A Facile Purification Procedure of Phospholipase D from Cabbage and its Characterization

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Abstract
Phospholipase D (PLD), an enzyme predestined for the preparation of new phospholipids, was isolated from cabbage and purified in a highly efficient way by using a combination of hydrophobic chromatography and a specific calcium effect. In the presence of calcium ions (50mM), PLD is bound from the crude enzyme solution to Octyl-Sepharose and subsequently selectively eluted by removing the calcium ions. The obtained enzyme is electrophoretically pure (95%), its molecular mass and isoelectric point were determined to be 87,000 Da and 4.7, respectively. The purified enzyme was kinetically characterized by use of mixed phosphatidylcholine-SDS micelles as well as the short-chain lecithins 1,2-dihexanoyl- and 1,2-diheptanoyl-sn-glycero-3-phosphocholine as substrates. A hyperbolic upsilon/[S]-characteristic was obtained for the mixed micellar system, whereas the upsilon/[S] curves of the short-chain lecithins reflect the dependence of velocity on the physical state of the substrate. A small velocity increase was observed up to a critical substrate concentration near the critical micelle concentration, from where the velocity increases hyperbolically.