0.5 ml of culture filtrate was taken and 0.5 ml of 2% casein
was added and incubated for 1 hour. After incubation, 0.1ml
of 10% trichloroacetic acid (TCA) was added and centrifuged.
To the 0.5 mL of supernatant, 2.5 mL of reagent A (2.4 g
sodium hydroxide, 5.8 g sodium carbonate and 200 mL
distilled water) and 0.75 mL of folin phenol reagent was
added to neutralize TCA and incubated for 20 minutes. The
readings were taken at 650 nm and the enzyme activity was
expressed per mL of filtrate. One unit of protease is that
amount of enzyme which produced an absorbance increase of
0.01 per hour under the assay conditions. The specific activity
of the enzyme is calculated as