Hydrolysis of steam-pretreated lignocellulose: synergism and adsorption for cellobiohydrolase I and endoglucanase II of Trichoderma reesei.

The mechanism of hydrolysis of cellulose is important for improving the enzymatic conversion in bioprocesses based on lignocellulose. Adsorption and hydrolysis experiments were performed with cellobiohydrolase I (CBH I) and endoglucanase II (EG II) from Trichoderma reesei on a realistic lignocellulose substrates: steam-pretreated willow. The enzymes were studied both alone and in equimolar mixtures. Adsorption isotherms were determined at 4 and 40 degrees C during 90-min reaction times. Both CBH I and EG II adsorbed stronger at 40 than at 4 degrees C. The time course of adsorption and hydrolysis, 3 min to 48 h, was studied at 40 degrees C. About 90% of the cellulases were adsorbed within 2 h. The hydrolysis rate was high in the beginning but decreased during the time course. Based on adsorption data, the hydrolysis and synergism were analyzed as function of adsorbed enzyme. CBH I showed a linear correlation between hydrolysis and adsorbed enzyme, whereas for EG II the corresponding curve leveled off at both 4 and 40 degrees C. At low conversion, below 1%, EG II produced as much soluble sugars as CBH I. At higher conversion, CBH I was more efficient than EG II. The synergism as function of adsorbed enzyme increased with bound enzyme before reaching a stable value of about 2. The effect of varying the ratio of CBH I:EG II was studied at fixed total enzyme loading and by changing the ratio between the enzymes. Only a small addition (5%) of EG II to a CBH I solution was shown to be sufficient for nearly maximal synergism. The ratio between EG II and CBH I was not critical. The ratio 40% EG II:60% CBH I showed similar conversion to 5% EG II:95% CBH I. Modifications of the conventional endo-exo synergism model are proposed.