Endoesterolytic activity in proteolytic extract of Asclepias curassavica

ORIGONE, ANABELLA; LIGGIERI, CONSTANZA; BARBERIS, SONIA

Estancia Grande - San Luis.

Congreso; XXXII Reunión Científica Anual de la Sociedad de Biología de Cuyo; 2014

Sociedad de Biología de Cuyo

Asclepias curassavica L. belongs to a milked plant family called Asclepiadaceae whose members produce latex usually containing proteases with pharmaceutical, medical and biotechnological applications. In our study we determinated the esterolytic activity of asclepain by the Silvertein´s method (1974), modified according to optimal conditions of the enzyme in order to select the most appropriated substrate for the synthesis of bioactive peptide in non aqueous media. Asclepain was tested against several N -alpha carbobenzoxy- p- nitrophenyl esters of some L- amino acids used as substrates. Assays were made at 40 ºC in Tris- HCl buffer 0.1 M (pH 8) containing Cys (20 mM) and 0.15, 0.3, 6.25 mM of each substrate in the reaction mixture. Liberation of p-nitrophenol was followed spectrometrically at 405 nm using a Cintra 6 UV-Visible spectrophotometer. To determinate the mmol of p-nitrophenolate produced during the reaction, a standard curve with p- nitrophenol (5 - 50 mM) in Tris- HCl buffer 0.1M (pH8) containing acetonitrile 5% was carried out. This enzyme expressed high activities with all amino acid derivates tested, showing low selectivity by basic, polar and not polar amino acids. According to this, asclepain offer a lot of possibilities for the substrate selection in order to carry out the desirable peptide synthesis.