Dissecting the role of carbohydrate binding modules in plant cell wall degradation

植物细胞壁包含地球上最丰富的有机碳源，并且其微生物降解为其组成糖具有相当大的生物学和工业重要性。事实上，光合固定碳的再循环对于食草动物营养、陆地和海洋微生物生态系统的维持以及几种植物病原体的宿主入侵至关重要。虽然攻击植物细胞壁的酶已经广泛用于几个基于生物技术的行业，包括造纸，纺织，洗涤剂和食品（动物和人类）部门，但这些生物催化剂的主要应用是将植物生物质转化为生物乙醇和其他形式的能源。植物细胞壁主要由一系列不同的多糖组成，这些多糖通过复杂的氢键网络相互作用。由于多糖之间广泛的相互作用极大地限制了攻击这种复合结构的糖苷水解酶和酯酶的电池的进入，因此它对生物降解是高度不稳定的。微生物植物细胞壁水解酶显示复杂的分子结构，其中催化模块附加到一个或多个非催化碳水化合物结合模块（CBM）。许多体外研究表明，通过结合到不溶性纯化的植物结构多糖，CBM使同源酶与其靶底物密切和长期的关联，导致催化的显着增强，因为在某种程度上，它们克服了“可及性问题”。有趣的是，申请人最近的研究表明，结构不同但对纯化的配体表现出相同特异性的CBM，在完整植物细胞壁的背景下识别其靶多糖的能力方面显示出高度显著的差异。植物中配体识别的这种变化可能反映了靶多糖与细胞壁其他组分的相互作用。因此，我们建议，不同的CBM的结合位点的拓扑结构是适应于识别其目标多糖在特定的细胞类型的特定生物体。迄今为止，对CBM在酶作用中的功能重要性的分析仅限于探索它们对纯化底物或简单的、高度加工的复合物的作用。鉴于CBM在植物中发挥的复杂靶向作用，这些模块在降解完整植物细胞壁中的功能重要性目前尚不清楚。虽然很明显，这些模块将通过增强酶底物接触来增加催化，但它们也可以在将显示互补活性的糖苷水解酶和/或酯酶组装成细胞壁中的并置中起作用，从而增强这些生物催化剂之间的协同相互作用。该提议将测试以下假设：细菌CBM多样性的生物学原理是：1）使同源酶能够接近位于不同植物细胞壁中的其靶底物，其中聚合物的背景将变化;以及2）将具有互补活性的酶募集到植物细胞壁的区域，其中生物催化剂之间的协同相互作用使降解过程最大化。该研究计划具有重要的生物学意义，因为该过程是食草动物，植物和微生物之间营养循环的组成部分。从工业的角度来看，这些数据将为设计用于产生新型糖苷水解酶和酯酶的策略提供信息和指导，这些酶对植物细胞壁的活性增加。这些酶在植物生物质的生物技术开发中具有相当大的工业用途，特别是在生物乙醇的产生中，而且在造纸、动物和人类饲料、洗涤剂和纺织品部门中。

The plant cell wall comprises the most abundant source of organic carbon on the planet and its microbial degradation to its constituent sugars is of considerable biological and industrial importance. Indeed, the recycling of photosynthetically fixed carbon is critical to herbivore nutrition, the maintenance of terrestrial and marine microbial ecosystems and host invasion by several phytopathogens. While the enzymes that attack the plant cell wall are already widely used in several biotechnology-based industries including the paper, textile, detergent and food (animal and human) sectors, the major application of these biocatalysts is the conversion of plant biomass into bio-ethanol and other forms of energy. The plant cell wall comprises predominantly of an array of different polysaccharides that interact with each other through complex hydrogen bonding networks. It is highly recalcitrant to biological degradation as the extensive interactions between the polysaccharides greatly restrict access to the battery of glycoside hydrolases and esterases that attack this composite structure. Microbial plant cell wall hydrolases display complex molecular architectures in which the catalytic module is appended to one or more non-catalytic carbohydrate binding modules (CBMs). Numerous in vitro studies have shown that by binding to insoluble purified plant structural polysaccharides, CBMs bring the cognate enzyme into intimate and prolonged association with their target substrate resulting in a significant potentiation of catalysis as, to some extent, they overcome the 'accessibility problem'. Intriguingly, recent studies by the applicants have shown that CBMs, which are structurally distinct but exhibit the same specificities against purified ligands, display highly significant differences in their capacity to recognise their target polysaccharides within the context of the complete plant cell wall. This variation in ligand recognition in planta likely reflects the interaction of the target polysaccharides with other components of the cell wall. Thus, we propose that the topology of the binding sites of different CBMs are adapted to recognize their target polysaccharides in specific cell types of specific organisms. To date the analysis of the functional importance of CBMs in enzyme action has been limited to exploring their role against purified substrates or simple, highly processed, composites. In view of the complex targeting role CBMs play in planta, the functional importance of these modules in degrading intact plant cell walls is currently unclear. While it is apparent that these modules will increase catalysis by enhancing enzyme substrate contact, they may also play a role in assembling glycoside hydrolases and/or esterases that display complementary activities into juxtapositions in the cell wall thereby potentiating the synergistic interactions between these biocatalysts. This proposal will test the hypothesis that the biological rationale for the diversity of bacterial CBMs is to 1) enable the cognate enzymes to access their target substrates located in different plant cell walls, where the context of the polymer will vary; and 2) to recruit enzymes with complementary activities to regions of the plant cell wall where the synergistic interactions between the biocatalysts maximise the degradative process. The research programme is of fundamental biological importance as the process is integral to the cycling of nutrients between herbivores, plants and microbes. From an industrial perspective the data will inform and direct strategies designed to generate novel glycoside hydrolases and esterases that display increased activity against plant cell walls. These enzymes would have considerable industrial utility in the biotechnological exploitation of plant biomass, particularly in the generation of bio-ethanol, but also in the paper, animal and human feed, detergent and textile sectors.