Thermobifida fusca cellulases exhibit increased endo-exo synergistic activity, but lower exocellulase activity, on cellulose-III
ACS Sustainable Chemistry & Engineering(2020)
Abstract
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.
MoreTranslated text
Key words
Cellulosic biofuels,Thermobifida fusca,Cellulases,Ammonia pretreatment,Cellulose allomorphs,Endo-exo cellulase synergy
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