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Enzyme Activity
Enzyme Activity
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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
2
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 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.
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INTRODUCTION
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:
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
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.
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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
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that the rate limiting step in enzymatic reactions is the 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.
Eqn.4
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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).
9
Figure 4: The enzyme-substrate reaction example.
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
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PROCEDURES
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.
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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.
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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.
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RESULTS
4.5
4
y = 4.3766x
3.5
Absorbance value
2.5
1.5
0.5
0
0 0.2 0.4 0.6 0.8 1 1.2
Concentration (g/L)
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
1
0.9
0.8
0.7
Absorbance value
0.6
0.5
y = -0.0819x + 0.8731
0.4
R = 0.1104
0.3
0.2
0.1
0
0 1 2 3 4 5 6 7 8 9 10
pH of starch solution
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pH Absorbance Glucose Concentration, Glucose Enzyme
OD X( g/ml) Released (mol) Activity, V
Enzyme activity vs pH
0.00E+00
0 1 2 3 4 5 6 7 8 9 10
-1.00E-07
-2.00E-07
-3.00E-07
Enzyme activity
-4.00E-07
-5.00E-07
-6.00E-07
-7.00E-07
-8.00E-07
-9.00E-07
pH
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c) Effect of temperature on the activity and stability of amylase enzyme
1.2
1
Absorbance value
0.8
0.6
0.4
0.2
0
0 10 20 30 40 50 60 70 80
Temperature
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Temperature, Absorbance OD Glucose Glucose Enzyme
Concentration Released Activity, V
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
-2.000E-07
-3.000E-07
Enzyme activity
-4.000E-07
-5.000E-07
-6.000E-07
-7.000E-07
-8.000E-07
-9.000E-07
Temperature
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d) Effect of substrate concentration on the activity and stability of amylase enzyme
0.16
0.14
0.12
Absorbance value
0.1
0.08
0.06
0.04
0.02
0
0 0.5 1 1.5 2 2.5 3 3.5
Substrate concentration
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Concentration Absorbance Glucose Glucose Enzyme
(%) OD Concentration Released Activity, V
0.5 0.112 -0.001477941 -8.21078E-06 -8.21078E-07
1.5 0.076 -0.001504412 -8.35784E-06 -8.35784E-07
2.0 0.016 -0.001548529 -8.60294E-06 -8.60294E-07
2.5 0.161 -0.001441912 -8.01062E-06 -8.01062E-07
3.0 0.032 -0.001536765 -8.53758E-06 -8.53758E-07
-8.1E-07
Enzyme activity
-8.2E-07
-8.3E-07
-8.4E-07
-8.5E-07
-8.6E-07
-8.7E-07
Concentration (%)
20
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
-1150000
-1160000 0 0.5 1 1.5 2 2.5
-1170000
-1180000
-1190000
-1200000
1/V
-1210000
-1220000 y = -14157x - 1E+06
-1230000
-1240000
-1250000
-1260000
1/S
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CALCULATION
Y-Values
8
7 y = 4.2325x - 1.6273
R = 0.9546
6
0
0 0.5 1 1.5 2 2.5
-1
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).
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I. How to determine the glucose concentration
X = concentration of glucose
Y = absorbance value
Y = 4.3766 x
Y
X = 4.3766 g/L
0.0256 g 1L
X= x 1000ml = 2.56 x 10-7 g/ml
L
= 1.42 x 10-9mol
= 1.42 x 10-10mol/min
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IV. Equation for Michaelis-Menten
Vmax [S]
V= Km + S
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 = 1.6273 = 0.6145
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
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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.
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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.
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REFERENCE
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.
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