Characterization of polyphenoloxidase (PPO) extracted from ‘Jonagored’ apple
Introduction
Polyphenoloxidase (PPO) activity has been extensively reported, by several authors, to be the main factor involved in apple browning (Nicolas, Richard-Forget, Goupy, Amiot & Aubert, 1994). Varietal difference in enzyme activity and susceptibility to browning is a well-known phenomenon. ‘Jonagored’ is a mutant of ‘Jonagold’ which is a controlled cross of ‘Jonathan’ with ‘Golden Delicious’ (Trillot, Masseron & Trovel, 1993). Although PPO has been investigated for many apple varieties, ‘Jonagored’ has not yet been studied. The characterization of the specific enzyme is necessary for a more effective means of controlling the enzymatic browning, or if a better understanding of the browning mechanism under specific storage conditions is required. Such information is important for the post-harvest handling of ‘Jonagored’ and, in particular, for minimal processing operations.
Two main problems are found in the optimization of the extraction conditions of PPO: the difficulty in obtaining full solubilisation of the membrane-bound PPO, and avoiding phenolic oxidation during and after extraction. The strength of PPO binding to membranes is variable. Therefore, in most cases, full extraction of the enzyme requires the use of a detergent such as Triton X100 Galeazzi and Sgarbieri, 1981, Zhou et al., 1993. The second problem arises from the simultaneous presence of quinones and their endogenous phenolic precursors in crude extracts of the enzyme. It is essential to minimize the formation of quinones, which may react with the enzyme, resulting in activity losses. Several methods have been described to prevent the reaction of phenols with PPO, including the use of phenol-binding agents such as soluble and insoluble polyvinyl pyrrolidone and polyvinilpolypyrrolidone (PVP and PVPP) (Galeazzi & Sgarbieri, 1981). Several investigators successfully used PVP in plant enzyme extraction due to its ability to bind to the phenols and, therefore in preventing phenol–protein interaction (Galeazzi & Sgarbieri, 1981).
The contribution of a given substrate to enzymatic discoloration depends on its concentration and on the nature of the other substrates present in the tissue. In apples, (+) catechin, (−) epicatechin and chlorogenic acid have been identified as substrates of PPO. The catechins were found to be oxidized more rapidly than chlorogenic acid, epicatechin contributing more to browning than the other two compounds. However, since the concentration of chlorogenic acid in apples is several times that of the catechins, its role in browning may be more important (Vámos-Vigyázó, 1981).
Polyphenoloxidase isoenzymes isolated from higher plants are able to oxidize a wide range of monophenols and o-diphenols with highly variable kinetic parameters, such as the maximum velocity (Vmax) and the Michaelis–Menten constant (Km), for different phenols. The Km is generally interpreted as a measure of affinity of the enzyme for the substrate. The affinity of plant PPO for the phenolic substrates is generally low (high Km values, 2–6 mM) (Nicolas et al., 1994). In some fruits, the best substrate of PPO is a compound not occurring as a phenolic constituent (Vámos-Vigyázó, 1981). Simple phenols such as catechol are commonly used in phenolase assays in spite of not always being found in association with the enzyme. However, the presence of a potential substrate does not ensure its role as an actual substrate in phenolase action leading to enzymatic browning (Vámos-Vigyázó, 1981).
As enzymatic browning of apple is pH dependent, this is important for the control of discoloration. Although PPO activity is negligible at the natural pH value of the fruit, it may be sufficient to cause browning (Vámos-Vigyázó, 1981). It is generally agreed that pH undoubtedly affects the Km value (Nicolas et al., 1994). Most previous studies have indicated that, although the optimum pH for the activity of PPO extracted from apple mitochondria is around 7, the PPO of the whole apple tissue has a maximum activity at pH between 4.5 and 5.5 Janovitz-Klapp et al., 1989, Trejo-Gonzaléz and Soto-Valdez, 1991, Richard-Forget et al., 1992, Zhou et al., 1993. Moreover, the enzyme seems to be relatively tolerant of acidic pH.
With the objective of subsequently controlling enzymatic browning mediated by PPO in this specific apple variety ‘Jonagored’, work was carried out to characterize the enzyme involved. Experiments were performed in order to optimize the extraction conditions of PPO (best ratio of extraction buffer/mass of fruit tissue, best PVP concentration, best pH of extraction) and to evaluate the affinity and specificity of the enzyme toward several substrates.
Section snippets
Plant material
Apples (cv. Jonagored) were grown at Estação Regional de Fruticultura e Vitivinicultura – Quinta de Sergude, Felgueiras, Portugal. The harvest was on 25 September. The fruit were stored in air at 4°C for 1–3 months until used in the experiments.
Characterization of apple maturity
The apples were initially characterised in terms of titratable acidity, pH and soluble solids content.
Aliquots (20–30 g) of apple juice from 10 crushed apple cubes were diluted with 250 ml of recently boiled water. Samples (25 ml) of the prepared juice
Results and discussion
Some chemical properties that enable the characterization of the degree of ripeness of the apples used in this experiment are in Table 1. The apples were at the ripe maturity stage at which they are normally used for consumption.
Acknowledgements
This research was funded by a JNICT scholarship (BD 2109/92-IF).
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