Proposal #: 1454379 Institution: University of Colorado at Boulder
The use of various renewable biomass materials as an alternate source of energy is an important focus of research today. Cellulose is an organic compound, a polysaccharide consisting of a linear chain of several hundred to many thousands of linked D-glucose units. Cellulose is an important structural component of the primary cell wall of among others, green plants and many forms of algae. For example, the cellulose content of wood is 40 to 50%. Cellulase is any of several enzymes produced chiefly by fungi, bacteria, and protozoans that catalyze cellulolysis, the decomposition of cellulose and of some related polysaccharides. Cellulases break down the cellulose molecule into monosaccharides ("simple sugars") such as beta-glucose, or shorter polysaccharides and oligosaccharides. The emergence of ionic liquids (ILs - salt-like substances that melt at ambient temperature) as alternative solvents for the dissolution of biomass presents considerable opportunities for the conversion of cellulosic materials. Of particular interest, with the appropriate selection of cation and anion, ILs can be rationally tuned to dissolve high concentrations of untreated crystalline cellulose. The formation of hydrogen bonds between the IL and cellulose disrupts the internal hydrogen bonding network that causes cellulose chains to pack tightly together. In addition to lowering the degree of crystallinity of cellulose, select ILs can loosen the matrix components within biomass that add to the overall recalcitrance of cellulose. Furthermore, due to the thermal stability and non-volatile nature of ILs, ILs constitute and environmentally attractive solution to the need for cleaner media for cellulose processing. Conventional solvents used to dissolve cellulose, which contain inorganic salts, acids, bases, and metal complexes, are largely toxic and environmentally polluting. However, the attractive properties of ILs are negated by the broad inactivation of cellulases in these solvents. Consequently, for ILs to be fully exploited for processing whole biomass, the solvent effects of ILs on cellulases needs to be mediated.
The aim of this integrated research and teaching project is to develop an approach to enhance the tolerance of cellulases to ionic liquids (ILs) for biomass processing via engineering enzyme charge. The research will specifically test the hypothesis that changing the surface charge of cellulases by site-directed mutagenesis can mediate interactions with ILs, which impact cellulase stability. As solvents for cellulosic materials, ILs present considerable opportunities due to their auspicious properties, including the unique capacity to solubilize large amounts of biomass. In this project, the impact of altering enzyme charge on the denaturation of cellulase by ILs will be directly probed by nuclear magnetic resonance spectroscopy and molecular dynamics (MD) simulations with unprecedented resolution. This impact will be investigated while using site-directed mutagenesis to alter the charge of the well-characterized endoglucanase EI from Acidothermus cellulolyticus, exoglucanase CbhA from Clostridium thermocellum, and beta-glucosidase JMB19063 GH3. Additionally, the role of surface electrostatics on the prevention of cellulase inhibition by insoluble matrix components in the conversion of whole biomass will be elucidated. The specific research objectives are to: 1) identify sites in EI, CbhA, and GH3 that are structurally perturbed by ILs by NMR and MD, 2) rationally design and test EI, CbhA, and GH3 variants with altered surface charge for improved tolerance to IL-induced inactivation, and 3) characterize the effect of site-directed charge mutations on non-productive lignin adsorption to EI, CbhA, and GH3. The educational objectives are to: 1) promote research opportunities for local high school students through involvement in Boulder Valley School District Research Seminar Program, 2) create opportunities for undergraduate research via growth of iGEM program, and 3) create and implement laboratory modules on biocatalysis for undergraduate curriculum.
