Comparative kinetic analysis of two fungal β-glucosidases

Background: The enzymatic hydrolysis of cellulose is still considered as one of the main limiting steps of the biological production of biofuels from lignocellulosic biomass. It is a complex multistep process, and various kinetic models have been proposed. The cellulase enzymatic cocktail secreted by Trichoderma reesei has been intensively investigated. beta-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. beta-glucosidase (BGL1) is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial beta-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188). Surprisingly, kinetic modeling of beta-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented beta-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1. Results: A comparative kinetic analysis of beta-glucosidase from Aspergillus niger and BGL1 from Trichoderma reesei, purified using a new and efficient fast protein liquid chromatography protocol, was performed. This purification is characterized by two major steps, including the adsorption of the major cellulases onto crystalline cellulose, and a final purification factor of 53. Quantitative analysis of the resulting beta-glucosidase fraction from T. reesei showed it to be 95% pure. Kinetic parameters were determined using cellobiose and a chromogenic artificial substrate. A new method allowing easy and rapid determination of the kinetic parameters was also developed. beta-Glucosidase SP188 (K-m = 0.57 mM; K-p = 2.70 mM) has a lower specific activity than BGL1 (K-m = 0.38 mM; K-p = 3.25 mM) and is also more sensitive to glucose inhibition. A Michaelis-Menten model integrating competitive inhibition by the product (glucose) has been validated and is able to predict the beta-glucosidase activity of both enzymes. Conclusions: This article provides a useful comparison between the activity of beta-glucosidases from two different fungi, and shows the importance of fully characterizing both enzymes. A Michaelis-Menten model was developed, including glucose inhibition and kinetic parameters, which were accurately determined and compared. This model can be further integrated into a cellulose hydrolysis model dissociating beta-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new beta-glucosidase activities.