The use of genome mining and protein engineering to generate novel xylan and cellulose degrading enzymes

在能源和化学工业中使用化石燃料已经站不住脚；它们是一种有限的资源，它们的使用会导致二氧化碳的排放，而二氧化碳是全球变暖的主要原因。因此，迫切需要找到可再生且不会对环境产生不利影响的液体燃料的替代来源。木质纤维素生物质是一种很有前途的生物燃料生产基质，因为它不是食物来源，比淀粉更丰富，而且它的使用是二氧化碳中性的。在生物燃料工业中使用木质纤维素生物质的一个重要限制是它对酶攻击的抵抗力。因此，作为木质纤维素生物质中的主要多糖，纤维素的化学性质很简单，但其高度结晶的结构使得作为水解酶的酶无法接近它。然而，最近的研究已经发现了新的酶，可以提高植物细胞壁解构的效率。因此，一些报告表明氧化酶在纤维素的结晶区域切割键，导致水解酶攻击的增加。木聚糖是木质纤维素生物质中主要基质多糖，其降解也取得了重大进展。人们普遍认为，木聚糖主链的降解需要在木聚糖酶攻击之前去除侧链。现在很明显，一组木聚糖酶不仅容纳侧链，而且实际上显示出对这些修饰的绝对需求。我们还表明，在生物技术上具有重要意义的阿拉伯糖葡聚糖酶的活性位点上引入新的功能是可能的，这些功能有助于从木聚糖中去除侧链。这种多功能酶的产生有可能简化分解植物细胞壁所需的生物催化剂，从而增加木质纤维素生物质作为生物燃料工业底物的经济潜力。在本项目中，我们将探索纤维素氧化酶、阿拉伯糖木聚糖酶和多功能阿拉伯糖木聚糖酶/木聚糖酶识别其靶底物的机制。这些数据将为进一步提高植物细胞壁降解催化工具箱的效率提供蓝图。

The use of fossil fuels in the energy and chemical industries is no longer tenable; they represent a finite resource and their use results in carbon dioxide emissions, which is a major cause of global warming. There is, therefore, an urgent need to find alternative sources of liquid fuels that are renewable and do not have an adverse effect on the environment. Lignocellulosic biomass is a promising substrate for biofuel production as it is not a food source, is more abundant than starch, and its use is carbon dioxide neutral. A significant limitation in the use of lignocellulosic biomass in the biofuel industry is its recalcitrance to enzyme attack. Thus, cellulose, the major polysaccharide in lignocellulosic biomass, is chemically simple but its highly crystalline structure makes it inaccessible to enzymes that act as hydrolases. Recent studies, however, have identified novel enzymes that could improve the efficiency of plant cell wall deconstruction. Thus, several reports have shown that oxidases cleave bonds in crystalline regions of cellulose, leading to increased access to hydrolase attack. Significant advances have also been made in the degradation of xylan, the major matrix polysaccharide in lignocellulosic biomass. It was widely believed that degradation of the main chain of xylan required the removal of side chains prior to attack by xylanases. It is now apparent that a cohort of xylanases not only accommodate side chains, but actually display an absolute requirement for these decorations. We have also shown that it is possible to introduce novel functionalities into the active site of biotechnologically significant arabinofuranosidases that assist in removing the side chains from xylan. The generation of such multifunctional enzymes has the potential to simplify the biocatalysts required to deconstruct plant cell walls, and thus increase the economic potential of lignocellulosic biomass as a substrate for the biofuel industry. In this project we will explore the mechanism by which cellulose oxidases, arabinoxylanases and multifunctional arabinofuranosidase/xylanases recognize their target substrates. The data will provide a blueprint for further enhancing the efficiency of the plant cell wall degrading catalytic toolbox.