Liu, Yuxin. Characterization of Thermobifida fusca cellulase activity on unnatural cellulose allomorphs. Retrieved from https://doi.org/doi:10.7282/t3-pfhf-yb94
DescriptionCellulases have traditionally been studied in the context of cellulose degradation to fermentable sugars, like glucose, for ethanol production. Cellulose recalcitrance to enzymatic degradation has long been considered as a critical technological barrier for commercialization of cellulosic biofuels. Recent research has led to the development of a cost-effective ammonia-based pretreatment method that allows modification of the crystalline allomorphic structure of cellulose from native cellulose-I to cellulose-III.* Chundawat and co-workers have already demonstrated that cellulose-III is up to five folds more digestible than native cellulose I by Trichoderma reesei derived fungal cellulase enzymes. † This improvement in fungal cellulase cocktail activity was shown to be largely arising due to enhanced endolytic-exolytic cellulase enzyme synergy on pretreated cellulose-III. Here, we explore the activity of cellulases secreted by a highly cellulolytic actinomycete called Thermobifida fusca. To the best of our knowledge, the work here signifies the first reported attempt to characterize the activity of T. fusca expressed cellulase cocktails and purified bacterial cellulases on pretreated cellulose-III. The four specific objectives that were completed to accomplish this goal were; (i) develop and optimize a cell culturing method for T. fusca growth to maximize recovered cellulase activity, (ii) characterize the activity of isolated crude T. fusca cellulase cocktails on native and ammonia-pretreated cellulose allomorphs, (iii) develop a protein purification protocol to isolate and purify T. fusca exocellulase (Cel6B) to electrophoretic homogeneity, and (iv) run enzyme synergism ‘re-start’ hydrolysis assays on isolated exocellulase Cel6B in concert with another bacterial endocellulase (CelE) to confirm if bacterial cellulases also show improved endo- exosynergistic activity on cellulose-III. Our results led further credence to our initial hypothesis that free cellulolytic enzyme cocktails, derived from either fungal or bacterial sources, show enhanced endo-exo synergy on pretreated cellulose-III. We also discuss future research directions and the relevance of the research to our understanding about how cellulases deconstruct various crystalline cellulose allomorphs in general.