Abstract
Aim:
The present study aimed to identify a negative modulator of lipolytic enzyme
fatty acid esterase (FAE) for exploring possibilities of increasing shelf
life of pearl millet flour through arresting in-situ hydrolysis of
lipids.
Methodology: FAE was partially purified from flour of pearl
millet hybrid HHB 234 by (NH4)2SO4
fractionation (30-60% saturation) and gel filtration chromatography using
Sephadex G-75. The enzyme was characterized for physico-chemical properties
viz., molecular mass, optimum pH, optimum temperature and thermal stability
and kinetic properties viz., Km value and effect of
modulators.
Results:
Crude extract contained 1008 units of activity and 421 mg proteins resulting
in to specific activity of 2.4 units mg-1 protein. The enzyme was
purified 10.7 fold with a recovery of 21.52% and specific activity of 25.7
units mg-1 protein by ammonium sulphate fractionation followed by
gel filtration. The molecular weight of purified enzyme preparation was 60
kDa, as determined by gel filtration through Sephadex G-75. The enzyme
exhibited maximum activity at pH 8.2 and 45°C. The enzyme was stable up to
60°C for 20 min and showed Km value of 0.65 µM for p-NPB. At 10 mM
concentration, Mg2+ and Zn2+ altered the activity
positively by 54% and negatively by 42% whereas EDTA, DTT, PMSF, ascorbic
acid inhibited the activity by 75, 68, 50 and 48%,
respectively.
Interpretation: Partially purified lipolytic enzyme FAE from
pearl millet flour was strongly inhibited by ascorbic acid. This novel
information might be useful in developing processing technologies for
improving shelf life of flour.
Key
words:
Characterization, Fatty acid esterase, Pearl millet, Purification, Shelf life
|