2.7 Preparation of crude protein extract and assay of
β-glucosidase enzyme
Extraction and assay of β-glucosidase enzyme from J. auriculatumflowers was carried out by the method described by Reuveni et al. (1999)
with slight modifications. Briefly, flowers were ground to powder with
the help of liquid nitrogen. The crude protein was extracted with an
extraction buffer comprising of 50 mM ascorbic acid, 50 mM Mes, 20 mM
DTT, 10 mM MgCl2, 4% PVPP, 3mM PMSF and the pH was
brought to 7.4 with the help of solid Tris. The slurry was centrifuged
at 12,000 r.p.m. for 20 min at 4°C. The supernatant obtained was used
for assay of β-glucosidase. The enzyme assay was carried out in 100 mM
Citrate buffer (pH 6). The cleavage of the substrate
p-nitrophenyl-β-D-gluco-pyranoside (pNGP) by crude enzyme to glycosyl
moiety was monitored spectrophotometrically at 400 nm. The increase in
absorbance was recorded for estimating the activity of β-glucosidase.