Improved Destaining and Antimicrobial Potential of Pepsin Cross-Linked HPMC-Se Nanoparticles

In this study, Hydroxypropyl methyl cellulose-stabilized selenium nanoparticles (HPMC-SeNPs) were successfully synthesized and utilized as a support for pepsin immobilization via glutaraldehyde crosslinking. Characterization through SEM, FTIR, and XRD confirmed their structural integrity, while zeta potential (- 12.61 mV) and DLS (PDI: 0.1818) indicated good colloidal stability and uniform size distribution. The immobilized pepsin (HPMC/Se-Pep) demonstrated high immobilization efficiency (81.25%) and yield (78.73%). The enzymatic system exhibited enhanced stability over a wider pH (2-6) and temperature range (20- $60~^{\circ }$ C), with improved kinetic parameters-lower K_m (0.16 mM) and higher V_max ( $0.94~\mu $ mol/min)-indicating stronger substrate affinity and better catalytic performance than free pepsin. Functional assays revealed significantly enhanced caseinolytic and antimicrobial activities, with inhibition zones of $22~\pm ~0.05$ mm (S. aureus) and $21~\pm ~0.12$ mm (E. coli), outperforming both native NPs and free enzyme. Notably, the immobilized pepsin achieved complete removal of blood stains within 30-40 min and retained 60.2% of its activity after five reuse cycles, confirming excellent operational stability. However, a gradual decline in enzymatic activity was observed after repeated reuse and prolonged storage, indicating the need for further optimisation to enhance long-term stability. These results demonstrate that HPMC-SeNPs are an effective platform for enzyme immobilization, offering improved performance, reusability, and multifunctionality. The developed system holds strong potential for applications in biocatalysis, textile processing, and healthcare industries.