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Student Publications
Author: Max Wellington
Title: o-DIPHENOLASE AND ITS ROLE IN THE
ENZYMATIC BROWNING IN FOODS
Area: Enzymology
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INTRODUCTION
The myriad of biochemical reactions
that occur in living systems are
nearly all mediated
by a series of proteinaceous,
biological catalysts known as
enzymes. Enzymes differ
from ordinary chemical catalysts in
several important respects namely:
(i) Higher reaction rates (10 6
-1012 times greater)
(ii) Milder reaction conditions �
(neutral pH; temperature below
100�C)
(iii) Greater reaction specificity
and
(iv) Capacity for regulation
Enzymes were first discovered in
1835 (-amylase) by Jacob Berzelius,
but their stereo-
chemical and functional elucidation
took some time and it was not until
1965 that the X-
ray crystallography of an enzyme,
Lysozyme, was available. Since then
however, nearly
some 2000 enzymes have been purified
and characterized to at least some
extent (Voet
and Voet, 1990).
Enzymes display immense specificity
for their respective substrates
which is achieved
through geometrically and physically
complimentary interactions which
permits enzymes
to be absolutely stereo-specific
both in binding substrates and
catalyzing reactions.
The regulation of enzymatic activity
in vivo can occur by
allosteric alteration of the active
site, by substrate binding affinity,
by substrate or product feedback
inhibition or by gene
regulation (Boyer, 1999).
The wide diversity of enzymes and
the rapidly growing number of newly
discovered
enzymes has led the International
Union of Biochemistry to adopt a
scheme for the
systematic functional classification
and nomenclature of enzymes. This
system is used
where possible in conjunction with
the informal or trivial name but
assumes fundamental
Classification
Type of Reaction Catalyzed
1. Oxidoreductases
Oxidation-reduction reactions
2. Transferases
Transfer of functional groups
3. Hydrolases
Hydrolysis reactions
4. Lyase
Elimination to form double bonds
5. Isomerase
Isomerization
6. Ligases
Bond formation with ATP hydrolysis
________________________________________________________________________
importance when ambiguity must be
minimized e.g. Peptidyl-L-amino acid
hydrolase has
classification number EC 3.4.17.1
[where EC signifies "Enzyme
Commision"; 3 indicates
the enzymes major class,
hydrolase(as in Table 1); 4 denotes
the subclass, peptide bonds;
17 designates its subclass,
carboxypeptidase and the fourth
number 1 is the enzymes
arbitrarily assigned serial number
in its subclass].
Enzymes are proteins and as such
their conformation is determined by
their amino acid
sequence or primary structure which
inevitably determines their
secondary and tertiary
structure. Enzymic structure plays
an important functional role as this
will determine the
steric conformation at the active
site of the enzyme and hence its
effective activity.
Enzyme activity is affected by any
or all of the following: Enzyme
Concentration,
Substrate Concentration, inhibitors,
pH an temperature and as a result
the characterization
of an enzyme usually involves an
assessment of its optimum
performance in relation to
these criteria as well as in terms
of their kinetics with regards to
models put forward by
Michaelis Menten . According to
Michaelis Menten, enzymatic activity
can me defined in
terms of the Michaelis constant Km
which is equal to the substrate
concentration which
gives half the maximum velocity (Datta
and Ottoway, 1976). The Km is
usually estimated
using the Lineweaver-Burke plot, a
reciprocal plot of the substrate
concentration on the
initial enzyme velocity where the
negative reciprocal of the x- axis
intercept yields the
Km of the enzyme (figure 1).
4
Figure 1.
Line-Weaver Burke Reciprocal Plot
of Substrate Concentration
versus Initial Velocity
Km could also be estimated from the
graph of initial velocity versus
Substrate
concentration or [S] but the
hyperbolic nature of the graph
(Figure 2) makes it difficult to
estimate the infinite [S] and
consequently Vmax (where Km =[S] at
� Vmax).
Figure 2. The effect of Substrate
Concentration on Initial Enzyme
Velocity
5
The Km value is also of
important in determining whether an
inhibitor is competitive
(inhibition nullified by increasing
substrate concentration) or
non-competitive (where
there is always inhibition even at
high substrate concentrations). For
a non-competitive
inhibitor the Km value for the
enzyme in the presence or absence of
the inhibitor remains
constant whereas for the competitive
Km is less.
Enzymes due to their protein nature
can be denatured by extremes of pH
and temperature.
The optimum pH and temperature of
the enzyme is the point where the
velocity or
activity is maximum at a given
substrate and enzyme concentration
(Figures 3 and 4)..
Figure 3. Effect of pH Change on
Enzyme Activity
6
Figure 4. Effect of Temperature
Change on enzyme Activity
With advances in biochemistry and
molecular biology it has now also
become the norm
for the molecular structure, gene
sequence and molecular catalytic
mechanism to be
elucidated before the enzyme is
deemed fully characterized.
This requires that the enzyme can
obtained in pure form (Shi et al,
2001; Ikediobi and
Obasuyi, 1982) and usually involves
the following sequence of steps.
First the tissue
from which the enzyme is to be
isolated from is grounded at very
cold temperature in a
stable buffer. Triton X-100 or some
other detergent is sometimes added
to facilitate the
fragmenting of cellular organelles
and membranes to release any bound
enzyme. The
resulting milieu is then centrifuged
to remove the particulates after
which the proteins in
the supernatant are precipitated
using ammonium sulfate. The desired
enzyme would
constitute just one in a mixture of
many proteins in the precipitate,
however, they usually
can be separated quite well based on
their molecular weights. The
ammonium sulfate can
be removed by dialysis and the
approximate molecular weight of the
enzyme can be
established using SDS-Polyacrylamide
gel electrophoresis. Testing of the
bands using
substrate to assay enzyme activity
should facilitate identification of
the desired enzyme.
The molecular weight can then be
determined by comparison to known
standard. The
7
use of column chromatography to
extract larger volumes of the enzyme
from the
supernatant based on the expected
molecular weight fraction can be
done and once a
sufficient quantity is obtained the
required biochemical and kinetic
data as well as X-ray
crystallographic and other
structural analytical data can be
obtained.
This research paper will examine the
distribution of o-diphenolase, its
structure, modus
operandi and economic
importance.
8
ACTUALIZATION
o-Diphenolase and its Role in
Enzymic Browning of Foods
o-Diphenolase also referred to as
polyphenoloxidase or catechol
oxidase which catalyze
the oxidation of catechols or
ortho-diphenols to orthoquinones has
been established to be
a copper containing protein (Robb
Figure 5. Oxidation of Catechol
Catalysed by o-Diphenolase
et al, 1965; Kidron et al, 1977,
Anosike and Ayaebene, 1982) strongly
related to both
tyrosinase and haemocyanin, which
all have a dinuclear copper complex
with histidine
ligands at the active site (Siegban,
2004). O-Diphenolases are ubiquitous
enzymes
capable of mediating or
participating in a number of
physiological processes. There is a
general dubiousness surrounding some
of the many functions associated
with o-
diphenolase, however its role in
enzymic browning has been long
established. O-
Diphenolase has six histidine
residues one of which is covalently
linked to a cysteine
molecule. The distance between the
copper atoms has been resolved by
X-ray studies to
range from about 2.5 to 2.9
Angstr�ms depending on whether it is
substrate bound or
free. The role which copper assumes
involves the binding of oxygen at
the active site
(Mayer and Harel, 1979; Fennol et
al, 2004 and Siegban, 2004). While
o-diphenolases
from animal issues are relatively
specific for tyrosine and dopa
(Mason, 1955), the fungal
and higher plant enzymes act on a
range of mono and diphenols (Mayer
and Harel, 1979;
Siegban, 2004; Fennol et al, 2004).
9
Figure 6. Crystal Structure of a
Plant Diphenolase
Extracted from Klabunde et al,
1998
Marked differences in both the level
of o-diphenolase activity and the
content of its
substrates have been observed
between cultivars of fruits (Matthew
and Parplia, 1971),
vegetables (Ben-Shalom et al, 1978)
and yams (Ikediobi and Obasuyi,
1982).
o-Diphenolase has been found to be
extensively a membrane bound enzyme.
Apart from
its location in chloroplasts,
diphenolases have been reported to
be located in
mitochondria, peroxisomes and
microsomes (Mayer and Harel, 1979).
The strength of
binding of o-diphenolases to
membranes appear to vary depending
on the tissues and the
stage of development of the plant
(Mayer and Harel,1979). In tobacco,
washing with
buffer suffices to release the
enzyme from chloroplast lamellae
(Hoffer, 1964). In most
cases more drastic conditions are
required for the solubilization of
membrane bound o-
diphenolases such as the use of
detergents e.g Triton X-100 (Hrel
et.al, 1964; Walker and
Hulme, 1966) and sodium dodecyl
sulfate (Yamaguchi et al, 1969)
In situ solubilization occurs
following exposure to certain stress
conditions (Volk et. al.,
1977) and also under more natural
conditions of ripening of fruits or
aging. Thus apple
10
(Harel et. al, 1966), grape ad
banana ( Mayer and Harel, 1979)
diphenolases become
increasingly soluble during fruit
ripening
The compartmentalization of phenolic
substrates of the enzyme, both in
special cells
(Mace, 1963) and within cells
(Roberts, 1962) have been reported.
This results in the
separation between the enzyme and
the bulk of its phenolic substrates
in situ.
The rise in diphenolase activity
which generally accompanies wounding
and stress has
been attributed to the de novo
synthesis of the enzyme (Hyodo and
Uritani, 1966). Other
researchers have attributed the rise
to activation of alredy existing
enzyme rather than re-
synthesis (Balasbrumani et al,
1971).
Many other roles have been ascribed
to diphenolase enzymes due to it's
activity response
to various stimuli and also by
virtue of it's location in the plant
cell. Some of these roles
are:
(i)
It has been correlated with fruit
formation in certain fungi and
bacteria
(Wilson, 1968; Leonard, 1973).
(ii)
It has been correlated with melanin
formation and as such has been
deemed to
play a role in cellular resistance
(Kuo and Alexander, 1967).
(iii)
Diphenolases have been suggested to
play a role in electron transport
(Kabowitz, 1938).
(iv)
Its presence in chloroplast
membranous structures have
implicated a possible
role in photosynthesis (Mayer and
Harel, 1979).
(v)
Its involvement in affecting the
regulation of plant growth has been
implied
(Gordon and Paley, 1961; Tomazowski
and Thieman, 1966).
(vi)
It has been implicated in rendering
seed coats impermeable to water
(Marbach
and Mayer, 1975).
Enzymic browning is one of the most
importantt color reactions that
affect foods. It is
catalyzed by diphenolase enzymes
which facilitate the conversion of
phenols to the
brown pigment melanin in an
oxidation reaction.
11
Figure 7. Formation of melanin
(Browning) from tyrosine. (From
Lerner, 1953).
Ikediobi and Obasuyi (1982) purified
the enzyme from yam and found the
molecular
weight to be 107,000 � 5400 with
temperature and pH optima of 25�C
and 6.8
respectively. Activity was
illustrated on catechol, chloregenic
acid, dopamine and
pyrogallol. The enzyme was found to
be inhibited strongly by
dithiothreitol,
diethyldithiocarbamate, potassium
cyanide, sodium metabisulfite,
2-mercaptoethanol
and L-cysteine. The rate for
catechol conversion in sweet
potatoes has been
measured to be 2.3 x 103S-1 (Baruah
and Swain, 1959) corresponding to a
rate-
limiting free energy barrier of
around 13 kcal/mol.
12
Figure 8. Comparison of
reactions catalysed by o-Diphenolase
and p-
Diphenolase. (From Walker, 1995).
Studies done on ripe banana
o-diphenolase show that dopamine is
the only significant
substrate in the browning reaction
of banana. The first reaction
results in the
orthohydroxylation of phenol and the
second, oxidation of the diphenol to
orthoquinone.
The remaining portion of the
reaction sequence involve
non-enzymic oxidations and
ultimate polymerization of indole
5,6-quinone to brown pigments
(Melanins) � as
schematized in figures 7 and 8.
Table 3. lists a number of phenols
found in fruits and vegetables.
Relatively few of these
serve as sustrate for diphenolase.
The most important are catechin, 3,4
dihydroxyphenylalanine (DOPA) and
tyrosine and the substrate
specificity varies
depending on the source of the
enzyme.
Table 2. Phenolic substrates of
Diphenolase in fruits, vegetables,
and seafoods.
Source
Phenolic substrates
Apple
chlorogenic acid (flesh), catechol,
catechin (peel), caffeic acid, 3,4-
dihydroxyphenylalanine (DOPA),
3,4-dihydroxy benzoic acid, p-cresol,
4-methyl
catechol, leucocyanidin, p-coumaric
acid, flavonol glycosides
13
Apricot
isochlorogenic acid, caffeic acid,
4-methyl catechol, chlorogenic acid,
catechin,
epicatechin, pyrogallol, catechol,
flavonols, p-coumaric acid
derivatives
Avocado
4-methyl catechol, dopamine,
pyrogallol, catechol, chlorogenic
acid, caffeic acid,
DOPA
Banana
3,4-dihydroxyphenylethylamine
(Dopamine), leucodelphinidin,
leucocyanidin
Cacao
catechins, leucoanthocyanidins,
anthocyanins, complex tannins
Coffee beans
chlorogenic acid, caffeic acid
Eggplant
chlorogenic acid, caffeic acid,
coumaric acid, cinnamic acid
derivatives
Grape
catechin, chlorogenic acid,
catechol, caffeic acid, DOPA,
tannins, flavonols,
protocatechuic acid, resorcinol,
hydroquinone, phenol
Lettuce
tyrosine, caffeic acid, chlorogenic
acid derivatives
Lobster
tyrosine
Mango
dopamine-HCl, 4-methyl catechol,
caffeic acid, catechol, catechin,
chlorogenic acid,
tyrosine, DOPA, p-cresol
Mushroom
tyrosine, catechol, DOPA, dopamine,
adrenaline, noradrenaline
Peach
chlorogenic acid, pyrogallol,
4-methyl catechol, catechol, caffeic
acid, gallic acid,
catechin, Dopamine
Pear
chlorogenic acid, catechol,
catechin, caffeic acid, DOPA,
3,4-dihydroxy benzoic acid,
p-cresol
Plum
chlorogenic acid, catechin, caffeic
acid, catechol, DOPA
Potato
chlorogenic acid, caffeic acid,
catechol, DOPA, p-cresol,
p-hydroxyphenyl propionic
acid, p-hydroxyphenyl pyruvic
acid, m-cresol
Shrimp
tyrosine
Sweet potato
chlorogenic acid, caffeic acid,
caffeylamide
14
Tea
flavanols, catechins, tannins,
cinnamic acid derivatives
Extracted from Marshall et al,
2002 (Structures of the listed
Phenolics can be found in the
Appendix)
Browning is desirable in some foods
e.g tea and coffee and in most
plants it has been
associated with pest and bacterial
resistance and wound healing .Some
have even
ascribed anticancer and antioxidant
properties to the melanins produced
during the
browning reaction (Marshall et al,
2000).
Projected increases in the fruit and
vegetable market for the future will
however not occur
if enzymatic browning is not
understood and controlled (Marshall
et al, 2000). It is
estimated that over 50 percent
losses in fruit occur as a result of
enzymatic browning
(Whitaker and Lee, 1995) and this
has increased interest in
understanding and controlling
diphenolase enzymes in foods.
Browning or melanosis has also been
observed during the
storage of some high value
crustaceans such as shrimp and
lobster connoting spoilage
(Otwell et al, 1992) and losses and
browning has been shown to adversely
affect flavor
and nutritional value of foods
(Marshall et al, 2002)..
Figure 9. Examples of enzymatic
browning in banana
Extracted from Marshall et al,
2000
Figure 10. Examples of enzymatic
browning in potato
15
Extracted from Marshall et al,
2000
CONTROL OF BROWNING
Browning does not occur in intact
plant cells due to vacuolar
separation of the phenolic
substrates from the enzyme which is
present in the cytoplasm. Cutting or
damage to the
tissue brings the enzyme and
substrate together resulting in the
observed brown
pigmentation which impacts both the
organoleptic and biochemical
characteristics of
fruits and vegetables (Marshall et
al, 2000).
The role of browning has been shown
to be mediated by several factors,
namely:
(i)
Tissue Diphenolase level [E]
(ii)
Tissue Phenolic content [S]
(iii)
pH
16
(iv)
Temperature and
(v)
Oxygen Availability
The control of browning consequently
can be effected through the
manipulation of these
factors (Marshall et al, 2002). Some
resulting methods of control
include:
(i)
The elimination of oxygen by vacuum
packing, immersion in liquid,
treatment
with reducing agents e.g. ascorbic
acid and antioxidants e.g butylated
hydroanisole (BHA).
(ii)
Inactivation of the enzyme by: (a)
Chelating the copper prosthetic
group of the
active site using EDTA, Sorbic Acid
or (b) denaturing with steam
treatment,
blanching, solar drying or freezing
or (c) Inhibition with cysteine,
honey,
heylresocinol etc.
(iii)
Removal of the enzyme e.g. from
juices by precipitation and
ultrafiltration
and
(iv)
Reducing Enzyme activity by
acidifying or lowering the pH e.g
Citric Acid
(Most diphenolases exhibit optimal
activity at pH 6.8).
Other non-conventional methods of
reducing enzymic browning of foods
include the use
of antienzymes or enzymes which
destroy some cofactor necessary for
the reaction e.g
some cleavage oxygenases (Kelly and
Prinkle, 1969); Catechol Transferase
(Prinkle and
Nelson, 1963) and Protease (Labuza
et al, 1992).
17
With the advent of recombinant DNA
technology, numerous amino acid
sequences of
diphenolase isozymes have been
deciphered using cDNA sequencing
techniques
(Marshall et al, 2000).
The inactivation of genes coding for
these enzymes using anti-sense RNA
specific for
diphenolase should lower the
browning reaction as it effectively
reduces diphenolase
gene expression and hence the
concentration of the enzyme in situ.
Anti-sense RNAs
were recently observed to
selectively block the gene
expression of other plant enzymes
such as polygalacturonase and
peroxidase in tomatoes (Marshall et
al, 2002).
Bachem et al (1994) determined that
the expression of diphenolase in
potatoes was
decreased through the use of
anti-sense cDNA.
It is hoped that though the use of
this technology that browning
resistant varieties maybe
developed to prevent or
significantly curtail the production
of Diphenolase.
CONCLUSION
18
In closing Diphenolases have been
shown to be ubiquitous enzymes which
can have
significant impact on the shelf life
and indeed the quality of fruits,
vegetables an certain
shell fish due to their facilitation
of enzymic browning. Enzymic
browning was shown to
be responsible for about 50% of
spoilage of fruits and vegetables
and therefore its control
can be of significant economic
impact to the global food supply.
Several method for its
control during the processing and
handling of foods were explored
bearing in mind that
treatments administered should not
affect product flavor, texture and
color.
The use of enzyme inhibitors,
reducing agents, anti-oxidizers,
heating, refrigerating, anti-
enzyme and anti-sense RNA technology
were all examined.
It is hoped that in the very near
future the use of anti-sense RNA
technology will be able
to see the production of food
varieties with a much reduced
propensities for enzymic
browning.
RECCOMENDATIONS:
1.
Study to quantify the economic value
of global food losses due to
enzymic browning.
2.
Elucidation of the genes for
diphenolase enzymes for all major
food
crops impacted by enzymic browning
to facilitate the use of anti-sense
RNA technology to reduce browning.
3.
Further explore the potential of
melanin in fighting cancer.
4.
Intensify research in food
technology to find novel yet
economical
ways to reduce enzymic browning in
foods accompanied by an
education drive to enlighten the
masses and other stakeholders.
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