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

在拟议的工作中，酶的专一性将被用来评估制造新型生物材料(如纳米纤维纤维素)或可发酵糖的潜力，这些可发酵糖可以进一步从生物质中加工成各种生物燃料和生物化学品。虽然纤维素降解的有效性通常是通过释放的可溶性糖量来评估的，但人们普遍认为，在整个酶介导的生物质/纸浆的修饰/解构过程中，限制速度的步骤是创造酶进入纤维素底物的途径。在大多数现实情况下，纤维素很可能总是与至少一些木质素和半纤维素组分密切相关。同时，典型生物质基质的纤维、原纤维和微原纤维结构将在限制纤维素的可获得性方面发挥作用。一个持续的挑战是找到一种可重复和可量化的方法来量化酶对纤维素成分的可及性，以及结合适当的酶鸡尾酒来修饰/解构纤维素纤维的前处理/制浆的作用。机械精炼将被评估为克服生物质顽固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，新闻纸通过机械精炼生产，在基础设施上投资了数十亿美元。然而，新闻纸是一个衰落的市场。*机械精炼已被证明通过外部原纤化和内部分层打开顽固的生物质结构。在拟议的工作中，各种纸浆，如北方漂白针叶木硫酸盐浆(NBSK)将得到不同程度的精炼。使用这些类型的底物，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性的可能影响，以及用于预测纤维素可获得性的方法。在以前的工作中，我们和其他研究人员使用了保水性、显微镜和纤维特性(如纵横比)等方法，作为预测纤维素可获得性的方法，从而预测酶介导的原纤化或水解性的有效性。然而，这些方法倾向于提供总价值，并且不能评估纤维内纤维素组织的微纤维水平的可能变化。在我们最近工作的基础上，我们将改进Simon‘s染色和碳水化合物结合模块(CBMS)“探针”方法，以更好地评估纤维素的可及性和酶介导的纸浆/生物质无定形形成背后的可能机制。CBMS的特异性使其成为研究纤维素底物微原纤维结构的理想探针。利用优化的前处理、酶鸡尾酒和新的和已建立的底物表征方法，我们希望在纤维、原纤维和微原纤维的结构水平上更好地了解纤维素的可及性，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.*****