The proteolytic activitywas determined according to the procedure of Arnon
[21] with slight modification. The reaction mixture contained 200 l of 50mM
cysteine–20mMEDTA (disodium salt), pH 8.0, 700 l 50mMTris–HCl buffer,
pH 8.0 and 100 l enzyme solution. The mixture was incubated at 37 ◦C for
5 min before starting the reaction by adding 1ml of 1% (w/v) casein solution.
After 10 min, the reactionwas stopped by adding 3ml of 5%(v/v) trichloroacetic
acid (TCA) and then cooled for 1 h. The reaction mixture was centrifuged, and
absorbance of the supernatant was measured at 275 nm. The reading was corrected
for a blank in which the enzyme was added after addition of TCA