Recent Advances in Chemistry of Enzymatic Browning: Coli
Recent Advances in Chemistry of Enzymatic Browning: Coli
Recent Advances in Chemistry of Enzymatic Browning: Coli
and vegetables that have undergone browning. Black spots in shrimp are caused by
PPO-catalyzed browning; the "browned" shrimp are not acceptable to the consumer
and/or they are down-graded in quality. PPO activity in plants is desirable in processing
of prunes, black raisins, black figs, zapote, tea, coffee and cocoa and it probably
protects plants against attack by insects and microorganisms (3).
PPO was first discovered by Schoenbein (4) in 1856 in mushrooms.
Subsequent investigations showed that the substrates for the enzyme are O2 and certain
phenols that are hydroxylated in the o-position adjacent to an existing -OH group
(Equation 1), further oxidized to o-benzoquinones (Equation 2) and then
nonenzymatically to melanins (brown pigments).
(1)
p-Cresol
Catechol
4-Methylcatechol
o-Benzoquinone
Millions of dollars are spent each year on attempts to control PPO oxidation; to date
none of the control methods are entirely successful. It is said that Napoleon offered a
sizable financial reward for the replacement of NaHS03, to which he was very
sensitive, in wines to prevent browning with an innocuous compound. To date, the
reward has not been claimed.
The objectives of this overview chapter are to provide a broad, general treatment
of the current knowledge of PPO, including structure and function, molecular biology,
biosynthesis and regulation, chemistry of formation of brown products and prevention
of browning, as well as suggestions of future research needs.
Structure, Function and Molecular Biology of PPO
Purification to homogeneity of the enzyme required before detailed structure and
function studies has been difficult, in large part because the required disintegration of
tissues leads to formation of 0-benzoquinones (first product formed); the obenzoquinones rapidly react non-enzymatically to form melanins, leading to
modifications of proteins, including PPO. Most of the earlier purification was done on
mushroom PPO, which occurs in multiple forms (isozymes and artifacts) with different
ratios of cresolase to catecholase activities. Mushroom PPO is a multi-subunit protein
which associates to give dimeric to octameric polymers. The purification of PPO from
2+
5,6-quinone from tyrosine for example with further polymerization to melanin and
reaction with nucleophiles, such as amino groups of proteins. The o-benzoquinones
can react covalently with other phenolic compounds by Michael addition, to give
intensely colored products that range from yellow, red, blue, green and black (72). oBenzoquinones also react with aromatic amines and thiol compounds, including those in
proteins, to give a great variety of products, including higher molecular weight protein
polymers (13).
The mechanism of action of N. crassa PPO has been extensively investigated
and there is a plausible and detailed theory explaining its catalytic activation. (Figure 1;
(14, 15)). The proposed mechanisms for hydroxylation (Equation 1) and
dehydrogenation (Equation 2) reactions with phenols probably occur by separate
pathways but are linked by a common deoxy PPO intermediate (deoxy in Figure 1).
The proposed mechanism of dehydrogenation, with intermediates, is shown in
Figure 1A. O2 is bound first to the two Cu(I) groups of deoxy PPO (deoxy) to give
oxy PPO in which the bond distance of O2 bound to the two Cu(II) groups is
characteristic of a peroxide (75). The two Cu(II) groups of oxy PPO then bind to the
oxygen atom of the two hydroxyl groups of catechol to form the 02*catecholPPO
complex.
The catechol is oxidized to 0-benzoquinone and the enzyme is reduced to met PPO.
Another molecule of catechol binds to met PPO, is oxidized to 0-benzoquinone and the
enzyme reduced to deoxy- PPO, completing the cycle.
The mechanism of 0-hydroxylation of a monophenol by PPO is shown in Figure
IB. In vitro, the reaction begins with met PPO (at about 11 o'clock on the A portion of
the diagram). Met PPO must be reduced by a reducing compound BH2 (Equation 1;
catechol is BH2) if a lag period is to be avoided, to give deoxy PPO. Deoxy PPO binds
O2 to give oxy PPO, the monophenol is bound to one of the Cu(II) groups via the
oxygen atom of the hydroxyl group to give the 02*monophenolPPO complex.
Subsequently, the 0-position of the monophenol is hydroxylated by an oxygen atom of
the O2 of the C^monophenolPPO complex to give catechol, which then dissociates to
give deoxy PPO, to complete the cycle. Only the first cycle of hydroxylation of a
monophenol requires starting at the Met PPO; all subsequent cycles begin with deoxy
PPO.
Inhibition of Enzymatic Browning
In theory, PPO-catalyzed browning of fruits and vegetables can be prevented by heat
inactivation of the enzyme, exclusion or removal of one or both of the substrates (O2
and phenols), lowering the pH to 2 or more units below the pH optimum, by reactioninactivation of the enzyme or by adding compounds that inhibit PPO or prevent melanin
formation. Hundreds of compounds have been tested as inhibitors of enzymatic
browning (16, 17).
Exclusion and/or separation of O2 and phenols from PPO prevents browning of
intact tissues; commercial utilization of these methods are being examined by numerous
researchers (18). Fruits and vegetables have "skins" (waxes, and other surface layers)
that exclude 62 as long as there is no damage to the skins. PPO is physically
compartmentalized from phenols in the intact cell. Commerically, O2 can be excluded
from or reduced in concentration in fruits and vegetables by controlled atmospheric
storage, packaging techniques, etc. Phenols can be removed from fruit and vegetable
juices by cyclodextrins or by treatment of cut surfaces with 02-impermeable coatings.
PPO activity can be decreased by modifying the pH; the pH optima of most PPO's are
near 6, although there are some exceptions.
Reducing compounds, such as ascorbate, sodium bisulfite and thiol compounds,
decrease browning by reducing the 0-benzoquinones back to 0-dihydroxyphenols or by
irreversible inactivation of PPO (79). Maltol does not inhibit PPO, but it prevents
browning by its ability to conjugate with 0-benzoquinones, while kojic acid is effective
in preventing browning by both reacting with PPO and with 0-benzoquinones (20).
Competitive inhibitors, such as benzoic acid and 4-hexyl-resorcinol, are useful in
controlling browning in some food products. 4-Hexylresorcinol is a very good
inhibitor of enzymatic browning of shrimp, apples and Irish potatoes.
Summary
Enzymatic browning due to PPO in our plant foods is controlled in the food processing
industry by use of ascorbate, sodium bisulfate and lowering the pH (addition of citric
acid for example). However, chemical control is not fail-safe, not acceptable to some
consumers and cannot be used to prevent browning in intact fruits and vegetables.
Through better understanding of the mechanism of action of PPO and its essential or
nonessential metabolic role(s) in plants, it is expected that genetic engineering
techniques will be important in preventing unwanted enzymatic browning. Breeders
have been working to decrease the level of PPO in apples, bananas, mushrooms,
peaches and other plants over many years. The genetic engineering approach provides a
more precise method of decreasing PPO expression, while retaining the desirable
genetic traits of plants. Its utility has already been demonstrated for preventing
browning in potatoes (77).
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