ABSTRACT
Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).
Subject(s)
Olea , Catechol Oxidase/genetics , Catechol Oxidase/chemistry , Hot Temperature , Electrophoresis, Polyacrylamide GelABSTRACT
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-PaPPO). Successful heterologous expression of PaPPO in Escherichia coli yielded substantial amounts of enzyme containing both copper ions in the catalytic active site. The expressed L-PaPPO 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-PaPPO, as they were oxidized much faster than the other identified phenolics.
ABSTRACT
The present research investigates the long term inhibition of enzymatic browning by inactivating the polyphenol oxidase (PPO) of apricot, using combinations of plant proteases and ascorbic acid (AA). The selected proteases were able to inactivate PPO at pH 4.5, with the degree of inactivation proportional to incubation time and protease concentration. Papain was the most effective protease, with 50⯵g completely inactivating PPO in less than one hour. AA prevented browning reactions that occur before or during PPO inactivation by protease. The combinations of AA/proteases were highly effective in vitro, where 2â¯mM AA/500⯵g proteases inhibited PPO activity completely over 24â¯h. The combination of AA/proteases was also effective in vivo, as treated apricot purees preserved their color (pâ¯<â¯0.0001, compared to untreated samples after 10â¯days of storage). The results demonstrate that AA/proteases combinations constitute a promising practical anti-browning method with feasible application in the food industry.
ABSTRACT
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 37â¯455.6 Da (Asp102 â 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.
