Process Biochemistry 47 (2012) 2434–2439 Contents lists available at SciVerse ScienceDirect Process Biochemistry jo u rn al hom epa ge: www .elsevier.com/locate/procbio Purification and biochemical properties of Hexaplex trunculus digestive lipase Zied Zarai ∗ , Madiha Bou Ali, Ahmed Fendri, Hanen Louati, Hafedh Mejdoub, Youssef Gargouri Laboratoire de Biochimie et de Génie Enzymatique des Lipases - Ecole Nationale d’Ingénieurs, B.P “1173” 3038 Sfax, Tunisia a r t i c l e i n f o Article history: Received 23 June 2012 Received in revised form 12 October 2012 Accepted 13 October 2012 Available online 23 October 2012 Keywords: Hexaplex trunculus Hepatopancreas Digestive lipase Purification Characterization a b s t r a c t Enzymes from fish and aquatic invertebrates have recently been characterized and their study has led to the emergence of some new applications of these classes of enzymes. However, very little is known about lipases from mollusks. A lipolytic activity was located in the marine snail digestive glands (hepatopan- creas), from which a marine snail digestive lipase named mSDL was purified. Pure mSDL has a molecular mass of about 70 kDa as determined by SDS/PAGE analysis. Unlike the known digestive lipases while acting at 37 ◦ C, the mSDL displayed its maximal activity on long and short-chain triacylglycerols at 50 ◦ C. A specific activity of 400 U/mg and 100 U/mg was obtained with TC4 or olive oil as substrate respectively. Only 25% of the maximal activity was measured at 37 ◦ C. Interestingly, neither colipase, nor bile salts were detected in the marine snail hepatopancreas, which suggests that colipase evolved in invertebrates simultaneously with the appearance of an exocrine pancreas and a true liver which produces bile salts. No similarity was found between the N-terminal amino acids sequence of the mSDL and those of the known digestive lipases. Altogether, these results suggest that the mSDL is a member of a new group of digestive lipases belonging to invertebrates. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction Lipases are hydrolytic enzymes (EC 3.1.1.3) catalyzing the hydrolysis of the water insoluble triglycerides into free fatty acids and glycerol and operate at the interface of the emulsified lipid substrates. Higher animal’s lipases are well-characterized [1–3]; in contrast, very little is known about lipases from lower ani- mal. A number of investigations on invertebrate lipases have been reported since the detection of a lipase activity in the gastric juice of some crustaceans (Homarus americanus) and mollusks (Aplysia californica) [4,5]. Recently, lipases have been isolated from diges- tive glands of lower animals, such as the scorpion [6] and the crab [7]. The purified enzymes showed no similarity with any known lipases. Within the phylum of mollusks, gastropods are by far the largest class. Many studies have described the morphology as well as the digestive system of snails [8,9]. In fact, the digestive glands (hepatopancreas) as well as the salivary gland of snails are the main source of digestive enzymes [9]. The digestive gland was shown to be composed of two main cell types, digestive and secretory cells. The digestive cells appeared to be concerned with the absorption and digestion of nutrients, while secretory cells produced digestive Abbreviations: mSDL, marine snail digestive lipase; SDL, scorpion digestive lipase; TPL, turkey pancreatic lipase; SXL, Staphylococcus xylosus lipase; NaTDC, sodium taurodeoxycholate; NaDC, sodium deoxycholate; PVDF, polyvinylidene difluoride; SDS, sodium dodecyl sulphate. ∗ Corresponding author. Tel.: +21 674675055; fax: +21 674675055. E-mail address: zaraizied@hotmail.fr (Z. Zarai). enzymes and calcareous concretions. Undifferentiated cells were scattered among these two main cell types [9]. The digestive gland or the hepatopancreas of gastropod mol- lusks, like the marine snail Hexaplex trunculus, is the main source of production of digestive enzymes. It is involved in the absorption of nutrients, food storage and excretion [10]. Digestive glands of gastropod mollusks are considered as an excellent model for food digestion and cell secretion by many authors [11]. Its primary roles are the synthesis and the secretion of digestive enzymes, swallow- ing and final digestion of the ingested food and subsequent uptake of nutrients. Many studies have attempted to purify peptidases and cellu- lases from snail hepatopancreas [12,13] but, to the best of our knowledge, only a few studies have been conducted on the lipolytic enzymes of snails. Lately, a digestive hepatopancreatic phospholi- pase from marine snail (H. trunculus) was purified in our laboratory [14]. Moreover, Amara et al. [15], purified a lipase from land snail (Eobania vermiculata). In this study, the marine snail hepatopan- creas represents the starting material to isolate a lipase. This paper reports the purification of a lipase from the marine snail hepatopan- creas to homogeneity. This lipase, tentatively named marine snail digestive lipase (mSDL), was characterized with respect to its bio- chemical properties. 2. Materials and methods 2.1. Biological material Marine snails (H. trunculus) were collected from the sea coast of Sfax, Tunisia. They were placed on ice and immediately transported, to the laboratory. The 1359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2012.10.004