Immobilization Techniques for Bacterial Proteases: Improving Stability, Activity and Application Potential

Abstract

Proteases are among the most important industrial enzymes and are widely incorporated into laundry detergent formulations because of their ability to hydrolyse proteinaceous stains. The present study investigated the laundry detergent application potential of a purified protease produced by Bacillus spp. through structural and kinetic characterization, immobilization, laundry detergent compatibility evaluation and washing performance analysis. Kinetic analysis using casein as the substrate revealed a Michaelis–Menten constant (Km) of 0.38 mg mL⁻¹ and a maximum reaction velocity (Vmax) of 128.29 U mL⁻¹, demonstrating high substrate affinity and catalytic efficiency. The protease was successfully immobilized in calcium alginate beads and it retained more than 80% of its initial activity in the presence of a laundry detergent, indicating good stability and compatibility. The washing performance of the proteasesupplemented laundry detergent was evaluated on cotton, polyester and silk fabrics stained with egg yolk and soy sauce. Optimization studies identified 1.0% protease concentration, 50°C and 30 min incubation as the optimal washing conditions. Under these conditions, stain-removal efficiencies ranged from 88% to 100%, significantly exceeding those achieved with laundry detergent alone (50–75%). Complete stain removal was observed for soy saucestained polyester fabric. Statistical analysis confirmed that fabric type, stain type, protease concentration, temperature and incubation time significantly influenced stain-removal efficiency (p < 0.05). SEM and FTIR analyses further verified the effective removal of stain residues and restoration of fabric morphology following enzymatic treatment. These findings highlight the potential of immobilized protease as an efficient, environmentally friendly additive contributing to greener household cleaning practices.