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

Phytochemistry (2010-11-23)
Gisela Palma-Orozco, Alicia Ortiz-Moreno, Lidia Dorantes-Alvarez, José G Sampedro, Hugo Nájera
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium metabisulfite, ReagentPlus®, ≥99%
Sigma-Aldrich
Sodium metabisulfite, puriss. p.a., ACS reagent, reag. Ph. Eur., dry, 98-100.5%
Sigma-Aldrich
Sodium metabisulfite, reagent grade, 97%
Sigma-Aldrich
4-Methylcatechol, ≥95%
Sigma-Aldrich
Sodium metabisulfite, puriss., meets analytical specification of Ph. Eur., BP, NF, FCC, E223, dry, 97-100.5%
Supelco
Sodium metabisulfite, analytical standard
Sigma-Aldrich
Sodium metabisulfite, tested according to Ph. Eur.
Sigma-Aldrich
Sodium metabisulfite, anhydrous, free-flowing, Redi-Dri, ReagentPlus®, ≥99%

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