Substrate factors that influence enzyme synergism during cellulose hydrolysis

To achieve effective cellulose hydrolysis requires the synergistic cooperation of various cellulases and accessory enzymes/proteins. Most previous synergism studies have used “model” cellulosic substrates, such as cotton or Avicel and have focused on the initial stages of hydrolysis. This earlier work also demonstrated that the extent of synergism is influenced by both the composition and concentration of “cellulase” mixture used and the nature of cellulosic substrate. To gain a better understanding of “cellulase synergism”, the actions of individual and combinations of cellulases (Cel7A, Cel6A, Cel7B, Cel5A), β-glucosidase (BG) and polysaccharide monooxygenase (AA9) were assessed on a “library” of pretreated lignocellulose at different enzyme loadings. It appeared that cellulose hydrolysis was mainly achieved by Cel7A, while other enzymes seemed to synergistically enhance the hydrolytic efficiency of Cel7A during hydrolysis. Enzyme synergism was most apparent when unsaturated concentrations of Cel7A were used. At the “reasonable”/low enzyme loading required for effective hydrolysis of cellulosic component within pretreated lignocellulose, the highest degree of synergism was observed between cellulases and BG. When the major substrate physicochemical characteristics were compared (lignin/xylan content, gross fiber characteristics, and cellulose accessibility/crystallinity/polymerization), best synergistic cooperation between cellulases occurred on the disorganized/accessible cellulose. The “boosting effect” of AA9 addition on the hydrolytic potential of cellulase mixture was most apparent on substrates with a higher degree of accessible crystalline, rather than amorphous cellulose. A “synergism model” based on the increased off-rate of processive Cel7A by other enzymes was used to better elucidate the influence of substrate characteristics and enzyme loading-dependent enzyme synergy.