2.3. Keratinase Activity Assay

The reaction mixture contained 1.0 mL of the cell-free supernatant + 0.01 g chicken keratin powder (prepared in 1 mL of 50 mmol citrate buffer pH 5.0). The mixture was incubated in a water bath at 50 °C for 60 min. The reaction was stopped by adding 2.0 mL 10% trichloroacetic acid (TCA). The resulting precipitate was separated by centrifugation at 10,000× g for 10 min. Then, 0.2 mL of the supernatant was diluted to 1.0 mL with purified water, and 5.0 mL of alkaline cupper reagent (sodium carbonate, 40 g; tartaric acid, 7.5 g; copper sulfate, 4.5 g and distilled. water, 1000 mL; final pH 9.9 ± 0.5) was added. Afterwards, 0.5 mL of the Folin–Ciocalteu reagent was added and the tubes were kept in the dark for 30 min to allow the blue color formation. Negative control was prepared by incubating the enzyme solution with 2 mL of 10% TCA without keratin. Absorbance was measured at 660 nm (UV-visible spectrophotometer; T80+; UK), using tyrosine as the standard. One unit of keratinolytic activity corresponds to the enzyme amount that releases 1 μmol tyrosine mL⁻¹ min⁻¹ under standard test conditions [24], according to the L-tyrosine standard curve (Equations (1)–(3)) [24].

Total protein content was measured with the method of [25], using bovine serum albumin (BSA) as the standard and the specific keratinase activity per mg protein was calculated.