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Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape

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Abstract

Polyphenol oxidase (PPO) was isolated from Thompson seedless grape (Vitis vinifera ‘Thompson Seedless’), and its biochemical characteristics were studied. The PPO showed activity to catechol and D, L-DOPA, but not towards monophenol l-Tyrosine, diphenols guaiacol and caffeic acid, and triphenols pyrogallic acid and gallic acid. Apparent Michaelis–Menten constant (K m) and maximum velocity of the reaction (V max) values were 45.0 ± 0.05 mM and 500.0 ± 15.3 OD400 nm/min for catechol, and 34.6 ± 0.03 mM and 384.6 ± 11.7 OD478 nm/min for D, L-DOPA, respectively. The obtained similar specificity values of V max/K m ratio of catechol and D, L-DOPA indicated their similar affinity to Thompson seedless PPO. The most effective inhibitor was l-cysteine, followed in decreasing order by ascorbic acid, sodium metabisulfite, EDTA, NaCl, and citric acid. It was discovered that metal ions of Mg2+ and Cu2+ increased, while Zn2+ and K+ reduced the PPO activity. Sugars showed inhibition on the PPO activity, with higher effect by sucrose and lower effect by fructose and glucose. Optimum pH and temperature for grape PPO activity were 6.0 and 25 °C with 10 mM catechol as substrate. The enzyme was heat stable between 10 and 25 °C, but showed significant activity loss at temperatures higher than 40 °C and completely inactivation at 70 °C for 10 min. Thermal inactivation of PPO showed a first-order kinetic with an activation energy (E a) of 146.1 ± 10.8 kJ/mol at pH 6.0.

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Acknowledgments

The authors acknowledge financial support from the Agriculture Department of China through project numbers of nycytx-30 and 201003021.

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Authors and Affiliations

  1. College of Food Science and Engineering, Northwest A&F University, Yangling, 712100, Shaanxi Province, China

    Yongju Zheng & Junling Shi

  2. Processed Foods Research Unit, USDA-ARS-WRRC, 800 Buchanan St., Albany, CA, 94710, USA

    Zhongli Pan

  3. Department of Biological and Agricultural Engineering, University of California, One Shields Avenue, Davis, CA, 95616, USA

    Zhongli Pan

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  1. Yongju Zheng
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Correspondence to Junling Shi.

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Zheng, Y., Shi, J. & Pan, Z. Biochemical characteristics and thermal inhibition kinetics of polyphenol oxidase extracted from Thompson seedless grape. Eur Food Res Technol 234, 607–616 (2012). https://doi.org/10.1007/s00217-012-1664-4

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  • DOI: https://doi.org/10.1007/s00217-012-1664-4

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