High enzyme cost is a major bottleneck for the economic production of bioethanol and other sugar-derived chemical products from lignocellulosic biomass. To reduce the cost, it is necessary to find novel enzymes of higher efficiency and to understand more fully which enzymes are important and why. To find more efficient endo-β1,4-glucanases (EGs), a key enzyme in cellulose depolymerization, I compared the activity of six homologous fungal EGs that come from two distinct sub-families of GH5. When included into an enzyme mixture, two EGs with additional mannanase activity gave higher glucose yields from pretreated corn stover when mannan, a known cellulase inhibitor, was present. Therefore, it is possible that endoglucanases that have activity on mannan as well as cellulose protect other cellulase mixtures from inhibition by mannan inhibitors by degrading them, thereby increasing overall biomass conversion efficiency. Lignin in lignocellulose is known to strongly inhibit the activity of cellulolytic enzymes, but such inhibition is largely reduced above pH 6. Unfortunately, current available Trichoderma reesei-based commercial enzymes lose much of their activity above pH 6. To find enzymes that can efficiently degrade cellulose at high pH, I purified and characterized an EG and a BG from the alkaliphilic fungus Cladorrhinum bulbillosum. Two C. bulbillosum cellulases showed higher activity than their T. reesei homologs between pH 6-8. Therefore, they may be useful as the basis of cellulase cocktails with better activity at higher pH.Lytic polysaccharide mono-oxygenases (LPMOs) significantly enhance enzyme efficiency in cellulose degradation, but the mechanism is not well understood. To understand how LPMO enhances cellulose degradation, in Chapter 4 I studied the molecular interaction of an LPMO (TrAA9A) and a cellobiohydrolase (TlCel7A) with bacterial microcrystalline cellulose (BMCC) in collaboration with Dr. Bo Song. Cellulose conversion by TlCel7A was enhanced 8% by TrAA9A. Atomic force microscope observation revealed that BMCC ribbons were split into fibrils with a smaller diameter after TrAA9A treatment. The dividing of the cellulose microfibrils occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone, while TlCel7A alone caused no separation. Therefore, TrAA9A may increase the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhance cellulose hydrolysis yield.
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