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 Vmax (10.7 ± 15 × 103 U/mg) and decreased Km (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.
ABSTRACT
The increase and dissemination of multi-drug resistant bacteria have presented a major healthcare challenge, making bacterial infections a significant concern. The present research contributes towards the production of bioactive subtilisin from a marine soil isolate Bacillus subtilis strain ZK3. Custard apple seed powder (raw carbon) and mustard oil cake (raw nitrogen) sources showed a pronounced effect on subtilisin production. A 7.67-fold enhancement in the production was evidenced after optimization with central composite design-response surface methodology. Subtilisin capped silver (AgNP) and zinc oxide (ZnONP) nanoparticles were synthesized and characterized by UV-Visible spectroscopy. Subtilisin and its respective nanoparticles revealed significant biological properties such as, antibacterial activity against all tested pathogenic strains with potential against Escherichia coli and Pseudomonas aeruginosa. Prospective antioxidant behavior of subtilisin, AgNP and ZnONP was evidenced through radical scavenging assays with ABTS and DPPH. Subtilisin, AgNP and ZnONP revealed cytotoxic effect against cancerous breast cell lines MCF-7 with IC50of 83.48, 3.62 and 7.57 µg/mL respectively. Characterizations of nanoparticles were carried out by Fourier transform infrared spectroscopy, scanning electron microscopy with energy dispersive X-ray, X-ray diffraction, thermogravimetric analysis and atomic force microscopy analysis to elucidate the structure, surface and thermostability properties. The study proposes the potential therapeutic applications of subtilisin and its nanoparticles, a way forward for further exploration in the field of healthcare.
