CHARACTERIZATION OF POLYPHENOL OXIDASE OF SOURSOP (Annona muricataL.) FRUIT AND A COMPARATIVE STUDY OF ITS INHIBITION IN ENZYME EXTRACT AND IN PULP CARACTERIZACIÓN DE POLIFENOLOXIDASA DE FRUTOS DE GUANÁBANA (Annona muricata L.) Y ESTUDIO COMPARATIVO DE SU INHIBICIÓN EN EXTRACTOS DE ENZIMA Y EN PULPA CARACTERIZACIÓN DE POLIFENOLOXIDASA DE FROITOS DE GUANÁBANA (Annona muricata L.) E ESTUDIO COMPARATIVO DA SÚA INHIBICIÓN EN EXTRACTOS DE ENZIMA E EN PULPA

Abstract

Crude enzyme extract containing polyphenoloxidase (PPO) was obtained from ripe soursop (Annona muricata L.) fruit by extraction with 0.2 M phosphate buffer (pH 7.5) containing 0.5 M NaCl, followed by precipitation with 20, 40, 60 and 80% saturation of ammonium sulfate and dialysis against distilled water at 4ºC. The maximum PPO activity was observed in the protein fraction obtained with 60% ammonium sulphate saturation. The effect of pH (3 to 8.8), temperature (20 to 60ºC) and chemical inhibitors (ascorbic acid, EDTA and SO 2 ) at several concentrations on the PPO activity was studied. The optimum pH and temperature for PPO activity were 7.5 and 32ºC, respectively. Among chemical inhibitors, ascorbic acid and SO 2 were almost equally effective, but EDTA showed very small inhibitory effect. 61.9 and 71.5% inhibition of PPO was achieved using 0.28 mM and 0.84 mM concentrations of ascorbic acid, while for 55.4 and 70.2% inhibition 0.78 mM and 2.34 mM concentrations of SO 2 were needed. Heating the enzyme extract at 70, 80, 90 and 95ºC for 6 s reduced 15.2, 49.5, 84.8 and 95.1% PPO activity, respectively. At the natural pH of pulp (4.3), SO 2 at concentration of 3.9 mM reduced specific activity of PPO from about 60 to about 8.4 U/mg protein in partially-purified enzyme extract (PPEE), while the specific activity of 22.5 U/mg protein was retained in the pulp. Heating PPEE at 80ºC at pH 4.3 (natural pH of pulp) for about 14 s decreased specific enzyme activity from 60.0 to about 11.0 U/mg and heating during 8 min decreased to 9.0 U/mg protein. ResumenSe obtuvo un extracto crudo de enzima conteniendo polifenoloxidasas (PPO) de frutos maduros de Guanábana (Annona muricata L.) por extracción con 0,2 M tampón fosfato (pH 7,5) conteniendo 0,5 M NaCl, seguido de precipitación con 20, 40, 60 y 80% de saturación de sulfato amónico y diálisis con agua destilada a 4ºC. El máximo de actividad de PPO se observó en la fracción de proteína obtenida con 60% de saturación sulfato amónico. Se estudió el efecto del pH (3 a 8,8), temperatura (20 a 60ºC) y inhibidores químicos (ácido ascórbico, EDTA y SO 2 ) a varias concentraciones, sobre la actividad de PPO. Los óptimos de pH y temperatura para la actividad de PPO fueron 7,5 y 32ºC, respectivamente. Mientras que para los inhibidores químicos, ácido ascórbico y SO 2 fueron casi igual de efectivos, pero EDTA mostró un bajo efecto inhibidor.Se obtuvo un 61,9 y 71,5% de inhibición de PPO con 0,28 mM y 0,84 mM de concentración de ácido ascórbico, mientras que con 0,78 mM y 2,34 mM de concentración de SO 2 obtuvo un 55,4 y 70,2% de inhibición, respectivamente. Calentando el extracto enzimático a 70, 80, 90 y 95ºC durante 6 s, se redujo a 15,2, 49,5, 84,8 y 95,1% la actividad de PPO, respectivamente. Al pH natural de la pulpa (4,3), SO 2 a la concentración del extracto enzimático 3,9 mM redujo la actividad específica de PPO desde 60 a 8,4 U/mg proteína en extracto enzimático parcialmente purificado (PPEE), mientras que en la pulpa la actividad específica se retuvieron 22,5 U/mg de pr...

Purification and biochemical characterization of polyphenol oxidase from soursop (Annona muricata L. ) and its inactivation by microwave and ultrasound treatments

“…(Tao et al, 2013). In partially pure PPO from soursop ascorbic acid showed 89% inhibition at 3.12 mM (Bora et al, 2004), while for PPO from atemoya 75% inhibition at 0.83 µM was observed (Rodrígues Cháves et al, 2011). Kojic acid in a concentration range of 1 to 4 mM has been observed to be a good antibrowning agent, in PPO from soursop it inhibited 100% at 10 mM as was the case for PPO from Manila mango and mamey (Palma-Orozco et al, 2014.…”

“…to 50°C, the optimum temperature being 28ºC; in the partially pure PPO from soursop activity was reported to be stable between 20 to 50ºC, with highest activity at 32ºC (Bora et al, 2004).…”

“…Rodrígues Cháves et al, 2011). In partially pure PPO from soursop, the optimum pH was 7.5(Bora et al, 2004).…”

Dye-decolourizing yeasts isolated from Las Yungas rainforest. Dye assimilation and removal used as selection criteria

“…Mn-dependent peroxidase (MnP) experiments were based on the oxidation of MBTH/DMAB followed by the increase in absorbance at 610 nm in a reaction mixture containing 0.07 MBTH, 1 mM DMAB, 0.3 mM MnSO 4 .7H 2 O, 0.05 mM H 2 O 2 in 100 mM succinateelactate buffer (pH 4.5) (Castillo et al, 1994). Finally, Tyrosinase (Tyr) measurements were based on catechol oxidation followed by the increase in absorbance at 420 nm in a reaction mixture containing 0.9 mM of cathecol in 50 mM phosphate buffer (pH 7.4) (Bora et al, 2004).…”

Evidence on manganese peroxidase and tyrosinase expression during decolorization of textile industry dyes by Trichosporon akiyoshidainum

“…Tyrosinase (Tyr, EC 1.10.3.1) was measured with 0.01% w v À1 pyrocatechol in 50 mM phosphate buffer, pH 7.4 (Bora et al, 2004).…”