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 Hg 2 + , while the activity loss was very small in the presence of Ca 2 + and Mg 2 + . The enzyme kinetic resulted in a of 103.89 U/mL and a of 3.71 mg/mL by nonlinear Michaelis–Menten regression. First‐order thermal inactivation showed k d value increasing from 0.000250 to 0.002900 min − 1 , E d (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.