解淀粉芽孢杆菌来源酸性木聚糖酶Bxyn酸稳定性和催化效率联结变化的分子控制机制

Xylanase is stable in acidic environment and has high catalytic efficiency, which is essential factor for its application in low pH industrial production. How to make acid stability and catalytic efficiency are effectively controlled is one of the important issues to achieve the large-scale industrial application of acid xylanase. In the work, the acid xylanase Bxyn from Bacillus amyloliquefaciens was studied. Homologous modeling and site-directed mutagenesis were used to determine the key sites of acid stability. The change of group conformation and reaction force of the key sites of acid stability in the catalytic hydrolysis process: substrate recognition, substrate hydrolysis and product release were analyzed. Around the key sites of acid stability, the key factors affecting the catalytic efficiency were comprehensively investigated and verified. Furthermore, the coupling change rule of acid stability and catalytic efficiency were explored and the relationship between the molecular structure of xylanase and the coupling change of acid stability and catalytic efficiency were revealed. The molecular control mechanism of the coupling change of acid stability and catalytic efficiency were clarified. It lays a theoretical foundation for the large-scale industrial application of acid xylanase.

木聚糖酶酸性条件下稳定且具有较高催化效率是其应用于低pH条件工业生产的必备要素，如何实现酸性木聚糖酶的酸稳定性和催化效率有效可控以贴合低pH生产条件工业领域成为解决酸性木聚糖酶实现大规模工业应用问题的重要课题之一。本项目以解淀粉芽孢杆菌来源酸性木聚糖酶Bxyn为研究对象，通过蛋白质同源建模及定点突变等技术确定酶分子酸稳定性关键位点及其位置，解析酸稳定性关键位点在催化水解过程（底物识别、底物水解和产物释放）中的基团构象及作用力变化，围绕酸稳定性关键位点对影响酶催化效率的关键因素进行综合考察并验证，探究酸稳定性和催化效率联结变化规律，揭示木聚糖酶分子结构与酸稳定性和催化效率联结变化相对应的关系，阐明酸稳定性与催化效率联结变化的分子控制机制，为实现酸性木聚糖酶大规模工业应用奠定有效的理论基础。

木聚糖酶酸性条件下稳定且具有较高催化效率是其应用于低pH条件工业生产的必备要素。本项目重点研究了木聚糖酶结构与其酸稳定性及催化效率的相互关联。已有的关于酸性木聚糖酶的耐酸机制认为邻近于催化活性位点的氨基酸残基与质子供体相互作用，质子供体的电离状态受到影响致使木聚糖酶酸稳定性发生一定的变化。而催化裂缝也是酶结合识别与水解底物的区域，裂缝中氨基酸残基的变化可能会触动分子作用力网络而影响到酶与底物的相互作用直至酶的催化效率。因此，在催化裂缝中进行分子改造探究酶结构与其酸稳定性及催化效率的关联不可行。通过蛋白同源建模，改造策略均集中于N-端，未直接触及木聚糖酶催化裂缝，在保证木聚糖酶催化效率的同时，探究N-端关联酸稳定性及催化效率的变化规律。重组木聚糖酶XynH和xynP经N-端替换所构建的突变体酸稳定性均有所下降，替换的氨基酸序列较原序列变短，同时易形成氢键的极性氨基酸变少，而GH11木聚糖酶的N端区域是其折叠起始处，该N-端替换使得酶结构可能变得松散，在低pH环境下更加不稳定。重组木聚糖酶xynT经N-端替换所构建的突变体酸稳定性增强，替换的原氨基酸序列中较大的苯环（Phe）被消除，可能进一步增加了N-端折叠的紧凑性，酶结构稳定性增强，对低pH环境适应性增强。据目前报道，木聚糖酶的N-端结构通常与酶的耐热性紧密相连，但该结构与酶的酸稳定性的关联却研究极少。木聚糖酶的N-端结构不仅与酶的热稳定性相关，对其酸稳定性也存在一定的影响。N-端结构的变化影响着木聚糖酶结构的稳定性，致使酶在低pH环境下的适应性改变。项目研究成果对实现酸性木聚糖酶的酸稳定性和催化效率有效可控以贴合低pH生产条件工业领域具有重要的理论指导意义。