Towards cellulosic ethanol: structure-function studies of glycoside hydrolases

这项建议中提出的实验计划代表了过去21年来在NSERC的主持下，我们实验室对糖苷水解酶结构-功能关系的研究的继续。根据我们的研究结果，我们假设厌氧瘤胃细菌琥珀酸纤维杆菌将其细胞表面的纤维素酶和木聚糖酶组织成纤维体样复合体，并释放一种可溶性因子来解稳结晶纤维素。为了验证这一假设，我们建议克隆和表达编码三种先前未知的木聚糖酶和纤维素酶的基因，以及假设的180 kDa和240 kDa支架。然后，我们将通过亲和层析和表面等离子体共振技术来研究这些蛋白质之间的相互联系。为了进一步研究产琥珀酸假单胞菌对纤维素的生物降解效率，我们将利用原子力和扫描隧道显微镜寻找产琥珀酸假单胞菌释放的一种因子，用于破坏结晶纤维素的稳定性。

The experimental plan presented in this proposal represents a continuation of studies on the structure-function relationship of glycoside hydrolases conducted in our laboratory over the past 21 years under the auspices of NSERC. Based on the results of our studies, we hypothesize that the anaerobic rumen bacterium Fibrobacter succinogenes organizes its cellulolytic and xylanolytic enzymes on its cell surface into cellulosome-like complexes and that a soluble factor is released to de-stabilize crystalline cellulose. To test this hypothesis, we propose to clone and express the genes encoding the three previously unidentified xylanases and cellulases, and the hypothetical 180 kDa and 240 kDa scaffoldins. Then, we will investigate the association, if any, of these proteins with one another by affinity chromatographic and surface plasmon resonance techniques. To further investigate the efficiency of cellulose biodegradation by F. succinogenes, we will initiate a search for a factor thought to be released by F. succinogenes for the destabilisation of crystalline cellulose using atomic force and scanning tunnelling microscopy.