Synthesis of Novel Alkaline Protease-Incorporated Hybrid Nanoflowers (atP@hNFs) and Their Applications in the Laundry Detergent Industry

In recent years, enzyme-inorganic hybrid nanoflowers (hNFs) have received considerable attention due to their superior stability, enhanced catalytic activity, and reusability. In this study, we developed protease-hybrid nanoflowers (atP@hNFs) and evaluated their washing performance against protein-based stains in laundry detergents. For this purpose, the free protease (atP) from the <i>Bacillus cereus</i> strain TNP13 was produced and used to synthesize protease-hybrid nanoflowers (atP@hNFs). Subsequently, the atP@hNFs were characterized by various methods. Finally, both atP and atP@hNFs were utilized as bioadditives in laundry detergent formulations. The optimum pH and temperature values were determined to be pH 9.0 and 45 °C for free atP, and pH 10.0 and 55 °C for atP@hNFs, respectively. The pH stability of atP@hNFs showed a 23.3% increase compared to the free enzyme. Both free atP and atP@hNFs maintained stability across a temperature range of 20 to 75 °C. In the zymogram analysis of free atP, six activity bands with molecular weights of 123, 84, 71, 63, 57, and 49 kDa were detected. atP@hNFs showed significant increases in their activities compared to free atP in the presence of various metal ions such as Ca²⁺, Co²⁺, Mg²⁺, and Zn²⁺. While atP@hNFs maintained all their stability in H₂O₂, they were about 67% more stable in SDS than free atP. The washing performance analysis revealed that the stain removal capacity of atP@hNFs was superior to that of free atP in removing protein-based stains. These findings suggest that the atP@hNFs are particularly effective in cleaning protein-based stains and are a valuable candidate for use as a biological additive in various laundry detergents.