INTERNATIONAL UNIVERSITY

BIOMEDICAL ENGINEERING DEPARTMENT (BME)

OCTOBER 22, 2021

BIOLOGY FOR BME

REPORT FOR
LABORATORY

GROUP 4

TRẦN ĐẠO QUANG

NGUYỄN THỊ QUỲNH NGA
NGUYỄN BÌNH PHƯƠNG NHI
ENZYMES - UNDERSTAND HOW
ENZYMES WORK

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TABLE OF CONTENTS
I. INTRODUCTION 4
A. PERPOSE 4
B. ENZYMES 5
1. Overview 5
2. Function 5
3. Mechanism of action 6
4. Special characteristics 7
C. THE EFFECT OF BROMELAIN ON GELATION OF GELATIN 8
1. Overview 8
a) Bromelain 8
b) Gelatin 9
2. The effect of bromelain on gelation of gelatin 9
II. CONTENT 10
1. The effect of bromelain on gelation of gelatin 10
a) Materials 10
b) Equipment 10
c) Procedure 10
2. The effect of temperature on bromelain’s activity 12
a) Materials 12
b) Equipment 12
c) Procedure 12
3. The effect of pH level on bromelain’s activity 14
a) Materials 14
b) Equipment 14
c) Procedure 14
III. ANSWERING QUESTION 17
1. QUESTION 1 17
2. QUESTION 2 23
3. QUESTION 3 30
IV. CONCLUSION 33
V. REFERENCES 34

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TABLE OF FIGURATION

FIGURE 14 1. TABLE II.1 11

FIGURE 24 2. TABLE II.2 13
FIGURE 34 3. TABLE II.3 15
FIGURE 46 4. TABLE III 2.2.a 27
FIGURE 56 5. TABLE III 2.2.b 29
FIGURE 67
FIGURE 77
FIGURE 88
FIGURE 99
FIGURE 109
FIGURE 1114
FIGURE 1217
FIGURE 1318
FIGURE 1419
FIGURE 1520
FIGURE 1621
FIGURE 1721
FIGURE 1822
FIGURE 1923
FIGURE 2025
FIGURE 2127
FIGURE 2231

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I. INTRODUCTION

A. PURPOSE

We learn about enzymes: definition, function, special properties, and mechanism of

action. In more details, we are going to explore more about bromelain enzyme. In terms
of bromelain enzyme, we do learn specifically where we can find such enzyme, how does
it work, or its characteristics. Finally, we will determine the effect of bromelain on
gelation of gelatin, the effect of temperature and pH level on the bromelain’s activity by
some experiments will be described carefully below.

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Figure 1: Amylase. Figure 2: Pepsin A.

Figure 3: Bromelain.

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B. ENZYMES

1. Overview:
Enzymes are composed of proteins folded into complex shapes; they are
present throughout parts the body. Playing role as biological catalysts, they can
help speed up (catalyze) metabolism, or the chemical reactions faster one million
of times in our bodies than it would have been without it. They build some
substances and break others down. All living things have enzymes. Our bodies
naturally produce enzymes. But enzymes are also presenting in manufactured
products and food. A substrate binds to the active site of an enzyme and is
converted into product. Once the products leave the active site, the enzyme is
ready to attach to a new substrate and repeat the process.

2. Function:
The digestive system: enzymes help the body break down larger complicated
molecules into smaller molecules, such as glucose, so that the body can use them
as energy.
Example: Amylase and other carbohydrase enzymes break down starch into
sugar or Lipase enzymes break down lipids (fats and oils) into fatty acids and
glycerol.
DNA replication: each cell in your body contains DNA. When a cell is dividing,
DNA needs to be copied. Enzymes help in this process by unwinding the DNA coils
and copying the information.
Example: DNA polymerase synthesizes the new DNA strand; also proofreads
and corrects some errors or Primase provides the starting point for DNA
polymerase to begin synthesis of the new strand.
Liver enzymes: the liver breaks down toxins in the body. To do this, it uses a
range of enzymes.
Example: Alkaline phosphatase (ALP), Alanine transaminase (ALT).
Enzyme can also help with building muscle, breathing, and nerve function.

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3. Mechanism of action:
The “lock and key” model was first proposed in 1894 by Emil Fischer. In this
model, an enzyme’s active site is a specific shape, and only the substrate will fit
into it, like a lock and key. This early model explains enzyme specificity but fails to
explain the stabilization of the transition state that enzymes achieve.

Figure 4: Enzyme’s lock and key model.

This model has now been updated and is called the induced-fit model –
proposed by Daniel Koshland in 1958. In this new model, Daniel Koshland
supposed that the active site changes shape as it interacts with the substrate. The
substrate does not simply bind to a rigid active site; the amino acid side - chains
that make up the active site are molded into the precise positions that enable the
enzyme to perform its catalytic function. Once the substrate is fully locked in and
in the exact position, the catalysis can begin.

Figure 5: Enzyme’s induced – fit model.

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4. Special characteristic:
Active site contains 3 to 12 amino acids and less hydrophobic amino acids.
Sensitive to temperature, pH level, and substrate concentration.
Enzymes are larger than substrates.
Lower the activation energy.
Required in very less amount compared to chemical catalysts.
Produces products using specific substrate.
Some are globular proteins, and few are RNA – based molecules.
Can be recycled or reused.
Forms an enzyme-substrate complex.
Function can be inhibited by inhibitors.
Enzymes are both intracellular and extracellular catalysts.
Some enzymes need coenzymes or cofactors.

Figure 6: Enzyme working process. Figure 7: Enzyme with cofactor.

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C. THE EFFECT OF BROMELAIN ON GELATION OF GELATIN

1. Overview:
a) Bromelain:
Bromelain is a proteolytic enzyme derived from the stem, fruit, and juice of the
pineapple plant that has protein-digesting properties. 
Bromelain may be used alone or in conjunction with other medications. People
use bromelain topically, to remove dead skin from burns, and orally, to reduce
inflammation and swelling — particularly of the nasal passages.
Bromelain is also used as a digestive aid, for osteoarthritis, and to reduce
soreness in aching muscles.

Figure 8: Pineapple and bromelain.

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b) Gelatin:
Gelatin is produced from the partial hydrolysis of collagen.
Collagen is the most abundant protein in the body. It is a key structural
component in many of our tissues, including our tendons and our bones.

Figure 9: Denaturation of collagen produces thermoreversible gelatin.

2. The effect of bromelain on gelation of gelatin:

Bromelain can break down the gelatin proteins into its amino acid
components, hence preventing it from setting.
After doing experiments, we can confirm that in different point of
temperature, pH level, or the purity of the pineapple samples we have, the
activity of Bromelain from such samples during the gelation of gelatin will be
affected partly or completely.

Figure 10: The effect of bromelain on gelation of gelatin.

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II. CONTENT

1. The effect of Bromelain on the gelation of Gelatin:

a) Materials:
Distilled water: 55ml.
Fresh pineapple juice: 3ml.
Canned pineapple juice: 3ml.
Power gelatin: 1 package.

b) Equipment:
Pipette (3ml): 3 pieces.
Beaker: 1 piece.
Test tube: 3 pieces.
Test tube rack: 1 piece.

c) Procedure:
Step 1: Prepare and label 3 test tubes as “Water”, “Fresh”, and “Canned”.
Step 2: Mix power gelatin with 50ml water in the beaker. After that, heat and
continuously stir the mixture at the appropriate temperature for 2 minutes.
Step 3: To each of the three test tubes, add warm gelatin.
Step 4: Use 3 separate pipettes to add 2ml of water, 2ml of fresh pineapple
juice, and 2ml of canned pineapple juice into the 3 pre-marked test tubes
(“Water”, “Fresh”, and “Canned”), stir the solutions in the test tube.
Step 5: Soak the test tubes in ice water for 5 to 10 minutes. Carefully observe
what happens in the test tube marked "Water" and compare the results with
the other 2 test tubes. After all, record the results in the notebook.
Step 6: Keep laboratory equipment and laboratory clean.

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THE EFFECT OF BROMELAIN ON GELATION OF GELATIN
TESTTUBE RESULT EXPLAIN
Gelatin solidifies

Water In room temperature, gelatin is solidified.

Gelatin does not solidify.

Because the pineapples contain

bromelain enzyme which has protein-
Fresh pineapple
digesting properties, the structure
juice
of gelatin will be broken and will not
solidify.

Gelatin solidifies. During production and storage process,

the enzyme Bromelain in pineapple was
decomposed by the environment and
temperature, so the gelatin solidified in
Canned canned pineapple juice.
pineapple juice On the other hand, canned pineapple
juice has mixed other ingredients and is
not 100% pineapple, so the enzyme
bromelain is very little or no to be able to
destroy the protein structure.
Table II.1: The effect of bromelain on gelation of gelatin.

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2. The effect of temperature on Bromelain’s activity:
a) Materials:
Distilled water: 55ml.
Fresh pineapple juice: 3ml.
Power gelatin: 1 package.

b) Equipment:
Pipette (3ml): 3 pieces.
Beaker: 1 piece.
Test tube: 3 pieces.
Test tube rack: 1 piece.

c) Procedure:
Step 1: Prepare and label test tube as “Water”, “Hot”, and “Cool”.
Step 2: Mix power gelatin with 50ml water in the beaker. After that, heat and
continuously stir the mixture at the appropriate temperature for 2 minutes.
Step 3: Add 2ml of water to the test tube was labeled “Water” and put in the
test tube rack.
Step 4: Add 2ml of fresh pineapple juice to the test tube was labeled “Cool”
and “Hot”, placed these test tubes in temperature room.
Step 5: Placed the test tube labeled “Hot” into a 70°C water bath for 5
minutes.
Step 6: After 5 minutes, carefully removed the test tube labeled “Hot” from
water bath.
Step 7: Add 3ml of warm gelatin to each test tube and then place three test
tubes in ice until the test tube labeled “Water” is solidified.
Step 8: Observe three test tubes, record the result of three test tubes.
Step 9: Keep laboratory equipment and laboratory clean.

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THE EFFECT OF TEMPERATURE ON BROMELAIN’S ACTIVITY

TEST TUBE RESULT EXPLAIN
Gelatin solidifies.

Water In room temperature, gelatin is solidified.

Gelatin solidifies.

Bromelain enzyme is affected by elevated

temperature. At elevated temperature,
Hot
Bromelain changes the structure, so it
does not break down gelatin.

Gelatin does notsolidify.

In cool temperature, gelatin is gel and

little solution. Bromelain enzyme is less
Cool active at room temperature, so the
phenomenon is that some gelatin does
not solidify.

Table II.2: The effect of temperature on bromelain’s activity.

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3. The effect of pH level on Bromelain’s activity:
a) Materials:
HCl (Acidic solution with low pH).
NaOH (Alkaline solution with high pH).
Gelatin solution.
Fresh pineapple juice.
Distilled water.

b) Equipment: Figure 11: pH scale.

Test tubes.
Test tube racks.
Pipettes (Do not use a pipette for both HCl solution and NaOH solution as it
will affect the purity of the reagent, leading to erroneous test results).

c) Procedure:
Step 1: First, we prepare and label test tubes number 1-6.
Step 2: Then, the pipettes were then carefully inserted into the test tubes by
the pipettes, respectively.
Test tube number 1: 2 mL of water.
Test tube number 2: 2 mL of fresh pineapple juice.
Test tube number 3: 1 mL HCl and 1 mL fresh pineapple juice.
Test tube number 4: 1 mL HCl and 1 mL water.
Test tube number 5: 1 mL NaOH and 1 mL fresh pineapple juice.
Test tube number 6: 1 mL NaOH and 1 mL water.
Step 3: Next, to mix the ingredients together in the test tube, shake gently
(around 3 minutes) and except for the gelatin tube.

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Step 4: After adding 3 mL of gelatin to each test tube and a test tube labeled
with "gelatin".
Step 5: Additionally, place all six test tubes in the ice bath and also the gelatin
test tubes until the "gelatin" tubes begin to solidify.
Step 6: At least the end of observing the test tubes and recording our results.
Before the end of class, we have cleaned laboratory equipment and labs in
accordance with regulations.

THE EFFECT OF pH LEVEL ON BROMELAIN’S ACTIVITY

TEST TUBE SOLUTION EXPLAIN
Appearance
This case is definitely solid because the water
2 mL of water solidification
will solidify at low temperatures become ice.

This case is definitely solid because, in the test

2 mL of fresh pineapple juice
1 mL HCl and 1 mL fresh
pineapple juice
1 mL HCl and 1 mL water
1 mL NaOH and 1 mL fresh
pineapple juice
Appearance
tube only fresh pineapple juice, the active
solidification
medium of bromelain is not affected by any
agent which will solidify at low temperature.
The pH for the enzyme bromelain to work well
is in the range of 5-8 depending on the
substrate, while the pH for the HCl is too low
Appearance
(about pH < 3.5), which strongly contribute to
solidification
the repressing toward the gelation of Gelatin
(components containing amino acids), so it can
inactive Bromelain and the liquid can become
a gel.
Since the concentration acidity of HCl has been
Invariable
diluted by water, it solidifies easily at low
temperatures.
Base solution containing NaOH (high pH, about
> 13) does not contribute to the forming of the
Invariable
gelation, so it polarizes the Bromelain into
amino acids, and it can active the Bromelain.
Thus, the liquid cannot become a gel.

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Since the concentration alkaline of NaOH has
Invariable been diluted by water, it solidifies easily at low
1 mL NaOH and 1 mL water
temperatures.

Table II.3: The effect of pH level on bromelain’s activity.

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III. ANSWERING QUESTION

QUESTION 1:
a) What is the activation energy of a reaction? What are
catalysts?
a.1) The activation energy of a reaction:
Firstly, the definition of "activation energy" is the smallest amount of
energy required to initiate a chemical reaction in chemistry and physics. In other
words, it is an energy barrier to a reaction. Additionally, it is denoted by Ea and
also it is expressed (unit) in kJ/mol. Moreover, activation energy is related to the
response rate so, slower reactions have greater activation energy, while quicker
reactions have lower activation energies. In fact, burning dry wood releases a lot
of energy, but firewood does not suddenly burst into flames. It only starts the
burning process when supplied with sufficient activation energy, and the
activation energy can be provided by a lighter.

Figure 12: This graph showing the activation energy of a

reaction. (Energy of activation ΔEa < ΔEa , due to the effect
of the enzyme on the substrate).

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a.2) Catalysts:
In science, a catalyst is defined as any material that increases the speed of a
process without it being consumed or permanently altered by the reaction. The
catalyst speeds up the process by reducing the activation energy. Especially, they
do not affect altering the chemical equilibrium between reactants and products.
In biology, enzymes are naturally occurring catalysts responsible for many
essential biochemical reactions, they speed up the rate of chemical reactions in a
cell or outside a cell.
In life, when we chew rice (rice has a very high starch content), salivary
amylase enzymes are secreted to hydrolyze polysaccharides and oligosaccharides
into monosaccharides. This is an ingredient that can be absorbed by the body.
However, the efficiency of salivary amylase activity is not as high as that of
pancreatic amylase, so pancreatic amylase activity in the small intestine is more
important. After amylase enzymes hydrolyze starch, the products formed are
glucose, maltose, maltotriose, and α - destrins.

Figure 13: The Enzymes speed up the breakdown of

food into nutrients in the body.

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b) How does enzyme, in general, work? What is it application in
the field of BME?
b.1) How does enzyme work?
Enzymes are ubiquitous in the body and are made up of protein folds with
complex shapes. Enzymes are biological catalysts that help speed up the process
of chemical reactions. Additionally, they create the right conditions for molecules
to collide, so activation energy is reduced, and chemical reactions occur faster. In
during work, the active site position of the enzyme will be adapted to the
substrate when interacting with each other. The catalysis just begin after the
substrate is fully locked and in the correct position. After that, the product is
formed and leave the active site, so the enzyme has completed its work cycle and
is ready to bind to a new substrate and start working again.

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Figure 14: The operation model of enzymes according to the

"lock and key" rule. (i.e., The induced-fit model).

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b.2) Enzyme’s application in the field of BME:
The first reason why biomedical engineers applied enzymes in machines,
drug manufacturing, and laboratories rooted in enzymes exist in all forms of life,
including people, plants, bacteria, and other species, and may thus be found
anywhere. Enzymes are required for the normal functioning of any living body.
Enzymes are catalyst naturally occurring proteins whose role is to accelerate and
improve the efficiency of a chemical reaction itself without being consumed in the
process. In other words, they catalyze a wide range of chemical processes
involved in growth, blood coagulation, healing, sickness, breathing, digestion,
reproduction, and a variety of other biological functions. Enzymes are the
biologically important catalysts that perform a variety of tasks in living beings.
Because enzymes have numerous advantages, biomedical engineering major has
applied them in medical devices to assist doctors in detecting and treating
illnesses. In fact, the machines with enzyme technology applications that have
been put into medical use today are machines enzymes immunoassay analyzer
(CLIA), the enzyme-linked immunosorbent assay (ELISA), etc.
Machines enzymes immunoassay analyzer (CLIA) is used to estimate
analytes that have extremely low concentrations in the blood such as
hormones, serological markers, and drugs.
The role enzymes are enzyme-specific substrate producing a
detectable signal in this test, they are labeled with antibodies
before reacting with antigen, which is mainly a change in the color of
the substrate.

Figure 15: Machines

enzymes immunoassay
analyzer (CLIA).

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Figure 16: Illustration of BIO-

FLASH system, a widely used CLIA
in the field of autoimmunity.

The enzyme-linked immunosorbent assay (ELISA) is a commonly used

laboratory test to detect antibodies in the blood and is also used for
HIV detection and quantitation of nucleic acids.

Enzymes are linked to a secondary antibody, which interacts with the

main antibody after being injected into the sample to indicate the
location of the protein. The activity of the conjugated enzyme is
evaluated by incubating it with the substrate to give a quantifiable
result. The highly specific antibody-antigen interaction is the most
significant component of the detection approach.

Figure 17: Machines enzyme-

linked immunosorbent assay
(ELISA).

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Figure 18: A sandwich immunoassay is used to measure the concentration of antigen.

The basic components of the test are antigen, labelled antibody, and substrate.

*ELISA is one of the most frequently used methods for serological

detection of the infection with about 1–5 h. ELISA, as the manual version of the
automated CLIA, so ELISA is cheap costs compared with CLIA, but it costs more
staff, time, and effort.
Especially, for ELISA-based COVID-19 detection through SARS-CoV-2
particular antibodies are injected into the well and bind to the immobilized viral
antigens. Primary antibodies bind to target antibodies, while a secondary
enzyme-linked tracer antibody reacts with a chromogen, causing a color shift.
The color intensity was linked to the concentration of antigen in the sample.

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Figure 19: ELISA method schematic for indirect detection of SARS-CoV-2.

QUESTION 2: What can be the factors that affect enzyme’s

activity? Carefully describe the procedure to conduct:
a) An experiment demonstrating the effect of temperature on
bromelain’s activity.
b) An experiment demonstrating the effect of pH level on
bromelain’s activity and explain how these experiments’
result is interpreted.

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2.1. Factors affecting enzymes activity:

The conditions used to assess an enzyme's activity would not be the same
as those chosen to measure the concentration of its substrate. There are five
types of factors that affect the rate at which enzymatic reactions proceed with
enzymes concentration, substrate concentration, inhibitors, pH, and temperature.
In addition, enzymes function best in specified temperature and pH ranges, thus,
when the allowable conditions are exceeded, the enzymes lose their ability to
bind to the substrate.

First, temperature and reaction rate are directly proportional to each

other. Therefore, when raising the temperature often speeds up the
reaction, whereas lowering the temperature slows it down. Extremely high
temperatures, on the other hand, can cause an enzyme to lose its denature
and stop operating. For example, when the influence of temperature on
pepsin enzyme activity was tested, the results revealed that pepsin
operated best at 30 °C. When the temperature was reduced to 22 °C, the
enzyme activity dropped dramatically.

Second, each enzyme has a preferred pH range. Changing the pH

over range will cause enzyme activity to slow down and are able to
denature when exposed to high pH levels. For instance, in the acidic
environment of the stomach, the enzyme pepsin has an optimum of pH 2.5
and a working range of pH 1-4. While the enzyme catalase can be found at
all key sites of H2O2 generation in the cellular environment, it has an optimal
pH of 9 and a working range of 7-11.

Furthermore, rising enzyme concentration will speed up the reaction

as long as there are substrates available for binding until all the substrates
have been bound, the process will not be able to speed up anymore as
there will be nothing for the new enzymes to bind. However, enzymes
become saturated when the substrate concentration is high.

Moreover, substrate concentration grew up accelerates the reaction

to a certain degree. Any increase in the substrate will have no influence on
the pace of reaction once all of the enzymes have bonded because the
available enzymes will be saturated and operating at their maximal rate.

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Finally, chemicals that change the catalytic function of an enzyme,
slowing down or possibly ending the catalytic process are enzyme
inhibitors. Based on the mechanism of action of inhibitors, enzyme
inhibition is classified into three groups such as competitive, non-
competitive, and substrate inhibition.

Figure 20: Flowchart showing the mechanism of action of three enzyme inhibitors.

2.2. The procedure to conduct experiment:

2.2.a) The testing effect of temperature on bromelain’s activity:
The pineapple fruit contains the most enzymes bromelain, which is a
protein. Since enzyme activity is limited by temperature, if the limit is surpassed,
the enzymes will be denatured, reducing their efficacy. As a result, this concept
serves as the foundation for this experimental design, in which rising temperature
causes denaturation of the enzymes contained in bromelain, diminishing its
catalytic efficacy. And it is also known that when heated over 80°C, the enzymes
in bromelain extract begin to denature. (Jutamongkon & Charoerein, 2010, p943-
948). Start the experiment with the preparation of the materials and equipment
as well as test tube racks, hot water baths (35 oC, 45oC, 50oC, 60oC and 90-100oC), a

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stirring rod, fresh pineapple juice, gelatin powder, a beaker, a thermometer, a
ruler, a razor, an electronic balance, and a water bath.
Step 1: The next step is preparation phase, place the gelatin and hot water
into a 250mL beaker and mix with a stirring rod until gelatin has dissolved,
then allow the gelatin to set in the cool room for a day and prepare before
one day begins this experiment.
Step 2: Then label the test tubes from 1-5.
Step 3: After that, cut gelatin becomes ten-part with 1.0 x 1.0 cm cubes by
using a razor blade and recording the weight of each cube into the record
table after weighing.
Step 4: In addition, adding 10 ml of fresh pineapple juice into each test
tube. Moreover, take test tubes 1-5 and heat them in a water bath for 10
minutes, at 35oC, 45oC, 50oC, 60oC, and 90oC, respectively.
Step 5: Next is to put tube "1" on the rack after taking it out of the pot and
letting this test tube cool to room temperature by using a thermometer.
Step 6: And also placing four test tubes from 2-5 in a bucket of crushed ice
may quicken the cooling process.
Step 7: Place allocated cubes of gelatin into respective tubes and allow to
sit for 20 minutes.
Step 8: Allow the gelatin capsules to dry completely after separating the
gelatin capsules from the juice in the test tubes and rinsing them with
distilled water.
Step 9: Finally, record their mass data and clean up the lab according to the
standards.

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RESULT TABLE
TEST
TEMPERATURE (OC) INITIAL WEIGHT (g) FINAL WEIGHT (g) WEIGHT LOSS (g)
TUBE

1 35oC 1.30 0.80 0.50

2 45oC 1.00 0.90 0.10

3 50oC 0.90 0.70 0.20

4 60oC 1.00 0.60 0.40

5 90oC 1.20 1.10 0.10

Control
Room temperature 0.80 0.60 0.20
tube

Figure 21: The result graph - Effect of temperature on enzymatic functioning in bromelain.

Table III 2.2.a: Result table.

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In conclusion, the results show that an increase in temperature applied to
the juice corresponds to a decrease in enzyme activity as described by the mass
loss of gelatin mass. This trend is illustrated in the chart and result table above. In
other words, the results of the above experiment support the theory that
temperature affects enzyme activity.

2.2.b) The test on the effect of pH level on bromelain activity:

The pineapple fruit contains the most enzymes bromelain, which is a
protein. Since enzyme activity is limited by pH concentration, if the limit is
surpassed, the enzymes will be denatured, reducing their efficacy. And also,
optimum conditions of bromelain use were found at pH 7. As a result, this
concept serves as the foundation for this experimental design, in which rising
temperature causes denaturation of the enzymes contained in bromelain,
diminishing its catalytic efficacy. To begin the experiment with the preparation of
the materials include H2SO4 (Acidic solution_ low pH), KOH (Alkaline solution_
high pH), distilled water, fresh pineapple juice, gelatin powder and equipment as
well as test tube racks, a water bath, a stirring rod, a beaker, a thermometer,
pipettes, an electronic balance and a water bath.
Step 1: The next step is preparation phase, place the gelatin and hot water
into a 250mL beaker and mix with a stirring rod until gelatin has dissolved,
then allow the gelatin to set in the cool room for a day and prepare before
one day begins this experiment.
Step 2: Label test tubes number 1-6.
Step 3: Then, the pipettes were then carefully inserted into the test tubes
by the pipettes, respectively.
Test tube “1”: 2 mL of water.
Test tube “2”: 2 mL of fresh pineapple juice.
Test tube “3”: 1 mL H2SO4 and 1 mL fresh pineapple juice.
Test tube “4”: 1 mL H2SO4 and 1 mL water.
Test tube “5”: 1 mL KOH and 1 mL fresh pineapple juice.
Test tube “6”: 1 mL KOH and 1 mL water.

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Step 4: Next, to mix the ingredients together in the test tube, shake gently
(~3 minutes) and except for the gelatin tube.
Step 5: After adding 3 mL of gelatin to each test tube and a test tube
labeled with "gelatin".
Step 6: Additionally, place all six test tubes in the ice bath and also the
gelatin test tubes until the "gelatin" tubes begin to solidify.
Step 7: At least the end of observing the test tubes and recording our
results. Before the end of class, we have cleaned laboratory equipment and
labs in accordance with regulations.

THE EFFECT OF pH LEVEL ON BROMELAIN’S ACTIVITY

TEST TUBE RESULT EXPLAIN
Appearance solidification This case is definitely solid because the water
2 mL of water
will solidify at low temperatures become ice.

This case is definitely solid because, in the test

2 mL of fresh pineapple Appearance solidification tube only fresh pineapple juice, the active
juice medium of bromelain is not affected by any
agent which will solidify at low temperature.

The pH for the enzyme bromelain to work well

is in the range of 5-8 depending on the
substrate, while the pH for the H2SO4 is too low
1 mL H2SO4 and 1 mL fresh Appearance solidification (about pH < 3.5), which strongly contribute to
pineapple juice the repressing toward the gelation of Gelatin
(components containing amino acids), so it can
inactive Bromelain and the liquid can become a
gel.

Since the concentration acidity of H2SO4 has

1 mL H2SO4 and 1 mL Invariable
been diluted by water, it solidifies easily at low
water
temperatures.

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Base solution containing KOH (high pH, about >

1 mL KOH and 1 mL fresh
pineapple juice
13) does not contribute to the forming of the
Invariable
gelation, so it polarizes the Bromelain into
amino acids, and it can active the Bromelain.
Thus, the liquid cannot become a gel.

Since the concentration alkaline of KOH has

Invariable been diluted by water, it solidifies easily at low
1 mL KOH and 1 mL water
temperatures.

QUESTION 3:
a) The experiment illustrates the activity of amylase enzyme:
Material:
Starch solution: 10ml.
Amylase solution.
Iodine reagent.

Equipment:
Pipette; Beaker; Spotting tiles; Cylinder.

Table III 2.2.b: The effect of pH level on bromelain’s activity.

Procedure:
Step 1: Prepare and label spotting tiles 1 minute apart.
Step 2: Put a few drops of starch solution on the dimples labeled as “0
minute”, then use Iodine reagent and see the starch turns blue-black.
Hence, we know that the solution contains starch.
Step 3: Measure by cylinder and put 10ml of starch solution into the test
tube.

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Step 4: Place the test tube containing starch solution in warm water (37
Celsius degree which the Amylase enzyme wok well) for 2 minutes. After
that, add 2ml of Amylase solution into this test tube.
Step 5: Every 1 minute, use a pipette to take a little of the solution in the
test tube into the previously marked spotting tiles which contain the iodine
reagent and watch the reaction happen.
Step 6: If a blue-black color appears, the starch solution will not be digested
by the Amylase enzyme. However, this solution is not a blue- black color,
the experiment has proven that the enzyme amylase breaks down starch
into simple sugars.
Step 7: Record result and keep laboratory and equipment clean.
In this experiment, negative control is step 2, we can use this result to
compare the color of other following experiments. Positive control is the
experiment at the 45th minute.

Figure 22: Result of the experiment.

b) The effect of temperature on Amylase’s activity:

Material:
Starch solution.
Amylase solution.

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Iodine reagent.
Distilled water.

Equipment:
Test tube; Test tube rack; A permanent marked; A Bunsen burner; Tripod;
Gauze mat; Beaker; Thermometer; Spotting tiles; Pipette; Stopwatch; Ice.

Procedure:
Step 1: Set up the water path stations add the water and the ice, turn on
the Bunsen burner, and wait until the water reaches the following
temperatures: 0oC, 20oC, 40oC, 60oC, 80oC.
Step 2: Place test tube in test tube racks, label the test tubes as “Starch”,
“Amylase” (5 pairs of test tubes).
Step 3: Add 5ml of starch solution into the “Starch” test tube and add 5ml
of Amylase into the “Amylase” test tube.
Step 6: We put each pair of test tubes respectively "Starch" and "Amylase"
into each water bath corresponding to each temperature and wait a few
minutes.
Step 7: During this time, we have dropped Iodine reagent to each spot on
the tile. After a few minutes combine the two solutions then use the
pipette to remove a drop of the Amylase and starch solution and place the
drop into Iodine.
Step 8: Observe the color drop initially and add a new drop every 30
seconds until there is no color change. Repeat steps for each temperature.
Step 9: Record result in notebook. Keep the laboratory and equipment
clean.
Experiments show that increasing the temperature increases the rate of
enzyme reaction (but the temperature should not be too high, amylase
enzyme works well at 37oC). Meanwhile, cold temperatures will slow
down the enzyme's reaction rate.

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IV. CONCLUSION

In summary, we have known that enzymes are composed of proteins with

complicated shapes. Enzymes are playing a very important role in our bodies. As
catalysts, enzymes help speeding up the metabolism and the reaction in our bodies.
Enzymes do take part in the digestion system, DNA replication, and liver. Hence, we
cannot live without enzymes. In terms of Bromelain – an enzyme – we know that this is
an enzyme that we can find in pineapples and after doing three different experiments to
determine exactly that whether the activity of bromelain during the gelation of gelatin
will be impacted by the pH level, or the temperature, we clearly confirm that such
factors can affect the bromelain’ activity in different level.
Furthermore, by doing such above experiments and learning many things about
enzymes in the laboratory, we can know more useful information and knowledge of
enzymes in general, and bromelain in particular.

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V. REFERENCE

https://chembam.com/resources-for-students/the-chemistry-of/
gelatin/
https://prezi.com/qlfq4g1fthyq/the-effect-of-bromelain-on-gelatin/
https://www.healthline.com/nutrition/pineapple-juice-benefits#1
https://www.youtube.com/watch?v=5lQbl_t2k3c&t=19s
https://drcuaban.com/bromelain/
https://www.biologyonline.com/dictionary/coenzyme
https://explore.globalhealing.com/what-are-coenzymes/
https://www.scientificamerican.com/article/exploring-enzymes/
https://en.wikipedia.org/wiki/Enzyme#/media/
File:Hexokinase_induced_fit.svg
https://my.clevelandclinic.org/health/symptoms/17679-elevated-liver-
enzymes
https://proteopedia.org/wiki/index.php/Bromelain
https://www.sciencedirect.com/topics/medicine-and-dentistry/pepsin-
a
https://www.medicalnewstoday.com/articles/319704#how-enzymes-
work
https://my.clevelandclinic.org/health/articles/21532-enzymes
https://youtu.be/FV7wDSggO_0 (Exercise 3b)

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https://youtu.be/kquaf6GPhpc (Exercise 3a)
https://youtu.be/OEwOYzWT8AI (Experiment 1)
https://youtu.be/5lQbl_t2k3c (Experiment 2)
https://www.khanacademy.org/science/ap-biology/cellular-
energetics/enzyme-structure-and-catalysis/a/activation-energy
https://www.livescience.com/45145-how-do-enzymes-work.html
Enzymes braking down food into nutrients Vector Image
(vectorstock.com)
https://www.medicalnewstoday.com/articles/319704#how-enzymes-
work
Enzyme Immunoassay - an overview | ScienceDirect Topics
An_Introduction_to_ENZYMES_122019-v6.pdf (worthington-
biochem.com)
https://www.ukessays.com/essays/sciences/enzymatic-functioning-
bromelain-pineapple-4509.php

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POINT FEEDBACK

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BIOLOGY FOR BME – LABORATORY

ENZYMES – UNDERSTAND HOW ENZYMES WORK
GROUP 4
THE END

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