Assay of protease activity
The proteolytic activity was determined at pH 6.0 by the casein
digestion method described previously [37]. In brief, 5ml of 1.2%
of casein solution in 0.05M phosphate buffer (pH 6.0) was added
1ml of enzyme solution, and the mixture was incubated at 35 ◦C
for 10min. After incubation, 5ml (0.44M) of tri-chloroacetic acid
(TCA) was added to quench the reaction, which was followed by
filtration. 2ml of the above filtrate was added 5 ml NaOH (0.28 N)
solution and 1.5ml phenol reagent (Folin-Ciocalteu phenol solution:
water = 1:2). After the mixture was kept at 35 ◦C for 15 min,
optical density (OD) at 660 nm was measured with a Hitachi spectrophotometer.
Ratio of the milk-clotting activity to proteolytic
activity is expressed as milk-clotting units (SU) per the OD660’s
obtained in the proteolytic measurements. Protein determination
was according to the method of Lowry et al. [38].