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不再可行;矿物燃料是一种有限的资源，其使用导致二氧化碳排放，这是全球变暖的一个主要原因。因此，迫切需要找到可再生且对环境没有不利影响的液体燃料的替代来源。木质纤维素生物质是用于生物燃料生产的有前景的底物，因为它不是食物来源，比淀粉更丰富，并且其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显著限制是其对酶攻击的抵抗力。因此，纤维素，木质纤维素生物质中的主要多糖，是化学上简单的，但其高度结晶的结构使其难以接近作为水解酶的酶。然而，最近的研究已经确定了可以提高植物细胞壁解构效率的新型酶。因此，一些报道已经表明，氧化酶在纤维素的结晶区域中切割键，导致水解酶攻击的增加。木质纤维素生物质中主要的基质多糖木聚糖的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，一群木聚糖酶不仅容纳侧链，而且实际上显示出对这些装饰的绝对需求。我们还表明，有可能将新的功能引入到生物技术上重要的阿拉伯呋喃糖苷酶的活性位点中，以帮助从木聚糖中去除侧链。这种多功能酶的产生具有简化解构植物细胞壁所需的生物催化剂的潜力，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。在这个项目中，我们将探索纤维素氧化酶，阿拉伯糖苷酶和多功能阿拉伯呋喃糖苷酶/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.