Polyphenol oxidase from apricot (Prunus armeniaca) (PaPPO) was purified in its latent form (L-PaPPO), and the molecular weight was determined to be 63 kDa by SDS-PAGE. L-PaPPO was activated in the presence of substrate at low pH. The activity was enhanced by CuSO4 and low concentrations (≤ 2 mM) of SDS. PaPPO has its pH and temperature optimum at pH 4.5 and 45 °C for catechol as substrate. It showed diphenolase activity and highest affinity toward 4-methylcatechol (KM = 2.0 mM) and chlorogenic acid (KM = 2.7 mM). L-PaPPO was found to be spontaneously activated during storage at 4 °C, creating a new band at 38 kDa representing the activated form (A-PaPPO). The mass of A-PaPPO was determined by mass spectrometry as 37 455.6 Da (Asp102 → Leu429). Both L-PaPPO and A-PaPPO were identified as polyphenol oxidase corresponding to the known PaPPO sequence (UniProt O81103) by means of peptide mass fingerprinting.

In this study, we investigate the effect of enzymatic browning on the phenolic composition of apricot in vivo and in vitro . The in vitro browning was caused by the recombinant latent apricot polyphenol oxidase (L- Pa PPO). Successful heterologous expression of Pa PPO in Escherichia coli yielded substantial amounts of enzyme containing both copper ions in the catalytic active site. The expressed L- Pa PPO was characterized with regard to its molecular mass (56531.3 Da), pH optimum (7.0), activation by SDS, and enzyme kinetics. LC-MS/MS was used to compare the phenolic profiles of brown and non-brown apricots. The browning reactions did significantly decrease total phenolics and antioxidant capacity (measured with DPPH and CUPRAC assays). Catechin, epicatechin, and B-type procyanidins were the individual phenolics most affected by browning, followed by chlorogenic and neochlorogenic acid. These phenolics are most likely the main endogenous substrates of L- Pa PPO, as they were oxidized much faster than the other identified phenolics.

HighlightsPlant protease preparations inactivate apricot polyphenol oxidase.The degree of inactivation is proportional to the contact time.Ascorbic acid inhibits polyphenol oxidase during the time of incubation.Ascorbic acid-Protease combinations inhibit enzymatic browning effectively.