新型双结构域木聚糖酶的分子改造和功能研究

项目名称： 新型双结构域木聚糖酶的分子改造和功能研究

项目编号： No.31300071

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 生物科学

项目作者： 王钱福

作者单位： 中国科学院上海生命科学研究院

项目金额： 24万元

中文摘要： 开发活性高、耐受性好的木聚糖酶是降低其在造纸和纤维素乙醇等领域应用成本的关键，对木聚糖酶中非催化结构域的结构和功能的解析有助于酶工程改造、拓展其应用。本课题的前期工作挖掘到一些新型高活性、pH适应范围宽的双结构域木聚糖酶，针对此类木聚糖酶热稳定性差的缺陷，拟通过结合蛋白定向进化技术进行改造，筛选热稳定性提高突变酶、确定热稳定性相关氨基酸序列，通过晶体结构和三维结构建模解析其热稳定性结构特征和分子机制；新型木聚糖酶中的Big2结构域和没有同源序列的新结构域，都是糖基水解酶中普遍存在且功能未知的结构域，本课题将联合结构域分离、交换、融合和linker优化、以及晶体结构解析，阐明非催化结构域的功能和结构、以及非催化结构域影响木聚糖酶特性的机制。本研究不仅可得到有应用价值的木聚糖酶，而且为解析木聚糖酶的热稳定性机制和糖基水解酶中非催化结构域的功能提供新的思路和方法，具有重要的理论意义和应用前景。

中文关键词： 木聚糖酶；定向进化；热稳定性；可溶性表达；

英文摘要： The xylanase is widely used in the paper-making and cellulose ethanol industries, and the key to reducing its application cost is developing high activity and tolerability enzyme. Many xylanase composes both the catalytic domain and non-catalytic domain, and some of them are domains of unknown function (DUF), which function should be illuminated to extending its application area. Some novel double-domain xylanase with high activity and wide pH tolerant were isolated, and we want to improve its thermal stability by protein directed evolution and determine the amino acid sequences and then analyze the structure feature and molecular mechanism related to thermostability. In this study, we will expound the mechanism of the effects of non-catalytic domain, such as Big2 and DUF which are widely existed in glycosyl hydrolases (GHs) family, on the characteristics of xylanase by domain separation, swapping, fusion and liker optimization and crystal structure analysis. This research will not only obtain xylanase with application value and also provide new idea and method for expound the thermostability mechanism of xylanase and the function of non-catalystic domain in GHs, which are significant to the theoretical research and practical application.

英文关键词： xylanase；directed evalution；thermostability；soluble expression；