Study of an extremophilic ß-endoglucanase

硫磺硫化叶菌（Sulfolobus solfataricus）的β-内切葡聚糖酶SSO 1949是一种具有优异性质的极端嗜热酶。这种酶在pH 1.8和80 °C下具有最高活性，并且在酸性条件下比在中性条件下更稳定。对于任何其他β-内切葡聚糖酶，还没有报道过这种嗜极特性的组合。对于从纤维素生物质生产可发酵糖，嗜酸性β-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，这最常用酸进行。在本申请中，我们希望研究形成SSO 1949的极低pH最佳值的基础的生物化学和结构细节，并且我们打算提高SSO 1949对结晶或酸溶胀纤维素的催化效率。我们将构建活性位点氨基酸发生改变的突变体，并研究突变体的活性。为了使氨基酸变化的影响合理化，SSO 1949的晶体结构将是有价值的，也是该项目的一部分。此外，我们将研究在酸性条件下的稳定性的SSO 1949展开实验，以了解在低pH值的酶的高稳定性。此外，我们将构建融合的催化结构域的SSO 1949与碳水化合物结合模块，以提高对纤维素的催化效率。最后，我们将随机产生突变酶，并筛选它们的酶活性增加。该应用程序结合了酶学的基础研究和应用研究，使用热和酸稳定的内切葡聚糖酶进行纤维素解聚。

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.