Purification and characterization of peroxidase from avocado (Persea americana Mill, cv. Hass)

• Published in: Journal of the science of food and agriculture 2014-07, Vol.94 (9), p.1844-1853
• Main Authors: Rojas‐Reyes, José O, Robles‐Olvera, Victor, Carvajal‐Zarrabal, Octavio, Castro Matinez, Claudia, Waliszewski, Krzysztof N, Aguilar‐Uscanga, María Guadalupe
• Format: Article
• Language: English
• Subjects: ascorbic acid; avocado; avocados; Biochemistry; catechol oxidase; Chromatography; copper; dithiothreitol; Enzyme Inhibitors - pharmacology; Enzyme kinetics; enzyme substrates; Enzymes; Food Preservation; Food science; Fruit - chemistry; Fruits; Gallates; Humans; Inhibition; ions; iron; isoelectric point; manganese; Mills; Molecular weight; Nutrition research; Oxidation-Reduction; Peroxidase; Peroxidases - isolation & purification; Peroxidases - metabolism; Persea - chemistry; Persea americana; Purification; SDS-PAGE; size exclusion chromatography; sodium azide; Symbols
• ISSN: 0022-5142; 1097-0010
• DOI: 10.1002/jsfa.6503

BACKGROUND: Avocado (Persea americana Mill, cv. Hass) fruit ranks tenth in terms of the most important products for Mexico. Avocado products are quite unstable due to the presence of oxidative enzymes such as polyphenol oxidase and peroxidase. The present study is to characterize the activity of purified avocado peroxidase from avocado in order to ascertain the biochemical and kinetic properties and their inhibition conditions. RESULTS: Purification was performed by Sephacryl S 200 HR gel filtration chromatography and its estimated molecular weight was 40 kDa. The zymogram showed an isoelectric point of 4.7. Six substrates were tested in order to ascertain the affinity of the enzyme for these substrates. The purified peroxidase was found to have low Kₘ (0.296 mM) and high catalytic efficiency (2688 mM⁻¹ s⁻¹) using 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid), optimum activity being reached at 51°C, pH 3.8. The addition of dithiothreitol, β‐mercaptoethanol, ascorbic acid, sodium azide, l‐cysteine and Tween‐20 had high inhibitory effects, while metals ions such as Cu⁺, Fe²⁺ and Mn²⁺ had weak inhibitory activity on purified avocado peroxidase. CONCLUSION: The purified avocado peroxidase exhibits high inhibition (Kᵢ = 0.37 µM) with 1.97 µM n‐propyl gallate using ABTS as substrate at 51°C, pH 3.8 for 10 min. © 2013 Society of Chemical Industry