Biochemical characterization of immobilized recombinant subtilisin and synthesis and functional characterization of recombinant subtilisin capped silver and zinc oxide nanoparticles

Abstract

This pioneering research explores the transformative potential of recombinant subtilisin, emphasizing its strategic immobilization and nanoparticle synthesis to elevate both stability and therapeutic efficacy. Achieving an impressive 95.25 % immobilization yield with 3 % alginate composed of sodium along with 0.2 M CaCl₂ indicates heightened pH levels and thermal resistance, with optimal action around pH 10 as well as 80 °C temperature. Notably, the Ca-alginate-immobilized subtilisin exhibits exceptional storage longevity and recyclability, affirming its practical viability. Comprehensive analyses of the recombinant subtilisin under diverse conditions underscore its adaptability, reflected in kinetic enhancements with increased V_max (10.7 ± 15 × 10³ U/mg) and decreased K_m (0.19 ± 0.3 mM) values post-immobilization using N-Suc-F-A-A-F-pNA. UV–visible spectroscopy confirms the successful capping of nanoparticles made of Ag and ZnO by recombinant subtilisin, imparting profound antibacterial efficacy against diverse organisms and compelling antioxidant properties. Cytotoxicity was detected against the MCF-7 breast cancer line of cells, exhibiting IC₅₀ concentrations at 8.87 as well as 14.52 µg/mL of AgNP as well as ZnONP, correspondingly, indicating promising anticancer potential. Rigorous characterization, including FTIR, SEM-EDS, TGA and AFM robustly validate the properties of the capped nanoparticles. Beyond therapeutic implications, the investigation explores industrial applications, revealing the versatility of recombinant subtilisin in dehairing, blood clot dissolution, biosurfactant activity, and blood stain removal. In summary, this research unfolds the exceptional promise of recombinant subtilisin and its nanoparticles, presenting compelling opportunities for diverse therapeutic applications in medicine. These findings contribute substantively to biotechnology and healthcare and stimulate avenues for further innovation and exploration.