To better elucidate the factors that influence enzyme accessibility (via pretreatment/amorphogenesis) to biomass/cellulose/pulps

在拟议的工作中，酶的特异性将用于评估制造新型生物材料（如纳米原纤化纤维素）或可发酵糖的潜力，这些糖可以进一步加工成各种生物燃料和生物化学品。虽然纤维素水解的有效性通常通过释放的可溶性糖的量来评估，但广泛认为生物质/纸浆的整体酶介导的改性/解构中的限速步骤是产生酶接近纤维素底物。在大多数实际情况下，纤维素很可能总是与至少一些木质素和半纤维素级分紧密结合。同时，典型生物质基质的纤维、原纤维和微纤维结构将在限制纤维素可及性方面发挥作用。一个持续的挑战是找到一种可重复的和可量化的方法来量化酶对纤维素组分的可及性以及预处理/制浆与适当的酶混合物组合以改性/解构纤维素纤维的作用。机械精炼将被评估为克服生物质不稳定性和开放生物质结构的一种方式。加拿大是新闻纸的主要生产国，通过机械精炼生产，在基础设施方面投资数十亿美元。然而，新闻纸是一个下降的市场。* 已显示机械精制通过外部原纤化和内部分层打开柠檬酸生物质结构。在所提出的工作中，各种纸浆，如北方漂白软木硫酸盐浆（NBSK）将被精制到不同程度。使用这些类型的基质，我们希望最大限度地减少木质素和半纤维素对原纤化和水解以及用于尝试预测纤维素可及性的方法可能产生的影响。在以前的工作中，我们和其他研究人员使用了保水性，显微镜和纤维表征等方法，如纵横比，作为预测纤维素可及性的方法，从而预测酶介导的原纤化或水解的有效性。然而，这些方法倾向于提供总值，并且不能评估纤维内纤维素组织的微原纤水平的可能变化。在我们最近工作的基础上，我们将改进Simon染色和碳水化合物结合模块（CBM）“探针”方法，以更好地评估纤维素可及性和酶介导的纸浆/生物质无定形背后的可能机制。CBMs的特异性使其成为研究纤维素底物微纤维结构的理想探针。使用优化的预处理，酶混合物和新的和成熟的底物表征方法，我们希望更好地了解纤维素在纤维，原纤维和微纤维结构水平的可及性，其结果有助于未来的纤维改性/解构应用。

In the proposed work, the specificity of enzymes will be used to assess the potential to make either novel biomaterials (such as nanofibrillated cellulose) or fermentable sugars that could be further processed to various biofuels and biochemicals from biomass. Although the effectiveness of cellulose hydrolysis is often assessed by the amount of soluble sugar released, it is widely acknowledged that the rate limiting step in the overall enzyme-mediated modification/deconstruction of biomass/pulp is creating enzyme access to the cellulosic substrate. In most realistic situations, it is highly probable that cellulose will always be in close association with at least some of the lignin and hemicellulose fractions. At the same time, the fiber, fibril and microfibril structure of a typical biomass substrate will play a role in limiting cellulose accessibility. An ongoing challenge has been to find a reproducible and quantifiable method to quantify enzyme accessibility to the cellulose component and the roles of pretreatment/pulping in combination with appropriate enzyme cocktails to modify/deconstruct cellulosic fibers. Mechanical refining will be assessed as one way of overcoming biomass recalcitrance and opening up the biomass structure. Canada is a major producer of newsprint, produced via mechanical refining, with billions of dollars invested in infrastructure. However, newsprint is a declining market. ***Mechanical refining has been shown to open up the recalcitrant biomass structure by external fibrillation and internal delamination. In the proposed work various pulps, such as northern bleached softwood Kraft pulp (NBSK) will be refined to various extents. Using these types of substrates we hope to minimise the possible influence of lignin and hemicellulose on both fibrillation and hydrolysis and the methods used to try to predict cellulose accessibility. In previous work we and other researchers have used methods such as water retention, microscopy and fiber characterisation, such as aspect ratio, as ways to predict cellulose accessibility and, consequently, the effectiveness of enzyme mediated fibrillation or hydrolysis. However, these methods have tended to provide gross values and are not able to assess possible changes at the microfibril level of cellulose organisation within the fibre. Building on our recent work we will refine the Simon's stain and Carbohydrate-binding modules (CBMs) “probes” methods to better assess cellulose accessibility and the possible mechanisms behind enzyme mediated amorphogenesis of pulps/biomass. The specificity of CBMs make them ideal probes to investigate the microfibril structure of cellulosic substrates. Using optimised pretreatment, enzyme cocktails and novel and established methods for substrate characterisation we hope to better understand cellulose accessibility at the fiber, fibril and microfibril structural levels, with the results contributing to future fibre modification/deconstruction applications.*****