SWISS GERMAN UNIVERSITY

CELL BIOLOGY LABORATORY REPORT

Subject
Lecturer
Instructor
Faculty/Class
Date of Experiment
Date of Lab. Report
Semester
Time of Experiment

Experiment:

Campus BSD City

Bumi Serpong Damai
Tangerang 15321 Indonesia

: Cell Biology Laboratory

: Dr.rer.nat. Maruli Pandjaitan
: Ms. Nani Pasaribu, M. Si; Ms. Sylvia Yusri, S. Si
: Life Science/LS 1 A
: 9 October 2013
: 23 October 2013
:1
: 14-00 17.00 pm

The Effect of Catecholase on a Cut Apple

Enzyme Concentration

NAME

: Felicia Melissa
Kristania Hadhiwaluyo
Jonathan Winarno
Nabila Putri

Group

: 4

Tel.
Fax.

+62 21 537 6221

+62 21 537 6201

sgu.info@sgu.ac.id
www.sgu.ac.id

I.

Objectives

To examine the factors that affects the activity of Catecholase enzyme on the
enzymatic browning of a cut apple.

To examine the effect of the increasing concentration of buffer solution (pH=7) to

the activity of the enzymes inside potato extract.

II.

Theoretical Background
Enzymes are proteins with a complex three-dimensional structure that speed up (as catalysts)
for the chemical reaction. They work by lowering energy of activation and make it occur
readily. Their ability to do is affected by physical and chemical conditions in the cell. For
every action, there is an equal and opposite reaction. In this case, factors that influence the
activity of an enzyme are called modulators. If modulators activate enzymes the reaction rate
catalyzed will significantly increase, but if the modulator inactivates enzymes the reaction
rate catalyzed will significantly decreased. Factors that affect enzymes activity include
temperature, pH, enzyme concentration, and substrate concentration on enzymes and other
proteins is one reason why these modulators are very strictly regulated by the body.

Temperature, a measure of the intensity of heat, is an important factor in the activity of

enzymes. The velocity of an enzymatic reaction is influenced by temperature. This is because
when molecules collide, the kinetic energy of the molecules can be converted into chemical
potential energy of the molecules. If the chemical potential energy of the molecules become
great enough, the activation energy of a reaction can be achieved and a change in chemical
state will result. Thus the greater the kinetic energy of the molecules in a system, the greater
is the resulting chemical potential energy when two molecules collide. As the temperature of
a system is increased it is possible that more molecules per unit time will reach the activation
energy. Thus the rate of the reaction may increase substrates collide. In addition, thermal
agitation causes protein molecules (enzymes) to denature ( breakdown of protein structures).
All enzymes have an optimal temperature at which reaction rates go fastest without
denaturing the enzyme pH, a measure of hydrogen ion concentration, is a second important
factor in the activity of enzymes.

Changes in pH can change the shape of the active site in an enzyme. Extremely high or low
pH concentrations usually result in complete loss of enzyme activity due to denaturation.
Hydrogen ions and or hydroxide ion concentration greatly influences the rate of reactions.

Besides that, enzyme concentration also affects the rate of enzymatic reactions. Without
enzymes, reactions can go very slowly, but with enzymes the rate is directly proportional to
the amount of enzyme that is present. In addition, these reactions will only increase if the
substrates present are in excess amounts. Cells can alter the amount of enzymes by
influencing their synthesis and breakdown. If enzyme synthesis exceeds breakdown, enzymes
accumulate and the speed of catalyzation increases, if enzyme breakdown exceeds synthesis,
the concentration of enzyme decreases along with reaction rate. In the event that the amounts
are equal there is still a turnover of enzymatic molecules.

Substrate concentration affects the rate of enzymatic reactions. At lower substrate

concentrations, the reaction rate is strictly proportional to the substrate concentration, but
once the substrate molecule concentrations increase to a maximum level, there are no more
binding sites available for them. This is called saturation, when enzymes catalyze as fast as
they can, and reaction rate reaches its maximum potential. Without substrate, enzymes cannot
function, and without the appropriate amount of substrate, the velocity of the reactions would
take place very slowly.

III.

Apparatus and Materials

Apparatus
- Bowl-shaped petri dish, 6
- Beaker glass, 100 cm3, 1
- Dropping pipette, 1
- Test tubes, 3
- Knife
- Mortar and pestle

Materials
- Cold, warm (40 0C), and normal temperature distilled water, H2O, pH = 7
buffer solution
- Potato 1
- Potato extract
- Washington apple, 1
- Lemon juice, pH < 7

IV.

Procedure

Experiment 1: Factors that affect the activity of Catecholase enzyme in a cut apple
1. Preparation of Liquid
1. Cold water was put in petri dish no 1
2. Lemon juice was put in petri dish no 2
3. Warm water was put in petri dish no 3
4. Distilled water (normal temperature) was put in petri dish no 4
5. No liquid was put in petri dish no 5 and 6
2. Preparation of Apple samples
1. The apple was cut into 6 pieces
2. Apple for petri dish no 1-4 and 6 was put into the petri dishes without
further treatment
3. Apple for petri dish no 5 was mashed with mortar and pestle, then it
was put in the petri dish
3. Preparation of Experiment Setup & Method of Investigation
After all the liquids and the apples were put in the petri dish, the condition of
the apples were checked every minute. The apples were in the petri dish for
30 minutes. We checked the submerged part of the apple.

Experiment 2: Amount of buffer solution (pH=7) and the enzymes activity of potato
extract
1. Preparation of Potato extract
1. The potato was cut into pieces and soaked into water in the blender.
2. After a couple of minute, the potato was blended
3. The extract was put in a beaker glass
2. Preparation of Experiment Setup & Method of Investigation
1. Every reaction tube got 10 drops of potato extract
2. Tube 1 got additional 25 drops of distilled water
3. Tube 2 got additional 15 drops of distilled water
4. Tube 3 got additional 5 drops of distilled water
5. The tubes are examined every 2 minutes for their color change

V.

Data

Experiment 1: Factors that affect the activity of Catecholase enzyme in a cut apple
TABLE 1.1

No. Of
Petri Dish

Type of Liquid

Cold distilled water, H2O

Lemon juice, pH < 7

Warm distilled water, H2O, 40 0C

Figures 1 - 6: Initial Condition of

Apples

Time
(minutes)
1
2
3
4
5
6
7

Distilled water, H2O, normal

temperature

Mashed apple with no liquid

Sliced apple with no liquid (a

controlled sample)

TABLE 1.2
No. Of Petri Dish
The level of browning that each apple samples undergoes changes at each time intervals
1
2
3
4
5
6
+
+
+
+
+
+
+
+
+
+
+
++
+
+
++
+
+++
+

8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++

++
++
++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++++
++++
++++
++++

++
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

+++
+++
++++
++++
+++++
+++++
+++++
+++++
++++++
++++++
++++++
++++++
++++++
++++++
++++++
+++++++
+++++++
+++++++
+++++++
+++++++
++++++++
++++++++
++++++++

TABLE 1.3
Figures 7-12: Final Condition of Apples after 30 Minutes

Petri dish 1
(Cold distilled water, H2O)

++
++
++
++
+++
+++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
+++++
+++++
+++++
+++++
+++++
+++++
+++++

Petri dish 2
(Lemon juice, pH < 7)

Petri dish 3
(Warm distilled water, H2O, 40 0C)

Petri dish 4
(Distilled water, H2O, normal temperature)

Petri dish 5
(Mashed apple with no liquid)

Petri dish 6
(Sliced apple with no liquid as a
controlled sample)

Experiment 2: Amount of buffer solution (pH=7) and the enzymes activity of potato
extract
TABLE 2.1

No. of
Test
Tubes

Drops of
Potato
Extract

Drops of Buffer
Solution
(Distilled water,
H2O, pH=7)

10

25

10

15

10

Figure 13: Experiment Setup

TABLE 2.2
No. Of
Test
Tubes

VI.

Time Minute(s)
The changes in color of the solution at each time intervals more +, the more
yellow is the solution
1

11

13

15

++

++

++

+++

+++

+++

+++

Discussion
Experiment 1: Factors that affect the activity of Catecholase enzyme in a cut
apple
According to the result of the investigation shown by the figures in the data table in the
previous pages, it was seen that the color of apple pieces would turn to red-brownish in
color at different rates. The main reason why the normal color of the apple turned into red-

brownish color is due to the presence of catechol inside the apple that able to turned into a
reddish-brown colored compound called benzoquinone in the reaction shown below:

Figure 14: The browning reaction of apple pieces

Catechol is a phenolic compound found in many plants. When plant tissues are injured
catechol is released and rapidly oxidized (Hart, Dr. M. C 2009) with the help of catechol
oxidase or catecholase enzyme to a compound called benzoquinone. This compound called
benzoquinone is known for its role as a natural antiseptic of the plants that are toxic to
bacteria and thus helps protect the plant from infection. Nonetheless, benzoquinone that is
formed by this reaction is also quickly converted to brown colored products, which is seen
by the brown-color of the surface of the apple pieces.

Factors that influences the rate of browning of apples

In this investigation it is proven that there are two out of several factors that influence the
activity of the catecholase enzymes thereby affecting the rate of the browning of the apples,
they are temperature and pH. Although in reality there are many kinds of factor that may
influence thus inhibit the activity of the enzyme in this investigation the effect of the
temperature and pH of the liquid plays a great contribution and display a clear impact to the
rate of the browning of the apple samples therefore will be discussed further in this
discussion section.

Firstly regarding the effect of the temperature, in this investigation the effect of the changes
in temperature to the rate of apple browning was monitored with the help of the three kinds
of temperature of distilled water, H2O, cold, hot, and normal. It was found out from the data
seen in table 1.2 and 1.3, that the apple that was located in a hotter temperature has a more
brown-color stained that occur in a faster rate, compared to the other apple samples in the
normal temperature water or even the cold one. The main reason why this was happened is
due to the fact that

Increasing the temperature will increase the rate of the enzyme activity as there is higher
kinetic energy that the molecules has. The high value of kinetic energy causes more
collision between enzyme and substrate molecules, making the reaction between them faster
thus forming more product at a shorter amount of time.

According to the figure of the graph of the

enzyme activity with temperature (figure
15), it is seen that the graph of he enzyme
tends to show a parabolic shape in general.
Meaning, the rate of the enzyme activity
will

increase

proportionally

as

the

temperature increased. However, as it

exceeds its optimum temperature in which
the enzyme has the rate of the enzyme
activity

will

decreased

rapidly.

Figure 15 - Graph of Temperature vs. Rate of Enzyme Activity

This

condition may happen if the apple pieces used in this first section of this investigation is put
in really hot or even boiling water. It is due to the fact that a really high temperature will
cause the conformational structure of the protein of the enzymes to be denatured, thus
changing the shape of active shape and hinders it to get into contact with the substrates.

Secondly, the factor that affects the rate of

the browning of the apples is the pH of the
liquid, where the apple is located. In this
first section of the investigation, two
different kinds of pH are used, one is a
neutral pH of 7 (in the normal temperature
distilled water, H2O) and the other one is
the one with pH below 7 or a quite acidic pH

Figure 16 - Graph of pH vs. Rate of Enzyme Activity

(lemon juice). In the data shown in the table 1.1, it is seen that there is no big difference in
the rate of the browning of the apple in both petri dish. In fact, the changes in pH should
affect the rate of browning of the apple as different types of enzymes have its very own
optimum pH values. It is seen from the figure 16, that at any pH that is located above or
below the Optimum will quickly cause a decrease in the rate of reaction, since more of the
enzyme molecules will have active sites whose shapes are not (or at least are less)
complementary to the shape of their substrates (Adam Day, S 2012). However the main

reason why there is no big difference in the rate of browning of the apple samples is mainly
due to the pH of the lemon juice (which is used as the acidic liquid sample) The pH of the
lemon juice in this investigation is no as acidic as it should be therefore the effect of the
changes in pH cannot be determined very clearly from the result of the investigation.

The other thing that influences the rate of the enzyme activity is the surface area.
Theoretically, the effect of surface area did not play a role that affect the rate of enzyme
activity however chemically speaking, changes in surface area actually plays a role on
affecting the rate of reaction. It is proven from the result of this experiment seen in table 1.1,
1.2, and 1.3 that the mashed apple pieces has the fastest rate compared to the controlled
apple sample that was sliced and was let into contact with oxygen, O2. This was happened
due to the fact that the increasing in surface area of the apple by mashing them causes more
particles, in this case the section of the apple tissues are exposed to the reactant which is
oxygen, O2. As a result there are more collision between the catechol in the apple tissues
with the oxygen, O2, in the air thus increasing the rate of oxidation reaction of catechol to
produce the brown-colored compound, benzoquinone.

Enzymatic browning of apples and how to prevent it

First and foremost, the word enzymatic browning is a process where a food particularly
fruits turned to brownish in color due to the enzymes that they contain. Enzymatic browning
itself happened when enzyme polyphenol oxidase catalyze the oxidation of phenols in the
fruit to form compounds called quinones (EdInformatics.com 1999) which then undergoes
polymerization reaction to polymerize itself forming melanin compounds, which responsible
for the brown pigment in enzymatic browning as its shown in the figure 17.

Figure 27 Polymerization of Quinones to form Melanin with the help of Phenolosidase

Theoretically, polyphenols compound can be classified into different categories such as

anthocyanins, flavonoids and non-flavonoids. Anthocyanins are pigments present in fruits,
flavonoids are occur in catechins, tannins and beverages and non-flavonoids are a
component of gallic acid in tea leaves (Sengupta, S 2010). Each of these polyphenols
compounds are unstable and when they are exposed to the oxygen, O2, in the air they are
catalyzed by the polyphenol oxidase enzyme, similar to the catecholase oxidase (catecholase
enzyme) of the apples, to undergoes series of biochemical reactions that form a brown
product, Melanin, as its end result. The product of this reactions itself also has its
advantages and disadvantages, as its advantages melanin is known to has antimicrobial
properties that prevents any infection and inflammation to the plant or fruits. Melanin also
has antibacterial, antioxidant and anticancer properties (Sengupta, S 2010) making these
renders food physiologically wholesome. However, in terms of the effect to the quality of
the product, the presence of this brown melanin pigment is considered to bring a bad effect
to the quality of the fresh fruit and vegetables, particularly apples and potatoes, and to
seafood such as shrimp.

The enzymatic browning that happened inside the apples itself is happened due to the
presence of several phenol compounds that are present in the peel and tissues of the apple.
These compounds are chlorogenic acid, caffeic acid, 3,4-dihydroxyphenylalanine (DOPA),
p-coumaric acid, flavonol glycosides, 4-methyl catechol, catechin, 3,4-dihydroxy benzoic
acid, p-cresol and leucocyanidin (Sengupta, S 2010) which are also substrates that react
with phenoloxidase which finally lead to formation of browning product.

Finally in order to minimize the effect of the enzymatic browning of the apples, the rate of
the enzyme activity of phenoloxidase has to be reduced and the oxygen must be minimized
from contacting with the fruits or vegetables. According to the theory, there are many ways
to minimize the effect of enzymatic browning such as by blanching, refrigeration, freezing,
changes in pH, dehydration, irradiation, high pressure treatment, ultrafiltration,
ultrasonication, treatment with supercritical carbon dioxide and addition of inhibitor.
However there are only several factors that may work for this investigation, which is by
using a very low or high temperature of liquid for the apples medium, a very low or high
pH, low temperature by refrigeration, and by limiting the contact of apples to the oxygen,
O2, by using vacuum packaging.
Firstly, a very low or high pH may work on this experiment because it will eventually slows
down the rate of enzyme activity of those phenoloxidase knowing that pH plays an
important role in influencing the enzyme activity. Secondly, refrigeration will work because
by lowering the temperature of its surrounding, it will lower the amount of kinetic energy
that the molecules has therefore limiting the possible collision between the substrates
(phenolic compounds of the apple) and the enzyme (phenoloxidase) which eventually
decrease the rate of the reaction, knowing the fact that at temperatures below 7 C the
phenoloxidase enzyme activity is inhibited (Food Info, 2013). Lastly the reason why
placing the apple samples inside a vacuum packaging (packaging with absence of oxygen,
O2) will work because by using vacuum packaging, it will limit the contact or will even
prevent the contact between the phenolic compounds of the apple with the oxygen, thereby
preventing the oxidation that triggers to activate the phenoloxidase enzyme.

Experiment 2: Amount of buffer solution (pH=7) and the enzymes activity of

potato extract
According to the result of the investigation shown by the figures in the data table in the
previous pages, it was seen that the color of the potato extract will have a higher yellow-

color intensity as the time increases with different rates, depending on the amount of buffer
solution (in this case normal distilled water, H2O) with a pH of 7 that is place along with the
potato extract in the test tubes. The main reason why each test tube has different intensities
is due to the fact that each test tube has different amount of buffer solution even though they
have the same amount of potato extract in it. The differences in the amount of buffer
solution placed inside the test tubes therefore follows the same principle of how enzymatic
browning in fruits such as apple is prevented, which is by controlling the pH.

Theoretically, there are many kinds of ways on how to control the pH of this investigation,
however to keep it simple, buffer solution (in a form of distilled water, H2O) is used. The
word buffer solution would mean a solution that resist changes in pH upon small adition
of acid or base. The presence of buffer solution in the test tubes inhibit the activity of the
enzyme of the potato extract in contact with the oxygen, O2, that may come into their
unclosed test tubes. Additionally the presence of this buffer solution also inhibits the
changes of pH of the potato extract that may occur knowing that changes in pH may affect
the overall enzyme activity. As a result, the more buffer solution that a test tube has, the
slower it takes for the potato extract to change its color intensity to a darker yellow in color.

VII.

Conclusion
Enzyme is basically a protein that speeds up a chemical reaction in a living organism. An
enzyme acts as catalyst for specific chemical reaction, converting a specific set of reactants
(called substrates) into specific products. There are several factors that affect enzyme activity
include temperature, pH, concentration of enzyme and substrate.
In this experiment we use catecholase (enzyme contained in some fruits and vegetables that
bind to the substrate Catechol) as an example. Catechol will react with oxygen and form
benzoquinone as the cause of browning color in fruits.
Catecholase work in a fast speed at room temperature (especially when we mashed it), in the
contrary it will slow down if the temperature is too cold. Besides temperature, pH is also
affect catecholase. The rate of catecholase activity is greatest at neutral pH (at distilled water),
while on the other hand if the pH is too acidic or too basic, it will denatured the enzyme and it
became inactive.

VIII.

References

Module Cell Biology

"BBC - GCSE Bitesize: Effect of surface area." BBC - Homepage. N.p., n.d. Web.
14 Oct.
2013.<http://www.bbc.co.uk/schools/gcsebitesize/science/add_ocr_gateway/chemica
l_economics/reaction3rev1.shtml>.

"Factors affecting Enzyme Activity | A Level Notes." A Level Notes. N.p., n.d. Web.
14 Oct. 2013. <http://alevelnotes.com/Factors-affecting-Enzyme-Activity/146>.

Sengupta, Saptakee. "Enzymatic Browning." Buzzle. N.p., 2010. Web. 14 Oct. 2013.
<http://www.buzzle.com/articles/enzymatic-browning.html>.

"What causes the browning of foods?." Science of Cooking. N.p., n.d. Web. 14 Oct.
2013. <http://www.scienceofcooking.com/browning_of_foods.htm>

Enzymatic Browning, Food Info, N.p., 2013. Web. 14 Oct. 2013.

<http://www.food-info.net/uk/colour/enzymaticbrowning.htm>

Dr. Marilyn C. Hart, Enzyme Activity, Minnesota State University Associate

Professor of Biological Sciences, 2009. Web. 14 Oct. 2013.
<http://www.intech.mnsu.edu/mhart/105%2009/Catechol%20oxidase.doc >