How do soluble enzymes from microbes degrade insoluble plant cell walls?

BBSRC DTP的主要战略主题：可再生资源和清洁生长的生物科学植物细胞壁由半纤维素包裹的纤维素原纤维组装而成。半纤维素木聚糖是木本生物质和禾本科植物中最重要和最丰富的半纤维素。最近的研究表明，木聚糖改变其构象为扁平的双折叠螺旋带，以结合到纤维素表面。木聚糖的这种功能形式是常规木聚糖酶无法接近的，因为它们需要可溶性三重螺旋形式来结合活性位点和水解。目前的木聚糖酶测定使用提取的木聚糖或已改变天然木聚糖构象的预处理的生物质。在自然界中，微生物遇到以双螺旋构象附着的木聚糖，这表明自然降解过程可能与测量的测定方法有些不同。在这个项目中，我们的目标是了解酶如何有效地降解固体天然底物。我们将使用全纤维素纳米纤维，其是在天然构象中用木聚糖包覆的纤维素。这些是最近在杜普利实验室分离出来的。我们将研究不同的木聚糖活性酶降解固态木聚糖涂层的能力。这样的酶可以包括木聚糖酶以及附加物释放酶如乙酰酯酶。我们将使用释放的糖的生物化学测定来研究活性，以及与整个综纤维素的降解相结合的基于液滴的测定。该项目可能涉及一些酶的发现，木聚糖降解途径的调查，以及一些酶的表征。

BBSRC DTP main strategic theme: Bioscience for renewable resources and clean growthPlant cell walls are assembled with cellulose fibrils coated with hemicelluloses. The hemicellulose xylan is the most important and abundant hemicellulose in woody biomass and in grasses. Recent studies have shown that xylan changes its conformation into a flat two-fold screw ribbon to bind to the surface of cellulose. This functional form of xylan is inaccessible to conventional xylanases, since they require a soluble three-fold screw form for binding to the active site and hydrolysis. Current xylanase assays use extracted xylan or pretreated biomass that has altered the natural xylan conformations. In nature, microbes encounter xylan attached in the two-fold screw conformation, suggesting the natural degradation process might be somewhat different to measured assays.In this project we aim to understand how enzymes can effectively degrade the solid natural substrate. We will use holocellulose nanofibrils, which are cellulose coated with xylan in the natural conformation. These have been isolated recently in the Dupree lab. We will study the ability of different xylan-active enzymes to degrade the xylan coatings in the solid state. Such enzyme could include xylanases and also appendage-releasing enzymes such as acetyl esterases. We will study the activity using biochemical assays of released sugars, and also droplet-based assays that are coupled to degradation of the entire holocellulose.The project could involve some enzyme discovery, investigation of xylan degradation pathways, and also some enzyme characterisation.