Biomedical Engineering Reference
In-Depth Information
above, the o -diphenolases are unusual because many also possess a monoxygenase
property which results in ortho hydroxylation of monophenols such as p -cresol,
p -coumaric acid, and tyrosine. Thus, it seems likely that many of the naturally
occurring diphenols, for example, chlorogenic acid and catechin, are formed from
the monophenol precursors. Sometimes polyphenol oxidase activity is determined
by reference to specific test substrates, e.g., chlorogenic acid oxidase, catechin
oxidase, or DOPA oxidase, but this does not necessarily imply the presence of
specific isoenzymes for each of the test substrates. Polyphenol oxidases are enzymes
now widely accepted as catalyzing two distinct, but related reactions, where the
products of the first reaction undergo dehydrogenation by the second enzyme cata-
lyzed reaction to produce quinones that then yield brown and black melanin pig-
ments. Therefore, the polyphenol oxidases are also classified as monophenol mon-
oxygenase, EC 1.14.18.1. However, there is no set ratio of monoxygenase activity
to diphenolase activity and the relative activities vary with the plant source. 125
The typical oxidation of polyphenols catalyzed by catechol oxidase is considered
as either a two-step reaction, starting with a monophenol, or a one-step reaction, in
which just a dihydroxyphenol is oxidized. The monophenol oxygenase activity may
be called cresolase activity and the enzyme for the dehydrogenation reaction has
been referred to as catecholase. Polyphenol oxidases are copper enzymes and the
oxidation reaction involves changes in valency of the transition metal which acts as
a single electron carrier:
PPO-2Cu + + O 2 + 2H + + 2e - = = = = = = = = = PPO-2Cu 2+ + H 2 O(9.1)
PPO-2Cu 2+ + 1/2 O 2 = = = = = = = = =PPO-2Cu + + H 2 O
(9.2)
The provision of two electrons to form the Cu(I) state of the enzyme from
reaction (9.2) is believed to be coupled to the reduction of oxygen to bring about
hydroxylation in reaction (9.1) with the reduction of one atom of oxygen to water.
Thus, the reactions are coupled with the recycling of electrons through oxidation
states I and II of the transition metal. Reaction (9.2) can be reversed by reduction
of the quinones with ascorbic acid or sulfite. The coupled reduction by ascorbic acid
will continue in fresh fruits and juices until oxidation to dehydroascorbic is complete,
at which stage browning will be observed. In fresh fruits and vegetables, ascorbic
acid delays the onset of enzymic browning to varying degrees depending on the
amount of ascorbic acid present. Consequently, the natural content of vitamin C, or
its deliberate addition, offers a useful commercial means for the control of enzymic
browning. Where this is desirable, as in the manufacture of buff-colored apple puree,
the oxidation may be allowed to commence before blanching. Quinones are very
reactive compounds and may form additional products with peptides and amino
acids resulting in a range of colored compounds commonly described as melanins.
All polyphenol oxidases are now thought to contain two atoms of Cu per mole which
have been suggested to exist in close proximity to each other at approximately 3.5 Å.
The oxy form of the enzyme, 125 where two electrons have been passed from the
2Cu(I) to O 2 to form 2Cu(II) peroxide, has been proposed as the active oxidizing
species ( Figure 9.3 ).
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