Title: Polyphenol oxidase activity of bananas

Introduction
Banana is a nutritious fruit with a pleasant flavour that is widely consumed throughout the
world. Its brownish colour is mainly attributed to the oxidation of phenolic compounds by an
enzyme called polyphenol oxidase. This enzyme catalyse the oxidation of different phenols to
the corresponding quinones which are highly reactive compounds that finally polymerize to
melanins. In previous years, polyphenol oxidase was isolated or extracted from various fruits
such as plum, litchi, apple, mango and grapes, the enzyme was extracted from these various
sources and studied under different physiochemical conditions such as pH, Temperature,
substrate concentration and these conditions were optimized for maximum enzymatic
activity. (Bensadoun and Keeton, 1980).

Apart from various plant sources, this enzyme is almost found in all living organisms
including animals and microorganisms. The catalytic action of polyphenol oxidase is
connected to undesirable browning and off-flavour generation in stored and processed foods.
On the other hand, polyphenol oxidase has been also shown to have some important
applications such as the use in the synthesis of valuable added products like the substituted
catechol. (Hancock, et al., 1996).

Aim:
To extract polyphenol oxidase enzyme from the banana fruit and to detect its presence and
measure its activity by looking at the effect it has on its substrate, catechol at 30 seconds time
intervals and then establish a linear progressive curve illustrating the relationship between
absorbance and time.

Materials and methods

The banana was carefully peeled and the fruit was mulled using a pestle in 0.1M acetate
buffer with a pH of 5.6 in a chilled mortar. About 4g of banana tissues were mulled in 8ml of
the buffer and the mixture was centrifuged on a picollo for one minute. The supernatant was
filtered through the glasswool and stored in a test tube on ice in an ice bucket, this served as
the enzyme preparation. The spectrophotometer was set to zero when the catechol-buffer was
placed in it. Amount of 0.25ml of the enzyme preparation was mixed with 2.25ml of
catechol-buffer in a test tube and the solution was immediately placed in a spectrophotometer,
then the absorbance was read at 450nm for every 30 seconds for five minutes.

Results

Table 1: represents the absorbance that was recorded at 450nm for every 30 seconds time
interval for five minutes during the enzyme activity.
Time (s)
0
30
60
90
120
150
180
210
240
270
300

Discussion

Absorbance (450nm)
0.343
0.363
0.386
0.409
0.432
0.457
0.482
0.507
0.533
0.560
0.582

Polyphenol oxidase is one of the most abundant occurring enzymes in nature and this enzyme
was extracted from the banana fruit. One of the physical methods was used in this practical to
disrupt the banana tissues and this was done by making use of the pestle and mortar with the
contents dispensed in an aqueous buffer. After mulling the fruit, the heterogeneous mixture
was centrifuged on a pocollo so that more dense components of the mixture migrate away
from the axis of the centrifuge, while less dense components of the mixture migrate towards
the axis and this caused the precipitate (pellet) to gather on the bottom of the tube and the
remaining solution called the supernatant containing an enzyme of interest was filtered and
stored in a test tube for further analysis. All these procedures were done in ice at very low
temperatures in order to keep an enzyme inactive since atmospheric oxygen was one of the
requirements for polyphenol oxidase to catalyse its reaction, so it was necessary to keep the
temperature lower during this process thereby keeping an enzyme inactive. (Glick, 1971).

The activity of this enzyme was monitored in the pH of about 5.6 using 0.1M acetate buffer
with 0.06M catechol buffer as the substrate, this was done because enzymes are amphoteric
in nature that is they have a large number of acid and basic groups, mainly situated on their
surfaces, therefore pH of the environment affect the charges on these groups and this affect
the total net charge of the enzyme as well as the reactivity of catalytic active site groups.
Each enzyme has an optimum pH and the theoretical value of the optimum pH of polyphenol
oxidase is around 6.5 which is slightly acidic hence buffer solution helps to stabilize the pH
during the reaction because some products may be acidic or basic. In this practical the
activity of polyphenol oxidase was monitored by looking at the effect it has on catechol and a
linear progressive curve was drawn illustrating the relationship between the absorbance and
time, the absorbance represent the appearance of the product or in other words we can say the
absorbance is directly proportional to the amount of product formed in the sense that a
coloured product was formed in which the colour of the substrate was colourless and after
five minutes the colour of the product was brown, therefore the concentrations were
measured by a spectrophotometer at every 30 seconds interval for five minutes in which
changes in absorbance corresponds to the colour changes. (Walker and Wilson, 1994).

This type of practical can be described as a continuous assay because cuvettes with an
enzyme reaction mixture were placed in the spectrophotometer and a change in absorbance of
a product formed was measured over time that is the reaction was occurring within the
cuvette., whereas in the discontinuous enzyme assay the reaction is stopped after a set period
of time and the absorbance is measured and the disadvantage of discontinuous enzyme assay
is that stopping the reaction requires a denaturing agent, such as heat, or severe pH change,
that will deactivate the enzyme by changing its chemical structure. From the progressive
curve plotted it can be seen that the absorbance increases with increasing time but the curve is
not starting at the origin, in theory progressive curves begin at the origin but in practical as
soon as the substrate is added to an enzyme, the enzyme will start catalysing the reaction such
that by the time the cuvette is placed in the spectrophotometer the reaction has already
started. (Gumport, et al., 1989).

Conclusion

Polyphenol Oxidase, one of the most abundantly occurring enzyme in nature was extracted
from the banana fruit for this study and its activity was determined by looking at the effect it
has on its substrate, catechol at 30 seconds interval for five minutes and it was observed that
the product formed is directly proportional to time.

References
Walker J and Wilson K, 1994, principles and techniques of practical
biochemistry, 4th edition, page 18 -20.
Glick D , 1971 , methods of biochemical analysis , volume 19
, page 12-14.
Gumport R, Jonas A and Mintel R, 1989, students companion to stryers
biochemistry, volume 1, page 129.
Bensadoun P and Keeton W, 1980, biological science, 3rd edition, page 86 -90.
Banowetz S, Jodi K and Hancock L, 1996, biochemistry and molecular
biology, 5th edition, page 400.