新型双结构域木聚糖酶的分子改造和功能研究

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.

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。

本课题利用蛋白定向进化手段对新型木聚糖酶XYL7进行改造，确定了氨基酸序列第51位和第242位对木聚糖酶XYL7热稳定性有重要影响。在此基础上，在XYL7中引入点突变，得到了两个热稳定性相对野生型有显著提高的突变子XYL7-TC和XYL7-TS。模拟建模分析新型木聚糖酶的结构特征和分子机制，确定疏水相互作用、氢键及芳香族相互作用的增加和改变共同造成了突变子热稳定性的增强。分析发现C端非催化结构域降低了XYL7的可溶性，对XYL7全长序列做截短表达后提高了木聚糖酶的可溶性蛋白表达量且酶学性质未受明显影响。在里氏木霉中成功表达木聚糖酶XYL7，促进了它的实际生产应用。本研究从碱性木聚糖酶出发，对其进行热稳定性的改造，大大提高了热稳定性及其对底物的降解，且在碱性环境（pH 7-11）均有较佳的酶活，这在造纸、纺织等行业有着良好的应用前景。