Crotalus venom as a source of biologically active enzymes for lysolecithin preparation

Abstract

Enzyme catalysed processes in industry are more and more numerous, as they have a number of advantages over conventional non-biological catalysts. Phospholipases A2 (PLA2s) are receiving a lot of attention due to their biotechnology potential. It is the family of enzymes most used for the enzymatic modification of lecithin. These enzymes act on glycerophospholipids, release the fatty acid from the 2-position of glycerol and thus lead to the formation of lysophospholipids. These molecules are excellent emulsifiers, particularly suitable for use in many industrial applications, such as food technology and the cosmetic and pharmaceutical industry. They are found in invertebrates (bees), mammals (bovine and porcine pancreas), and some microorganisms and in the snake venoms. The aim of this work was to evaluate the use of Crotalus durissus terrificus venom (rich in PLA2 enzymes) for lysolecithins production to be used later in the formulation of safe and effective emulsions with industrial potential. The crude lecithins extract (CLE) were carried out by solvent extractions from egg yolk (20 g): first step with ethanol (96%), then, the soluble fraction was treated with acetone; finally the precipitated was dried and weighed. Lecithins present in CLE were detected by two test: a) precipitation with saturated solution of cadmium chloride 2% in ethanol, and b) by the relative mobility on thin layer chromatography (TLC), on silica gel 60 F254 using acetone / hexane (1: 3) v / v; Cl3CH / MeOH / acetic acid / water (50: 25: 8: 2) v / v as mobile phase and Dragendorff´s reagent as developer solution. Lysolecithins (crude lysolecithins extract, CLyE) were obtained by the action of Crotalus durissus terrificus venom (1 mg /ml PBS, 2 ml) on CLE (30 mg/ml), during 30 min, 37°C. The lysofosfolipids presents in CLyE were detected by: a) TLC and b) hemolytic test, by measuring of absorbance at 530 nm of supernatant solution after incubation (30 min, Tamb) of lysolecithins solution with erythrocytes suspension. The results showed that:- solvents extraction was effective to obtain a solid extract (0,32 g) rich in lecithins. An abundant white precipitate with cadmium solution and a spot with Rf 0,47 by TLC assay confirmed the lecithins presence.- C.d. terrificus venom was capable of hydrolyze phospholipids from CLE; obtaining an extract rich in lysolecithins (CLyE). This extract exhibited hemoltytic activity and the spot in TLC showed a low retention factor value (Rf 0,20), typical of lysolecithins. These preliminary results demonstrate the ability of crotalic venom to produce lysolecthins capable to disrupt the eritrocitary membrane, as “strong" detergents do. Further studies are required to assess the potential use of these biomolecules to produce lysolecithins able to be used in industry as emulsifiers and detergents.