A Serine Protease-Producing Bacillus pumilus PUMMD23 Isolated from Agricultural Soil: Molecular Characterization and Biotechnological Potential

<title>Abstract</title> <p> BACKGROUND Microbial serine proteases are important biotechnological enzymes, accounting for nearly 60% of industrial enzyme applications. They are widely used in detergent, pharmaceutical, textile, food, and waste management industries. Soil microbiota is valuable sources of enzyme-producing bacteria. Among them, <italic>Bacillus pumilus</italic> is preferred because of its high enzyme productivity and generally recognised as safe (GRAS) status. This study aimed to isolate <italic>B. pumilus</italic> PUMMD23 from soil for industrial biotechnology applications. METHODS Soil samples collected from Southern India were stored at 4°C. One gram of soil was suspended in sterile water, serially diluted to 10⁻⁶, and plated on nutrient agar and actinomycetes agar. Isolates were purified by the T-streak method. Protease production was screened using skim milk agar. The isolates were characterized by Gram staining, biochemical tests, PCR, and BLAST analysis. RESULTS The soil samples contained diverse microorganisms. The selected isolate formed yellow colonies and appeared as Gram-positive rods in chains, indicating Bacillus. Clear zones on skim milk agar confirmed protease production. PCR and biochemical analyses identified the isolate as Bacillus <italic>pumilus</italic> PUMMD23. Casein hydrolysis after 48 hours at 27°C further confirmed extracellular protease production. CONCLUSION <italic>Bacillus pumilus</italic> PUMMD23 produces a serine protease with strong extracellular activity. Its proteolytic potential indicates possible applications in detergents, pharmaceuticals, food processing, and environmental biotechnology. Molecular characterization supports its future development as an industrial biocatalyst. </p>