Enzymatic recovery of nano-silver from used X-ray films by alkaline protease from sp. VITGSS4.

Seventeen halotolerant bacteria were isolated from the Muthupettai mangroves, Tamil Nadu, India, with eight exhibiting protease production. The most potent isolate, VITGS4, identified as sp. via polyphasic taxonomy, yielded 470 U mL. Response surface methodology (RSM) optimized protease production by Box-Behnken Design (BBD) using casein (5.5% w/v), pH 7.5, and 9.5 days incubation, achieving 282 U mL. The recovered protease was partially purified through acetone precipitation (50% acetone), followed by dialysis, and its purity was estimated through HPLC (high pressure liquid chromatography). Enzyme kinetics revealed a Km of 0.347 µM, a Vo of 0.464 µM min, a Vmax of 3.167 µM min, and a Kcat of 0.0002 min. The enzyme was identified as a halo-thermo-alkaline serine protease, optimally active at pH 8 and 45°C, with activity significantly inhibited by Pb and Hg and enhanced by Zn (95%). Notably, PMSF strongly inhibited protease activity, indicating a serine protease. This protease was successfully employed to recover 726 mg of silver slurry (537 µg mL silver) from X-ray films. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of silver (2.2% in the analyzed region), while zeta potential (-26.35 mV) and hydrodynamic diameter (89.94 nm) analyses indicated stable silver nanoparticles. These results demonstrate the potential of marine actinobacteria-derived proteases for efficient silver recovery, offering promising applications in therapeutic and industrial fields.