All the purification procedures were performed at room temperature
since the enzyme activity was stable at room temperature.
Approximately, 450 mL of supernatant was collected through above
procedure. Finely grind ammonium sulfate (NH4)2SO4 of protein
precipitation grade was added to the supernatant with gentle
stirring until the solution reached 60% saturation. The solution was
centrifuged at 15,000 g for 10 min, and the supernatant was
collected. More (NH4)2SO4 was added to the supernatant until the
solution reached 70% saturation. The solution was centrifuged and
precipitate was dissolved in 6 mL of 50 mM TriseHCl buffer
(pH 8.0). This product was designated as crude enzyme extract
(CEE) and stored at 80 C until use.