UNIVERSITI TEKNOLOGI MARA

FAKULTI KEJURUTERAAN KIMIA

BIOPROCESS ENGINEERING LABORATORY

NAME AND MATRIC NO : MUHAMMAD FAISAL BIN NOOR JAMIN

(2016239034)

GROUP :2

EXPERIMENT : LAB 5 (INVESTIGATION ON ENZYME ACTIVITY

AND KINETICS)

SEMESTER :5

PROGRAMME CODE : EH220 5E

SUBMIT TO : MDM 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. Appendices 5
TOTAL 100

Remarks:
Checked by: Rechecked by:
……………………………......... ……………………………..

Date: Date:

1
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 & 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 λ=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.

2
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 they’ve turned brown, the
reaction has been completed. This is why brown bananas taste sweeter than their green counterpart.

3
Objectives

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

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 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 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 V 0 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.

4
 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;

5
 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-Burk plot. The main advantage of Lineweaver- Burk plot is to
determine the Vmax more accurately, which can only be approximated from a simple graph of V0 versus
S.

Figure 4.2: Lineweaver-Burk plot. (Nelson, D.L et. al, n.d)

6
The enzyme α Amylase can catalyze the hydrolysis of internal α -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.

α Amylase
Starch Maltose + glucose

Figure 4.3: The enzyme-substrate reaction example. (vlab.amrita.edu, 2011)

7
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

8
Procedure

i. Preparation of 2% Starch Solution

a) 4 g of soluble starch is mixed in approximately 50 ml of cold water.

b) While stirring, the slurry is added to approximate 100 ml of gently boiling water in a large beaker.
c) Then the final volume of 200ml is topped up and mix well.

ii. Effect of pH on the activity and stability of amylase enzyme.

a) Five test tubes is labelled with pH 5, 6, 7, 8 and 9. In each tube, 1 mL of 2% starch solution is placed
and 1 mL of the appropriate buffer is added (at corresponding pH) to each tube.
b) Five additional clean test tubes is added and 2 mL of amylase solution was put in each tube.
c) All 10 tubes is placed in the 37°C 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 37°C 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.

iii. Effect of temperature on the activity and stability of amylase enzyme.

a) One test tube is labelled with 30ºC. In the tube, 1 mL of 2% starch solution and 1 mL of pH=7 buffer is
placed to the tubes.
b) Additional clean test tub is added and 2 mL of amylase solution is put in the tube.
c) Both tubes is placed in the 30°C water bath for about 5 minutes to allow the temperature to equilibrate.
d) The contents of the amylase is poured test tube into starch test tube and mix them on vortex mixer.
e) Return the tubes to the 30°C water bath.
f) Let the hydrolysis reaction proceeded for exactly 10 minutes.
g) The amylase activity is determined using the method given in Appendix 1.
h) Step a-g is repeated at 4 different temperatures ranging from 30-70 ºC.
i) Graph of temperature vs. amylase activity is plotted.

9
iv. Effect of substrate concentration on the activity of amylase enzyme.
a) Starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and 3.0% w/v) is prepared as the substrate.
b) Each tube is labelled with starch concentration and place 1ml of each starch solution into the test tubes.
c) 1 mL of pH=7 buffer is added to the tubes.
d) Five additional clean test tubes is added and put 2 mL of amylase solution in each tube.
e) All tubes is placed in the 37°C water bath for about 5 minutes to allow the temperature to equilibrate.

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 37°C 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 λ=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.

Appendix 2 (Glucose Standard Curve Preparation)

a. The standard solutions of glucose is prepared at five different concentrations ranging from 0-
1000mg/L by serial dilution.
b. 1 ml of each glucose solution is added in test tubes.
c. 1 ml of DNS reagent is added in each tube and mix for few seconds on vortex mixer.
d. The test tubes is placed in water bath (T=100°C) for 10 min and then left to cool at room

10
Results
The obtained results that being recorded from the experiment of investigation on enzyme activity and
kinetics.

Glucose Standard Curve

Table 7.1: The values of absorbance optical density (OD) at five different concentration of glucose
Glucose Absorbance Optical Density
Concentration (OD)(nm)

0.8 0.066
1 0.14
1.2 0.46
1.4 0.702
1.6 0.9
1.8 1.24

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.

Absorbance Optical Density vs Glucose

Concentratiom
1.4
Absorbance Optical Density (OD)(nm)

1.2 y = 0.181x
R² = 0.9087
1
0.8
0.6
0.4
0.2
0
1 2 3 4 5 6
-0.2
Glucose concentration

Figure 7.1: The standard curve of absorbance optical density (OD) (nm) against glucose concentration
(g/L)

11
The effect of pH to enzyme activity

Table 7.2: The values of absorbance optical density (OD) at five different pH Values

pH Absorbance Glucose Glucose Released Enzyme Activity, V

Optical Density Concentration, X (mol) (mol/min)
(OD)(nm) (g/mL)
5 0.52 2.87293E-06 1.59469E-08 1.59469E-09

6 0.564 3.11602E-06 1.72963E-08 1.72963E-09

7 0.584 3.22652E-06 1.79096E-08 1.79096E-09

8 0.474 2.61878E-06 1.45362E-08 1.45362E-09

9 0.636 3.51381E-06 1.95043E-08 1.95043E-09

The table 7.2 above showed the reading of absorbance optical density (OD) (nm) which affected by five
different pH Value.

Absorbance Optical Density (OD) (nm) vs pH

value
0.7
Absorbance Optical Density (OD) (nm)

0.6
0.5
0.4
0.3
0.2
0.1
0
0 2 4 6 8 10
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.
12
 Table 7.3: The values of enzyme activity at different pH

pH Absorbanc Glucose Glucose Enzyme

value e reading concentration released activity, V
(nm) , X (g/mL) (mol) (mol/min)
-6
5 2.6 0.0015 8.26×10 8.26×10-7
80
6 5.17 0.0029 1.59×10-5 1.59×10-6
0
7 2.62 0.0015 8.09×10-6 8.09×10-7
5
8 2.54 0.0014 7.80×10-6 7.80×10-7
6
9 2.35 0.0013 7.26×10-6 7.26×10-7
0

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 Activity against pH value

2.5E-09
Enzyme Activity, V (mol/min)

2E-09

1.5E-09

1E-09

5E-10

0
5 6 7 8 9
pH value

Figure 7.3: The effect of enzyme activity against pH values

Based on the observation, the graph that plotted in the figure 7.3 showed the higher values of enzyme
activity was occurred at pH 6 or optimum pH. The enzyme is more reproducible during the optimum
pH.

13
 The effect of temperature on the activity and stability of amylase enzyme

Table 7.4: The values of absorbance optical density (OD) at four different temperatures (ͦ C).

Temperature Absorbance Glucose Glucose Released Enzyme Activity,

(ᴼC) Optical Density Concentration, X (mol) V (mol/min)
(OD)(nm) (g/mL)
30 0.587 3.24309E-06 1.8E-08 1.8E-09

40 0.615 3.39779E-06 1.89E-08 1.89E-09

50 0.69 3.81215E-06 2.12E-08 2.12E-09

60 0.811 4.48066E-06 2.49E-08 2.49E-09

70 0.573 3.16575E-06 1.76E-08 1.76E-09

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.

A Graph of absorbance Optical Density (OD)

against Temperature (ᴼC)
0.9
Absorbance Optical Density (OD)(nm)

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
30 40 50 60 70
Temperature (ᴼC)

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.

14
 Table 7.5: The values of enzyme activity at four different temperatures

Temperature Absorbance Glucose Glucose Released Enzyme Activity,

(ᴼC) Optical Concentration, X (mol) V (mol/min)
Density (g/mL)
(OD)(nm)
30 0.587 3.24309E-06 1.8E-08 1.8E-09

40 0.615 3.39779E-06 1.89E-08 1.89E-09

50 0.69 3.81215E-06 2.12E-08 2.12E-09

60 0.811 4.48066E-06 2.49E-08 2.49E-09

70 0.573 3.16575E-06 1.76E-08 1.76E-09

The table 7.5 above showed the effect of four different temperatures on the values of enzyme activity for
amylase enzyme.

Enzyme activity, V vs Temperature (ᴼC)

3E-09
Enzyme Activity, V (mol/min)

2.5E-09

2E-09

1.5E-09

1E-09

5E-10

0
30 40 50 60 70
Temperature (ᴼC)

Figure 7.5: The values of enzyme activity at four different temperatures

The graph that plotted as shown in the figure 7.5 showed the values of enzyme activity that affected by
the four different temperatures at range between 30 ͦ C to 70 ͦ C only. The values of enzyme activity kept
increasing as the enzyme is more reproducible under the optimum temperature that not exceeds 70 ͦ
C. The higher temperature at which the enzyme is operating at is well above 100oC, and then thermal
deactivation can occur which this condition also known as denaturation.

15
 The effect of substrate concentration on the activity of amylase enzyme

substrate Absobance Glucose Glucose Enzyme 1/V 1/S

concentration Reading concentration Released Activity
0.5 0.767 4.23757E-06 2.35217E- 2.35217E- 425139737.9 2
08 09
1.5 0.76 4.1989E-06 2.3307E- 2.3307E- 429055498.7 0.666667
08 09
2 0.72 3.9779E-06 2.20803E- 2.20803E- 452891915.3 0.5
08 09
2.5 0.575 3.1768E-06 1.76336E- 1.76336E- 567099441.7 0.4
08 09
3 0.511 2.8232E-06 1.56709E- 1.56709E- 638125594.9 0.333333
08 09

The table 7.6 above showed the effect of five different substrate concentrations on the values of enzyme
activity for the amylase enzyme.

Graph of enzyme activity vs substrate

concentration
2.5E-09
Enzyme Activity, V (mol/min)

2E-09

1.5E-09

1E-09

5E-10

0
0 0.5 1 1.5 2 2.5 3 3.5
Substrate Concentration (%)

Figure 7.6: The values of enzyme activity against substrate concentration

Based on the observation, the graph that plotted in figure 7.6 showed the effects of substrate concentration
against the values of enzyme activity. The values kept decreasing since the amount of substrate
concentration kept increasing as well. This condition showed that the enzyme activity a bit slow or being
inhibited by the increasing of substrate concentration.

16
 A graph of 1/V against 1/S
7

4
1/V

3
y = -0.8182x + 5.6622
2 R² = 0.354

0
0 0.5 1 1.5 2 2.5
1/S

Figure 7.7: The values of 1/V that affected by the 1/S

Overall the graph that plotted in figure 7.7 showed the 1/S can affect the values of 1/V. Based on the
observation, the 1/V values were keeping decreased as the 1/S values were keeping longer. The equation
that being presented in this graph is 𝑦 = 3.8889𝑥 + 1.2327 and 𝑅2 = 0.4161.

17
Calculation

Determination of glucose concentration X

From the standard curve of glucose that has been plotted, the liner equation of the curve is presented
as:

Y = 0.181X

Where X = protein concentration and Y = absorbance reading. Therefore, to calculate protein

concentration,
𝑌
X=
0.181

0.760
X = 0.181

4.204 1𝐿 1 𝑥 10−3
X= x 1000𝑚𝐿 x
𝐿 𝑚𝑖𝑙𝑙𝑖

X = 4.204x10-6

Determination of glucose released (mol)

MW of glucose = 180.1559 g/mol; Volume of enzyme (amylase) = 1 mL

4.204x10−6 g/mL
Moles of glucose released (mol) = x 1 mL
180.1559 𝑔/𝑚𝐿

Moles of glucose released (mol) = 2.33 x 10-8 mol

Determination of enzyme activity, V (mol/min)

Duration of hydrolysis reaction: 10 minutes

𝑚𝑜𝑙 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑
Enzyme Activity (mol/min) = 𝐻𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒

2.33 x 10−8 mol

Enzyme Activity (mol/min) = 10 𝑚𝑖𝑛

Enzyme Activity (mol/min) = 2.33 x 10-9 mol/min

18
Equation for Michaelis-Menten
𝑉𝑚𝑎𝑥 [𝑠]
V= 𝐾𝑚+𝑆

Double reciprocal:
1 𝐾𝑚 1 1
= 𝑉𝑚𝑎𝑥[𝑆] 𝑆 + 𝑉𝑚𝑎𝑥
𝑉

1
y – axis = 𝑉

1
x – axis = 𝑆

1
Intercept = 𝑉𝑚𝑎𝑥

𝐾𝑚
Slope = 𝑉𝑚𝑎𝑥

From graph, the linear equation obtained is:

𝑦 = −0.8182𝑥 + 5.6622

Finding value of Vmax, maximum enzyme activity:

1
= 5.622
𝑉𝑚𝑎𝑥

𝑉𝑚𝑎𝑥 = 0.178 𝑚𝑜𝑙/𝑚𝑖𝑛

Finding value of Km, Michaelis Constant

𝐾𝑚
= −0.8182
𝑉𝑚𝑎𝑥

𝐾𝑚 = (−0.8182)(0.178) = −0.146

19
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 won’t
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 don’t 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 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 60 oC 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

20
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.

Figure 9.1: Effect of pH on reaction rate (Anonymous, n.d)

In this experiment, the pH is increases from pH of 5 up to pH 9. But, starting from pH 6, the

enzyme activity decreases from 1.59×10-6 for pH 6 to 8.09×10-7 for pH 7. After that, the enzyme activity
decrease until pH 9 with value of 7.26×10-7. Based on this result of experiment, the optimum pH that is
with highest velocity is sample pH 6 with velocity 0.2488. The lowest velocity is pH 1.
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.

21
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,

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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 David

L. Nelson, Michael M. Cox , Lehninger principles of biochemistry, 4th edition.

vlab.amrita.edu,. (2011). Effect of Substrate Concentration on Enzyme Kinetics. Retrieved 15 October 2015,
from vlab.amrita.edu/?sub=3&brch=64&sim=1090&cnt=1
Talamond, Pascale, Michel Noirot, and Alexandre De Kochko. “The Mechanism of Action of α- amylase
from Lactobacillus Fermentum on Maltooligosaccharides.”Journal of Chromatography B (2005):
42-47. Science Direct. Web.

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Appendix

Figure 12.1: Absorbance Optical Density Figure 12.2: The effect of substrate
(OD) of Glucose concentration

Figure 12.3: The effect of pH values Figure 12.4: The effect of temperatur e

Figure 12.5: Spectrophotometer Figure 12.6: Test tube

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