UNIVERSITI TEKNOLOGI MARA FAKULTI KEJURUTERAAN KIMIA

REACTION ENGINEERING LABORATORY

(CHE506)

NAME : NUR FATIHAH BINTI ABDUL RAHMAN

STUDENT NO. : 2016249808
GROUP : EH220 5E (GROUP 3)
EXPERIMENT : LAB 8 INVESTIGATION ON ENZYMES AND
KINEMATICS______________________________
DATE PERFORMED : 30TH NOVEMBER 2018
SEMESTER :5
PROGRAMME / CODE : EH220
SUBMIT TO : MADAM NURUL ASYIKIN MD ZAKI

No. Title Allocated Marks (%) Marks

1 Abstract/Summary 5
2 Introduction 5
3 Aims 5
4 Theory 5
5 Apparatus 5
6 Methodology/Procedure 10
7 Results 10
8 Calculations 10
9 Discussion 20
10 Conclusion 10
11 Recommendations 5
12 Reference 5
13 Appendix 5
TOTAL MARKS 100

Remarks:

Checked by : Rechecked by:

---------------------------- --------------------------

Date : Date :
1.0 ABSTRACT
This experiment was carried out to study the effects of temperature, pH and substrate
concentration on enzyme activity. The 2% starch solution was handled with different
temperature (30⁰C, 40⁰C, 50⁰C and 60⁰C), pH (5, 6, 7, 8, 9) and substrate concentration (0.5,
1.0, 1.5, 2.0, 2.5). The objectives for this experiment are to determine the effects of temperature
on the enzymatic activity and changes in the enzyme concentration of an enzyme-catalyzed
reaction, to describe the relationship between substrate concentration and the maximum
velocity of an enzyme and to an estimation of Michaelis-Menten parameters, effects of pH and
temperature on enzyme and kinetics of inhibition. From the experiment, the optimum pH for
amylase is at pH 7, the optimum temperature was 50⁰C. For the substrate concentration, the
optimum value cannot be determined due to some error. Hence, the Vmax also fail to be
determined.

2.0 INTRODUCTION
To figure out the mechanism of an enzyme-catalyzed reaction is to determine the rate
of reaction and its response with the parameters such as temperature, pH and substrate
concentration. In this experiment, we can observe how those parameters affect the enzyme
activity.
Michaelis-Menten model is one of the easier and best-known approaches to enzyme
kinetics. It takes the form of an equation relation reaction velocity to substrate concentration
for a system where substrate, S binds reversibly to an enzyme E to form enzyme-substrate
complex ES and produce a product, P and also free enzyme, E. From the Michaelis-Menten
equation, the Vmax represents the maximum velocity that archived by the system at maximum
concentration, Km.
Enzymes are biological molecules (typically proteins) that significantly speed up the
rate of virtually all of the chemical reactions that take place within cells.
They are vital for life and serve a wide range of important functions in the body, such as aiding
in digestion and metabolism.
Some enzymes help break large molecules into smaller pieces that are more easily
absorbed by the body. Other enzymes help bind two molecules together to produce a new
molecule. Enzymes are highly selective catalysts, meaning that each enzyme only speeds up a
specific reaction. Enzymes are a complex tertiary or quarternary shape and catalyze the reaction
by forming a complex form that known as enzyme substrate complex at a specific region called
active site.
 Enzyme + Substrate → enzyme-substrate complex → enzyme + product
Amylase is a family of enzymes that degrade starch which is polymers of glucose into a
smaller disaccharide (maltose). Amylase is depended on the breaking bonds in starches,
polysaccharides, and complex carbohydrates into simple form to absorb chemical sugars.

3.0 OBJECTIVE
i. To determine the effects of temperature on the enzymatic activity and changes in an
enzyme concentration of an enzyme-catalyzed reaction.
ii. To describe the relationship between substrate concentration and the maximum
velocity of an enzyme.
iii. To estimate the Michaelis-Menten parameters, the effect of pH and temperature.

4.0 THEORY
Enzymes act as a biological catalyst by speeding up the rate of a reaction without
changes in the overall process. The long chain amino acids were bound together by peptide
bonds. In all living cells has enzymes which control the metabolic process which convert
nutrients into energy and new cells. It will also help to break down the food material to the
simplest form. The reactants of enzymes namely known as substrates. Each of enzymes has
specific function and character which act on certain substrates to produce certain products.
Enzyme kinetics is where we study their mechanism of an enzyme-catalyzed reaction by
investigating the rate of reactions and their parameters.
For the temperature parameter, as the temperature increase, the molecule will react more
and produce more kinetic energy. This will increase the chances of a successful collision hence,
the rate of reaction increase. The temperature will increase until reached its optimum
temperature, then above the temperature, the enzyme's structure will begin to break down to
gain more kinetic energy.
 Moving on to the next parameter is pH which enzyme works within a small pH range.
There is an optimum pH due to pH can make and break intra- and intermolecular bonds changes
the shapes of enzymes and its effectiveness.

Reviewing on the substrate concentration, sbstrate concentration is one of the crucial

parameters that affect the rate of reaction. During enzyme-substrate reaction, the initial
velocity, Vo gradually increasing as the concentration of substrate increase until reached a
certain point. A graph of the corresponding velocity versus substrate concentration is plotted.
It can be noticed that the concentration of substrates increases as the velocity increase.
Leonor Michaelis and Maud Menten assumed that the enzymes first combine
reversibly with its substrate to form an enzyme-substrate complex in a relatively fast reversible
step then ES complex breaks down into the free enzymes and product.
 The relationship between substrate concentration, substrate, and initial velocity of the
enzyme, Vo has the same general shape for most enzymes. The equation can be derived with:

5.0 APPARATUS/MATERIAL
- Alpha Amylase Enzyme - Falcon tube rack
- Starch solution - Falcon tube
- pH buffer solution - Label stickers
- DNSA Reagent - Vortex mixer
- Beaker - Measuring cylinder
- Cuvette - Water bath
- Schott bottle - Thermometer
- Spectrometer - Hot plate

6.0 METHODOLOGY/PROCEDURE
PROCEDURES
A) Preparation of 2% Starch Solution
1. 4g of soluble starch is mixed with approximately 50ml of cold water.
2. The slurry about 100ml of gently boiling water was added while stirring in a large beaker.
3. The beaker is top up with the final volume of 200ml and well-mixed.

B) Effect of pH on the activity and stability of amylase enzyme

1. Five test tubes with pH 5, 6, 7, 8, 9 were labeled. 1mL of 2% starch solution was added in
each test tubes. 1mL of appropriate buffer (at corresponding pH) was added to each test tube.
2. Five new clean test tubes were taken and 2mL of amylase solution was added in each test
tubes.
3. All 10 test tubes were placed in the 37⁰C water for 5 minutes to allow the temperature to
equilibrate.
4. The content of each amylase test tube was poured into each test tube and mixed them on a
vortex mixer.
5.The tubes were returned to the 37⁰C water bath.
6. The hydrolysis reaction proceeded for 10 minutes.
7. The amylase activity was determined using the method in Appendix 1.
8. A graph pH vs enzymatic activity was plotted.

C) Effects of temperature on the activity and stability of amylase enzyme

1. One test tube with 30⁰C was labeled. 2% of the starch solution and 1mL of pH=7 buffer was
placed into the tube.
2. One additional test tube was taken and 2mL of amylase solution was added into the test tube.
3. Both test tubes were placed in the 30⁰C water bath for 5 minutes to allow the temperature to
equilibrate.
4. The contents of the amylase test tube were poured into a starch test tube and were mixed
with a vortex mixer.
5. The tube was returned back to a 30⁰C water bath.
6. The hydrolysis reaction proceeded for 10 minutes.
7. The amylase activity was determined by using the method in Appendix 1.
8. The steps i-vii was repeated with 4 different temperature from 30-70⁰C.
9. A graph of temperature vs amylase activity was plotted.

D) Effects of substrate concentration on the activity of amylase enzyme

1. The starch solution of varying concentration (0.5, 1.0. 1.5, 2.0, 2.5) was prepared as the
substrate.
2. The tubes with substrate concentrations were labeled and placed 1mL of each starch solution
into the test tubes.
3. 1mL of pH=7 buffer was added to the test tubes.
4. Five additional clean test tubes were taken and 2mL of amylase solution was added in each
test tube.
5. All the test tubes were placed in 37⁰C water bath for about 5 minutes to allow the temperature
to equilibrate.
6. The content of each amylase test tube was poured into the starch test tube and mixed on a
vortex mixer.
7. The tube was returned back to a 30⁰C water bath.
8. The hydrolysis reaction proceeded for 10 minutes.
9. The amylase activity was determined by using the method in Appendix 1.
10. A graph of starch concentration vs amylase activity was plotted.
E) Appendix 1 (Demonstration of enzyme activity)
1. After 10 minutes hydrolysis reaction, the reaction was stopped by adding 4mL of DNSA
reagent.
2. Boiled for 10 minutes and left to cool at room temperature.
3. The absorbance of the samples was measured at λ=540 nM.
4. The absorbance value was compared with the glucose standard curve prepared to obtain the
glucose concentration.
5. The enzyme activity was calculated.

F) Appendix 2 (Glucose standard curve preparation)

1. The standard solutions of glucose at 5 different concentrations ranging from 0-1000mg/L
were prepared.
2. 1mL of each glucose solution was added in the test tubes.
3. 1mL of DNSA reagent was added in each test tubes and mix for a few seconds on a vortex
mixer.
4. The test tubes were placed in a water bath (100⁰C) for 10min and left to cool at room
temperature.
5. The absorbance of the samples was measured at λ=540 nM.
6.The standard curve of absorbance vs glucose concentration was plotted.
7.0 RESULT AND CALCULATIONS

Effect on pH activity and stability of amylase enzyme

pH Absorbance Glucose Glucose Enzyme

(nM) Concentration, Released (mol) Activity, V
X (g/mL) (mol/min)
5 5.20 4.34 x 10-6 2.41x10-8 2.41x10-9
6 5.64 4.70 x 10-6 2.61x10-8 2.61x10-9
7 5.84 4.87 x 10-6 2.70x10-8 2.70x10-9
8 6.36 5.30 x 10-6 2.94x10-8 2.94x10-9

Absorbance Optical Density vs pH

6.6
Absorbance Optical Density , OD

6.4
6.2
6
5.8
5.6
5.4
5.2
5
4.5 5 5.5 6 6.5 7 7.5 8 8.5
pH

FIGURE 1

Enzyme activity vs pH
3.00E-09
2.90E-09
Enzyme activity

2.80E-09
2.70E-09
2.60E-09
2.50E-09
2.40E-09
2.30E-09
4.5 5 5.5 6 6.5 7 7.5 8 8.5
pH
 FIGURE 2
Effect of temperature on the activity and stability of amylase enzyme

Temperature (⁰C) Absorbance Glucose Glucose Enzyme

(nM) Concentration, X released activity, V
(g/mL) (mol) (mol/min)
30 0.5870 4.90 𝑥 10−7 2.72 𝑥 10−9 2.72 𝑥 10−10
40 0.6150 5.13 𝑥 10−7 2.85 𝑥 10−9 2.85 𝑥 10−10
50 0.6900 5.76 𝑥 10−7 3.20 𝑥 10−9 3.20 𝑥 10−10
60 0.8110 6.76 𝑥 10−7 3.75 𝑥 10−9 3.75 𝑥 10−10
70 0.5730 4.78 𝑥 10−7 2.65 𝑥 10−9 2.65 𝑥 10−10

Absorbance Optical Density vs Temperature

0.85
Absorbance Optical Density ,OD

0.8

0.75

0.7

0.65

0.6

0.55

0.5
20 30 40 50 60 70 80
Temperature

FIGURE 3
 Enzyme activity vs pH
4.00E-10

3.80E-10

3.60E-10

3.40E-10

3.20E-10

3.00E-10

2.80E-10

2.60E-10
25 30 35 40 45 50 55 60 65 70 75

FIGURE 4

Effect on substrate concentration on the activity and stability of amylase enzyme

Concentration Absorbance Glucose Glucose Enzyme 1/V 1/S

(%) Reading Concentration, released activity, V
(nM) X (g/mL) (mol) (mol/min)
0.5 7.67 6.40 𝑥 10−6 3.55 𝑥 10−8 3.55 𝑥 10−9 281690140.8 2
1.0 7.60 6.34 𝑥 10−6 3.52 𝑥 10−8 3.52 𝑥 10−9 284090909.1 1
1.5 7.20 6.01 𝑥 10−6 3.34 𝑥 10−8 3.34 𝑥 10−9 299401197.6 0.666667
2.0 5.75 4.80 𝑥 10−6 2.66 𝑥 10−8 2.66 𝑥 10−9 375939849.6 0.5
2.5 5.11 4.26 𝑥 10−6 2.36 𝑥 10−8 2.36 𝑥 10−9 423728813.6 0.4
 Absorbance Optical Density vs Substrate Concentration
9
8
7
6
5
4
3
2
1
0
0 0.5 1 1.5 2 2.5 3

FIGURE 5

Enzyme activity vs Substrate concentration

3.80E-09
3.60E-09
3.40E-09
3.20E-09
3.00E-09
2.80E-09
2.60E-09
2.40E-09
2.20E-09
2.00E-09
0 0.5 1 1.5 2 2.5 3

FIGURE 6
 1/V vs 1/S
450000000

430000000

410000000

390000000

370000000

350000000

330000000
y = -7E+07x + 4E+08
310000000 R² = 0.4927
290000000

270000000

250000000
0 0.5 1 1.5 2 2.5

FIGURE 7

Glucose standard preparation curve

Concentration Glucose (mg/L) Absorbance (nM)

0.8 0.0660
1.0 0.1400
1.2 0.4600
1.4 0.7020
1.6 0.9000
1.8 1.2400
 Absorbance Optical Density vs Standard Concentration
Glucose
1.4
Absorbance Optical Density, OD(nM) 1.2 y = 1.1989x - 0.9738
R² = 0.9816
1

0.8

0.6

0.4

0.2

0
0.7 0.9 1.1 1.3 1.5 1.7 1.9
-0.2
Glucose concentration (mg/L)

FIGURE 9
Sample Calculations
i) To determine the glucose concentration, X (g/mL)

Based on the glucose standard curve above, the equation of the curve that is obtained:
Y = mX
Y = 1.1989x
while X is the protein concentration and Y indicates the absorbance reading.
To calculate protein concentration based on data in Table 1, for pH
𝑌
X = 1.1989
5.20 1𝐿 1×10−3
= 1.1989 × 1000 𝑚𝐿 × 𝑚𝑖𝑙𝑙𝑖

= 4.36x10-6 g/mL

ii) To determine the amount of glucose released (mol)

MW of glucose = 180.1559 g/mol

Volume of amylase enzyme = 1 ml
Moles of glucose released (mol)
𝑔
𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 ( )
𝑚𝐿
= × 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑒𝑛𝑧𝑦𝑚𝑒 (𝑚𝐿)
𝑀𝑊 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
𝑔
4.36 𝑥 10−7
𝑚𝑙
= 𝑔 × 1𝑚𝐿
180.1559
𝑚𝑜𝑙

= 2.41 × 10-9 mol

 iii) Determine of enzyme activity, V (mol/min)
Duratin of hydrolysis : 10 minutes

𝑚𝑜𝑙 𝑚𝑜𝑙 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑

𝐸𝑛𝑧𝑦𝑚𝑒 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 ( )=
min ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
2.41 ×10−9
= 10 𝑚𝑖𝑛

= 2.41 x 10-10 min

iv) Michaelis – Menten :
𝑉 [𝑆]
V = 𝑉 𝑚𝑎𝑥 + 𝑆
𝑚𝑎𝑥

1 𝐾𝑚 1 1
Double Reciprocal: = +𝑉
𝑉 𝑉𝑚𝑎𝑥 𝑆 𝑚𝑎𝑥

y = -7E+07x + 4E+08
1
= 4𝑥10−8
𝑉𝑚𝑎𝑥
V max = 2.5x10-9
The Km Michaelis constant,
𝐾𝑚
= - −7𝑥10−7
𝑉𝑚𝑎𝑥

Km = (-7x 10-7) × ( 2.5x10-9) = 1.75x10-15

8.0 DISCUSSION

Enzymes are proteins that act as a catalyst for biological reactions used to speed up
the chemical reactions. This occurs by lowering the activation energy of a reaction. All
biochemical reactions are catalyzed by enzymes and have their own optimum activity at a
neutral pH and body temperature. Enzymes are also specific that will act on one substrate
only and the substrate will into the active site
Theoretically, during the catalysis, the first step is the substrate (S) binds to the enzyme (E)
to form the enzyme-substrate complex (ES). This equilibrium reaction will favor by the high
concentration of enzyme and/or substrate. After the substrate was bound, the reaction takes
place when the product was released.
The first objective for this experiment is to determine the factors that effect on the enzymatic
activity. Enzyme activity is the ability of enzyme that catalyzes a specific reaction under
specific conditions. For this experiment, the factors that effect on enzyme activity are
temperature, pH and substrate concentration. The glucose standard curve was plotted as shown
in Figure 8. Based on Figure 8, the linear equation was y = 1.1989x - 0.9738. Hence, to find
the protein concentration for each factor, the absorbance reading (y)/gradient.
The enzyme activity for each factor was determined from the concentration of glucose moles
of glucose released. pH gives a different effect on structure and enzyme activity. Theoretically,
for amylase optimum pH is at 7. According to Figure 2, it showed that the graph decrease
gradually, enzyme should be at its peak of ph 7 but then there might be some error during the
experiment.
Next, the effect of enzyme activity with temperature. Based on the plotted graph, the higher
the optical density (OD), the higher the temperature. When the temperature is very high, it will
be denatured thus the production of product decrease.
Theoretically, as the substrate concentration increase, the enzyme activity will increase until
reached its optimum concentration then decrease back. However, based on figure 7.6, the
substrate decrease then increase back after concentration 1. At Figure 5 and 6, we can see that
the optimum substrate value cannot be determined. This happens due to an error that may occur
during the experiment were conducted.
Lastly, the next objective of this experiment is to describe the relationship between substrate
concentration and the maximum velocity of an enzyme. As the concentration of substrate
increases, the rate of reaction also increases until the point of saturation occurs. When it reached
the maximum point, the rate has achieved its maximum and there is no free enzyme to bind
with the substrate and all active sites of enzymes are bound to the substrate. After the point
have been achieved, increasing the concentration do not have any effect. The maximum for
each enzyme is usually given by Km value. From the experiment, the Km value was -0.18.
Theoretically, the Lineweaver Burke Plot should linearly increase, but from the experiment that
has been conducted it show that it decreases linearly. However, based on Figure 7.7, the best
fit line was decrease linearly as the Vmax cannot be determined. It shows from the calculation
the Vmax is 7.07 × 10−6 mol/min. Hence, there may be an error during the experiment were
carried out such as that the temperature water bath did not achieve 37⁰C to allow the solution
to react.

9.0 CONCLUSION

From the result of this experiment shows that catalyze functions of pH, substrate
concentration and temperature give effects on the enzyme of activity. The optimum pH was 7
where the catalase became more efficient and able to carry out its function. After it reached its
optimum pH of the enzyme, the solution will become more acidic and the enzymes present will
start to denature and not able to function accurately.
At higher and lower temperature, the absorbance and enzyme activity should be at the lowest
value. The optimum temperature for the alpha-amylase enzyme from the experiment is 50⁰C.
The absorbance or enzyme activity start to increase until reached its optimum temperature,
50⁰C then the enzymes start to denature.
For the substrate, theoretically, the enzyme activity should be increasing as the substrate
increase until reached its maximum point before start to denature. However, from the result that
has been obtained, the optimum substrate cannot be determined due to an error. Hence,
according to Michaelis-Menten kinetics, the Vmax and Km cannot be identified due to the data
obtained was fluctuated.
10.0 RECOMMENDATION

There are errors that occur during the experiment were conducted. Hence, some
precaution must be taken before handling these experiment. Firstly, wash hand properly using
soap and wear gloves before handling the culture which may expose to health. Make sure the
gloves were disinfected with ethanol before handling the culture. Next, avoid the parallax error
during measure the volume of reagent or solution using the measuring cylinder. This may affect
the concentration of the solution and interrupt the absorbance optical density (OD) values.
Then, ensure the cuvette has been wiped cleanly to prevent any scratch that may affect the
spectrophotometer reading on absorbance optical density (OD). Ensure the water bath
temperature has been reached at 37⁰C and the solution is quite sticky before proceeding to the
next step.

11.0 REFERENCES

1. Retrieved on 19 October 2018 from:

https://www.integrativepro.com/Resources/Integrative-Blog/2018/Digestive-
Enzymes-Amylase-Protease-Lipase
2. Retrieved on 19 October 2018 from:
https://www.chemguide.co.uk/organicprops/aminoacids/enzymes.html
3. Retrieved on 20 October 2018 from:
http://vlab.amrita.edu/?sub=3&brch=64&sim=1090&cnt=1
4. Retrieved on 20 October 2018 from:
https://www.hygrozyme.com/resources/temperature-enzyme-activity/
5. Retrieved on 25 October 2018 from:
https://biology.stackexchange.com/questions/16388/why-does-ph-have-an-effect-
on-enzymes