Selected fungal cultures were first established in 30 mL of
McDox broth and incubated for 5 days at 120 rpm. After incubation,
100 lL of broth (crude enzyme) was pipetted into 2 mL
microcentrifuge tubes. To this, 100 lL of Tris HCl (pH 7.2),
200 lL of 0.04 M asparagine and 100 lL of sterile distilled
water (SDW) were added. The mixture was incubated at
37± 2 C for 1 h. After incubation, 100 lL of 1.5 M Trichloroacetic
Acid (TCA) was then added to stop the enzymatic
reaction. This was followed by pipetting 100 lL of the mixture
into fresh tubes containing 750 lL SDW and 300 lL of Nessler’s
reagent [18] and incubated at 28 ±2 C for 20 min, after
which the absorbance of the samples was measured at 450 nm.
One unit of asparaginase is expressed as the amount of enzyme
that catalyzes the formation of 1 lmol of ammonia per minute
at 37 ±2 C