CELL BIOLOGY LABORATORY REPORTSubject : Cell Biology LaboratoryLecturer : Dr.rer.nat. Maruli PandjaitanInstructor : Ms. Nani Pasaribu, M. Si; Ms. Sylvia Yusri, S. SiFaculty/Class : Life Science/LS 1 ADate of Experiment : 9 October 2013Date of Lab. Report : 23 October 2013Semester : 1Time of Experiment : 14-00 – 17.00 pm

Experiment:The Effect of Catecholase on a Cut AppleEnzyme Concentration

NAME:

Felicia Melissa Kristania HadhiwaluyoJonathan WinarnoNabila Putri

Group : 4

S W I S S G E R M A N U N I V E R S I T Y

Campus BSD CityBumi Serpong DamaiTangerang 15321 – Indonesia

Tel.+62 21 537 6221Fax. +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 1.1No. Of

Petri Dish Type of Liquid Figures 1 - 6: Initial Condition of Apples

1 Cold distilled water, H2O

2 Lemon juice, pH < 7

3 Warm distilled water, H2O, 40 0C

4 Distilled water, H2O, normal temperature

5 Mashed apple with no liquid

6 Sliced apple with no liquid (a controlled sample)

Time (minutes)

TABLE 1.2No. Of Petri Dish

The level of browning that each apple samples undergoes – changes at each time intervals1 2 3 4 5 6

1 - - - - - -2 - - - - - -3 - - - - - -4 - + - + + -5 - + + + + +6 - + + + ++ +7 - + ++ + +++ +

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)

Figure 13: Experiment Setup

1 10 25

2 10 15

3 10 5

TABLE 2.2

No. Of Test

Tubes

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

yellow is the solution

1 3 5 7 9 11 13 15

1 - - - - + + + ++

2 - - - - + ++ ++ +++

3 - - - - + +++ +++ +++

VI. Discussion

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

appleAccording 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. 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

(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

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

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

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 it’s shown in the figure 17.

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

thocyanins, flavonoids and non-flavonoids. “Anthocyanins are pigments present in fruits,

flavonoids are occur in catechins, tannins and beverages and non-flavonoids are a compo-

nent 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 disadvan-

tages, 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 pres-

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

ence 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, ultrasonica-

tion, 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 apple’s 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 impor-

tant 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 en-

zyme activity is inhibited” (Food Info, 2013). Lastly the reason why placing the apple sam-

ples 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 be-

tween the phenolic compounds of the apple with the oxygen, thereby preventing the oxida-

tion that triggers to activate the phenoloxidase enzyme.

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

potato extractAccording 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 en-

zyme 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 ben-

zoquinone 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 af-

fect 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/chemical_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 >