Viscera from S. basilisca were wash

Viscera from S. basilisca were washed with distilled water then
with buffer A (10 mM Tris–HCl buffer, pH 8.0). The cleaned viscera
(40 g) were defatted by homogenisation with 80 mL cold acetone
for 30 s in a tissue homogeniser. The homogenate was filtered
and the acetone-insoluble material was washed several times with
cold acetone and then dried overnight at room temperature. The
acetone dried powder was then suspended in buffer A in a ratio
of 1:10 (w/v) and stirred continuously for 3 h at 4 C. The mixture
was centrifuged at 8500g for 30 min at 4 C. The pellet was discarded
and the supernatant was collected and used as the crude
protease extract.