P35: Product inhibition of five Hypocrea jecornia cellulases

Pretreated cellulosic biomass may be converted to fuel ethanol through either separate- or simultaneous saccharification and fermentation. A key parameter for the evaluation of these two approaches is the degree of inhibition of cellulolytic enzymes exerted by their products cellobiose and glucose. Yet, quantitative information on individual cellulases hydrolyzing insoluble cellulose remains scarce. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Thricoderma reesei, Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3). The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC₅₀=19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the high sensitivity to glucose. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC₅₀=60-80 mM), and weakly inhibited by glucose (IC₅₀=350-380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose).