Enhanced and Optimized Production of Cellulase Using Trichoderma harzianum through the Utilization of Pretreated Lignocellulosic Wastes: Purification, Characterization, and Industrial Applications.
Abstract
In the current study, various Aspergillus and Trichoderma strains were explored to produce cellulase using a variety of agricultural wastes as cheaper substrates. Among the tested strains, Trichoderma harzianum exhibited the highest enzyme activity (84.0 FPU/mL) when grown on alkali-pretreated sugarcane bagasse after purification. Out of three substrates, pretreated sugarcane bagasse consistently resulted in higher cellulase activity (42.05 FPU/mL) as compared to wheat straw (26.5 FPU/mL) and wheat bran (16.7 FPU/mL) across all tested strains due to its high cellulose/hemicellulose content and low lignin levels in proximate analysis. Central Composite Design Response Surface Methodology was employed in Minitab software to optimize solid-state fermentation parameters, achieving 76.2 FPU/mL activity in T. harzianum at pH 5.5, 28°C, 68% moisture, 0.5 mL inoculum, 5 g substrate with 5 days' incubation time as compared to Aspergillus fumigatus (57.2 FPU/mL). Stepwise ammonium sulfate precipitation (60%-80%) followed by dialysis and gel chromatography increased specific activity to 84.00 FPU/mg, with a purification fold of 5.51 and a 22.0% yield, whereas SDS-PAGE further confirmed enzyme homogeneity. The purified cellulase exhibited maximum relative activity at 50°C and pH 4 in the characterization study. The activity was strongly inhibited by Hg2 +, while the activity loss was very small in the presence of Ca2 + and Mg2 +. The enzyme kinetic resulted in a V max of 103.89 U/mL and a K m of 3.71 mg/mL by nonlinear Michaelis-Menten regression. First-order thermal inactivation showed kd value increasing from 0.000250 to 0.002900 min- 1, Ed (72.5 kJ/mol), and z (28.3°C). These results indicated that the optimized and purified enzyme still had high catalytic activity toward carboxymethyl cellulose (CMC). Industrial applicability was demonstrated through bio-polishing of cotton fabric, resulting in improved surface smoothness and a 3.8% weight loss, comparable to that of standard cellulase. Compatibility with various commercial detergents showed the highest level of activity, increasing from 69% to 97% in Surf Excel. This finding offers a cost-effective design for enzyme production using large amounts of lignocellulose byproducts, which can meet industry needs while also managing the disposal of agricultural wastes, etc.