A Model Explaining Declining Rate in Hydrolysis of Lignocellulose Substrates with Cellobiohydrolase I (Cel7A) and Endoglucanase I (Cel7B) of Trichoderma reesei

Abstract

It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

“…Obstacle-based kinetic interpretations have been repeatedly suggested for cellobiohydrolases (12,20,25,(37)(38)(39) and recently also for processive endoglucanases (40), and the present results support the general validity of this approach. We emphasize, however, that obstacles have not yet been directly identified and that their structural origin remains obscure.…”

Development and validation of a stochastic molecular model of cellulose hydrolysis by action of multiple cellulase enzymes

“…In the previous model, the CBH action could only produce cellobiose during cellulose hydrolysis. However, glucose formation during cellulose hydrolysis by CBH action has been observed by some researchers (Eriksson et al 2002;Medve et al 1998), and was also observed in our experiments (discussed later in the "Results and discussion" section). Therefore, the model was modified to include glucose formation in addition to the cellobiose.…”

Origin of Initial Burst in Activity for Trichoderma reesei endo-Glucanases Hydrolyzing Insoluble Cellulose

“…This implies that upon addition to the substrate, the EG will perform many (10s or 100s) endo-processive runs at a turnover rate that is over 20 s Ϫ1 for TrCel7B. It follows that unlike earlier interpretations for exo-cellulases (11,13,14,66), the early slowdown for the enzymes studied here is not necessarily linked to processivity. As the process progresses, an increasing population of the enzyme gets randomly trapped in unproductive positions for extended periods, and the turnover rates consequently begins to fall.…”