Determination of Enzyme (Cellulase from Trichoderma reesei) Kinetic Parameters in the Enzymatic Hydrolysis of H2SO4- Catalyzed Hydrothermally Pretreated Sugarcane Bagasse at High-Solids Loading

Abstract

Enzymatic kinetic studies of celulignin material obtained from acid-catalyzed hydrothermally pretreated sugarcane bagasse at high-solids loading (S / 25%), acid concentration (A) equal to 1.0; 2.0 and 3.0% w/v and pretreatment time (t^P) equal to 30; 90 and 150 min were carried out using the synergistic action of cellulase from T. reesei and cellobiase from Aspergillus niger.
The kinetic experiments were carried out at 50 ^oC, 150 rpm and 3 h with substrate (water insoluble solids after pretreatment - WIS) loading range between 30 g_substrate/L_sol and 100 g_substrate/L_sol, and mixed with cellulase (at 5.0; 15.0; 30.0 and 60.0 FPU cellulase/_gsubstrate) and ß-glucosidase (at 33.0 IU ß-glucosidase/g_substrate) enzymes. A Michaelis-Menten dependence on the cellulase from T. reesei concentration and substrate concentration plots were obtained based on experimental data. The rate constant, K_m and maximum reaction rate attainable, V_max were determined to characterize the cellulase-catalyzed reaction.
Results showed that the cellulase from T. reesei behavior was highly specific for each WIS fraction obtained under a combination of [A-S-t^P] where V_max varies greatly as enzyme (cellulase) concentration rises. For example, V_max values when considered a substrate obtained at [1.0% w/v - 25% - 30 min] and [3.0% w/v - 25% - 30 min] using 5.0 FPU cellulase/_{gsubstrate(WIS)} and [1.0% w/v - 25% - 90 min] and [3.0% w/v - 25% - 90min] using 60.0 FPU cellulase/_{gsubstrate(WIS)} were 5.96±0.27 g_GLC/L_SOLh; 2.90±0.05 g_GLC/L_SOLh; 5.16±0.18 g_GLC/L_SOLh; 7.48±0.81 g_GLC/Lh, respectively. Dependence on the K_m of enzyme-catalyzed reaction exhibited a considerably variation with the substrate nature and the enzymatic kinetic conditions establishing that K_m for the cellulase from T. reesei –substrate complex was between 54.81 g_GLC/L_SOL and 209.99 g_GLC/L_SOL