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.

Enzymatic degradation of cellulose into soluble, and hence more easily utilizable, sugars constitutes a central step in the utilization of renewable plant biomass for production of fuels and base chemicals. Cellulose degaradtion is a critical factor limiting the efficiency and economic viability of the whole process. It has therefore been a main theme of research and technology development in the field (biotechnology and renewable energy) which is considered to have high relevance and broad implications. Cellulose is a structurally complex, solid biomaterial which is characterized by an outstanding recalcitrance against chemical or enzymatic degradation. Despite extensive efforts across numerous disciplines over many decades the mechanistic principles of enzymatic conversion have remained, t a certain extent in particular areas, fundamentally elusive. Core problem of the experimental study of the action of cellulose-degrading enzymes (cellulases) is that biocatalytic processes take place, and so have to be observed, directly at the solid surface of cellulose. Additionally, the reaction involve a rather broad range of length scales, from nano- to micrometeres, in which processes need to be analyzed dependent on time. To gain deepened understanding of the biological process in a holistic manner represents a key goal of fundamental importance in the field and it is widely appreciated that new strategies of enhancing the efficiency of enzymatic conversion of cellulose might be obtained thus. This FWF project has used an interdisciplinary approach, combining expertises from physics and biochemistry/biotechnology, to elucidate with atomic force microscopy in a laterally and time resolved manner the action of cellulases on the surface of cellulose. Enzymes have been visualized at single molecule resolution. Significant method developments concerning the substrate preparation and dynamic analysis under near natural reaction conditions were key to the study of the relevant biological problems. Fundamentally new insights into the dynamic mode of action of individual cellulases, alone or in synergistic combination with other cellulases, were obtained. The specificity of fungal cellulases regarding the surface morphology of the cellulose substrate to be attacked was directly analyzed and so revealed for the first time. Based on the data from time-resolved visualization a new approach of modeling cellulose surface degradation by cellulases was developed. It builds on the so- called cellular automata concept and its successful validation of a model against dynamic experimental evidence presented a major advance. Besides the classical cellulases, which hydrolytically degrade cellulose, oxidatively cellulose degrading enzymes and cellulose binding proteins that lack enzymatic activity were characterized in their interaction with and effect on the cellulose surface. Fundamentally new mechanistic discoveries were made thus.