Colonies of the selected isolate were transferred to 250 mL
Erlenmeyer flasks containing 50 mL of the optimized production
medium and incubated at 40°C under orbital shaking (200 rpm).
These cultures were used to inoculate 1 L flasks containing 250 mL
of the same medium and cultivated under the same conditions.
Samples (1 mL) were withdrawn at 2 h intervals up to 48 h for the
measurement of growth and alkaline protease activity. The samples
were centrifuged at 6000 × g at 4°C, and the pellets obtained were
washed twice using Tris buffer (pH 8), and resuspended in 1 mL
of the same buffer. Absorbance was measured at 600 nm against
Tris Buffer (pH 7) as blank, and was reported as growth of the
bacterium. The cell-free supernatant was filtered through a 0.45-μm
pore-size membrane filter, and the alkaline protease activity was
measured as described above. The triplicate of each period was taken
to calculate the growth and enzyme activity and the mean values
were reported.