Purification and partial biochemical characterization of polyphenol oxidase from mamey ( Pouteria sapota)

• Published in: Phytochemistry (Oxford) 2011, Vol.72 (1), p.82-88
• Main Authors: Palma-Orozco, Gisela, Ortiz-Moreno, Alicia, Dorantes-Álvarez, Lidia, Sampedro, José G., Nájera, Hugo
• Format: Article
• Language: English
• Subjects: Ascorbic Acid - pharmacology; Biological and medical sciences; Catechol; Catechol Oxidase - antagonists & inhibitors; Catechol Oxidase - isolation & purification; Catechol Oxidase - metabolism; Catechols - metabolism; Chemical constitution; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fruit - enzymology; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Mexico; Molecular Weight; Plant physiology and development; Polyphenol oxidase; Pouteria - enzymology; PPO purification; Pyrogallol; Pyrogallol - metabolism; Sapotaceae; Sapote mamey ( Pouteria sapota); Sulfites - pharmacology; Thermodynamics
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2010.10.011

In this study, two isoenzymes of PPO from mamey fruit were obtained by chromatographic column separation; PPO 1 was obtained before the KCl gradient while PPO 2 was obtained during the salt gradient change. While a long shelf life for fruit products is highly desired, enzymatic browning is the main cause of quality loss in fruits and is therefore a main problem for the food industry. In this study polyphenol oxidase (PPO), the main enzyme responsible for browning was isolated from mamey fruit ( Pouteria sapota) and characterized biochemically. Two isoenzymes (PPO 1 and PPO 2) were obtained upon ammonium sulfate precipitation and hydrophobic and ion exchange chromatography; PPO 1 was purified up to 6.6-fold with 0.28% yield, while PPO 2 could not be characterized as enzyme activity was completely lost after 24 h of storage. PPO 1 molecular weight was estimated to be 16.1 and 18 kDa by gel filtration and SDS–PAGE, respectively, indicating that the native state of the PPO 1 is a monomer. The optimum pH for PPO 1 activity was 7. The PPO 1 was determined to be maximum thermally stable up to 35 °C. Kinetic constants for PPO 1 were K m = 44 mM and K m = 1.3 mM using catechol and pyrogallol as substrate, respectively. The best substrates for PPO 1 were pyrogallol, 4-methylcatechol and catechol, while ascorbic acid and sodium metabisulfite were the most effective inhibitors.