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 (tP) 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 gsubstrate/Lsol and 100 gsubstrate/Lsol, 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/gsubstrate) 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, Km and maximum reaction rate attainable, Vmax 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-tP] where Vmax varies greatly as enzyme (cellulase) concentration rises. For example, Vmax 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 gGLC/LSOLh; 2.90±0.05 gGLC/LSOLh; 5.16±0.18 gGLC/LSOLh; 7.48±0.81 gGLC/Lh, respectively. Dependence on the Km of enzyme-catalyzed reaction exhibited a considerably variation with the substrate nature and the enzymatic kinetic conditions establishing that Km for the cellulase from T. reesei –substrate complex was between 54.81 gGLC/LSOL and 209.99 gGLC/LSOL.