2.5.3. Protein Estimation and Protease Activity Assay

The concentration of protein (mg/mL) was estimated following Lowry et al. (1951) and determined as a measure of its absorbance at 700 nm after each step of the enzyme purification process, using bovine serum albumin (BSA) as a reference [42]. Protease activity of the enzyme (U/mL) was assessed as per standard protocol by Cupp-Enyard, [43] with modifications, as pH and temperature of the assay were optimized wherein the enzyme was incubated at different pH (6–12) with temperature ranging 10–90 °C. Thus, at each purification process as well as for characterization of enzyme, the protease activity was measured at a standardized pH, 8 and temperature, 70 °C during this study.

The activity was assessed by incubating 5 mL of casein (0.65% w/v) prepared in 0.1 M Tris-HCl buffer (pH 8.0) with 1 mL of the enzyme at 70 °C for 30 min. This reaction was stopped by adding 5 mL of 10% (w/v) trichloroacetic acid (TCA) and incubated for 10 min at 37 °C followed by 15 min centrifugation at 10,000 rpm (4 °C). To 1 mL of supernatant collected in a fresh tube, 2.5 mL sodium carbonate (0.4 M) and 0.5 mL Folin–Ciocâlteu reagent (1 N) were added, gently mixed and incubated at 37 °C for 30 min. The absorbance was measured at 660 nm. For calculations, one-unit enzyme activity was interpreted as the amount of enzyme required to hydrolyze the casein to release 1 μmol tyrosine mL⁻¹ min⁻¹ under the standard conditions of the assay. A standard tyrosine curve was used to determine the number of tyrosine equivalents released. The specific activity of the sample was calculated by dividing the enzyme units (U mL⁻¹) by the overall protein content (mg mL⁻¹). Thus, the sample activity was denoted in units (U mL⁻¹) while the specific activity was designated as enzyme activity per mg of protein and expressed as (U mg⁻¹).