PRACTICAL REPORT ENZYME TECHNOLOGY

MINISTRY OF EDUCATION AND TRAINING
NONG LAM UNIVERSITY – HO CHI MINH CITY
FACULTY: CHEMICAL ENGINEERING AND FOOD TECHNOLOGY
----

PRACTICAL REPORT
ENZYME TECHNOLOGY
Instructor: Nguyen Minh Xuan Hong

GROUP 6 - MEMBERS
Đỗ Hồng Ánh Mai
Đỗ Thị Yến Ly
Nguyễn Thu Hiền
Võ Nguyễn Thục Trinh
Bùi Thiên Lộc

14th March 2022 - Ho Chi Minh city


TABLE OF CONTENT
TABLE OF FIGURES ......................................................................................................... iii
Lab 1: ENZYME KINETICS ........................................................................................ 1
Experiment 1.1 Effect of enzyme concentration on enzyme activity ................................... 1
1. Abstract .......................................................................................................................... 1
2. Background .................................................................................................................... 1
3. Materials and Methods .................................................................................................. 1
4. Results............................................................................................................................ 2
5. Discussion ...................................................................................................................... 3
Experiment 1.2 Effect of substrate concentration on enzyme activity.................................. 4
1. Abstract. ......................................................................................................................... 4
2. Background .................................................................................................................... 4

3. Materials and Methods .................................................................................................. 4
4. Results............................................................................................................................ 5
5. Discussion ...................................................................................................................... 6
Experiment 1.3. Specificity of enzyme ................................................................................. 7
1. Abstract .......................................................................................................................... 7
2. Background .................................................................................................................... 7
3. Materials and Methods .................................................................................................. 7
4. Results............................................................................................................................ 8
5. Discussion ...................................................................................................................... 9
Experiment 1.4. Effect of temperature on enzyme activity ................................................ 11
1. Abstract ........................................................................................................................ 11
2. Background .................................................................................................................. 11
3. Materials and Methods ................................................................................................ 11
4. Results.......................................................................................................................... 13
5. Discussion .................................................................................................................... 15
Experiment 1.5: Effect of pH on enzyme activity............................................................... 16
1. Abstract ........................................................................................................................ 16
2. Background .................................................................................................................. 16

i


3. Materials and Methods ................................................................................................ 16
4. Results.......................................................................................................................... 18
5. Discussion .................................................................................................................... 19
Experiment 1.6: Effect of Inhibitor and Activator on enzyme activity............................... 20
1. Abstract ........................................................................................................................ 20
2. Background .................................................................................................................. 20
3. Materials and Methods ................................................................................................ 20
4. Results.......................................................................................................................... 20

5. Discussion .................................................................................................................... 21
Lab 3: Enzyme protease .............................................................................................. 23
Experiment 3.3: Effect of enzyme bromelain on meat tenderizing. ................................... 23
1. Abstract ........................................................................................................................ 23
2. Background .................................................................................................................. 23
3. Materials and Methods ................................................................................................ 23
4. Results.......................................................................................................................... 24
5. Discussion .................................................................................................................... 26
Lab 4: Enzyme pectinase............................................................................................. 27
Experiment 4.1: Determination of pectinase activity using color-metric method. ............. 27
1. Abstract ........................................................................................................................ 27
2. Background .................................................................................................................. 27
3. Materials and Methods ................................................................................................ 27
4. Results.......................................................................................................................... 28
5. Discussion .................................................................................................................... 30
Experiment 4.2. Application of pectinase in clarification of apple juice. ........................... 31
1. Abstract ........................................................................................................................ 31
2. Background .................................................................................................................. 31
3. Materials and methods ................................................................................................. 31
4. Results.......................................................................................................................... 32
5. Discussion .................................................................................................................... 32
References ................................................................................................................... 33

ii


TABLE OF FIGURES
Figure 1. The effect of enzyme amylase concentration on enzyme activity. .......... 2
Figure 2. The relationship between enzyme concentration and reaction time. ....... 3
Figure 3. The effect of substrate concentration on enzyme activity. ...................... 5

Figure 4. The relationship between starch concentration and reaction time. .......... 6
Figure 5. Fehling solution (mixed) and three prepared test tubes. .......................... 8
Figure 6. Results of three test tubes after keeping in hot water for 1-3 min and
cool down. ........................................................................................................................... 9
Figure 7. Test tubes were prepared at three different assay temperatures. ........... 13
Figure 8. Drops of each solution were disappeared in black blue color. .............. 14
Figure 9. Relationship between temperature and reaction time ............................ 14
Figure 10. Test tubes prepared at three different pH with the enzyme solution ... 17
Figure 11. Test tubes prepared at three different pH with buffer instead of enzyme
solution .............................................................................................................................. 17
Figure 12. Drops of each solution with Lugol solution until all reactions have
been completed .................................................................................................................. 18
Figure 13. The chart shows the relationship between pH and reaction time ........ 19
Figure 14. The results of three tubes with Lugol solution. .................................... 21
Figure 15. Meat samples before the experiment. .................................................. 24
Figure 16. Meat samples before the experiment. .................................................. 24
Figure 17. Meat samples after the experiment. ..................................................... 25
Figure 18. Meat samples after the experiment. ..................................................... 25
Figure 19. Meat samples after the experiment. ..................................................... 26
Figure 20. Samples of two test tubes which 5ml Anthrone in 2.5 ml diluted
filtrate................................................................................................................................. 28
Figure 21. The initial value of the spectrophotometer. ......................................... 29
Figure 22. The value of test tube 1 is measured. ................................................... 29
Figure 23. The value of test tube 2 is measured. ................................................... 30
Figure 24. Diagrammatic representation of different steps involves in apple fruit
juice. .................................................................................................................................. 31
Figure 25. Results of four test tubes for observation and discussion. ................... 32

iii

Lab 1: ENZYME KINETICS.

RATE OF REACTION

Experiment 1.1 Effect of enzyme concentration on enzyme activity.
1. Abstract.
Enzymes are catalysts which lower the activation of chemical reactions, thus
making them happen more rapidly. In this lab, enzyme kinetics are examined utilizing
various experimental techniques, including measurements of temperature. The purpose of
this lab is to investigate the impact of enzyme concentration on enzyme activity. If the
concentration of the enzyme is increased, the rate of the reaction will be increased as well.
The dependent variable is the rate of the reaction.
2. Background.
Enzymes are catalysts that lower the activation of chemical reactions, thus making
them happen more rapidly. Enzyme kinetics is the study of catalytic reactions, or reaction
rate, which occurs in the presence of enzymes under varying conditions, specificities, and
mechanisms such as the proximity
effect, orientation effect, catalytic
effect, and energy effect; the studies
are
conducted
under
assorted
circumstances, such as temperature,
pH, and component concentrations in
correlation to reaction rates. For
example,
enzyme
concentration

directly relates to reaction rates
whereby an increase in enzyme
concentration will also increase the
rate of the reaction in a linear
ENZYME CONCENTRATION
relationship. (as seen in the above
graph on the left).
In experiment 1, different
amounts of enzyme and the same amounts of substrate were put in a test tube. Then those
were observed using Lugol solution to see how fast they reacted to produce the product.
3. Materials and Methods.
 Materials
- 1% starch solution
- Buffer solution (distilled water)
- Lugol solution
- Amylase enzyme solution
 Laboratory tools
1


- Test tubes
- Pipette
- Test tube stands
- Sticker notes
*Methods:
- Assemble and label clean 5 test tubes to prevent contamination or premature
reaction from occurring.
- Measure enzyme amylase and starch then distribute chemicals as follows:
Tube 1
Tube 2

Tube 3
Tube 4
Tube 5
Distilled water
1 ml
0.9 ml
0.8 ml
0.5 ml
0 ml
1% Enzyme
0 ml
0.1 ml
0.2 ml
0.5 ml
1 ml
1% Starch
1 ml
1 ml
1 ml
1 ml
1 ml
[ Enzyme]
0%
0.05%
0.1%
0.25%
0.5%
- Check the reaction with Lugol solution after each 30 seconds until all reactions
have completed. Record the time.
4. Results.

[ Enzyme]
0%
0.05%
0.1%
0.25%
0.5%
Reaction
No react
5.36 mins
4.11 mins
3.18 mins
2.32 mins
time (min)

Figure 1. The effect of enzyme amylase concentration on enzyme activity.

2


Effect of enzyme concentration
0.60%

[ Enzyme]

0.50%
0.40%
0.30%
0.20%
0.10%
0.00%

0

1

2

3

4

5

6

[ Reaction time]

Figure 2. The relationship between enzyme concentration and reaction time.
5. Discussion.
It was found that as the concentration of enzyme increased, the speed of reaction
increased and the reaction time decreased. As there was a higher amount of enzyme, the
substrate had more chances to bind to the amylase active sites and reacted more at once,
therefore increasing the rate of reaction.
Thus, the higher amount of enzyme is, the more effective its activity is. Higher
levels of enzyme mean there will be a higher turnover rate of the product.

3


Experiment 1.2 Effect of substrate concentration on enzyme activity.
1. Abstract.

As the concentration of the substrate was increased, the rate of the reaction also
increased for reasons similar to the concentration of the enzyme. The amount of substrate
increased there were more substrate molecules to bond with the active site of the amylase
enzyme. Different amounts of starch and the same amount of enzyme amylase were put in
a test tube, then were observed using Lugol solution to show the goal of the experiment is
how the effectiveness of amylase activity under effect of substrate concentration according
to the speed of the reaction.
2. Background
This lab provides changing of starch concentrations affect the rate of reaction of
enzyme amylase reaction. Controlling this factor in a cell is one way that an organism
regulates its enzyme activity and so its metabolism. As we know, enzymes will work best
if there is plenty of substrates. As the concentration of the substrate increases, so does the
rate of enzyme activity. However, when the enzymes become saturated and no more
substrates can fit at any one time, the rate of enzyme activity does not continue to increase
indefinitely.
In experiment 2, different amounts of enzyme and substrate were put in a test tube.
Then those were observed using Lugol solution to see how fast they reacted to produce the
product.
3. Materials and Methods
 Materials
- 1% starch solution
- Buffer solution (distilled water)
- Lugol solution
- Amylase enzyme solution
 Laboratory tools
- Test tubes
- Test tube stands
- Pipette
- Sticker notes
 Method

- Assemble and label clean 5 test tubes to prevent contamination or premature
reaction from occurring.
- Measure enzyme amylase and starch then distribute chemicals as following:
4


Distilled
water
1% Starch
1% Enzyme
[ Enzyme]

Tube 1
1.8 ml

Tube 2
1.6 ml

Tube 3
1.3 ml

Tube 4
0.8 ml

Tube 5
0.3 ml

0 ml
0.2 ml
0%

0.2 ml
0.2 ml
0.1%

0.5 ml
0.2 ml
0.25%

1.0 ml
0.2 ml
0.5%

1.5 ml
0.2 ml
0.75%

- Check the reaction with Lugol solution after each 30 seconds until all reactions
have completed. Record the time.
4. Results
[ Starch]
0%
Reaction
No react
time (min)

0.1%
1.04 mins

0.25%

2.38 mins

0.5%
4.08 mins

Figure 3. The effect of substrate concentration on enzyme activity.

5

0.75%
7.34 mins


Effect of substrate concentration
0.80%
0.70%

[ Starch]

0.60%
0.50%

0.40%
0.30%
0.20%
0.10%
0.00%
0

1

2

3

4

5

6

7

8

[ Reaction time]

Figure 4. The relationship between starch concentration and reaction time.
5. Discussion
Likewise, it was found that as the concentration of starch was increased, the speed
of reaction increased. When there is more substrate, there will be just as much enzyme,
also making the rate of the reaction increase. But in the case of experiment 1.2, the amount
of enzyme is kept the same for each tube, a higher amount of starch does not have enough
enzyme to react by the maximum velocity, so the rate of reaction was decreasing and the
reaction time was longer.

6


Experiment 1.3. Specificity of enzyme

1. Abstract
The goal of this experiment is to examine the specificity of the enzyme through the
Fehling test reaction of enzyme amylase with starch, saccharose, and distilled water.
Fehling's solution can be used to distinguish aldehyde vs ketone functional groups.
Aldehydes give a positive result, but ketones do not react unless they are α-hydroxy
ketones. So, Fehling's test is given by reducing sugars. Experiment 1.3 was conducted to
observe the three test tubes to compare the colors, the time it takes for reaction happened.
2. Background
Specificity is a property of the enzyme and describes how restrictive the enzyme is
in its choice of the substrate; a completely specific enzyme would have only one substrate.
For a substrate to bind to the active site of an enzyme it must fit in the active site and be
chemically attracted to it.
The specificity of each enzyme is reflected in the point: Each enzyme only acts on
a certain substrate. For example, amylase only hydrolyzes polysaccharides but does not
affect disaccharides.
3. Materials and Methods
* Materials
- 1% saccharose solution
- 1% starch solution
- Fehling's solution
- Amylase solution
* Laboratory tools
- Test tubes
- Test tube stands
- Pipette
- Sticker notes
* Method
- Prepare 3 test tubes. Add 0.5 mL of 1% enzyme amylase into each tube. Then add
1mL of 1% starch into tube 1, 1mL of 1% saccharose into tube 2, and 1mL of distilled
water into tube 3. Keep 3 test tubes into a water bath at 37oC in 5 minutes.

7


Figure 5. Fehling solution (mixed) and three prepared test tubes.
- Fill each test tube with 1 mL of the mixed Fehling solution and shake vigorously.
Then put these test tubes in hot water for 1-3 minutes until they change colors then take
out and cool down. Observe and compare the results.
4. Results
Tube 1 which contains starch solution forms a brick-red color first. While the test
tube containing saccharose solution only appeared a very light brick-red color. Tube 3
which contains distilled water does not react.

8


Figure 6. Results of three test tubes after keeping in hot water for 1-3 min and cool down.
After leaving three test tubes at room temperature for a while, the brick-red color
gradually darkened in tubes 1 and 2.
5. Discussion
When the enzyme amylase is added to the starch solution, the starch is hydrolyzed
to produce a small amount of reducing sugar, which is then broken down by the enzyme
into a quantity of dextrin. When Fehling is added and heated, the cleavage process becomes
increasingly stronger, producing more dextrin, resulting in a darker brick-red color. Dextrin
reacts with the Fehling test to produce a brick-red precipitate because it is a combination
of polymers of D-glucose units linked by α- (1 4) or α- (1 6) glycosidic linkages.
Enzyme amylase is responsible for hydrolyzing starch. In the presence of amylase,
a sample of starch will be hydrolyzed to shorter polysaccharides, dextrins, maltose, and
glucose, The extent of the hydrolysis depends on how long it is allowed to react-if the
starch is hydrolyzed completely, the resulting product is glucose. We will test for the

presence or absence of starch in the solutions using Fehling solution. In the
monosaccharide molecule, the group -CHO, -C=O have reducing properties so they
9


eliminate Cu, Fe, … When boiling with Fehling solution will give the red precipitate of
𝐶𝑢2 O because the monosaccharide reduces Cu(OH)2 to Cu2O.
When enzyme amylase is added to a test tube containing saccharose solution at room
temperature, it will not be able to cleave because it lacks of reducing properties (it does not
contain 1-4 glucoside bonds). As a result, the enzyme will not be able to cleave at room
temperature, and there will be no Fehling's reagent phenomenon. When the test tube was
heated to boiling, the enzyme broke down a little amount of saccharose and converted it to
reducing sugar. As a result, the tube takes longer to turn brick-red in color and the color is
lighter than the tube containing starch. The explanation for this is that saccharide does not
react with Fehling because it lacks of a free ketose or aldehyde group. This reaction
happens only when sucrose has been hydrolyzed to fructose and glucose.
The tube containing distilled water does not react because enzyme amylase does not
react with distilled water.

10


Experiment 1.4. Effect of temperature on enzyme activity
1. Abstract
This experiment is aimed to examine the effect of temperature on enzyme activity
at three different assay temperatures (0oC, 30oC, 90oC). Then plot the relationship between
temperature and reaction time. The results obtained show the optimal temperature and how
each temperature affected enzyme activity.
2. Background
Enzymes are proteinaceous in nature and catalyze the chemical reaction in

biochemistry. Enzymes are responsible for speeding up reactions and are mostly
synthesized in living cells.
At higher temperatures, the enzymes are denatured, while at a lower temperature,
the enzymes are deactivated.
As the temperature increases so does the rate of enzyme activity. Optimum activity
is reached at the enzyme's optimum temperature. A continued increase in temperature
results in a decrease in activity as the enzyme's active site changes shape. It is now
denatured. Each enzyme has a temperature that it works optimally in.
3. Materials and Methods
* Materials
- 1% starch solution
- Amylase enzyme solution
- Buffer solution (distilled water)
- Lugol solution
* Laboratory tools
- Test tubes
- A pot
- Test tube stands
- Pipette 1mL, and pipette 5mL
- Cooking stove
* Methods
At 0oC
- Prepare 3 test tubes
+ Tube 1: use a 5mL pipette to take 1mL of 1% starch solution into the test tube.
+ Tube 2: use 1mL Pipette to take 0.1mL of diluted amylase solution into the test
tube.
+ Tube 3: use a 5mL Pipette to take 1mL of 1% starch solution into the test tube.
- Put these test tubes in an ice water pot at 0oC for about 5 minutes.
- After that, take them out and mix test tubes 1 and 2 together, shake well. Besides
that, put 0.1mL of distilled water (buffer) instead of the enzyme into test tube 3, shake well.

11


- Put 1 drop of the mixture above the case on the glass, then for every 30 seconds,
put 1 drop of Lugol to that drop of the mixture. Repeat until the blue color does not appear
anymore then stop. Do the same for tube 3.
At 30oC
- Prepare 3 test tubes
+ Tube 1: use a 5mL pipette to take 1mL of 1% starch solution into the test tube.
+ Tube 2: use 1mL Pipette to take 0.1mL of diluted amylase solution into the test
tube.
+ Tube 3: use a 5mL Pipette to take 1mL of 1% starch solution into the test tube.
- Leave these test tubes at room temperature for about 5 minutes.
- After that, take them out and mix test tubes 1 and 2 together, shake well. Besides
that, put 0.1mL of distilled water (buffer) instead of the enzyme into test tube 3, shake well.
- Put 1 drop of the mixture above the case on the glass, then for every 30 seconds,
put 1 drop of Lugol to that drop of the mixture. Repeat until the blue color does not appear
anymore then stop. Do the same for tube 3.
At 90oC
- Prepare 3 test tubes
+ Tube 1: use a 5mL pipette to take 1mL of 1% starch solution into the test tube.
+ Tube 2: use 1mL Pipette to take 0.1mL of diluted amylase solution into the test
tube.
+ Tube 3: use a 5mL Pipette to take 1mL of 1% starch solution into the test tube.
- Heat these test tubes in a hot pot at 90oC for about 5 minutes.
- After that, take them out and mix test tubes 1 and 2 together, shake well. Besides
that, put 0.1mL of distilled water (buffer) instead of the enzyme into test tube 3, shake well.
- Put 1 drop of the mixture above the case on the glass, then for every 30 seconds,
put 1 drop of Lugol to that drop of the mixture. Repeat until the blue color does not appear

anymore then stop. Do the same for tube 3.

12


Figure 7. Test tubes were prepared at three different assay temperatures.
4. Results
Temperature
Tube
Starch + enzyme

0oC

30oC

90oC

500s

105s

30s

13


Figure 8. Drops of each solution were disappeared in black blue color.

Figure 9. Relationship between temperature and reaction time

14


5. Discussion
From the figure 9, for the mixed tube containing starch and enzyme, we can see that
o
at 0 C, the hydrolysis time of amylase is the longest because, at low temperatures, the
number of successful collisions between the enzyme and substrate is reduced because their
molecular movement decreases lead to slow down the reaction. At 30oC, the hydrolysis
time of amylase is relatively fast because this is the suitable temperature range for the
enzyme to be slightly active. At 90oC, the hydrolysis time of amylase is the fastest because
this enzyme has an optimum temperature from 85-97oC, and is still highly active at 100oC.
Besides that, this enzyme is derived from the bacterium Bacillus licheniformis, active at
90oC, has the ability to liquefy starch and reduce the viscosity of gelatinization by randomly
hydrolyzing starch, glycogen, and other products in α-1,4 glycosidic. So that is why even
at high temperatures such as 90oC, enzyme amylase is still active and has the fastest
hydrolysis time.

15


Experiment 1.5: Effect of pH on enzyme activity
1. Abstract
The purpose of this experiment was to determine the effect of pH on the activity of
the enzyme amylase. In this experiment, we have used buffer solutions with the pHs of 4,
6, and 8. According to the reaction time of enzyme amylase at each different pH, we can
know the optimum pH for enzyme amylase activity, and then we will compare it with the
pH of the enzyme in production. The influence of pH may involve several different types
of effects because enzymes are proteins with many ionic groups also present in the active
site. Proteins are stable only within a relatively limited pH range most often near neutrality.

2. Background
Enzymes are affected by changes in pH. The most favorable pH value-the point
where the enzyme is most active – is known as the optimum pH. Extremely high or low
pH is also a factor in the stability of enzymes. As with activity, for each enzyme, there is
also a region of pH optimal stability. For many enzymes, this corresponds to pH values of
around 7. For pepsin, which is active in the stomach, the optimum pH is 2 (the pH of the
stomach). Trypsin, which is active in the small intestine, has an optimum pH of 8 that
matches the pH of the small intestine. The optimum pH for the enzymatic activity of
amylase ranges from 6 to 7. Above and below the range, the reaction rate reduces as
enzymes get denatured.
3. Materials and Methods
* Materials:
- 1% Starch solution
- Buffer at different pH (4, 6, 8)
- 1% Enzyme amylase solution
- Distilled water
- Lugol solution
- Test tube
* Methods:
- Prepare 3 tubes containing 0.5 ml of 1% starch solution
- Add 2 ml of buffer at different pH (4, 6, 8) to each tube and shake well

16


- Then add 0.1 ml of 1% enzyme solution to each tube.

Figure 10. Test tubes prepared at three different pH with the enzyme solution
- Be sure to run a control set of three starch tubes with 0.1 ml buffer (distilled water) instead
of enzyme solution for each pH (4, 6, 8)

Figure 11. Test tubes prepared at three different pH with buffer instead of enzyme solution

17


- Check the reaction with Lugol solution after every 30 seconds until all reactions
have been completed. Record the time. Plot the relationship between pH and reaction time.
4. Results
pH
Reaction
time t (s) of tube:
Starch + enzyme

4

6

8

522s

98s

453s

Figure 12. Drops of each solution with Lugol solution until all reactions have been
completed

18

Figure 13. The chart shows the relationship between pH and reaction time
5. Discussion
- As can be seen from the chart, at pH = 6, the reaction time hydrolyzed of enzyme
amylase is shortest, which means that enzyme amylase is most active. At pH = 4 and pH =
8, the reaction time is too long so amylase is less active.
- Our stomach has a high level of acidity which causes amylase to denature and
change its shape. So, the amylase does not function once it enters the stomach. Similarly,
a pH level that is too high/alkaline or too low/acidic can also denature enzymes and stop
amylase enzyme activity. pH = 4 is acidic and pH = 8 is alkaline, therefore amylase is less
active in these tubes.
- Each enzyme will have an optimal pH. In this optimal pH value, the enzyme will
react the most. Thus, at pH = 6, it performs best and has maximum activity, which indicated
that the optimum pH of the enzyme was 6.0. Enzymatic reactions have an optimal pH, and
the enzyme activity decreases when the pH is above or below the optimal pH. Because the
pH of the solution affects the binding ability of the active center of the enzyme and the
substrate, the binding ability and activity are the highest at the optimal pH.

19


Experiment 1.6: Effect of Inhibitor and Activator on enzyme activity
1. Abstract
The goal of this experiment is to observe how inhibitors and activators affect
enzyme amylase activity. To observe the activity of enzyme amylase during the starch
hydrolysis, it is affected by the activator substances or inhibitors, we use NaCl, CuSO4,
and distilled water in this experiment. From that, we can determine which substance is an
inhibitor, which is an activator substance.
2. Background

Just as enzymes act as a catalyst to biochemical reactions, enzyme activity can be
affected by other molecules: inhibitors are molecules that decrease enzyme activity and
activators are molecules that increase activity. Enzyme inhibitors are compounds that
modify the catalytic properties of the enzyme and therefore, slow down the reaction rate,
or in some cases, even stop the catalysis. Such inhibitors work by blocking or distorting
the active site. Enzyme activators are chemical compounds that increase the velocity of
enzymatic reactions. The binding of an activator to a regulatory site stabilizes the shape
that has the functional active site. Amylase will increase in the environment with NaCl,
vice versa CuSO4 will inhibit the effect of this enzyme.
3. Materials and Methods
* Materials:
- 1% enzyme amylase solution
- 1% NaCl
- 1% CuSO4
- Distilled water
- 1% starch solution
- Lugol solution
* Methods:
Prepare 3 tubes as follows:
Tube 1: 0.5 ml of 1% amylase solution and 0.5 ml of 1% NaCl
Tube 2: 0.5 ml of 1% amylase solution and 0.5 ml of 1% CuSO4
Tube 3: 0.5 ml of 1% amylase solution and 0.5 ml of distilled water
Add 1 ml of 1% starch solution to each tube. Keep it at room temperature for 1-3
minutes. Then check the reaction with Lugol solution. Observe and compare the results.
4. Results
Tube 1: Only appear the color of Lugol solution,
Tube 2: Purple-blue color

20

Tube 3: Color of Lugol solution,

Figure 14. The results of three tubes with Lugol solution.
5. Discussion
Enzyme inhibitors, activators can also affect amylase enzyme activity.
Tube 1: Do not appear purple color characteristic indicates that the starch has been
completely hydrolyzed by amylase enzyme so when to add Lugol solution, the tube only
appears the color of Lugol solution. Thus, the appearance of NaCl as a catalyst increases
starch degradation of the enzyme amylase because the chloride ions activate the reaction.
NaCl is an activator.
If the concentration of sodium chloride increases, the rate of enzyme activity will
gradually slow down because high amounts of sodium chloride disrupt the bonds and
structure of the active site. This then leads to denaturation and the starch is no longer able
to bind to some of the active sites. Enzyme activity will gradually slow down as more and
more enzymes become denatured and finally stop. Thus, such a low concentration is
selected as the most suitable, to investigate the activating property of Cl- ions.
Tube 2: At room temperature, starch has torsion-type amylose molecules with
perforated springs, the starch absorbs Iodine in Lugol solution for special purple-blue. The
amylase was used to hydrolyze the alternate glycosidic bonds in starch. By doing this it
meant that the starch, a polysaccharide was broken down into maltose, a disaccharide.
However, copper sulfate would interfere with starch for the active sites and so prevent the
21