FWF - Enzymatischer Zellulaseabbau - Visualizing Enzymatic Cellulose Degradation in Situ

Enzymatic hydrolysis of cellulose is key for the production of second generation biofuels which represent a long-standing leading theme in the field of sustainable energy. Despite the wealth of knowledge about cellulase structure and function, the elusive mechanism by which these enzymes disintegrate the complex structure of their insoluble substrate - which is the gist of cellulose saccharification - is still unclear. Overcoming cellulose recalcitrance therefore constitutes a central aim in biofuels development. To investigate the structural dynamics occuring during enzymatic cellulose degradation we capitalize on the fact that the substrate is solid: in situ visualization of enyzmatic action using atomic force microscopy (AFM) is the microscopic method of choice. AFM has the necessary resolution to even observe single enzymes as well as the overall effect of many enzymes. Moreover, it renders visualization of the enzymatic breakdown of cellulose possible without destroying substrate or enzymes. Also, measurements in liquids are possible when using a so-called liquid cell. In a preliminary study we characterized the structural dynamics of the action of a complete Trichoderma reesei cellulase system on a nano-flat cellulose preparation using AFM in a time-resolved manner. We observed that as a first step in substrate disintegration, elongated fissures emerge which develop into coniform cracks as disintegration continues. The dynamics of crack morphology reflects the interplay between surface degradation inside and outside of the crack and it is conceivable how hindered diffusion leads to product inhibition and loss of cooperative interaction between the enzymes, thus transiently limiting cellulase activity inside the growing crack. In the project proposed here, we aim at elucidating the mechanism by which single cellulases such as cellobiohydrolase I and II and endoglucanases interact with and degrade their substrate by using in situ visualization of the cellulases and their action on a nano-flat substrate specimen by means of AFM. Besides these non-complexed systems we want to determine how bacterial cellulosomes from Clostridium thermocellum degrade cellulose. Additionally, we plan at investigating the interaction of sole cellulose binding domains with cellulose. To this end our laboratory, which has a long research history in enzymatic cellulose degradation, closely cooperates with the Institute for Electron Microscopy (FELMI-ZfE) at Graz University of Technology. We expect that in project we will answer fundamental questions: how does enzymatic cellulose disintegration proceed on a structural level; what are the roles of the different exo- and endoenzymes; what are the limiting / rate retarding factors in cellulose hydrolysis and how can they possibly be overcome; how are non-hydrolytic components of the cellulases involved in substrate disruption; what is the influence of substrate characteristics (cristallinity, presence of microstructures, changing surface, etc.) on cellulase action; and how is the substrate changing over time during hydrolysis. This project will give a deep insight into the structural dynamics occuring during the elusive process of saccharification of the insoluble cellulose by cellulases thus opening up new strategies to make biofuel production from lignocellulosic feedstock economic.