Impaired desorption provides a mechanistic link for activity loss during enzymatic hydrolysis of biomass

Activity loss and desorption of cellobiohydrolase1 (CBH1) were studied under three conditions of practical significance: (1) mechanical shaking, (2) in the presence of an inhibitor, and (3) in the presence of palladium. Strong effect of mechanical shaking was observed at 300 rpm, with 70% of activity loss and only about 15% of CBH1 desorption following 24 hours of incubation, compared to almost complete desorption and little mechanical inactivation of CBH1 at 150 rpm. In the presence of 20 mM cellobiose inhibitor, only about 65% of CBH1 desorbed after 24 hours. Desorption was about 25% less after being inhibited with 0.25 M GdnHCl compared to that in the absence of the inhibitor, implying enzyme returns extremely slowly to the bulk solution in the presence of inhibitors and is unable to find a new binding site to start another hydrolysis cycle immediately. When 50 and 162.5 μM of the metal component palladium were added to the reaction, activity dropped about 70% and 63%, and less than 5% of enzyme desorbed and returned to solution. These results suggest that the inability of bound enzymes to desorb from substrate is a mechanistic link for enzyme activity loss under conditions of duress.