UC Davis

Cellulose is the most abundant biopolymer in the world and has been the focus of biorefining efforts for decades. Its relatively simple structure of b-1,4-linked glucose monomers provide a plentiful source of monomeric sugars that can be converted to fuels and platform chemicals in the biochemical industry. However, in practice, full hydrolysis of cellulose is difficult to achieve due to the hierarchical structure that in turn makes it very recalcitrant. An alternative to completely breaking down cellulose into simple sugars for fermentation, is to produce cellulose nanocrystals. Cellulose nanocrystals are highly crystalline, recalcitrant nanoparticles isolated from cellulose that have received emerging interest in the advanced biomaterial industry due to their unique properties. Current production methods of cellulose nanocrystals are less than desirable, ultimately impeding their full market potential.Using cellulase enzymes to isolate cellulose nanocrystals from cellulose offers a unique opportunity in a producing high-value, environmentally-friendly biomaterial in a sustainable, energy- conscience way. Previous research has primarily used endoglucanase enzymes to isolate cellulose nanocrystals from cellulosic substrates because their cleft-shaped active site promotes preferential hydrolysis of glycosidic bonds within the amorphous regions of cellulose while preserving the crystalline regions. However, despite optimally targeted hydrolysis, yields of cellulose nanocrystals from endoglucanase hydrolysis are low. Yields of enzymatically produced cellulose nanocrystals can be increased by understanding both the enzyme-related factors and substrate-related factors that affect enzyme-efficiency, yet very few studies have done so. The overall goal of this research was to investigate how cellulase structural features and cellulase- cellulose interactions influence cellulose nanocrystal yields. I hypothesized that cellulose nanocrystal yields will vary based on the degree of cellulase-cellulose interactions, with the enzymes exhibiting stronger interactions resulting in higher yields. The first objective in this work explored the relationship between the structural features of endoglucanase enzymes and the resulting yields of cellulose nanocrystals (Chapter 3). The structural features of six endoglucanase enzymes distributed between four glycosidic hydrolase families were explored and compared to cellulose nanocrystal yields, to identify which features may be beneficial for activities resulting in cellulose nanocrystals. The results of this study indicated that enzymes with wider, more flexible active sites resulted in higher yields of cellulose nanocrystals (and soluble sugars), suggesting the importance of active site structure for cellulose nanocrystal production. Additionally, the endoglucanase that did not have a carbohydrate binding module (CBM) exhibited lower cellulose nanocrystal yields and lower soluble sugar yields, again, highlighting the importance of a CBM for cellulose nanocrystal production. The second objective explored the synergistic interactions between lytic polysaccharide monooxygenase (LPMO) and endoglucanase enzymes to understand how these interactions were affecting substrate accessibility and the subsequent production of cellulose nanocrystals. This work utilized the best performing endoglucanase from Chapter 3 (NS 51137) and a C1/C4-active LPMO from Thermoascus aurantiacus (TaAA9A, PDB: 2YET) to produce cellulose nanocrystals. The results of this study demonstrated substantial increases in cellulose nanocrystal yields when the two enzymes were simultaneously present, resulting in high degree of synergy (DS) values (DS between 1.4 and 5). Additionally, the resulting synergy curves between the LPMO and endoglucanase revealed an exoglucanase-endoglucanase synergistic relationship (exo-endo synergy). We propose that the exo-endo shaped synergy curves produced by this study suggests the LPMO assists endoglucanase in the production of cellulose nanocrystals by increasing the accessibility of the EG to previously inaccessible, crystalline cellulosic substrate through oxidative cleavage. This synergistic relationship was further confirmed through soluble sugar analysis and assessing enzyme-substrate interactions via productive CBH binding capacity measurements. The results of this dissertation enhance our understanding on enzymatic hydrolysis processes that result in cellulose nanocrystals, and provide direction for future research in enzyme engineering, scalability, and downstream processing of large-scale cellulose nanocrystal production. Future research in these directions will contribute to the development, advancement, and economic viability of sustainable cellulose nanocrystal production.