1.

ABSTRACT

Enzymes are catalysts which lower the activation of chemical reactions, thus making
activity happen more rapidly (Adam C. 2006). 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, study the relationship between substrate
concentration and the maximum velocity of an enzyme, and obtain the Michaelis-Menten
parameters, effect of pH and temperature on enzyme activity and kinetics of inhibition.
The 2% starch solution and amylase solution as enzyme solution was prepare first to be
mix for study the effects of all parameters in objective of this experiments. From the
results of experiment, the optimum pH for this enzyme (amylase) is 7 based on Figure
7.1. Next, optimum temperature for amylase is about 60 , and amount of the enzyme
is kept constant and the substrate concentration is then gradually increased, as the
reaction velocity will increase until it reaches a maximum. Lastly, from Figure 9.3 and
Figure 9.4 calculation based on Michaelis-Manten constant, the maximum velocity, V max
for amylase is 0.4647 mol/min. Meanwhile, the Michaelis constant,K m is 1.2291
g.min/mol.mL.

2.0

INTRODUCTION

Amylase is digestive enzymes which hydrolyze glycosidic bonds in starch to glucose,

maltose, maltotriose and dextrin (Singh et. al., 2011). They have a lot of potential
applications in both food and pharmaceutical industries. Amylases are produced by
microorganisms using submerged and solid state fermentation. However, solid state
fermentation is more convenient because of usage of cheap substrate, high yields and
avoids substrate inhibition.
In order for cells to function properly, the activity of enzyme is crucial. Enzyme catalyzes
reactions metabolically. The enzyme reacts in an intracellular environment, it also
allowed for the control and stabilization of the process. The behavior of enzyme in
response to different concentrations, which referred to as enzyme kinetics, includes the
basic characteristics of each type of enzyme. This behavior is responsible for most of the
reaction occurred in biological systems (McCarty P. 2012).

The unique amino acids sequence is the reason behind the specific reaction of each
enzyme. Each enzyme has its different structure and the active site also has specific
shape. This allows only certain out of thousands of compound present in the cell that it
can interact with (Nelson D. 2008).The activity of enzyme can be interfered by blockage
of the active site and this also impedes the efficiency of the enzyme.
In food industry, amylase is commonly used enzyme to catalyze reaction. The main
advantages of amylase are that easy control of pH and temperature to achieve optimum
enzyme activity. Bacterial amylases liquefy starch at higher temperatures. They are used
to produce glucose and maltose syrups and to replace malted grain for brewing (Fellows
P., 2000).
Hydrolysis is a common mechanism used by enzyme to break chemical bond. In this
mechanism, amylase hydrolyzes linkages to liquefy starch and produce maltose. A
molecule of water is split during the hydrolysis and the OH- and H+ ions binds to the
exposed ends of the broken starch polymer.

Figure 2.0 Hydrolysis of starch by amylase

3.0

OBJECTIVES
1. To determine the effects of temperature on the enzymatic activity and changes in
enzyme concentration of an enzyme-catalyzed reaction.
2. To study the relationship between substrate concentration and the maximum
velocity of an enzyme.
3. To obtain the Michaelis-Menten parameters, effect of pH and temperature on
enzyme activity and kinetics of inhibition.

4.0

THEORY
2

In a kinetic study of enzyme, the effect of velocity for a certain reaction changes is to be
determined when concentration of substrate varies in the presence of a constant
concentration of an enzyme. Therefore, it is important to measure the initial velocity (V 0)
as to obtain the Vmax. If we plot V0 against substrate concentration [S], we will see the
following curve, called a Michaelis-Menten curve, shown below (Micheal S. & Fikret K.
2002).
Kinetics of simple enzyme-catalyzed reactions is often referred to as Michaelis-Menten
kinetic. The Michealis-Menten curve was shown in the figure below (Fogler H., 2006).
The reciprocal of Michaelis Menten become the Lineweaver Burk plot.

Figure 3.0 Michaelis-Menten curve

Michealis- Menten,

Lineweaver Burk plot,

Figure 3.1 Lineweaver-Burk plot

V 0=

V max X [S]
K m+[S ]

K +[ S]
1
= m
V 0 V max X [S ]

Where
V0 or Vi= Initial velocity (moles/time)
[S] = Substrate concentration
Vmax = Maximum velocity
Km = Michaelis Menten constant

There are several factors that are especially important in determining the enzymes
shape and these are closely regulated both in the living organism and in laboratory
experiments to give the optimum or most efficiency enzyme activity. The factors are
temperature, pH, salt concentration and other small molecules (Fogler H., 2006)
However, in this experiment, the factors involved are the temperature, pH and substrate
concentration. Variations in the pH of the medium result in changes in the ionic form of
the active site. Hence, explaining the existence of optimal pH and temperature in
enzyme activity. The effect of pH and temperature on enzyme activity is shown in the
figures below (Harvey B. and Douglas C., 1997).

Figure 3.2 Typical pH profile of enzyme activity

Figure 3.3 Typical temperature profile of enzyme activity

5.0

APPARATUS AND MATERIALS

APPARATUS
1. Beaker
2. Measuring cylinder
3. Cuvette
4. Falcon tube rack
5. Falcon tube
6. Micropipette and tips
7. Label sticker
8. Schott bottle
9. Vortex mixer
10. Spectrophotometer
11. Hot plate
12. Digital balance

Figure 5.0 Spectrophotometer

MATERIALS
1. Alpha amylase enzyme
2. Starch
3. pH buffer solution (pH 4-9)
4. DNSA reagent
5. Water bath (37C)

Figure 5.1 Vortex mixer

*Other equipment can be found in appendix

6.0

PROCEDURES
A. PREPARATION OF 2% STARCH SOLUTION
5

1) 4g of soluble starch is mixed in approximately 50ml of cold water.

2) The slurry is added to approximately 100ml of gently boiling water while stirring in
a large beaker.
3) Then, the final volume of 200ml is top up and it is mixed well.
B.
1)
2)
3)
4)

EFFECT OF PH
Five test tubes are labeled with pH 5,6,7,8 and 9.
In each tube, 1ml of 2% starch solution is placed.
1ml of appropriate buffer is added at corresponding pH to each tube.
Five additional clean test tubes are prepared and 2ml of amylase solution is

added in each tube.

5) All ten tubes are 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.
1)
2)
3)
4)

EFFECT OF TEMPERATURE
A test tube is labeled with 30C.
In the tube, 1ml of 2% starch solution is placed.
1ml of buffer with pH 7 is added to each tube.
An additional clean test tube is prepared and 2ml of amylase solution is added

the each tube.

5) Both tubes are placed in the 30C water bath for about 5 minutes to allow the
temperature to equilibrate.
6) The content of the amylase test tube is poured into the starch test tube and they
are mixed on vortex mixture.
7) The tubes are returned to the 30C 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) The steps 1-9 are repeated with different temperatures ranging from 40C until
70C.
11) A graph of temperature versus amylase activity is plotted.
D. EFFECT OF SUBSTRATE CONCENTRATION
1) Starch solutions of varying concentration (0.5, 1.5, 2.0, 2.5, and 3.0% w/v)are
prepared as the substrate.
2) Each test tubes are labeled with starch concentration and 1ml of starch solution
is added into each test tube.
3) 1 ml of buffer (pH 7) is added into the test tubes.
4) Five additional clean test tubes are prepared and 2ml of amylase solution is
added in each tube.
6

5) All the tubes are 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 starch concentration versus enzyme activity is plotted.
Appendix 1 (Demonstration of enzyme activity)
1. The reaction is stopped after 10 minutes (the time of hydrolysis reaction) by
adding 4ml of DNS reagent.
2. The samples are boiled for 10 minutes and then they are cooled to room
temperature.
3. The absorbance of each sample is measured by using spectrophotometer at =
540nm.
4. The absorbance value is compared with glucose standard curve that has been
prepared to obtain the glucose solution.
5. The enzyme activity (the amount of glucose formed in reaction mixture per unit
time) is calculated.
Appendix 2 (Glucose standard curve preparation)
1. Standard solutions of glucose are prepared at five different concentrations
ranging from 0 mg/L until 1000mg/L by serial dilution.
2. 1ml of each glucose solution is put into the test tubes.
3. 1ml of DNS reagent is added in each test tube and they are mixed for a few
seconds on vortex mixer.
4. The test tubes are placed in water bath (100C) for 10 minutes and cooled at
room temperature.
5. The absorbance of each sample is measured by using spectrophotometer at =
540nm.
6. The standard curve of absorbance versus glucose concentration is drew. The
graph is in straight line for absorbance less than 0.7.

7.0

RESULTS

APPENDIX 1
a) Effect of pH on the activity and stability of amylase enzyme
7

pH

Absorbance,
OD (nm)

Glucose

Glucose

Enzyme

concentration

released

activity, V

(mol)
4.975110-7

(mol/min)
4.975110-8

0.242

, X (g/mL)
8.963010-5

0.084

3.111110-5

1.726910-7

1.726910-8

0.145

5.370410-5

2.980910-7

2.980910-8

0.069

2.555610-5

1.418510-7

1.418510-8

0.382

1.414810-5

7.853310-7

7.853310-8

Enzyme activity against pH

9
8
7
6
5
Enzyme activity, V (10-8 mol/min) 4
3
2
1
0
4

10

pH

Figure 7.1: Graph of pH versus Enzyme Activity

b) Effect of temperature on the activity and stability of amylase enzyme

Temperature

Absorbance,

(C)

OD (nm)

Glucose

Glucose

Enzyme

concentration,

released (mol)

activity, V

30

0.039

X (g/mL)
1.444410-5

40

0.961

3.559310-4

-8

8.017710

1.975710-6

(mol/min)
8.017710-9
1.975710-7
8

50

1.798

6.659310-4

3.696410-6

3.696410-7

60

2.844

1.053310-3

5.846810-6

5.846810-7

70

0.389

1.440710-4

7.997210-7

7.997210-8

Enzyme Activity against Temperature

70
60
50
40
Enzyme activity (10-8 mol/min) 30
20
10
0
20

30

40

50

60

70

80

Temperature (C)

Figure 7.2: Graph of Temperature versus Amylase Activity

c) Effect of substrate concentration on the activity and stability of amylase

enzyme

Substrate

OD

Glucose

Glucose

Enzyme

conc

reading

conc, X

released

activity, V

1/V

(%)
0.5

(nm)
0.131

(g/mL)
4.8510-5

(mol)
2.6910-7

(mol/min)
2.6910-8

3.71107

2.00

1.0

0.031

1.1510

-5

-8

-9

1.5710

1.00

1.5

0.276

2.0
2.5
3.0

1/S

6.3710

6.3710

1.0210-4

5.6710-7

5.6710-8

1.76107

0.67

0.284

1.0510-4

5.8410-7

5.8310-8

1.71107

0.50

0.181

6.7010

-5

-7

3.7210

-8

0.40

5.9410

-4

3.3010

-7

0.33

1.604

3.7210

-6

3.3010

2.6910
3.0310

Enzyme Activity against Substrate Concentration

7
6
5
4
Enzyme activity (10-8 mol/min) 3
2
1
0
0

0.5

1.5

2.5

3.5

Substrate concentration (%)

Figure 7.3: Graph of Substrate Concentration versus Amylase Activity

1/V against 1/[S]

18
16
14
12
10
1/V 8
6
4
2
0
0.2

f(x) = 2.65x + 2.15

0.4

0.6

0.8

1.2

1.4

1.6

1.8

2.2

1/[S]

Figure 7.4: Lineweaver-Burk Plot

10

Maximum enzyme activity, Vmax = 0.4647 mol/min

Michaelis constant, Km = 1.2291 g.min/mol. mL
APPENDIX 2
Glucose concentration (mg/L)

Absorbance, OD (nm)

200

0.357

400

0.249

600

0.633

800

3.016

1000

2.922

Absorbance vs Glucose concentration

3.5
3
2.5

f(x) = 0x

2
Absorbance, OD 1.5
1
0.5
0
100 200 300 400 500 600 700 800 900 1000 1100
Glucose concentration (mg/L)

Figure 7.5: Standard Curve of Absorbance versus Glucose Concentration

11

8.0

CALCULATIONS

Sample calculations
Effect of substrate concentration on the activity and stability of amylase enzyme
When substrate concentration (%) = 1.0
Molecular weight of glucose, MWglucose = 180.1559 g/mol
Volume of amylase = 1 mL
Hydrolysis reaction time = 10 mins
Standard curve equation (from figure 7.5), y = 0.0027x
y : OD reading

x : glucose concentration
3

Glucose concentration, x =

0.0027
1L
10

=
y
1000 mL milli

Mole of glucose released =

X
V amylase
MW glucose
g
mL
1.0 mL
g
180.1559
mol

1.1510-5 g/mL

1.15 105
Moles of glucose released =

Enzyme activity, V =

= 6.3710-8 mole

moles of glucosereleased
hydrolysis reactiontime

12

Enzyme activity, V =

6.37 108 mole

10 mins

=6.3710-9 mole/min

Determination of Michaelis-menten parameters

V=

V max [ S ]
K m +[S]

Double reciprocal:

1 Km 1
1
=
+
V V m [ S] V max
y = m x +

y-axis :

1
V

x-axis :

1
[S ]

Slope, m =

Km
Vm
1

y-intercept =

V max

From figure 7.5, the linear equation obtained is:

y = 2.6451 x + 2.152

1
y-intercept, V max

= 2.152

13

Vmax =

1
=0.4647 mol/min
2.152

Slope, m =

Km
Vm

Km
Vm

= 2.6451

= 2.6451

Km = 2.6451 (0.4647) = 1.2291 g.min/mol. mL

9.0

DISCUSSION

Enzymes, proteins that act as catalysts, are the most important type of protein. Catalysts
speed up chemical reactions and can go without being used up or changed. Without
enzymes, the biochemical reactions that take place will react too slowly to keep up with
the metabolic needs and the life functions of organisms.
There are four factors that will have an effect on the structure of an enzymes active site,
the activity of the enzyme, and the rate of the reaction in which the enzyme is involved.
The four factors that can affect the activity of an enzyme include temperature, pH,
enzyme concentration, and substrate concentration. However, for this experiment, only 3
parameters are tested which are temperature, pH and substrate concentration.

14

Enzyme activity against pH

10
8
6
Enzyme activity, V (10-8 mol/min)

4
2
0
4

10

pH

Figure 7.1: Graph of enzyme activity against pH

Enzymes are affected by changes in pH. The most favourable pH value the point where
the enzyme is most active. In other words, each enzyme has an optimum pH at which it
works best. However, extremely high or low pH values generally result in complete loss
of activity for most enzymes. At very high or very low pH's, these bonds within the
enzyme can be disrupted, and it can lose its shape. If it loses its shape, the active site
will probably be lost completely. This is essentially the same as denaturing the protein by
heating it too much.
Based on Figure 7.1, the optimum pH for this enzyme (amylase) is 7. Compared to
theory, the graph should have a bell shape. However, for this experiment, the only bell
shape occur was during pH 6 to pH 8. There might be errors occur during the reading of
pH 5 and pH 9 which supposed the value should be lower than pH 6 and pH 9
respectively.

15

Enzyme Activity against Temperature

80
60
Enzyme activity (10-8 mol/min)

40
20
0
20

30

40

50

60

70

80

Temperature (C)

Figure 7.2: Graph of Enzyme activity against temperature

Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the
temperature is raised. In the case of enzymatic reactions, this is complicated by the fact
that many enzymes are adversely affected by high temperatures. Over a period of time,
enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5C
or below is generally the most suitable. Some enzymes lose their activity when frozen.
The temperature at which the rate is fastest is called the optimum temperature for that
enzyme. Different enzymes have different optimum temperatures. Some enzymes, for
example, in organisms known as thermophiles or extremophiles are capable of working
at temperatures like 80C or even higher. The optimum temperature for a particular
enzyme varies depending on how long it is exposed to the higher temperatures.
Enzymes can withstand temperatures higher than their normal optimum if they are only
exposed to the higher temperatures for a very short time.
As shown in Figure 2, the reaction rate increases with temperature to a maximum level,
then abruptly declines with further increase of temperature. Therefore, the optimum
temperature for amylase is about 60

16

Enzyme Activity against Substrate Concentration

40
30
Enzyme activity (10-8 mol/min)

20
10
0
0

0.5

1.5

2.5

3.5

Substrate concentration (%)

Figure 7.3: Graph of enzyme activity against substrate concentration

1/V against 1/[S]

20
15
1/V

10
5

f(x) = 2.65x + 2.15

0
0.2

0.4

0.6

0.8

1.2

1.4

1.6

1.8

2.2

1/[S]

Figure 7.4: Lineweaver-Burk plot

It has been shown experimentally that if the amount of the enzyme is kept constant and
the substrate concentration is then gradually increased, the reaction velocity will
increase until it reaches a maximum. After this point, increases in substrate
concentration will not increase the velocity.
It is theorized that when this maximum velocity had been reached, all of the available
enzyme has been converted to ES, the enzyme substrate complex. This point on the

17

graph is designated as

V max . Using this maximum velocity and equation, Michaelis

developed a set of mathematical expressions to calculate enzyme activity in terms of

reaction speed from measurable laboratory data.
Michaelis constants have been determined for many of the commonly used enzymes.
The size of Km tells us several things about a particular enzyme. A small

Km

indicates that the enzyme requires only a small amount of substrate to become
saturated. Hence, the maximum velocity is reached at relatively low substrate
concentrations. A large

Km

indicates the need for high substrate concentrations to

achieve maximum reaction velocity.

For very, very low substrate concentrations, the graph is almost a straight line which
indicate that the rate is proportional to the substrate concentration. But as concentration
increases, increasing the concentration even more has less and effect. This is because,
the reaction has reaches its maximum rate. Therefore, increasing the concentration any
more makes no difference to the rate of the reaction.
The reason for this is actually fairly obvious. After a certain concentration of substrate is
reached, every enzyme molecule present in the mixture is working as fast as it can. If
the substrate concentration is increased any more, there aren't any enzyme molecules
free to help the extra substrate molecules to react.
As shown in Figure 9.3, Figure 9.4 and calculation based on Michaelis-Manten constant,
the maximum velocity,
constant,

10.0

V max

for amylase is 0.4647 mol/min. Meanwhile, the Michaelis

K m is 1.2291 g.min/mol.mL.

CONCLUSION

As a conclusion, the experiment was successfully conducted as all the objectives of the
experiment had been achieved which are to determine the effects of temperature on the
18

enzymatic activity and changes in enzyme concentration of an enzyme-catalysed

reaction, to describe the relationship between substrate concentration and the maximum
velocity of an enzyme, and to estimate Michaelis-Menten parameters, effect of pH and
temperature on enzyme activity and kinetics of inhibition. At pH 9, the enzyme is most
active with the amount of enzyme activity which is 7.853310 -8 mol/min. This is called as
optimum pH. For temperature, enzyme is most active at 600C with the highest amount of
enzyme activity which is 5.846810 -7 mol/min. For substrate concentration, the enzyme
activity increase when the substrate concentration increase and the enzyme become
most active at concentration of 3.0%. For Michaelis-Menten parameters which is V max
and Km, the values are 0.4647 mol/min and 1.2291 g.min/mol. mL respectively. A small
Km shows that the enzyme needs only a small amount of substrate to become
saturated. So, the maximum velocity is reached at relatively low substrate
concentrations.

11.0

RECOMMENDATIONS

1. Before taking the absorbance reading, the cuvette must be wiped as clean as
possible to prevent any scratch that would affect the spectrophotometer reading.
2. Shake the cuvette with vortex shaker before recording the OD to get more
accurate reading.
3. Prepare appropriate stand for cuvette to avoid any accident and spillage.
4. Do the zeroing before taking any OD value to obtain more accurate result.
5. Close the beaker containing water bath and test tubes to maintain the water bath
temperature.
6. Make sure the position of eye is perpendicular to the reading during measuring
the volume using beaker to avoid error.

12.0

REFERENCES

1. Clark, D. S., & Blanch, H. W. (1996,1997) . Biochemical engineering. Marcel Decker.

2. Fellows, P.J., (2000). Food processing technology: Principles and practice.
Woodhead Publishing Limited.
3. Fogler, H. S. (2006). Elements of chemical reaction engineering. New York: Prentice
Hall.
4. McCarty, P. L. (2012). Environmental biotechnology: principles and applications. Tata
McGraw-Hill Education.
19

5. Nelson, D. L., Lehninger, A. L., & Cox, M. M. (2008). Lehninger principles of

biochemistry. Macmillan.
6. Retrieved November 13, 2016 from
users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Carbohydrates.html
7. Shuler, M. L., & Kargi, F. (2002). Bioprocess engineering. New York: Prentice Hall.
8. Singh, S., Sharma, V., Soni, M. L., & DAS, S. (2011). Biotechnological applications of
industrially important amylase enzyme. International Journal of Pharma and Bio
Sciences, 2, 486-496.
9. Adam, C. (2003). Enzymes Kinetics Lab- The Relationship Between Enzyme and
Substrate Concentrations and Rates of Reaction.

13.0

APPENDICES

Figure 13.0 Digital balance

Figure 13.1 Beaker, water bath, and test tube

Figure 13.2 Cuvette with DNS reagent+sample

Figure 13.3 Hot plate

20