Title: Polyphenol oxidase activity of bananas

Introduction
An enzyme is a protein molecule that is a biological catalyst that speed up the rate of reaction
without itself being part of the products at the end of the reaction. Like most proteins, they
are synthesized by the ribosomes in the cell and they have some most remarkable
characteristics such as they are regulated from a state of low activity to high activity and vice
versa and also most enzymes act specifically with only one reactant called a substrate, to
produce products. The activity of enzymes is strongly affected by changes in pH and
temperature. Each enzyme works best at a certain pH and temperature, its activity decreases
at values above and below that point due to denaturation. For enzymes, denaturation can be
defined as the loss of enough structure rendering the enzyme inactive.( Banowetz , et al.,
1996).

Polyphenol oxidase is the enzyme that is found in almost all living organisms including
plants, animals and microorganisms. In plants, it is involved in defence mechanism. When a
plant gets a bruise or cut, certain phenolic compounds are oxidized in the presence of
oxygen to form a polymeric structure which prevents microbial contamination and its
catalytic action is connected to undesirable browning and off-flavour generation in stored and
processed foods.( Zhao ,et al.,2005)

Aim:
To examine how the temperature and pH level changes the activity of polyphenol oxidase and
also to determine the narrow range of temperature and pH values at which polyphenol
oxidase exhibits its optimum activity.

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 8g of banana tissues were mulled in 16ml
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.
pH optimum
Amount of 0.5ml of the enzyme preparation was mixed with 4.5ml of catechol buffer in test
tubes with different pH values ranging from (0.1M acetate pH 3.7 ; 4.8 ;5.6 ; 0.1M
phosphate pH 6.4 ; to 7.0) , the test tubes containing the substrate enzyme mixture were
incubated for five minutes at room temperature. Soon after the incubation process they were
placed in boiling water for one minute and the absorbance for each of the mixtures was read
using a spectrophotometer at 450nm which was calibrated using the catechol buffer.
Temperature optimum
Amount of 0.5ml of the enzyme preparation was mixed with 4.5ml of catechol (0.1M
acetate pH 5.6) in five different test tubes. Test tube number one was incubated for five
minutes in the fridge immediately after incubation it was placed in a boiling water for one
minute and its absorbance was recorded, followed by test tube number two which was
incubated at room temperature for five minutes again and placed in boiling water for one
minute and its absorbance was recorded the procedure was repeated for the rest of the test
tubes at, 37C, 50C and 70 C.

Results
Table 1: A tabular presentation of absorbance recorded at 450nm using different pH values.
pH
3.7
4.8
5.6
6.4
7.0

Absorbance (450nm)
0.808
2.231
1.682
1.300
1.254

Table 2: A tabular presentation of absorbance recorded at 450nm at different temperatures.

Temperature (C )
4
Room temperature (24)
37
50
70

Absorbance (450nm)
0.595
0.897
0.908
1.329
0.620

Discussion
Each enzyme has an optimum temperature at which it performs best.Most enzymes are active
between 10-40oC,the effect of temperature and pH on the enzymatic activity of polyphenol
oxidase was determined by measuring absorbance in varying temperatures and pH. The
temperature was ranging from 4C to 70C using catechol as a substrate (0.06M).From the
graph of absorbance against temperature drawn above it can be observed that at extreme
temperatures that is 4 C and 70C the absorbance value recorded is small indicating low
activity of polyphenol oxidase, this is because these extreme temperatures cause the native
folded structure of proteins to uncoil into random configuration. As a result, the protein loses
its biological enzymatic activity and this condition is referred to as denaturisation. The graph
produced a bell-shaped curve with the highest peak at 50C indicating the optimum
temperature for enzymatic activity. At 4C, enzymatic reaction of polyphenol oxidase occurs
slowly due to lack of energy and heat. As the temperature increases, its enzymatic also
increases up until the optimum temperature.( Gumport , et al.,1989).

Most enzymes are active only over a narrow pH range and have an optimal pH, at which
reaction is the fastest. An increase or decrease in pH also causes denaturation in enzymes,
thereby affecting their activity. From fig 2 above is a graph of pH versus absorbance at pH
3.7, the enzyme is in a too acidic environment to function. As pH decreases, certain amino
acids like aspartate and glutamate are protonated, causing them to lose their net negative
charge which consequently denatures the enzyme. From the graph it can also be noted that
the curve has the highest peak at pH 4.8 which suggest that the enzyme had the maximum
activity in the pH range 4.8-5.6, however in literature the optimum pH of polyphenol oxidase
is around 6.5. Deviation of the results obtained from the expected value can be attributed to
human error such as inaccuracies in measurement and timing during the experiment.( Alicia ,
et al.,2002)

In general, the activity of enzymes may be markedly changed by any alteration in pH, which
in turn, alters electrical charges on the enzyme. Changes in charge affect the ionic bonds that
contribute to the enzymes tertiary and quaternary structure, thereby changing the proteins
conformation and activity. Thus, pH-activity relationship of enzymes is dependent on the
amino acid side chains present in the enzyme.( Talwar and Srivastava , 2006).

Conclusion
Several factors affect the activity of enzymes. Among these are the temperature and pH. At
optimum levels of these factors, enzymes perform their function best. Optimum temperature
and pH differ from one enzyme to another.

References

Alicia N, Norman R, Jones F, 2002, principles and techniques of biochemistry, 4th

edition, page 82.
Banowetz S, Jodi K and Hancock L, 1996, biochemistry and molecular biology, 5th
edition, page 400 -405.
Gumport R, Jonas A and Mintel R, 1989, students companion to stryers biochemistry,
volume 1, page 126-130.
Talwar G and Srivastava L, 2006, biochemistry and human biology,3rd edition, page
96.
Zhao H, Moore J, Volkov A, and Arnold F, 2005, Methods for Optimizing Industrial
Enzymes by Directed Evolution, 2nd edition page 597-604.