University of Santo Tomas – Legazpi

College of Health Sciences

Bachelor of Science in Medical Technology

ENZYMES

Experiment No. 3

Dealca, Patricia Andreana L.

Usero, Jessa Marie T.

Lui, Paula V.

Pequina. Quenndhee D.

Manalo, Renan Kyle P.

ABSTRACT

The purpose of this laboratory experiment is to investigate effect of temperature,

effect of the pH, substrate concentration, enzyme concentration, and the presence of

the inhibitor on the effectiveness and the rate of the enzyme using baker’s yeast as a

main component for enzyme extraction. Using the Dinitrosalicylic (DNS) Colorimetric

Method, this method will reduce the sucrose assay of an enzyme. If the concentration of

an enzyme increases, the rate of the reaction increases as well. If the temperature of an

invertase increases on a given temperature, the rate of the reaction will also increase. If
a pH of an invertase increases, the rate of the reaction will also increase. The

dependent variable is the rate of the reaction and the independent variable is the

enzyme concentration, the temperature, the pH, and the substrate.

Objectives:

At the end of this laboratory report, the students would be able to:

 To extract invertase from baker’s yeast,

 To determine the effects of the changes in Ph and temperature on the reaction

rates of an enzyme catalyzed reactions, and

 To describe the specific action of the enzyme catalase, also including the

substrate and the products of the reaction.

INTRODUCTION various factors such as temperature,

pH, cofactor, activators and inhibitors.

Living organisms are composed

of complex but coordinated system of Enzymes are proteins, so they

chemical reactions. Enzymes are are very sensitive to the changes of pH,

proteinaceous catalysts that speed up each enzyme has its optimum range for

the rate of a biochemical reaction. They pH where it will be most active. This is

reduce the activation energy that is the result of the effect of pH on a

essential for starting any type of combination of factors:

chemical reaction. With a low energy

1.Binding of enzymes
requirement for activation, the reaction
2.Catalytic activation of the enzymes
takes place faster. The overall

performance of an enzyme depends on 3.Ionization of the substrate

4.Variation of protein structure Enzymes accelerate reactions

but are themselves not subject to

The initial rates for many
change. Thousands of different
enzymatic reactions exhibit bell-shaped
enzymes can be found within a cell and
curves as a function of pH as shown in
each has a unique three-dimensional
the example:
structure that determines its function.

Like most chemical reaction,

the rate of an enzyme-catalyzed

reaction increases as the temperature is

raised. A ten degrees centigrade rise in

temperature will increase the activity of

most enzymes by 50% to 100 %. The

Figure 1. Enzymatic Activity vs. pH

The bell-shaped shows the stability of

enzymes during the alteration of pH.

Meaning, Low pH results to slow

reaction rate but too high pH shows the

same result because of the enzymatic

loss. The peak of the “bell”

Figure 2. Enzymatic Activity vs.
demonstrates the “best” pH suitable for
Temperature
the enzyme that it reaches the

maximum reaction rate of the enzymatic activity peak at a specific temperature

activity. unique to the enzyme. This is known as

the optimum temperature (the

temperature at which an enzyme is First, prepare a seven-test tube.

maximally active. Over a period of time Each test tube has assigned Inverted

the enzymes will be deactivated at even Sugar Standard Solution, mL and a

moderate temperature. Distilled H2O, mL.

Test Blan 1 2 3 4 5 6
Tube k
No.
PROCEDURE Sugar 0 0.1 0.3 0.5 0.8 1.0 1.2
Standar 0 0 0 0 0 0
d
This chapter present the Solution,
mL
procedures applied in order to meet the Distilled 2.50 2.4 2.2 2.0 1.7 1.5 1.3
H2O, mL 0 0 0 0 0 0
experiment’s purpose. It explains the
Table 1. Measurement of Sugar
procedures and various methods,
Standard Solution and Distilled Water
reagents and materials used in the
After putting all the solution in the
experiment.
test tube, a 0.50 mL of 0.05 M acetate
Reducing Sugars Assay Using the
buffer solution is added to each test
Dinitrosalicylic (DNS) Colorimetric
tube, and mix well. 2 mL of the DNS
Method
reagent is added in the test tube, after

we cover all the tubes to prevent

evaporating. In the hot plate for about

10 minutes we water bath all the test

tube to develop a characteristic red-

brown color. In an ice water bath, all the

Figure 3. Final result of the Reducing
test tubes are set aside to cool. A 5 ml of
Sugars Assay Using the Dinitrosalicylic
distilled water is added to each tube and
(DNS) Colorimetric Method
mix it. The Absorbance is measured at of distilled water to each tube and mix

540 nm. well.

Effect of Temperature on Invertase Prepare another test tube for

Activity reagent blank containing 0.1 mL of the

denatured enzyme, 1 mL of 0.03 M

Set a water bath with a
sucrose, 1.4 mL of distilled water and
temperature of 20 ̊ C, 30 ̊ C, 50 ̊ C,60
0.50 mL of 0.05 M acetate buffer
̊C,70 ̊C and 90 ̊C. Prepare six test tubes
solution with pH 4.7. Incubate the test
that contain 1mL of 0.03 M sucrose, 1.4
tube for 5 minutes at room temperature.
mL of distilled water and 0.50 mL of 0.05
Repeat the step of adding 2 mL of DNS
M acetate buffer solution with pH 4.7.
reagent. Immerse the test tubes in 95 ̊ C
Incubate the test tubes separately for 5
water bath for 10 minutes to develop a
minutes in each of the water baths. After
characteristic of red brown color. The
incubating, add 0.1 mL of enzyme
test tubes should be covered to prevent
solution to tubes and incubate the test
the solvent from evaporating. Cool the
tubes for another 5 minutes. Test tube
test tubes in an ice water bath, add 5 mL
should not be removed in its respective
of distilled water to each tube and mix
water baths. Then, add 2 mL of DNS
well.
reagent. Immerse the test tubes in 95 ̊ C

water bath for 10 minutes to develop a Lastly, measure the absorbance at 540

characteristic of red brown color. The nm. Determine the amount of invert

test tubes should be covered to prevent sugar produced in each tube reaction

the solvent from evaporating. Cool the using the invert sugar standard curve

test tubes in an ice water bath, add 5 mL constructed in first experiment. Plot the
temperature against the amount of Add 0.10 mL of the enzyme solution and

invert sugar. add 1.4 mL of distilled water both to

each test tube and mix thoroughly.

Incubate the said test tubes in a 60 C

Effect of pH on Invertase Activity
water bath for 5 minutes. Next, add 1

mL of the 0.03 M sucrose and incubate

the reaction solution in a 60 C for 5

minutes then add 2 mL of DNS reagent.

To develop a characteristic red-brown

color, immerse the test tubes in a boiling

water bath for 10 minutes. Remember to

Figure 4. Final Result of the Invert
cover all the tubes with marbles to
Sugar
prevent the solvent from evaporating. In
Prepare six numbered test tubes. Add
an ice water bath, cool the test tubes.
0.50 mL of the appropriate 0.1 M buffer
Add 5 mL of distilled water to each test
solution as described below and label
tubes and mix it well. Afterwards,
the tubes accordingly.
prepare another set of six test tubes for

Test 1 2 3 4 the reagent blank. Each tube should

Tube
No. contain 0.10 mL of the denatured
pH of 3 5 7 10.6
the enzyme, 1.4 distilled water and 0.5 mL
Buffer
Solutio of the corresponding buffer solution. Mix
n
Table 2. pH of the Buffer Solution per it thoroughly and for 5 minutes, incubate

Test Tube the test tubes in a 60 C water bath.

Repeat the same procedure: from (mmol/L)

adding 1 mL of the 0.03 sucrose up to Blank

Reagent
cooling the test tubes and adding 5 mL 1 23.8329
2 7.6929
of distilled water to each.
3 205.2945
In a spectrophotometer, measure the 4 29.2631
5 13.2740
absorbance at 540 nm.determine the
6 112.5273
amount of invert sugar produced Table 3. Sucrose Assay using the

(expressed in mmol/L) in each test tube Dinitrosalicylic Colorimetric Method

reaction applying the invert sugar For the reduction of sugars assay

standard curve constructed in the prior using the dinitrosalicylic (DNS)

experiments. Finally, plot the pH against colorimetric method we come up with

the amount of the invert sugar obtained.

PRESENTATION OF RESULTS

This portion elaborate the results

of the experiment obtained by the

students.

Reducing Sugars Assay Using the

Dinitrosalicylic (DNS) Colorimetric

Method

Test Amount
Tube No. Invert Sugar
the followingresults: 6th test tube has the amount of invert

sugar of 112.5273 mmol/L.

Effect of Temperature on Invertase

Activity

In order to determine the effect of

temperature on invertase activity, the

Plot A540 students made use of different

250 temperature with 20 ̊ C, 30 ̊ C, 50 ̊ C,60
200
150 ̊C,70 ̊ C and 90 ̊ C water bath to obtain
100
the result of red brown characteristic.
50
0 The test tubes containing 0.03 M
Bl a nk 1 2 3 4 5 6
Amount Inverted Sugar (mol /L) sucrose, 1.4 mL of distilled water, 0.50

mL of 0.05 M acetate buffer solution with

Figure 3. Amount of Invert Sugar
pH 4.7 are used to obtain the result and
(mmol/L)
proceed in getting the concentration of
The amount of invert sugar which
each sample through Cyan The results
is expressed is (mmol/L) for the blank
are indicated below:
test tube is at 0 mmol/L, while for the 1st
Temperature Amount Invert
test tube it is at 23.8329 mmol/L, 2nd Sugar Produced
(mol/L)
test tube has 7.6929 mmol/L, 3rd test
Reagent 384.3427 mmol/L
tube has 205.2945 mmol/L, 4th test tube Blank
20 67.4259 mmol/L
has 29.2631 mmol/L, the 5th test tube 30 152.9527 mmol/L
50 155.6678 mmol/L
has 13.2740 mmol/L and the last and
60 169.9977 mmol/L
70 353.5711 mmol/L
90 163.8132 mmol/L
Table 4. Effect of temperature on

Invertase Activity

Figure 5. Test tube 1 in 20 ̊C

Figure 10. Test Tube 6 in 90 ̊C

Figure 6. Test Tube 3 in 50 ̊C

The invertase activity of the

samples and reagent blank are shown in

the Table 1. Effect of temperature on

Invertase Activity and Figures 1 to 7.

After cooling the test tubes in an ice

bath and adding distilled water, it results

to a red brown characteristic. At 20 ̊ C

the test tube no. 1 obtained a 67.4259

Figure 7. Test Tube 4 in 60 ̊ mmol/L, test tube no.2 at 30 ̊ C obtained

a 152.9527 mmol/L, test tube no. 3 at 50

̊C obtained 155.6678 mmol/L, test tube

no. 4 at 60 ̊ C obtained 169.9977

Figure 8. Reagent Blank mmol/L, test tube no. 5 at 70 ̊C obtained

Figure 9. Test Tube 5 in 70 ̊C 353.5711 mmol/L, test tube no.6 at 90 ̊C

obtained 163.8132 mmol/L and the

reagent blank obtained 384.3427 optimum temperature and test tube no.

mmol/L. The amount of inverted sugar 5 obtained the highest optimum

obtained, consisting 1mL of 0.03 M temperature.

sucrose, 1.4 mL of distilled water, 0.50

Effect of pH on Invertase Activity
mL of 0.05 M acetate buffer solution with
The data obtained in this experiment are
pH 4.7, 2 mL of DNS reagent and 5 mL
shown below:
of distilled water are processed in the

CYANsmart Biochemistry Analyzer.

Figure 9. Optimum pH of Active Enzyme

Figure 8. Graph of Temperature on

Invertase Activity
Figure 10. Optimum pH of the
The table and the graph shown
Denatured Enzyme
above represent the invertase activity
Amount of Invert
occur in the samples with the given
Sugar Produced
temperature in each test tubes. The test
(mmol/L)
tube no.1 at 20 ̊ C obtained the lowest pH Active Denatured
Enzyme Enzyme
3 132. 4383 139.2262 INTERPRETATION OF RESULTS
5 63.8057 40.8779
7 21.7211 164.0546
Reducing Sugars Assay Using the
10.6 39.3695 177.8415
Table 5. Amount of Invert Sugar Dinitrosalicylic (DNS) Colorimetric
Produced (mmol/L) by the Ae and De at Method
Different pH
In Figure 3. Amount of Invert

Amount of Invert Sugar Produced (mmol/L) by the Ae and De at Different pH Sugar (mmol/L) and in Table 3. Sucrose
200
180 177.84 Assay using the Dinitrosalicylic
160 164.05
139.23
140
132.43 Colorimetric Method the results are
120
100
80 obtained based on the invert sugar
60 63.81
40 40.88 39.37 standard solution (mL), it shows that on
20 21.72
0
pH 3 pH 5 pH 7 pH 10.6 the test tube no. 3 its enzyme
Active Enzyme Denatured Enzyme
concentration increases, but on test

Figure 11. Graph of pH on Invertase tube no.4, the enzyme concentration

Activity decreases. Lastly, on the test tube no.6

the enzyme concentration slightly

As shown in the presentation,
increases.
enzymes are affected by the changes in
Effect of Temperature on Invertase
pH. The graph shows the optimum pH
Activity
value which the active enzyme has

reach its maximum rate of reaction is In Table 1. Effect of temperature

132.4383, while the denatured enzyme’s on Invertase Activity, we obtain the

optimum pH was graphed 177.8415. results for the following test tubes in a

The results are both obtained using a given set of temperature: Test tube no. 1

spectrophotometer. at 20 ̊ C results to 67.4259 mmol/L, Test

tube no.2 at 30 ̊ C results to 152.9527 temperature a sudden drop results in a

mmol/L, Test tube no. 3 at 50 ̊ C results belle shaped curve. Since invertase

to a 155.6678 mmol/L, Test tube no. 4 at refer as tertiary structure enzyme that

60 ̊C results to a 169.9977 mmol/L, Test when exposed to high temperature

tube no. 5 at 70 ̊ C results to a 353.5711 undergo denaturation. The denatured

mmol/L, Test tube no.6 at 90 ̊C results to proteins results to a drastic drop of the

a 163.8132 mmol/L and the reagent rate of reaction.

blank results to a 384.3427 mmol/L.

Effect of pH on Invertase Activity

The results are obtained based

An invertase is a carbohydrate-
on temperature that gives the reaction
digesting enzyme that catalyzes the
rate of the samples. The variation of the
breakdown of sucrose (common sugar)
invertase activity observed different
which gives the mixture of glucose and
peaks of concentration. At 70 ̊ C the test
fructose. It is derived from baker’s yeast.
tube no. 5 shows the highest activity
The DNS method was then
where the enzyme most effective and in
applied in this experiment wherein the
Figure 8. Graph of Invertase Activity
presence of free carbonyl functional
shows the sudden drop of the rate of
group or the reducing sugar is tested.
reaction from the highest optimum
This DNS only binds to reducing sugars
temperature which is the test tube no.1
such as glucose but it cannot bind to
at 20 ̊ C. It is shown that temperature
non-reducing sugars like the sucrose.
increases the rate of reaction and the
After the denaturation of the solution
enzyme activity. Nevertheless, when the
developed its red-brown color due to
rate reaction reaches its optimum
DNS reagent. At lowest pH, in this
experiment pH 3, of both active and enzyme has reach its maximum rate of

denatured enzyme is where the red- reaction is pH 3 at 132.4383, while the

brown color is emphasized the most. denatured enzyme’s optimum pH is pH

The DNS reagent acted as a chromogen 10.6 at 177.8415.

or a substance that is readily converted

into a dye that produces a colored

solution.

Using the spectrophotometer, the

optimum pH value which the active

REFERENCES:

Raymundo. A. K. (1999). Laboratory manual for molecular biology 201.1. Los Banos,

Laguna: Institute of Biological Sciences, College of Arts and Sciences, University of the

Philippines

The Biochemistry Faculty. (1980). A laboratory manual for biochemistry. Quezon City:

C.A.S. University of the Philippines.

College of the Redwoods & Tidewater Community College. (n.d.). Biology I Laboratory

Manual. Retrieved from https://courses.lumenlearning.com/suny-

biolabs1/chapter/enzymes/.

3,5-Dinitrosalicylic acid. (2019, January 28). Retrieved from

https://en.m.wikipedia.org/wiki/3,5-Dinitrosalicylic_acid.

Invertase. (2019, September 23). Retrieved from

https://en.m.wikipedia.org/wiki/Invertase.

www./doc/58234422/Formal-Report-Experiment-3-Enzymes

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