Enzimas inmovilizadas del veneno de cascabel para la producción de lisolecitinas

MARIA DEL ROSARIO ALONSO; LAURA C. LEIVA; ECHEVERRÍA, SILVINA M.; FUSCO LUCIANO S

Buenos Aires

Jornada; XXIII Annual meeting of the argentinean biology society; 2022

Enzymes are highly effective catalysts produced by living things and are required in very low concentrations.These characteristics make them suitable for use in industrial, food, cosmetic and pharmaceutical applications, among others.The NEA are found species that are of interest for offering a secretion rich in enzymes that may have industrial applicability, such is the particular case of the rattlesnake (Crotalus durissus terrificus), which is characterized by its venom having a high content of phospholipase A2 (PLA2).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 and surfactants suitable for use in many industrial.Taking into account that the catalysts must remain in the reaction medium when the catalysis products are removed, the objective of this work was to immobilize Crotalus durissus terrificus venom (rich in PLA2) and evaluate its use on a source rich in lecithins for the production of lysolecithins. The venom was immobilized on CNBr-activated Sepharose TM 4B at room temperature, with magnetic stirring for 24hs.The amount of venom retained was assessed by absorbance measurement (UV-280nm). The PLA2 activity of the venom was determined by colorimetric assay with phenol red. The crude lecithin extract (CLE) was obtained from egg yolk (20 g)by solvent extractions:first step with ethanol (96%),and then, the soluble fraction was treated with acetone; finally, the precipitate was dried in an oven at 37ºC.Lysolecithins were obtained by means of an enzymatic reaction under batch type system with ELs and PLA2 previously immobilized in 10mM phosphate buffer, for 2 hs under agitation at 37ºC.The initial sample (ELs) as the liquid phase after the contact of the ELs with the immobilized enzyme was subjected to TLC on silica gel 60 F254 using petroleum ether/ethyl ether/acetic acid (90:10:1 v/v/v ) and chloroform/methanol/water (65:35:3 v/v/v) as mobile phases. Developed with Dragendorff´s reagent. The stains obtained were analyzed by densitometry (ImageJ software),and from the areas of each peak the relative percentage content was estimated of lecithins and lysolecithins present in the different samples.It was immobilized to 99,64% of the protein content present in the initial solution of the venom.On the other hand, although lysolecithins are present in a small proportion in the initial lecithin sample, treatment with immobilized PLA2 raised lysophospholipids by 20% compared to the initial sample. These preliminary findings will contribute to the use of matrices with immobilized enzymes for the production of lysolecithins with potential biotechnological use.