Purification, and characterization of detergent-compatible serine protease from Bacillus safensis strain PRN1: A sustainable alternative to hazardous chemicals in detergent industry

Owing to vast therapeutic, commercial, and industrial applications of microbial proteases microorganisms from different sources are being explored. In this regard, the gut microbiota of Monopterus cuchia were isolated and examined for the production of protease. All the isolates were primarily and secondarily screened on skim milk and gelatin agar plates. The protease-positive isolates were characterized morphologically, biochemically, and molecularly. Out of the 20 isolated strains,6 belonging to five different genera viz. Bacillus, Priestia, Aeromonas, Staphylococcus, and Serratia demonstrated proteolytic activity. Bacillus safensis strain PRN1 demonstrated the highest protease production and, thus, the largest hydrolytic clear zones in both skim milk agar (15 ± 1 mm) and gelatin (16 ± 1 mm) plates. The optimized parameters (time, pH, temperature, carbon, nitrogen) for highest protease activity and microbial growth of B. safensis strain PRN1 includes 72 h (OD₆₀₀ = 0.56,1303 U/mL), pH 8 (OD₆₀₀ = 0.83, 403.29 U/mL), 40 °C (OD₆₀₀ = 1.75, 1849.11 U/mL), fructose (OD₆₀₀ = 1.22, 1502 U/mL), and gelatin (OD₆₀₀ = 1.88, 1015.33 U/mL). The enzyme was purified to homogeneity using salt-precipitation and gel filtration chromatography. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified enzyme was a monomer of a molecular weight of ∼33 kDa. The protease demonstrated optimal activity at pH 8 and 60 °C. It was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), demonstrating that it belongs to the serine-proteases family. The compatibility of the enzyme with surfactants and commercial detergents demonstrates its potential use in the detergent industry. Furthermore, the purified enzyme showed antibacterial and blood-stain removal properties.