Towards cellulosic ethanol: structure-function studies of glycoside hydrolases

本实验方案是在NSERC的资助下，对本实验室21年来糖苷水解酶结构-功能关系研究的延续。 基于我们的研究结果，我们假设厌氧瘤胃细菌琥珀酸纤维杆菌在其细胞表面上将其纤维素分解酶和木聚糖分解酶组织成纤维素体样复合物，并且释放可溶性因子以使结晶纤维素去稳定。 为了验证这一假设，我们建议克隆和表达编码三种以前未鉴定的木聚糖酶和纤维素酶以及假设的180 kDa和240 kDa支架蛋白的基因。 然后，我们将研究协会，如果有的话，这些蛋白质与另一个亲和层析和表面等离子体共振技术。 进一步研究F.因此，我们将开始寻找一种被认为是由F.使用原子力和扫描隧道显微镜，研究了琥珀酸酯用于结晶纤维素的去稳定化。

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.