Thermobifida fusca cellulases exhibit increased endo-exo synergistic activity, but lower exocellulase activity, on cellulose-III

Cellulose recalcitrance toward saccharification is a barrier for low-cost biofuels production. Ammonia-based pretreatments can alter the native cellulose-I allomorphic state to form an unnatural cellulose-III allomorph that is less recalcitrant toward enzymatic hydrolysis. Here, we characterize the hydrolytic activity of a thermophilic cellulolytic microbe, Thermobifida fusca, derived cellulase on cellulose-III. Up to 2-fold improved activity was observed for homologously expressed T. fusca cellulase enzymes on cellulose-III. Surprisingly, T. fusca exocellulases like Cel6B alone had lower activity on cellulose-III. We hypothesized that increased activity of T. fusca cellulases on cellulose-III arises mostly due to enhanced endocellulase activity and improved synergism between endo/exocellulases. Representative T. fusca endocellulase (Cel5A) and exocellulase (Cel6B) were heterologously expressed in Escherichia coli, purified, and systematically characterized for synergistic activity on cellulose-III. Hydrolytic activity assays confirmed increased activity of Cel5A on cellulose-III and improved endo/exo synergistic activity for various combinations of Cel6B/Cel5A. We finally conducted a two-step restart hydrolysis assay to also confirm if increased endoactivity results in a endo-treated cellulose-III that is amenable toward increased Cel6B activity. This work provides a mechanistic basis for increased synergistic cellulase activity on cellulose-III and provides a rationale for focusing future T. fusca enzyme engineering efforts toward potentially rate-limiting exocellulases like Cel6B.