The filamentous fungus Trichoderma reesei produces two potent cellobiohydrolases, CBHI and CBHII. The two enzymes have different structures but both have active sites located in enclosed tunnels containing constellations of amino acids that interact with each other and the substrate. Our earlier studies and comparisons of the enzymes with homologous cellulases provide narrow structural constraints for proposed catalytic mechanisms and suggest residues whose mutation will alter pH-dependencies. Our objective is examine and engineer the catalytic mechanisms of T. reesei CBHI and CBHII by state of the art methods in molecular and structural biology and bio-organic chemistry. A detailed model of the interaction of a cellulose chain with these enzymes will be generated by crystallographic analysis of mutant enzymes in complex with natural and modified oligosaccharides. Novel mechanistic probes will be used to establish still unknown details of the mechanisms of the wt enzymes, and to delineate changes of mechanism in the engineered proteins. An exhaustive search of interactions in the active site, carried out by making a range of systematically modified inhibitors and probes, together with complementing mutations in the enzymes will lead to iterative improvement of the active site. This will allow us to raise the pH optima of the two enzymes. Cellulolytic enzymes are already used in e.g. textile and paper industry. Genetic engineering, when combined with structural biology and a solid background in enzymology provides powerful means for learning and improving the enzyme function. In this proposal, all these aspects are included making possible significant advances in the understanding and engineering the catalytic mechanisms of cellulases. Since the same catalytic principles apply to a wide range of other glycosidases which also share similar three-dimensional structures and active site topologies, the results of this work have a wide application potential in enzymatic modification of carbohydrates. Better understanding of these enzyme mechanisms provides new product ideas for the existing biotech industries and may well lead to the development of completely new industries. The results will help European industry to utilise renewable resources in the development of novel biodegradable products and processes, and in future production of renewable energy and raw materials.
Funding SchemeCSC - Cost-sharing contracts
M60 1QD Manchester
751 24 Uppsala