TY - JOUR
T1 - Purification and characterization of peroxidase from avocado (persea americana mill, cv. hass)
AU - Rojas-Reyes, José O.
AU - Robles-Olvera, Victor
AU - Carvajal-Zarrabal, Octavio
AU - Castro Matinez, Claudia
AU - Waliszewski, Krzysztof N.
AU - Aguilar-Uscanga, María Guadalupe
PY - 2014/7
Y1 - 2014/7
N2 - 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.
AB - 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.
KW - Avocado
KW - Chromatography
KW - Peroxidase
KW - SDS-PAGE
UR - http://www.scopus.com/inward/record.url?scp=84901827211&partnerID=8YFLogxK
U2 - 10.1002/jsfa.6503
DO - 10.1002/jsfa.6503
M3 - Artículo
C2 - 24288244
AN - SCOPUS:84901827211
SN - 0022-5142
VL - 94
SP - 1844
EP - 1853
JO - Journal of the Science of Food and Agriculture
JF - Journal of the Science of Food and Agriculture
IS - 9
ER -