PURIFICATION OF PPO FROM Prunus persica (L.) var. nectarine

TELLO, JESICA; GASSULL, ESTELA

Otro; XXXIV Reunión Anual de la Sociedad de Biología de Cuyo; 2016

Polyphenoloxidases (PPO) are widely distributed in fruits and vegetables, and is important to characterize their activity in order to facilitate their control during processing. The enzyme extraction was conducted following the procedure of Gauillard and Richard-Forget with some modification. The enzyme extract obtained was purified by a chemical and a physical methods and enzyme activity was evaluated by measuring the variation of absorbance (A) at 400 nm, which is the length of the maximum absorbance of the reaction product (using 50 𝜇L of 2.737 M of 4-Methylcatechol as substrate and 100 𝜇L of enzyme in 3mL of solution buffer 5.5). The first purification method, consisted of precipitating the enzyme with (NH4)2SO4 at two concentrations, 35% w/v and 70% w/v. After purification with 35% (NH4)2SO4 activity of the extract was 2.14E-01 A/min mL E, whereas using 70% (NH4) 2SO4, the activity was 8.00E-03 A/mL min E. The enzyme purified by the second method exhibits lower activity values respect to the first, due to a possible inhibition of the enzyme. The physical method consisted of ultrafiltration, centrifuging the extract in Centriplus tubes with a 30 kDa membrane cut-off. Activities of 1.72 A/ min mL E for purified enzyme and 2.27E-01 ΔA/min mL E for corresponding residue were obtained, indicating that a large part of sugars and amino acids present in the extract with molecular weights less than 30 kDa, pass through the membrane, while the enzyme is retained in the supernatant. This result is expected, according to the molecular weights reported for different isozymes PPO, ranging from 50 kDa to 120 kDa. Considering the extract without any purification showed an activity of 1.42E-03 ΔA/mL min E, we conclude that the most effective method to purify the extract was the physical method.