Partial purification and characterization of fatty acid esterase from pearl millet

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