Application of bioactive peptides (BAPs) is promising due to their potential antimicrobial, antioxidant, agonistic, and ACE inhibition properties. To achieve a stable and active peptide at relatively high pH and temperatures by microbial fermentation, a wide variety of microorganisms need to be explored from diverse habitats, and compost is the excellent source. In an attempt to isolate potent protease-producing bacteria, gelatin-supplemented DM agar medium was used. Out of 140 pure cultures, initial protease production selects isolate D3L/1 (26 U/mL), and 16S rDNA sequencing confirmed it as Bacillus subtilis. Protease production was increased to 55.55 U/mL, with pH 7.5, 1% glucose, 1% casein, 1% ammonium sulfate, for 96 h of fermentation, at 37 °C under 140 rpm of shaking. Ion-exchange, and size-exclusion chromatography, 30 KDa protease was purified up to 4.1-fold (specific activity 3448.62 U/mL; 67.66% yield). The enzyme was active under broad temperatures (60 °C optimum), organic solvents, and pH variations. A total of 5% H2O2 can only reduce 40% of enzyme activity. However, 1 mM, Fe2+, and Cu2+ increased enzyme activity by five times. Soy hydrolysis (SPI) byD3L/1 protease produces bioactive compound (<3 KDa), which confirmed the peptide bond in the far UV region (205 nm, 215 nm, 225 nm, and 280 nm). The compound was ineffective towards Serratia marcescens but active against Escherechia coli (47%), Staphylococcus aureus (28%), and Pseudomonas aeruginosa (12%).
