2.5. Protease assay

Protease activity was detected in terms of tyrosine content according to Lowry's method (Lowry, Rosebrough, Farr, & Randall, ¹⁹⁵¹). 250 μl of the protease solution was mixed with 250 μl 2% casein solution (50 mM sodium acetate buffer, pH 6.0) in 50°C water for 10 min. Then, the Folin reagent was added, and the mixed solution was detected at 680 nm. One unit of protease activity was defined as the amount of enzyme capable of hydrolyzing to 1 μg of tyrosine per min.