开系统研究，为壳聚糖酶的催化机理及壳寡糖酶法制备相关研究提供了理论基础，具有重要的理论意义和应用价值。

功能性低聚糖是国内外食品营养与健康领域的研究热点之一。壳寡糖是一种重要的功能性低聚糖，在调节肠道菌群、提高机体免疫力和抗真菌等方面具有广阔的应用前景。然而由于天然壳聚糖酶的内切型反应机制，目前特定聚合度区间壳寡糖的制备还存在诸多难题，造成壳寡糖单体构效关系不明确，限制了壳寡糖的进一步研究和应用。本项目以特定聚合度区间壳寡糖酶法定向制备为目标，利用生物信息学手段挖掘得到了两种具有应用潜力的壳寡糖酶，通过X-射线晶体法解析得到了两种新型GH46家族壳聚糖酶的蛋白质结构及其与壳四糖、壳五糖的复合物结构，阐明了GH46家族壳聚糖酶催化凹槽底物识别的分子机制及变构催化反应机制。基于结构信息对壳聚糖酶进行理性设计改造及结构域融合改造，优化酶解产物聚合度分布，阐明了影响壳聚糖酶产物聚合度差异的分子机制，提升目标壳聚糖酶稳定性的同时实现了壳聚糖酶的一步纯化-固定化。建立了基于固定化酶反应器、酶膜耦合反应器的不同聚合度区间壳寡糖产物制备策略。本研究从结构、功能及应用方面对壳聚糖酶展开系统研究，为壳聚糖酶的催化机理及壳寡糖酶法制备相关研究提供了理论基础，具有重要的理论意义和应用价值。

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