Comparative kinetic analysis of two fungal β-glucosidases

<p>Abstract</p> <p>Background</p> <p>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 <it>Trichoderma reesei </it>has been intensively investigated. β-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. β-glucosidase (BGL1) is very poorly secreted by <it>Trichoderma reesei </it>strains, and complete hydrolysis of cellulose often requires supplementation with a commercial β-glucosidase preparation such as that from <it>Aspergillus niger </it>(Novozymes SP188). Surprisingly, kinetic modeling of β-glucosidases lacks reliable data, and the possible differences between native <it>T. reesei </it>and supplemented β-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1.</p> <p>Results</p> <p>A comparative kinetic analysis of β-glucosidase from <it>Aspergillus niger </it>and BGL1 from <it>Trichoderma reesei</it>, 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 β-glucosidase fraction from <it>T. reesei </it>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. β-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 β-glucosidase activity of both enzymes.</p> <p>Conclusions</p> <p>This article provides a useful comparison between the activity of β-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 β-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new β-glucosidase activities.</p