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