UNIVERSITI TEKNOLOGI MARA

FAKULTI KEJURUTERAAN KIMIA

REACTION ENGINEERING LABORATORY (CHE 506)
STUDENT NAME :MUHAMMAD IKHWAN BAHARUDDIN
(2015856728)
MIRDZA FAROUK B MURHAN MUKHOYIDDIN
(2015636698)
PUTRI ANI NARISA BT AHMAD BAKHTIAR
(2015636856)
SITI AMIRA BT DZULKIFLI (2015655796)
SITI MARYAM BT MOHD RAMZI (2015694934)
GROUP :EH2205F
EXPERIMENT :INVESTIGATION ON ENZYME ACTIVITY &

Allocated Marks
No. Title Marks
(%)
1 Abstract/Summary 5
2 Introduction 5
3 Aims 5
4 Theory 10
5 Apparatus 5
6 Methodology/Procedure 10
7 Results 10
8 Calculations 10
9 Discussion 20
10 Conclusion 10
11 Recommendations 5
12 Reference / Appendix 5
TOTAL MARKS 100

Remarks:
Checked by:
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Date:

TABLE OF CONTENT

No Page
1 Abstract 3
 2 Introduction 4
3 Objectives 5
4 Theory 6
5 Apparatus & Materials 10
6 Procedures 11
7 Results 14
8 Calculation 22

9 Discussion 24
10 Conclusion 25
11 Recommendations 26
12 References 27
13 Appendices 28

ABSTRACT

Enzyme kinetics is the study of how biological catalysts increase the

reaction rate in biochemical reactions. This experiment is conducted to
investigate the effect of temperature and pH on enzyme activity. The 2%
starch solution was treated with different temperature, pH and substrate
concentration. Data of the absorbance were collected using UV-
spectrometer at wavelength=540nm. The kinetics of an enzyme-catalyzed

2
reaction can be studied when the concentration of the enzyme is small
compared to the concentration of the substrate. It can be seen that
different temperature, pH and substrate concentration leads to different
enzyme activity and absorbance value. The optimum pH for amylase is
around pH 6.0 to pH 7.0. As for temperature, the enzyme activity is
highest around 37oC. Meanwhile, for the substrate concentration, no
accurate result was obtained as the graph fluctuated due to some errors
that occurred while conducting the experiment. Based on the results of
this experiment, it shows the activity of an enzyme is affected by its
environmental conditions, such as pH, temperature, substrate
concentration and enzyme concentration. Changing these alter the rate of
reaction caused by the enzyme.

INTRODUCTION

Enzyme is a substance produced by a living organism that acts as a

catalyst to bring about a specific biochemical reaction. It increases the
speed of a chemical reaction without themselves undergoing any

3
permanent chemical change. They are neither used up in the reaction nor
do they appear as reaction products. Enzyme activity is a measure of the
quantity of active enzyme present and is thus dependent on specific
conditions. Several factors affect the rate at which enzymatic reactions is
temperature, pH, enzyme concentration, substrate concentration, and the
presence of any inhibitors or activators

The activity of enzyme is important for the proper functioning of

cells. In the context of energy flow in living organism, enzyme catalyzes
most reactions in metabolic pathways. The behavior of enzyme in
response to different concentrations of the reaction chemicals (both
substrates and products) comprises the basic characteristics of each type
of enzyme. This behavior, referred to as enzyme kinetics which is
responsible for most of the reaction occurred in biological systems.

The Michaelis-Menten model is the one of the simplest and best-

known approaches to enzyme kinetics. It takes the form of an equation
relating reaction velocity to substrate concentration for a system where a
substrate S binds reversibly to an enzyme E to form an enzyme-substrate
complex ES, which then reacts irreversibly to generate a product P and to
regenerate the free enzyme E. This system can be represented
schematically as follows:

The Michaelis-Menten equation for this system is:

4
 Here, Vmax represents the maximum velocity achieved by the
system, at maximum (saturating) substrate concentrations. KM, the
Michaelis constant, is the substrate concentration at which the reaction
velocity is 50% of the Vmax. [S] is the concentration of the substrate S. This
is a plot of the Michaelis-Menten equations predicted reaction velocity as
a function of substrate concentration, with the significance of the kinetic
parameters Vmax and KM graphically depicted. Figure 1 show the Michaelis-
Menten curve.

Figure 1: Michaelis-Menten Curve.

OBJECTIVES

1. Determination of the effects of temperature on the enzymatic

activity and changes in enzyme concentration of an enzyme-
catalysed reaction.
2. Describe the relationship between substrate concentration and the
maximum velocity of an enzyme.
3. Estimation of Michaelis-Menten parameters, effect of pH and
temperature on enzyme activity and kinetics of inhibition.

5
THEORY

Enzymes are protein molecules that act as biological catalysts by

increasing the rate of reactions without changing the overall process. They
are long chain amino acids bound together by peptide bonds. Enzymes
are seen in all living cells and controlling the metabolic processes in which
they converted nutrients into energy and new cells. Enzymes also help in
the breakdown of food materials into its simplest form.

The reactants of enzyme catalysed reactions are termed as

substrates. Each enzyme is quite specific in character, acting on a
particular substrates to produce a particular products. The central
approach for studying the mechanism of an enzyme catalysed reaction is
to determine the rate of the reaction and its changes in response with the
changes in parameters such as substrate concentration, enzyme
concentration, pH, temperature etc. This is known as enzyme kinetics.

One of the important parameters affecting the rate of a reaction

catalysed by an enzyme is the substrate concentration. During enzyme
substrate reaction, the initial velocity V0 gradually increases with
increasing concentration of the substrate. Finally a point is reached,
beyond which the increase in V0 will not depend on the substrate
concentration. When we plot a graph with substrate concentration on the
X axis and corresponding velocity on Y-axis. It can be observed from the
graph that as the concentration of the substrate increases, there is a
corresponding increase in the V0. However beyond a particular substrate
concentration, the velocity remains constant without any further increase.
This maximum velocity of an enzyme catalysed reaction under substrate
saturation is called the Vmax, maximum velocity.

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 Figure 2: Graph of initial velocity against substrate concentration

Michaelis Menten Equation

Leonor Michaelis and Maud Menten postulated that the enzyme first
combines reversibly with its substrate to form an enzyme-substrate
complex in a relatively fast reversible step:

Eqn.1
In the next step, this ES complex is breaks down in to the free enzyme and
the reaction product, P:

Eqn.2

Since the second step is the rate limiting step, the rate of overall
reaction must be proportional to the concentration of the ES that reacts in
the second step. The relationship between substrate concentration,
substrate and Initial velocity of enzyme, V0 has the same general shape
for most enzymes (it approaches a rectangular hyperbola). This can be
expressed algebraically by the Michaelis-Menten equation. Based on their
basic hypothesis that the rate limiting step in enzymatic reactions is the

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breakdown of the ES complex to free enzyme and product, Michaelis and
Menten derived an equation which is:

Eqn. 3

The necessary terms in this reaction are S, V0, Vmax, and Km

(Michaelis constant). All these terms can be measured experimentally.

Lineweaver Burke Plot

In 1934, Lineweaver and Burke made a simple mathematical
alteration in the process by plotting a double inverse of substrate
concentration and reaction rate.

Eqn.4

For enzymes obeying the Michaelis-Menten relationship, the double

reciprocal of the V0 versus S from the first graph, yields a straight line.
The slope of this straight line is KM /Vmax, which has an intercept of
1/Vmax on the 1/V0 axis, and an intercept of -1/KM on the 1/[S] axis. The
double-reciprocal presentation, also called a Lineweaver-Burke plot. The
main advantage of Lineweaver-Burke plot is to determine the Vmax more
accurately, which can only be approximated from a simple graph of V0
versus S.

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 Figure 3: Linewearver-Burke plot

The enzyme Amylase can catalyse the hydrolysis of internal -1,

4-glycosidic bond present in starch with the production of reducing sugars.
In the study of substrate concentration on enzyme kinetics, the enzyme is
kept constant whereas the concentration of Starch is taken in increasing
order. As the substrate concentration increases, the amount of products
produced in every successive tube also increases.

This was explained by Michealis and others that an enzyme

catalysed reaction at varying substrate concentrations is diphasic i.e. at
low substrate concentration the active sites on molecules (enzyme) are
not occupied by substrate and the enzyme rate varies with substrate
molecules concentration (phase1). As the number of substrate molecules
increases, the enzyme attains the saturation level, since there is no more
reaction sites remaining for binding. So the enzyme can work with full
capacity and its reaction rate is independent of substrate concentration.
(Phase II).

This Enzyme substrate reaction can be determined by measuring

the increase in reducing sugars using the 3, 5 Dinitro salicylic acid
reagent. In an alkaline condition, the pale yellow coloured the 3, 5- dinitro
salicylic acid undergo reduction to yield orange coloured 3- amino -5-
nitrosalicylic acid. The absorbance of resultant solutions is read at 540nm.

9
The intensity of colour depends on the concentration of reducing sugars
produced.
Starch Amylase Maltose+Glucose

Figure 4: The enzyme-substrate reaction example.

APPARATUS AND MATERIALS

APPARATUS MATERIALS
Beaker Alpha amylase enzyme
Measuring cylinder Starch
Cuvette pH buffer solution (pH 4-9)
Falcon tube rack DNSA reagent
Falcon tube Water bath
Micropipette and tips
Label sticker
Schott bottle
Vortex mixer
Spectrophotometer
Hot plate

PROCEDURES

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A. PREPARATION OF 2% STARCH SOLUTION
1) 4g of soluble starch was mixed in approximately 50ml of cold water.
2) The slurry is added to approximately 100ml of gently boiling water
while stirred in a large beaker.
3) Then, the final volume of 200ml was added and mixed well.

B. EFFECT OF PH
1) Five test tubes were labeled with pH 5,6,7,8 and 9.
2) 1ml of 2% starch solution was added into each tube.
3) 1ml of appropriate buffer was added at corresponding pH to each
tube.
4) Five additional clean test tubes were prepared and 2ml of amylase
solution was added in each tube.
5) All ten tubes then placed in the 37C water bath for about 5 minutes
to allow the temperature to equilibrate.
6) The content of each amylase test tube is poured into each starch
test tube and they are mixed on vortex mixture.
7) The tubes are returned to the 37C water bath.
8) The hydrolysis reaction is left to proceed for exactly 10 minutes.
9) The amylase activity is determined by using the method given in
Appendix 1.
10) A graph of pH versus enzyme activity is plotted.

C. EFFECT OF TEMPERATURE
1) A test tube was labeled with 30C.
2) 1ml of 2% starch solution was placed into the test tube
3) 1ml of buffer with pH 7 was then added to each tube.
4) An additional clean test tube was prepared and 2ml of amylase
solution was then added into the each tube.
5) Both tubes were placed in the 30C water bath for about 5 minutes
to allow the temperature to equilibrate.
6) The content of the amylase test tube was then poured into the
starch test tube and they were mixed on vortex mixture.
7) The tubes were returned to the 30C water bath.
8) The hydrolysis reaction was left to proceed for exactly 10 minutes.
9) The amylase activity was determined by using the method given in
Appendix 1.
10) The steps 1-9 were repeated with different temperatures
ranging from 40C until 70C.
11) A graph of temperature versus amylase activity was plotted.

11
 D. EFFECT OF SUBSTRATE CONCENTRATION
1) Starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and
3.0% w/v) were prepared as the substrate.
2) Each test tubes were labeled with starch concentration and 1ml of
starch solution was added into each test tube.
3) 1 ml of buffer (pH 7) was added into the test tubes.
4) Five additional clean test tubes were prepared and 2ml of amylase
solution was then added in each tube.
5) All the tubes were placed in the 37C water bath for about 5
minutes to allow the temperature to equilibrate.
6) The content of each amylase test tube was then poured into each
starch test tube and then mixed on vortex mixture.
7) The tubes were returned to the 37C water bath.
8) The hydrolysis reaction is left to proceed for exactly 10 minutes.
9) The amylase activity was determined by using the method given in
Appendix 1.
10) A graph of starch concentration versus enzyme activity was
plotted.

Appendix 1 (Demonstration of enzyme activity)

i. The reaction was stopped after 10 minutes (the time of hydrolysis
reaction) by adding 4ml of DNS reagent.
ii. The samples were boiled for 10 minutes and then cooled to room
temperature.
iii. The absorbance of each sample was measured by using
spectrophotometer at = 540nm.
iv. The absorbance value was then compared with glucose standard
curve that has been prepared to obtain the glucose solution.
v. The enzyme activity (the amount of glucose formed in reaction
mixture per unit time) was calculated.

Appendix 2 (Glucose standard curve preparation)

i. Standard solutions of glucose were prepared at five different
concentrations ranging from 0 mg/L until 1000mg/L by serial
dilution.
ii. 1ml of each glucose solution was added into the test tubes.
iii. 1ml of DNS reagent was added in each test tube and mixed for a
few seconds on vortex mixer.

12
iv. The test tubes are then placed in water bath (100C) for 10 minutes
and cooled at room temperature.
v. The absorbance of each sample was measured by using
spectrophotometer at = 540nm.
vi. The standard curve of absorbance versus glucose concentration is
drew and the graph is in straight line for absorbance less than 0.7

RESULTS

a) Glucose standard curve

No Concentration (g/L) Absorbance OD

1 0.2 2.168
2 0.4 3.012
3 0.6 3.257
4 0.8 3.34
5 1.0 3.364

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Absorbance value vs concentration of glucose
f(x) = 4.38x

Graph of absorbance value against concentration of glucose

14
b) Effect of pH on the activity and stability of amylase enzyme

pH Absorbance OD
5 0.029
6 0.914
7 0.465
8 0.029
9 0.062

Absorbance value vs pH

f(x) = - 0.08x + 0.87

R = 0.11

4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5

pH of starch solution

15
 Graph of absorbance value against pH of solutions

pH Absorbanc Glucose Glucose Enzyme

e OD Concentration, Released Activity, V
X( g/ml) (mol)
5 0.029 -0.0015087 -8.38156 -8.38156E-
x10-6 07
6 0.914 -0.0006237 -3.4649 x10- -3.4649E-07
6

7 0.465 -0.0010727 -5.95934 -5.95934E-

x10-6 07
8 0.029 -0.0015087 -8.38156 -8.38156E-
x10-6 07
9 0.062 -0.0014757 -8.19823 -8.19823E-
x10-6 07

16
 Enzyme activity vs pH
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5

pH

Graph of enzyme activity against pH of solutions

17
c) Effect of temperature on the activity and stability of amylase enzyme

Temperature (C) Absorbance OD

30 0.124
40 1.1254
50 0.915
60 0.595
70 0.486

Absorbance value vs Temperature

 Graph of absorbance value against temperature of solution (C)

Temperatur Absorbanc Glucose Glucose Enzyme

e, e OD Concentrati Released Activity, V
on
30 0.124 -0.001469 -8.162E-06 -8.162E-07
40 1.1254 -0.000732 -4.071E-06 -4.071E-07
50 0.915 -0.0008875 -4.931E-06 -4.931E-07
60 0.595 -0.001123 -6.238E-06 -6.238E-07
70 0.486 -0.001202 -6.683E-06 -6.683E-07

Enzyme activity vs Temperature

Graph of enzyme activity against temperature

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

Concentration (%) Absorbance OD

0.5 0.112
1.0 0.076
2.0 0.016
2.5 0.161
3.0 0.032

Absorbance value vs substrate concentration

 Graph of absorbance value against substrate concentration

Concentratio Absorbanc Glucose Glucose Enzyme

n (%) e OD Concentrati Released Activity, V
on
0.5 0.112 - -8.21078E- -8.21078E-
0.001477941 06 07
1.5 0.076 - -8.35784E- -8.35784E-
0.001504412 06 07
2.0 0.016 - -8.60294E- -8.60294E-
0.001548529 06 07
2.5 0.161 - -8.01062E- -8.01062E-
0.001441912 06 07
3.0 0.032 - -8.53758E- -8.53758E-
0.001536765 06 07
Enzyme activity vs Concentration (%)

Graph of enzyme acitvity % concentration

1/S 1/V
2 -1217910.448
0.667 -1196480.938
0.500 -1162393.162
0.400 -1248342.682
0.333 -1171291.866
 1/V vs 1/S

f(x) = - 14157.24x - 1188241.17

Graph of 1/V against 1/S

CALCULATION

Concentra Absorba Concentra Glucos Enzyme 1/V 1/S

tion nce OD tion of e Activity, x109
substrate, glucose, release V (min/
S (%) X (g/ml) d (mol/min) mol)
(mol)
0.5 0.112 2.56 x 10-7 1.42 x 1.42 x 10- 7.04 2
10-9 10
 1.5 0.076 1.736 x 10-5 9.64 x 9.64 x 10-9 0.10 0.667
10-8
-6
2.0 0.016 3.66 x 10 2.03 x 2.03 x 10-9 0.49 0.5
10-8
2.5 0.161 3.68 x 10-6 2.04 x 2.04 x 10-9 0.49 0.4
10-8
3.0 0.032 7.31 x 10-6 4.06 x 4.06 x 10-9 0.25 0.333
10-8

Y-Values

f(x) = 4.23x - 1.63

R = 0.95

To determine all the parameters, the calculations are based on

glucose standard curve which is by using the linear equation
obtained from the graph of absorbance value against the
concentration of glucose solution (y = 1.36x + 2.2122).

I. How to determine the glucose concentration

X = concentration of glucose
Y = absorbance value
 Y = 4.3766 x
Y
X= g /L
4.3766
0.0256 g 1L
X= x = 2.56 x 10-7 g/ml
L 1000 ml

II. How to determine the number of moles of glucose

released

Mw of glucose, C6H12O6 = 180 g/mol

Volume of enzyme (amylase) =1ml
Concentration of glucose
Number of moles of glucose released = x Volume
M w of glucose
of enzyme
2.56 x 107 g /ml
= x 1ml
180 g/mol
= 1.42 x 10-9mol

III. How to determine the enzyme activity

Hydrolysis reaction time = 10 minutes

Number of moles of glucose released
Enzyme activity =
hydrolysis reactiontime
9
1.42 x 10 mol
=
10 minutes
= 1.42 x 10-10mol/min

IV. Equation for Michaelis-Menten

 V max [S ]
V=
Km+ S
Rearrange based on linear equation:
1 Km 1 1
= +
V V max S V max
Km
Gradient of the graph =
V max
The linear equation obtained from 1/V versus 1/S is y = 4.2325x -1.6273
Neglect the negative sign at y-intercept,
1
Maximum enzyme activity, Vmax = =0.6145
1.6273
Michaelis-Menten Constants, Km = 4.2325 V max =4.2325 0.6145=2.602

DISCUSSION

The objective of this experiment were to determine the effects of

temperature on the enzymatic activity and changes in enzyme
concentration of an enzyme-catalyzed reaction.To study the relationship
between substrate concentration and the maximum velocity of an
enzyme.To obtain the Michaelis-Menten parameters, effect of pH and
temperature on enzyme activity and kinetics of inhibition. For this
experiment , spectrophotometer was used to measure the absorbance in
sample.

For the first experiment , different pH buffer solution was used to

determined the effect of pH on enzyme activity.Table 7.1 show the
absorbance value in each pH solution. By referring at pH 5, the value of
absorbance was the lowest (0.029) and it was the highest at pH 6 (0.914).
Base on theory ,the optimum pH of the enzyme was 6.0 to 7.0. The
amylase retained more than 50% of its original activity between pH 4.6
and 6.8. The enzyme was stable over a wide pH range. More than 50%
residual activity was obtained between pH 4.7 and 9.0. The enzyme was
not stable below pH 3.5 or above pH 10.0. Its shows that some mistake
have occurred during the experiment .
The second experiment was conducted to determined the effect of
temperature on enzyme activity. In vitro activity of human salivary alpha-
amylase showed the optimum pH and temperature at 7.0 and 37 degrees
C, respectively. The result shows the reading of absorbance value at
different temperature of starch solution containing amylase . By referring
at temperature 40 degree C the value of absorbance was the highest
(1.1254) and it was the lowest at temperature 30 degree C (0.124). From
the value, its shows that the amylase activity start to become inactive
when the temperature higher than 40 degree C. At 50 degree C ,the value
increase drastically due to mistake happen when reading the absorbance
value.
Furthermore, based on the results the difference in the amount of
substrate concentration affects the enzyme activity as the reading of
absorbance obtained varied at each concentration. However, due to some
reasons and mistakes occurred during the experiment, the values are not
stable

CONCLUSION

The data shown on the graph from the experiment shows that catalase
functions of pH, substrate concentration and temperature give different
effect on enzyme activity. Table (b) shows that from pH 5 to pH 6, the
enzyme activity increases and at pH 7 the enzyme activity start to
decrease. The enzyme activity keep on decreasing when the pH is 8,
however experienced a slight increase at pH 9. When the enzyme activity
increase the absorbance reading also increase. By looking at Graph (b),
one can see that as the pH of the solution rose to a pH of 6, catalase
became more efficient and was able to better carry out its function. These
results help support the idea that as a solution becomes more acidic than
the optimum pH of an enzyme, the enzymes present in the solution will
denature, and in turn will not be able to function properly. This will result
in lower reaction rates, which is shown in Graph (b).

At very high and very low temperatures we expected the

absorbance or enzyme activity be low. The highest absorbance should
have appeared at around 37C, because most human enzyme activity
occurs at body temperature. In this case using enzyme amylase, Graph (c)
shown that the enzyme activity had the highest absorbance at 40C and
after, started to decrease as the temperature increased. This proved to us
that when the temperature is very high, it will be denatured thus the
production of product decreased.

For the substrate concentration, we could not obtain an accurate

result due to some errors occurred when performing the experiment as
the graph fluctuated as the concentration increased. The information
gathered throughout this experiment is very useful for the future. This
experiment has shown that enzymes must have certain environmental
conditions present in order for them to function properly. With this
knowledge, one can successfully perform experiments using enzymes in
the future by making sure that the environmental conditions present are
optimum for the enzyme that is being used.

A limitation of the procedure was that we were unable to test for the
presence of catalase in the extract before beginning the experiment. If we
were able to test for the presence of catalase in the extract, we could
have ensured that the decomposition of hydrogen peroxide resulted from
enzyme catalysis and not from the natural spontaneous decomposition of
the chemical. Instead, we were forced to assume that catalase was
present in the extract, an assumption that may, or may not have, been
correct.

RECOMMENDATIONS
 1. In order to prevent contamination from the surrounding to directly attach
to the culture, the experiment must be carried out under laminar flow.
2. Buffer solution must be prepared carefully, else the readings for the
reaction rate will be erroneous.
3. Auto hydrolysis of enzyme can result if the enzyme solution is prepared
before the substrate solution.
4. Always ensure the cuvette is wiped cleanly in order to prevent anything
that would affect the result of reading from the spectrophotometer on
absorbance optical density (OD) values.
5. Hand must be washed properly using appropriate soap after handling the
culture as it can affect the health.
6. Parallax error must be avoided during the measuring volume, as it can
affect the concentration of solution which indirectly interrupts the
absorbance optical density (OD) values.

REFERENCE

1. Worthington Biochemical Corporation (2013). Introduction to enzyme.

Retrieved on November 19, 2016 from http://www.worthington-
biochem.com/introbiochem/effectsph.html

2. Anonymous, Effect of pH on enzyme. Retrieved on November 19, 2016

from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm

3. Talamond, Pascale, Michel Noirot, and Alexandre De Kochko. The

Mechanism of Action of -amylase from Lactobacillus Fermentum on
Maltooligosaccharides. Journal ofChromatography B (2005): 42-
47.Science Direct.Web.

4. Anonymous, (n.d). Effect of pH on enzyme. Retrieved on November 19,

2016
from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm