- Characterization of natural cellulosomes and the selected substrate. CellulosomePlus will produce the basic components of natural cellulosomes as well as other lignocellulosic enzymes and characterize the hydrolysis (by the cellulosome, its components and by the DCs (Designer Cellulosomes) to be developed in this project) of the substrate of interest: organic fraction of municipal solid waste, OFMSW. We will focus on studying the relevant catalytic activities, physicochemical properties (thermodynamic, kinetic and mechanical stabilities), atomic and supramolecular structure, and interactions of the various cellulosomal components. An important (but not indispensable for the main objective of the project) step is to develop a standard enzymatic assay for performance comparisons. The knowledge obtained will provide adeeper understanding of the architecture, nanomechanics and catalytic properties of cellulosomes, and the logic behind their construction.
- Modelling the cellulosome for in silico knowledge integration. Multi-scale modelling (from atomic to supramolecular levels) will provide crucial support for the synthesis, self-assembly and characterization tasks, supplying detailed structural and energetic information that will aid in the design and interpretation of the experiments. Model outputs will provide new information on the conformation-driven and dynamic catalytic properties, together with mechanical stabilities, which will provide new ideas for experiments, in a synergistic feedback loop towards the production of optimized DCs.
- Rational design and mass production of DCs. The integration of the acquired knowledge from 1 and 2 into DCs and their subsequent screening will provide us with a platform to test the goal of constructing final DCs (carrying both cellulosomal and noncellulosomal components) optimized for the degradation of the selected industrial substrate, and validated at the laboratory-scale. Our final goal is to obtain higher activity as assessed by comparing the rates of hydrolysis and final sugar yield to those of commercial enzymatic cocktails based on grams of enzymes/grams of biomass
Schematic representation depicting a functional model of a cellulosome and the interaction of its component parts with the cellulose substrate (Czjzek et al, 2012. Methods in Enzymology).
Further efforts will be invested into generalizing these findings to a wider range of lignocellulosic substrates of agricultural, industrial and forest interest using a high throughput screening method of lignocellulolytic enzymes that will be applied in this project. Moreover, to ensure the commercial success of our project we have included in the consortium three industrial partners that, among other tasks, will evaluate the feasibility of DC mass production and its large-scale use (including proof of concept high-volume scale).