Study of an extremophilic ß-endoglucanase

硫酸根中的ß-内切葡聚糖酶SSO1949是一种性能优异的嗜极酶。该酶在pH 1.8和80℃时活性最高，在酸性条件下比在中性条件下更稳定。这种嗜极性的组合在其他ß-内切葡聚糖酶中还没有报道。对于从纤维素生物质中生产可发酵糖，嗜酸型ß-内切葡聚糖酶可能具有很大的优势，因为原料生物质必须进行预处理，而预处理通常是用酸进行的。在这个应用中，我们想要研究SSO1949的生化和结构细节，这些细节构成了SSO1949极低pH值的基础，我们打算提高SSO1949对结晶或酸胀纤维素的催化效率。我们将构建活性位点发生氨基酸变化的突变体，并研究突变体的活性。为了使氨基酸变化的影响合理化，SSO1949的晶体结构将是有价值的，也是本项目的一部分。此外，我们将通过展开实验研究SSO1949在酸性条件下的稳定性，以了解该酶在低ph下的高稳定性。此外，我们将构建SSO1949的催化结构域与碳水化合物结合模块的融合，以提高对纤维素的催化效率。最后，我们将随机产生突变酶，并对其进行酶活性增加的筛选。该应用程序将酶学的基础研究与使用耐热和耐酸稳定的ß-内切葡聚糖酶进行纤维素解聚的应用研究相结合。

The ß-endoglucanase SSO1949 from Sulfolobus solfataricus is an extremophilic enzyme with outstanding properties. This enzyme has its highest activity at pH 1.8 and 80 °C, and it is more stable under acidic conditions than at neutrality. Such a combination of extremophilic properties has not been reported for any other ß-endoglucanase. For the production of fermentable sugars from cellulosic biomass, an acidophilic ß-endoglucanase could be of great advantage since the raw biomass must be pretreated which is most commonly done with acids. In this application we want to investigate the biochemical and structural details which form the basis of the extremely low pH optimum of SSO1949 and we intend to improve the catalytic efficiency of SSO1949 against crystalline or acid-swollen cellulose. We will construct mutants with amino acid changes at the active site and study the activity of the mutants. To rationalize the effects of amino acid changes, a crystal structure of SSO1949 would be valuable and is also part of this project. Moreover we will study the stability of SSO1949 under acidic conditions with unfolding experiments in order to understand the high stability of the enzyme at low pH. Furthermore we will construct fusions of the catalytic domain of SSO1949 with carbohydrate binding modules in order to improve the catalytic efficiency against cellulose. Finally we will randomly generate mutant enzyme and screen them for increased enzymatic activity. The application combines basic research in enzymology with applied research for the use of heat- and acid-stable ß-endoglucanase for cellulose depolymerisation.