Isolation, Screening and Characterization of Protease Producing Bacteria: Comparative Analysis of Solid-State and Submerged Fermentation Methods

Proteases are among the most widely used industrial enzymes, with significant applications across food, detergent, leather, and pharmaceutical industries. This study reports the isolation of protease-producing bacteria from a protein-rich environment, with Bacillus subtilis I19 (GenBank Accession: PV942511) showing the highest activity. The culture was optimized under submerged fermentation (SmF) conditions with shaking at 35 °C and pH 7–9. The enzyme retained activity for up to three days, indicating good stability for industrial applications. The enzyme was purified using ammonium sulphate precipitation, dialysis, ion exchange, and gel filtration chromatography, resulting in a 4.5-fold purification with a final specific activity of 23.49 U/µg. SDS-PAGE revealed a prominent band at ~20 kDa, typical of Bacillus proteases. In SSF, finger millet (ragi) stem supplemented with skimmed milk powder yielded maximum protease levels (5673.2 U/g), indicating a novel substrate for protease production through Bacillus strains. These findings highlight B. subtilis I19 as a promising candidate for cost-effective protease production using agro-residues, contributing to sustainable enzyme biotechnology.