Effect of cellulase mole fraction and cellulose recalcitrance on synergism in cellulose hydrolysis and binding.

Elucidating the molecular mechanisms that govern synergism is important for the rational engineering of cellulase mixtures. Our goal was to observe how varying the loading molar ratio of cellulases in a binary mixture and the recalcitrance of the cellulose to enzymatic degradation influenced the degree of synergistic effect (DSE) and degree of synergistic binding (DSB). The effect of cellulose recalcitrance was studied using a bacterial microcrystalline cellulose (BMCC), which was exhaustively hydrolyzed by a catalytic domain of Cel5A, an endocellulase. The remaining prehydrolyzed BMCC (PHBMCC) was used to represent a recalcitrant form of cellulose. DSE was observed to be sensitive to loading molar ratio. However, on the more recalcitrant cellulose, synergism decreased. Furthermore, the results from this study reveal that when an exocellulase (Cel6B) is mixed with either an endocellulase (Cel5A) or a processive endocellulase (Cel9A) and reacted with BMCC, synergism is observed in both hydrolysis and binding. This study also revealed that when a "classical" endocellulase (Cel5A) and a processive endocellulase (Cel9A) are mixed and reacted with BMCC, only limited synergism is observed in reducing sugar production; however, binding is clearly increased by the presence of the Cel5A.