Purification and Characterization of a Dark Red Skin Related Dimeric Polyphenol Oxidase from Huaniu Apples

Abstract

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 Km value for catechol (0.60 mM), and the best substrate was 4-methylcatechol, with a Vmax of 526.32 U mg−1 protein. EC is a suitable natural substrate, with a Km value of 1.17 mM, and 55.27% of the Vmax/Km 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.

Recent Advances Regarding Polyphenol Oxidase in Camellia sinensis: Extraction, Purification, Characterization, and Application

“…PPO in Pueraria lobata was purified [80], and its molecular weight was determined to be 21 kDa via SDS-PAGE. The molecular weight of PPO from Huaniu Apples [81] was determined to be 140 kDa using native-PAGE and SDS-PAGE, but on the basis of urea-SDS-PAGE, it was found to be 61 kDa, which indicates that it may be a dimer. The high abundance of the PPO homodimer suggests that it may be involved in proanthocyanidins polymerization, which leads to the formation of the dark-red skin of apples.…”

Optimization of the Fermentation Conditions of Huaniu Apple Cider and Quantification of Volatile Compounds Using HS-SPME-GC/MS

“…Red Delicious) and cultivated in the province of Gansu, China. Renowned as a distinguished apple brand, Huaniu apples are comparable to American Red Delicious apples and Japanese Fuji apples [ 1 ]. They are primarily cultivated in Tianshui City, situated in Gansu Province, China, and have gained recognition as a distinctive local agricultural product.…”