Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples
The distinct dark-red skin of Huaniu apples renders them attractive to customers. However, the mechanism that leads to the development of the color of the fruit is unclear. In this study, we found that compared with red Fuji (a bright-red apple cultivar), Huaniu apples had higher contents of (−)-epi...
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MDPI AG
2022-06-01
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| Series: | Foods |
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| author | Bin Liu Xianfang Zhou Haiyan Guan Xuequn Pang Zhaoqi Zhang |
| author_facet | Bin Liu Xianfang Zhou Haiyan Guan Xuequn Pang Zhaoqi Zhang |
| author_sort | Bin Liu |
| collection | DOAJ |
| description | The distinct dark-red skin of Huaniu apples renders them attractive to customers. However, the mechanism that leads to the development of the color of the fruit is unclear. In this study, we found that compared with red Fuji (a bright-red apple cultivar), Huaniu apples had higher contents of (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-gallocatechin gallate (GCG), and procyanidins (PCs) B2 and C1 in the peel, which implies that the polymerization of the flavanols and PCs may be correlated with the dark-red skin of the fruit. Using EC as a substrate, we purified an enzyme from Huaniu peel. We performed protein sequencing and discovered that the enzyme was a polyphenol oxidase (PPO). The molecular weight of the enzyme was approximately 140 kDa, which we estimated by native-PAGE and SDS-PAGE, while it was 61 kDa by urea-SDS-PAGE, from which we discovered that the PPO was a dimer. We observed the lowest <i>K</i><sub>m</sub> value for catechol (0.60 mM), and the best substrate was 4-methylcatechol, with a <i>V</i><sub>max</sub> of 526.32 U mg<sup>−1</sup> protein. EC is a suitable natural substrate, with a <i>K</i><sub>m</sub> value of 1.17 mM, and 55.27% of the <i>V</i><sub>max</sub>/<i>K</i><sub>m</sub> of 4-methylcatechol. When we used EC as a substrate, the optimum temperature and pH of the PPO were 25 °C and 5.0, respectively. In summary, we purified a dimeric PPO from Huaniu apples that showed high activity to EC, which might catalyze the polymerization of flavanols and PCs and lead to the dark-red color development of the fruit. |
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| spelling | doaj.art-35176dfce6ed45b1ac8e0b5cc7d416b92023-11-23T16:39:06ZengMDPI AGFoods2304-81582022-06-011112179010.3390/foods11121790Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu ApplesBin Liu0Xianfang Zhou1Haiyan Guan2Xuequn Pang3Zhaoqi Zhang4State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruit and Vegetables/Engineering Research Center for Postharvest Technology of Horticultural Crops in South China, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruit and Vegetables/Engineering Research Center for Postharvest Technology of Horticultural Crops in South China, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruit and Vegetables/Engineering Research Center for Postharvest Technology of Horticultural Crops in South China, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruit and Vegetables/Engineering Research Center for Postharvest Technology of Horticultural Crops in South China, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources/Guangdong Provincial Key Laboratory of Postharvest Science of Fruit and Vegetables/Engineering Research Center for Postharvest Technology of Horticultural Crops in South China, South China Agricultural University, Guangzhou 510642, ChinaThe distinct dark-red skin of Huaniu apples renders them attractive to customers. However, the mechanism that leads to the development of the color of the fruit is unclear. In this study, we found that compared with red Fuji (a bright-red apple cultivar), Huaniu apples had higher contents of (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-gallocatechin gallate (GCG), and procyanidins (PCs) B2 and C1 in the peel, which implies that the polymerization of the flavanols and PCs may be correlated with the dark-red skin of the fruit. Using EC as a substrate, we purified an enzyme from Huaniu peel. We performed protein sequencing and discovered that the enzyme was a polyphenol oxidase (PPO). The molecular weight of the enzyme was approximately 140 kDa, which we estimated by native-PAGE and SDS-PAGE, while it was 61 kDa by urea-SDS-PAGE, from which we discovered that the PPO was a dimer. We observed the lowest <i>K</i><sub>m</sub> value for catechol (0.60 mM), and the best substrate was 4-methylcatechol, with a <i>V</i><sub>max</sub> of 526.32 U mg<sup>−1</sup> protein. EC is a suitable natural substrate, with a <i>K</i><sub>m</sub> value of 1.17 mM, and 55.27% of the <i>V</i><sub>max</sub>/<i>K</i><sub>m</sub> of 4-methylcatechol. When we used EC as a substrate, the optimum temperature and pH of the PPO were 25 °C and 5.0, respectively. In summary, we purified a dimeric PPO from Huaniu apples that showed high activity to EC, which might catalyze the polymerization of flavanols and PCs and lead to the dark-red color development of the fruit.https://www.mdpi.com/2304-8158/11/12/1790Huaniu applepolyphenol oxidasedimerprocyanidinspolymerization |
| spellingShingle | Bin Liu Xianfang Zhou Haiyan Guan Xuequn Pang Zhaoqi Zhang Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples Foods Huaniu apple polyphenol oxidase dimer procyanidins polymerization |
| title | Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples |
| title_full | Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples |
| title_fullStr | Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples |
| title_full_unstemmed | Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples |
| title_short | Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples |
| title_sort | purification and characterization of a dark red skin related dimeric polyphenol oxidase from huaniu apples |
| topic | Huaniu apple polyphenol oxidase dimer procyanidins polymerization |
| url | https://www.mdpi.com/2304-8158/11/12/1790 |
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