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
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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
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
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
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.
OBJECTIVES
5
THEORY
6
Figure 2: Graph of initial velocity against substrate concentration
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
7
breakdown of the ES complex to free enzyme and product, Michaelis and
Menten derived an equation which is:
Eqn. 3
Eqn.4
8
Figure 3: Linewearver-Burke plot
9
The intensity of colour depends on the concentration of reducing sugars
produced.
Starch Amylase Maltose+Glucose
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
10
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.
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
13
Absorbance value vs concentration of glucose
f(x) = 4.38x
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
pH of starch solution
15
Graph of absorbance value against pH of solutions
16
Enzyme activity vs pH
4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5
pH
17
c) Effect of temperature on the activity and stability of amylase enzyme
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
CALCULATION
Y-Values
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
DISCUSSION
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).
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