Purification and characterization of avocado (Persea americana) polyphenol oxidase by affinity chromatography.

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.

Purification of damson plum polyphenol oxidase by affinity chromatography and investigation of metal effects on enzyme activity.

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.