2.4. Screening of Endophytes for L-

2.4. Screening of Endophytes for L-Asparaginase Activity.
The plate assay method of Gulati et al. [22] was
adopted to screen fungal endophytes for L-asparaginase
activity on modified Czapek Dox’s (MCD) agar medium
(glucose—2.0 g/L, L-asparagine—10 g/L, potassium dihydrogen
phosphate (KH2PO4)—1.52 g/L, potassium chloride
(KCl)—0.52 g/L, magnesiumsulphate (MgSO4
⋅7H2O)—
0.52 g/L, copper nitrate (CuNO3
⋅3H2O)—0.001 g/L, zinc
sulphate (ZnSO4
⋅7H2O)—0.001 g/L, and ferrous sulphate
(FeSO4
⋅7H2O)—0.001 g/L) adjusted to a pH of 6.2. Phenol
red indicator (0.009%) was prepared from a stock solution of
2.5% of the dye in ethanol.The control plates were prepared
withMCDmediumdevoid of asparagine (instead containing
KNO3—0.001 g/L as the nitrogenous source) and phenol
red indicator to check the ability of test fungi to grow in
the medium. The mycelial plugs from four different fungi
were inoculated on MCD agar medium marked into four
quadrants. The plates were incubated at 27∘C for five days.
The colonies exhibiting pink zones were inoculated onMCD
agar medium plates to confirm the activity of enzyme prior
to estimation.
2.5. Enzyme Estimation by Nesslerization. The positive isolates
were cultured in MCD broth medium incubated at
30∘C in orbital shaker (GeNei, Bangalore) set at 120 rpm
for five days. L-asparaginase was estimated by Nesslerization
as described by Imada et al. [23]. The reaction mixture
containing 0.5mL of 0.04M L-asparagine, 0.5mL of 0.5M
Tris HCl buffer (pH 8.2), 0.5mL of enzyme, and 0.5mL
distilled water was incubated at 27∘C for 30min. 0.5mL of
1.5M trichloroacetic acid (TCA) was added to each reaction
tube to stop the reaction. 0.1mL was drawn from the above
reaction mixture tube to another tube to which 3.7mL of
distilledwater and 0.2mLofNessler’s reagentwere added and
incubated for 20 min. The optical density was read at 450nm
using UV-Visible spectrophotometer (TPL Technology Pvt.
Ltd., Bangalore). Blank tubes were prepared by adding the
enzyme after the addition of TCA. One international unit
(IU) of L-asparaginase is the amount of enzyme needed to
liberate one