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 Ne