RESUMO
Polyphenol oxidase (PPO) enzyme was purified from avocado (Persea americana) by ammonium sulfate precipitation 0-80%, dialysis and affinity chromatography. Characterization studies were performed with catechol (0.10 M, pH: 7.2, 37 °C), 4-methyl catechol (0.10 M, pH: 6.0, 37 °C), pyrogallol (0.02 M, pH: 8.5, 5 °C), chlorogenic acid (0.20 M, pH: 6.8, 10 °C) and caffeic acid (0.20 M, pH: 8.5, 10 °C), respectively. Vmax and KM values were determined for catechol (15789.96 U*mL-1*min-1, 10 mM), 4-methyl catechol (6768.40 U*mL-1*min-1, 2 mM), pyrogallol (6802.72 U*mL-1*min-1, 4 mM), chlorogenic acid (1377.97 U*mL-1*min-1, 14.29 mM) and caffeic acid (2567.24 U*mL-1*min-1, 5 mM). PPO was purified as 147.73-fold and 154.00-fold by Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose-6B-L-Tyrosine-p-aminobenzoic acid, respectively. 4B isolated PPO gave two bands at 35 and 50 kDa in SDS-PAGE while visible and slightly visible bands at 50-70 kDa and 100 kDa in Native-PAGE. 6B isolated PPO gave bands as distinctively at 50 kDa and unclearly at around 35 kDa in SDS-PAGE while visible and slightly visible bands at 50-70 and 100 kDa in Native-PAGE. The synthesis of original 6B-affinity gel and no any study found in literature on affinity purification of avocado PPO show importance of our study.
Assuntos
Persea , Persea/metabolismo , Pirogalol , Catecol Oxidase , Ácido Clorogênico , Ácido 4-Aminobenzoico , Catecóis , Cromatografia de Afinidade , Guaiacol , Tirosina , Concentração de Íons de Hidrogênio , Cinética , Especificidade por SubstratoRESUMO
Polyphenol oxidase (PPO) was firstly purified from damson plum as a high antioxidant source. PPO was treated by 0-80% ammonium sulfate precipitation and dialysis. Characterization results were determined for catechol, 4-methyl catechol, pyrogallol and caffeic acid as 0.05 M/pH: 7.2/25 °C; 0.2 M/pH: 4.5/10 °C; 0.01 M/pH: 6.8/5 °C, and 0.2 M/pH: 8.5/10 °C, respectively. Vmax and KM values were calculated for same substrates as 17,219.97 U/(mL*min) and 11.67 mM; 7309.72 U/(mL*min) and 5 mM; 12,580.12 U/(mL*min) and 3.74 mM; 12,100.41 U/(mL*min) and 6.25 mM, respectively. Catechol gave the highest Vmax value among substrates. Affinity purification was performed by using Sepharose 4B-L-Tyrosine-p-aminobenzoic acid and Sepharose 6B-L-Tyrosine-p-aminobenzoic acid. Single bands were approximately observed at 50 kDa for each affinity sample in SDS-PAGE and Native-PAGE. 93.88 and 10.46 purification-folds were obtained for PPO by reference Sepharose-4B and original Sepharose-6B gels. Metal effects upon PPO activity were also investigated due to the importance of enzymatic browning in foods. Cu+2 activation and Fe+2 inhibition were observed with a final metal concentration of 1 mM at 219.66 and 43.18%, respectively. PPO purification from damson plum by affinity chromatography, its characterization, stability evaluation by statistically, and effects of metal ions on damson plum PPO have not been investigated in the literature.