Isolation, Purification and Characterization of Phospholipase D from Karpolah Almond (Prunus Dulcis) of Ziarat Balochistan, Pakistan.

Abstract

Phospholipase D allows the release of phosphatidic acid on hydrolysis of phospholipids which is an important intermediate of different signaling reactions and pathways, including resistance response in plants and carcinogenicity in mammals. In this study Phospholipase D was isolated, purified, and characterized from Karpolah almond (Prunus dulcis) of District Ziarat, Balochistan. Phospholipase D activity was observed in crude extract, in acetone precipitates and in purified sample using Bergmeyer Gavehn technique with slight modifications. The results showed 1246±0.02, 1007±0.03 and 987±0.01 U of activity in crude extract, acetone precipitates and in purified sample, respectively. The maximum extraction time for the activity of PLD was observed as 60 minutes. Utilizing Octyle-Sepharose gel and Hydrophobic Interaction Chromatography, the enzyme was isolated with the aid of Ca⁺² ion effect. In first fraction the enzyme was strongly bound with Octyl residue in the presence of 50mM of Ca⁺²ion and hence no activity was observed. When the concentration of Ca⁺² ion was lowered from 50 to 30mM, the loosely bounded proteins left the column in different fractions. Finally, by removing Ca⁺²ion from the column using 0.1 M EDTA, Phospholipase D was eluted and displayed highest activity. The purification of enzyme was confirmed by SDS-PAGE, which showed a molecular weight of 54kDa. The optimum temperature for that purified enzyme was observed as 50°C and at optimum pH as 6. Phospholipase D showed a stability of 40-90mM for Ca⁺² ion which confirms that this purified enzyme is Ca⁺² ion dependent.