Professional Documents
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GROUP
: 5
EXPERIMENT
DATE PERFORMED
: 29 SEPTEMBER 2015
SEMESTER
: 5
PROGRAMME CODE
: EH242 5E
SUBMIT TO
No.
1.
2.
3.
4.
5.
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13.
Title
Abstract/Summary
Introduction
Aims
Theory
Apparatus
Methodology/Procedure
Results
Calculations
Discussion
Conclusion
Recommendations
Reference
Appendices
TOTAL
Allocated Marks
5
5
5
5
5
10
10
10
20
10
5
5
5
100
Marks
Remarks:
Checked by:
Rechecked by:
.........
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Date:
Date:
1
Table of Contents
NO.
TITLE
PAGES
Abstract
Introduction
Objectives
Theory
Apparatus
Methodology/Procedure
Results
11
Calculations
18
Discussion
20
10
Conclusion
22
11
Recommendations
23
12
Reference
23
13
Appendices
24
Abstract
Enzymes are protein catalysts that speed up chemical reaction in living organisms. This
investigation tested the effects of temperature and pH has on enzyme activity. The 2% starch
solution was treated with different temperature (30 C, 40 C, 50 C and 60 C), pH (5,6, 7, 8 and
9) and the substrate concentration (0.5%, 1.5%, 2.0%, 2.5% and 3.0%). Data was collected by
determine the enzyme activity and the absorbance value at =540 nm. The objective for this
experiment is determination of the effects of temperature on the enzymatic activity and changes
in enzyme concentration of an enzyme-catalyzed reaction. It is also describe the relationship
between substrate concentration and the maximum velocity of an enzyme. After conducting the
experiments we can see that different temperature, pH and substrate concentration give different
enzyme activity and the absorbance value. From the pH experiment we can see the optimum pH
for amylase is at pH 6, the optimum temperature we fail to determine due to some error while
conducting the experiment while the higher substrate concentration the lower the enzyme activity.
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.
Introduction
Cells function largely because of the action of enzymes. Life is a dynamic process that involves
constant changes in chemical composition. These changes are regulated by catalytic reactions,
which are regulated by enzymes. At one time, the cell was actually conceived of as a sac of
enzymes. It was believed that if we knew all of the reactions and their rates of action, we could
define the cell, and indeed, life itself. Few biologists continue to think of this as a simple task, but
we know that life as we know it could not exist without the function of enzymes. Ideally, we would
examine enzymes within an intact cell, but this is difficult to control. Consequently, enzymes are
studied in vitro after extraction from cells.
Enzymes are protein molecule that acts as biological catalysts. Without changing of the
overall process, they increase the rate of reactions. Enzymes are long chains of amino acids
bound together by peptide bonds. Besides that, they are seen in all living cells and controlling the
metabolic processes in which they converted nutrients into energy and new cells. Other than that,
enzymes also help in the breakdown of food materials into its simplest form. The reactants of
enzyme catalyzed reactions are termed substrates and 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-catalyzed 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 and known as enzyme kinetics. The substrate
concentration, is one of the important parameter that affecting the rate of a reaction that catalyzed
by an enzyme. However, studying the effects of substrate concentration is elaborated by the fact
that during the course of an in vitro reaction, substrate changes due to the conversion of substrate
to product. In this experiment we can see how substrate concentration, pH and temperature effect
the enzyme activity.
Amylase is a type of enzyme. Amylase has an active site organized in subsites, each of
which accommodates a glucose residue (Talamond, Noirot & de Kochko, 2005). It breaks down
starch to glucose, giving food that sweet taste. An example of amylase in the natural world is in
bananas. When they are green, the amylase has yet to break down the starch, but by the time
theyve turned brown, the reaction has been completed. This is why brown bananas taste sweeter
than their green counterpart.
Objectives
Describe the relationship between substrate concentration and the maximum velocity of
an enzyme.
Theories
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 catalyzed 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 enzymecatalyzed 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 catalyzed 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 catalyzed reaction
under substrate saturation is called the Vmax , Maximum velocity.
Figure 4.1: Graph of initial velocity against substrate concentration (Nelson, D.L et. al, n.d)
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 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 doublereciprocal presentation, also called a Lineweaver-Burk plot. The main advantage of LineweaverBurk plot is to determine the Vmax more accurately, which can only be approximated from a
simple graph of V0 versus S.
The enzyme Amylase can catalyze 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 where as 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
catalyzed 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 salycilic acid reagent. In an alkaline condition, the pale yellow colored
the 3, 5- dinitro salicylic acid undergo reduction to yield orange colored 3- amino -5-nitrosalicylic
acid. The absorbance of resultant solutions is read at 540nm. The intensity of color depends on
the concentration of reducing sugars produced.
Amylase
Starch
Maltose + glucose
Apparatus
1. Alpha Amylase enzyme
2. Starch
3. pH buffer solution (pH 4-9)
4. DNSA Reagent
5. Beaker
6. Measuring cylinder
7. Cuvette
8. Falcon tube rack
9. Falcon tube
10. Micropipet and tips
11. Label sticker
12. Schott bottle
13. Vortex mixer
14. Water bath
15. Spectrophotometer
16. Hotplate
Procedure
c) All 10 tubes is placed in the 37C water bath for about 5 minutes to allow the temperature to
equilibrate.
d) The content of each amylase test tube is poured into each starch test tube and mixed on vortex
mixer.
e) The tubes is returned to the 37C water bath.
f) The hydrolysis reaction is proceed for exactly 10 minutes.
g) The amylase activity is determined using the method given in Appendix 1.
h) Graph of pH vs. enzyme activity is plotted.
10
f) The content of each amylase is poured test tube into starch test tube and mix them on vortex
mixer.
g) Return the tubes to the 37C water bath.
h) Let the hydrolysis reaction proceed for exactly 10 minutes.
i) The amylase activity is determined using the method given in Appendix 1.
j) Graph of starch concentration against amylase activity is plotted.
Appendix 1 (Demonstration of Enzyme Activity)
a. After 10 minutes (the time of hydrolysis reaction), the reaction is stopped by adding 4 ml of
DNS reagent.
b. Then it is boiled for 10 minutes and then left to cool to room temperature.
c. The absorbance of the samples is measured at =540 nm.
d. The absorbance value is compared with glucose standard curve prepared to obtain the glucose
concentration.
e. The enzyme activity is calculated.
Note: Enzyme activity is the amount of glucose formed in reaction mixture per unit time.
11
Glucose
Absorbance Optical
No.
Concentration (g/L)
200
0.378
400
0.668
600
0.801
800
1.224
1000
2.052
The data on the table 7.1 above is used to plot the standard curve of absorbance optical density
(OD) (nm) against glucose concentration (g/L). These obtained results were recorded based on
the observation through the spectrophotometer which already set up at 540 nm.
y = 0.0018x
R = 0.89
1.5
1
0.5
0
200
400
600
800
1000
Concentration (g/L)
Figure 7.1: The standard curve of absorbance optical density (OD) (nm) against glucose
concentration (g/L)
The effect of pH to enzyme activity
Table 7.2: The values of absorbance optical density (OD) at five different pH Values.
No.
1.
pH Value
2.680
12
2.
5.170
3.
2.625
4.
2.546
5.
2.350
The table 7.2 above showed the reading of absorbance optical density (OD) (nm) which affected
by five different pH Value.
pH Value
Figure 7.2: The effect of absorbance Optical Density (OD) (nm) reading at different pH Values
The plotted graph as shown in figure 7.2 showed the five different pH Values which affect the
absorbance Optical Density (OD) (nm) reading. The absorbance Optical Density (OD) (nm)
reading was rapidly increasing from the pH 5 until pH 6 which known as the optimum pH for the
amylase enzyme and the absorbance Optical Density (OD) values is 5.170 nm.
Table 7.3: The values of enzyme activity at different pH
pH
Absorbance
Glucose
Glucose
Enzyme
value
reading (nm)
concentration,
released (mol)
activity, V
X (g/mL)
5
2.680
0.0015
(mol/min)
8.2610-6
8.2610-7
13
5.170
0.0029
1.5910-5
1.5910-6
2.625
0.0015
8.0910-6
8.0910-7
2.546
0.0014
7.8010-6
7.8010-7
2.350
0.0013
7.2610-6
7.2610-7
The table 7.3 above showed the calculated values that obtained according to the effect of five
different pH values against the reading of the absorbance optical density (OD) (nm).
Enzyme 0.000001
activity, V
0.0000008
(mol/min)
0.0000006
0.0000004
0.0000002
0
0
10
pH
No.
Temperature ( C)
14
1.
30
2.630
2.
40
5.350
3.
50
7.290
4.
60
8.070
The table 7.4 above showed the effect of four different temperatures on the reading of the
absorbance optical density (OD). The temperatures that required are in the range between 30 C
to 70 C only.
40
50
60
Temperature ( C)
Figure 7.4: The reading of absorbance Optical Density (OD) (nm) against temperature ( C)
The plotted graph in figure 7.4 showed the reading of absorbance Optical Density (OD) (nm) kept
increasing continuously against four different temperature which not exceed 70 C. The values of
absorbance Optical Density (OD) is more higher if the surrounding temperature not almost at
boiling temperature.
Table 7.5: The values of enzyme activity at four different temperatures
Temperature
Absorbance
Glucose
Glucose
Enzyme
(C)
reading (nm)
concentration,
released
activity, V
X (g/mL)
(mol)
(mol/min)
15
30
2.63
0.0015
8.3310-6
8.3310-7
40
5.32
0.0030
1.6710-5
1.6710-6
50
7.29
0.0041
2.2810-5
2.2810-6
60
8.07
0.0045
2.5010-5
2.5010-6
The table 7.5 above showed the effect of four different temperatures on the values of enzyme
activity for amylase enzyme.
Enzyme
activity, V 0.0000015
(mol/min) 0.000001
0.0000005
0
0
10
20
30
40
50
60
70
Temperature (C)
Substrate
Absorbanc
Glucose
Glucose
Enzyme
1/V
1/S
concentratio
e reading
concentration
released
activity, V
n (%)
(nm)
, X (g/mL)
(mol)
(mol/min)
0.5
4.06
0.0023
12.27x10-6
12.27x10-7
8.15105
2.00
1.5
3.39
0.0019
10.55x10-6
10.55x10-7
9.48105
0.67
2.0
3.26
0.0018
9.991x10-6
9.991x10-7
1.00105
0.50
2.5
2.66
0.0015
8.326x10-6
8.326x10-7
1.20105
0.40
3.0
2.18
0.0012
6.661x10-6
6.661x10-7
1.50105
0.33
The table 7.6 above showed the effect of five different substrate concentrations on the values of
enzyme activity for the amylase enzyme.
4
2
0
0
0.5
1.5
2.5
3.5
17
y = 3.8889x + 1.2327
R = 0.4161
1/V 6
4
2
0
0.33
0.83
1.33
1.83
2.33
1/S
Calculation
1. Determination of glucose concentration, X (g/mL)
From the standard curve of glucose that had been plotted as shown in figure 7.1, the linear
equation of the curve is presented as:
= 0.0018
Where; X = protein concentration and Y = absorbance reading. Therefore, to calculate protein
concentration,
=
0.0018
2.660
0.0018
1477.78
1
1 103
1000
= 0.0015 g/mL
18
( )
0.0015
Moles of glucose released (mol) =
1
180.1559
(/) =
8.326 106
10
[]
+
Double reciprocal;
1
1
1
=
+
=
19
= 1.2327
= 0.8112 mol/min
Finding value of Km, Michaelis constant
= 3.8889
Discussion
As the concentration of substrate increases, the rate of reaction also increases until the
point saturation occurs. It means as you increase the concentration, rate keeps increasing and
then one point comes when the maximum rate is achieved and there is no free enzyme to bind
with substrate and all the active sites of enzyme are bound to the substrate. So after that point,
increasing the concentration wont have any effect. The maximum for each enzyme is usually
given by Km value (michealis menten graph or the other one called Lineweaver burke plot). The
Km value is the rate constant or it can be explained as how much substrate concentration is
required by an enzyme to reach to the half of maximum rate or velocity of enzyme. Each enzyme
has different Km values. Wherever the Vmax occurs and it intersects the curve drawn for substrate
concentration and velocity (or rate of reaction), that point is the saturation point or maximum
substrate concentration to have maximum rate of the reaction.
From the graph, we can see the results follow what is stated in theory. As the concentration
increase, the enzyme activity decrease. This is because when there is too much of substrate, the
enzyme dont have enough space to growth. So, to get optimum production of product, we need
to provide balance amount of substrate and enzyme
The enzyme reaction have effect with change of temperature. Based on the plotted graph,
as the temperature increase, the absorbance Optical Density (OD) also increase. Enzyme usually
20
have its limit on temperature. When the temperature is very high, it will be denatured thus the
production of product decrease. From this experiment we can see that amylase enzyme still can
grow up to 60oC but the reaction of enzyme becomes slower. If this experiment is proceed with
higher temperature maybe the enzyme activity will decrease because starting from 50oC the
enzyme activity not have much increase. This is because the enzyme start to denatured.
pH can give several effect on structure and activity of an enzyme. For example, pH can
have an effect of the state of ionization of acidic or basic amino acids. Acidic amino acids
have carboxyl functional groups in their side chains. Basic amino acids have amine functional
groups in their side chains. If the state of ionization of amino acids in a protein is altered then the
ionic bonds that help to determine the 3-D shape of the protein can be altered. This can lead to
altered protein recognition or an enzyme might become inactive.
Changes in pH may not only affect the shape of an enzyme but it may also change the
shape or charge properties of the substrate so that either the substrate cannot bind to the active
site or it cannot undergo catalysis.
The most favorable pH value - the point where the enzyme is most active - is known as
the optimum pH.
21
There is fluctuation of value of absorbance that give the fluctuation of value of velocity
might because of the sample is contaminated. The other factor might be the time for taking reading
of absorbance for every pH sample is not fixed. Besides that, while taking reading of absorbance,
the water vapor from condensation of the sample is still there, so, it might affect the absorbance
reading.
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 7.3 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 and 9. When the enzyme activity
increase the absorbance reading also increase. By looking at figure 7.2 and figure 7.3, 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 figure 7.2 and 7.3.
At very high and very low temperatures we expected the absorbance or enzyme activity
be low. The highest absorbance should have appeared at room temperature, because most
human enzyme activity occurs at room temperature but in this case using enzyme amylase, we
still cannot find the optimum temperature as we can see in figure 7.4 and figure 7.5 the graph did
not shows any decreasing in enzyme activity. For me I think maybe there is an error during
conducting experiment or maybe the amylase have high optimum temperature.
For the substrate concentration, we can see in figure 7.6 that as the percent of substrate
concentration increase, the enzyme activity increase.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,
22
we were forced to assume that catalase was present in the extract, an assumption that may, or
may not have, been correct.
Recommendations
This experiment must be carried out under the laminar flow hood for sterility to prevent
any contamination from the surrounding directly attached to the culture.
Properly, wash hand with plenty of water and appropriate soap after handling the culture
which can expose to the health.
Worn the appropriate gloves that provided and disinfect the work area with Ethanol (70%
ethanol for swabbing for sterility) before handling the culture.
Avoid the parallax error to be occurring during the measuring volume or amount of reagent
and solution by using provided apparatus. This can affect the concentration of solution
which indirectly interrupts the absorbance optical density (OD) values.
The amylase is easier to expose against the surrounding contaminants especially through
the air.
Always ensure the cuvette must be wiped cleanly to prevent any scratch that would affect
the spectrophotometer reading on absorbance optical density (OD).
Dispose of all contaminated materials after taking the reading of absorbance optical density
(OD) by using the spectrophotometer in appropriate containers.
Reference
Anonymous, (n.d). Effect of pH on enzyme. Retrieved on October 15, 2015
from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm
from
Lactobacillus
Fermentum
on
Maltooligosaccharides.Journal
of
23
Appendix
25