Production of surfactant-stable keratinolytic protease from B. subtilis ES5 and its application as a detergent additive

Microorganisms capable of producing keratinase from Ethiopian traditional leather tanning environments have not been well studied. The objective of this study was to isolate, screen, and identify bacteria that produce keratinase in this natural environment and enhance its production for detergent additive applications. Isolate ES5 exhibited the highest diameter of clear zone (34.0 ± 0.00 mm) on skim milk agar (SMA). Maximum keratinase production was achieved at an incubation temperature, medium pH, incubation period, inoculum size, and feather concentration of 40 °C, 8, 48 h, 2%, and 9 g/L, respectively. Among the seven factors evaluated, incubation period, temperature, and feather concentration significantly influenced keratinase production using Plackett-Burman Design (PBD) statistical optimization. Response surface methodology (RSM) based optimization revealed a P-value of <0.0001 for the model, suggesting the importance and potential applicability of the model in keratinase production. Overall, statistical optimization revealed a keratinase yield of 317.60 ± 1.21 U/ml, demonstrating a 1.50-fold increase. Crude keratinase showed optimum activity at pH 8 and a temperature of 45 °C. This enzyme also showed the highest stability to Ariel detergent with retained activity of 95.45 ± 3.20%. A maximum cleaning performance of the enzyme of 96.41 ± 0.19 was achieved at temperature, shock time and detergent concentration of 45 °C, 15 min and 7 mg/mL respectively. The production of keratinase in a short period of time using inexpensive substrate demonstrates potential biotechnological applications in various industries. Crude keratinase from B. subtilis ES5 showed high stability and compatibility with surfactants and detergents and was considered a promising detergent additive.