Polyphenol oxidase (PPO; also polyphenol oxidase i, chloroplastic), an
enzyme involved in
fruit browning, is a
tetramer that contains four atoms of copper per molecule.[1]
The
amino acidtyrosine contains a single phenolic ring that may be oxidised by the action of PPOs to form o-quinone. Hence, PPOs may also be referred to as
tyrosinases.[5]
In plants, PPO is a
plastidic enzyme with unclear synthesis and function. In functional chloroplasts, it may be involved in oxygen chemistry like mediation of
pseudocyclic photophosphorylation.[14]
A mixture of monophenol oxidase and catechol oxidase enzymes is present in nearly all plant tissues, and can also be found in bacteria, animals, and fungi. In insects, cuticular polyphenol oxidases are present[16] and their products are responsible for
desiccation tolerance.
Grape reaction product (2-S glutathionyl caftaric acid) is an oxidation compound produced by action of PPO on
caftaric acid and found in wine. This compound production is responsible for the lower level of browning in certain white wines.
Plants make use of polyphenol oxidase as one in a suite of chemical defences against
parasites.[17]
Inhibitors
There are two types of inhibitor of PPO, those competitive to oxygen in the copper site of the enzyme and those competitive to phenolics.
Tentoxin has also been used in recent research to eliminate the PPO activity from seedlings of higher plants.[18]Tropolone is a grape polyphenol oxidase
inhibitor.[19] Another inhibitor of this enzyme is
potassium metabisulfite.[20] Banana root PPO activity is strongly inhibited by
dithiothreitol and
sodium metabisulfite,[21] as is banana fruit PPO by similar sulfur-containing compounds including
sodium dithionite and
cysteine, in addition to
ascorbic acid (vitamin C).[22]
Assays
Several assays were developed to monitor the activity of polyphenol oxidases and to evaluate the inhibition potency of polyphenol oxidase inhibitors. In particular,
ultraviolet/visible (UV/Vis) spectrophotometry-based assays are widely applied.[23] The most common UV/Vis spectrophotometry assay involves the monitoring of the formation of
o-quinones, which are the products of polyphenol oxidase-catalysed reactions, or the consumption of the substrate.[24] Alternative spectrophotometric method that involves the coupling of
o-quinones with
nucleophilic reagents such as 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) was also used.[25] Other techniques, such as activity staining assays with the use of
polyacrylamide gel electrophoresis,[26]tritium-based radioactive assays,[27] oxygen consumption assay,[28] and
nuclear magnetic resonance (NMR)-based assay were also reported and used.[29]
Enzymatic browning
Polyphenol oxidase is an enzyme found throughout the plant and animal kingdoms,[30] including most fruits and vegetables.[31] PPO has importance to the food industry because it catalyzes enzymatic browning when tissue is damaged from bruising, compression or indentations, making the produce less marketable and causing economic loss.[30][31][32] Enzymatic browning due to PPO can also lead to loss of nutritional content in fruits and vegetables, further lowering their value.[10][30][31]
Because the substrates of these PPO reactions are located in the
vacuoles of plant cells damaged mainly by improper
harvesting, PPO initiates the chain of browning reactions.[32][33] Exposure to oxygen when sliced or pureed also leads to enzymatic browning by PPO in fruits and vegetables.[31] Examples in which the browning reaction catalyzed by PPO may be desirable include avocados, prunes, sultana grapes, black tea, and green coffee beans.[10][31]
In mango
In mangoes, PPO catalyzed enzymatic browning is mainly caused by sap burn which leads to skin browning.[citation needed]Catechol oxidase-type PPO is located in the chloroplasts of mango skin cells and its phenolic substrates in the vacuoles. Sap burn is therefore the initiating event of PPO in mango skin, as it breaks down cell compartments.[33] PPO is located in mango skin, sap and pulp, with highest activity levels in skin.[31]
In avocado
PPO in avocados causes rapid browning upon exposure to oxygen,[10] a multistep process involving oxidation reactions of both
monophenols and polyphenols, resulting in
o-quinone products subsequently converted irreversibly into brown
polymericpigments (
melanins).[34]
In apple
Present in the chloroplasts and mitochondria of all parts of an apple,[31] PPO is the major enzyme responsible for enzymatic browning of apples.[35] Due to an increase in consumer demand for pre-prepared fruits and vegetables, a solution for enzymatic browning has been a targeted area of research and new product development.[36] As an example, pre-sliced apples are an appealing consumer product, but slicing apples induces PPO activity, leading to browning of the cut surfaces and lowering their esthetic quality.[36] Browning also occurs in apple juices and purees when poorly handled or processed.[37]
Apricot as a climacteric fruit undergoes fast post-harvest
maturation. The
latent PPO form can spontaneously activate during the first weeks of storage, generating the active enzyme with a molecular weight of 38 kDa.[39]Ascorbic acid/
protease combinations constitute a promising practical anti-browning method as treated
apricot purees preserved their
color.[40]
In potato
Found in high concentrations in potato
tuber peel and 1–2 mm of the outer
cortex tissue, PPO is used in the potato as a defense against insect predation, leading to enzymatic browning from tissue damage.[citation needed] Damage in the skin tissue of potato tuber causes a disruption of cell compartmentation, resulting in browning. The brown or black pigments are produced from the reaction of PPO
quinone products with
amino acid groups in the tuber.[32] In potatoes, PPO genes are not only expressed in potato tubers, but also in leaves,
petioles, flowers and roots.[32]
In walnut
In walnut (Juglans regia), two different genes (jr PPO1 and jr PPO2) encoding polyphenol oxidases have been identified. The two
isoenzymes prefer different
substrates, as jr PPO1 shows a higher activity towards
monophenols, whereas jr PPO2 is more active towards
diphenols.[41][42]
Hemocyanin is homologous to the phenol oxidases (e.g.
tyrosinase) since both enzymes sharing type copper active site coordination. Hemocyanin also exhibits PPO activity, but with slowed kinetics from greater
steric bulk at the active site. Partial denaturation actually improves hemocyanin's PPO activity by providing greater access to the active site.[46]
Aureusidin synthase is homologous to plant polyphenol oxidase, but contains certain significant modifications.
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