International Journal of Biological Macromolecules 42 (2008) 380–385 Phospholipase D from Allium sativum bulbs: A highly active and thermal stable enzyme Hafeeza Khatoon, Sariya Talat, Hina Younus ∗ Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Near JNMC, Aligarh 202002, India Received 30 April 2007; received in revised form 28 January 2008; accepted 28 January 2008 Available online 7 February 2008 Abstract This is the first report on the identification and partial characterization of phospholipase D (EC 3.1.4.4) from Allium sativum (garlic) bulbs (PLD GB ). The enzyme shares the phenomenon of interfacial activation with other lipolytic enzymes, i.e. the hydrolytic rate increases when the substrate changes to a more aggregated state. The enzyme activity is highly temperature tolerant and the temperature optimum was measured to be 70 ◦ C. PLD GB unlike many plant PLDs exhibited high thermal stability. It was activated further after exposure to high temperatures, i.e. 80 ◦ C, indicating that the enzyme refolds better upon cooling back to room temperature after short exposure to thermal stress. The activity of PLD GB is optimum in 70 mM calcium ion concentration and the enzyme is activated further in the presence of phosphatidyl-4,5-bisphosphate (PIP 2 ). PLD GB exhibited both hydrolytic and transphosphatidylation activities, both of which appear to be higher than those of PLD from cabbage leaves (PLD CL ). © 2008 Elsevier B.V. All rights reserved. Keywords: Phospholipase D; Allium sativum; Activity; Stability 1. Introduction Phospholipase D (PLD) is implicated in a number of cel- lular processes such as transmembrane signaling, intracellular protein trafficking, secretion, alteration in cell morphology and motility [1,2]. PLD catalyzes two reactions: the hydroly- sis of phospholipids and the transfer of phosphatidyl groups to various alcohols, the so-called transphosphatidylation reac- tion. The transphosphatidylation activity of PLD is utilized commercially for the synthesis of rare natural phospholipids, e.g. phosphatidylserine and phosphatidylglycerol, and novel artificial phospholipids [3–5]. Artificial phospholipids have applications in both pharmaceutical and cosmetic industries [6]. The transphosphatidylation reaction is usually performed in biphasic systems consisting of water-insoluble organic sol- Abbreviations: BCA, bicinchoninic acid; GDH, glutamate dehydrogenase; PC, phosphatidylcholine; PE, phosphatidylethanol; PIP 2 , phosphatidylinositol- 4,5-bisphosphate; PLD, phospholipase D; PLD CL , PLD from cabbage leaves; PLD GB , PLD from garlic bulbs. ∗ Corresponding author. Tel.: +91 571 2720388; fax: +91 571 2721776. E-mail address: hinayounus@rediffmail.com (H. Younus). vents. The thermostability of PLD is important when it is used as a biocatalyst in these systems. Although PLDs from plants (preferable cabbage) and micro-organisms have been applied to synthesize phospholipids in laboratory and industrial scale for many years [7,8], those exhibiting broader substrate speci- ficity and higher thermostability are desired in the industrial setup. However, there are only few reports on the thermosta- bility of PLD [9]. A mutant PLD from Streptomyces sp. with an enhanced thermostability has also been generated recently [10]. Despite the importance of PLDs in basic research and biocatalytic application, knowledge regarding their molecular properties, particularly their tertiary structure and conforma- tional stability, is very limited. PLDs from different sources show differences in their properties, i.e. in their activity and sta- bility. So it becomes important to explore new sources of PLD for identifying some useful preparations, i.e. those that possess high catalytic activity and stability. We have recently identified a highly active and stable PLD from Brassica juncea seeds [11]. While investigating the enzyme activities present in the medici- nal plant garlic, we identified another highly active and thermal stable PLD in garlic bulbs (PLD GB ) and have partially charac- 0141-8130/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2008.01.007