FACULTY OF CHEMICAL ENGINEERING

BIOPROCESS ENGINEERING LAB(CBE661)

NAME AND MATRIC NO

: NIK ABD HAFIIDZ BIN NIK ABDUL MALEK

(2014688106)

GROUP
PROGRAM
LAB NO./TITLE OF EXPERIMENT

: 6
: EH2425E
: INVESTIGATION ON ENZYME ACTIVITY AND KINETICS

DATE PERFORMED
DATE OF SUBMISSION
LECTURER

: 29 SEPTEMBER 2015
: 13 OCTOBER 2015
: PN SUHAILA BINTI MOHD SAUID

No.
1

Content
Abstract

Allocated Marks
5

Introduction

Aims/Objectives

Theory

10

Apparatus

Methodology/Procedure

10

Results

10

Calculations

10

Discussion

20

10 Conclusion

10

11 Recommendation

12 Reference/Appendices

TOTAL

Marks Obtained

100

Remarks:
1

Checked by:

Date:

............
(PN SUHAILA BINTI MOHD SAUID)

1.0

ABSTRACT

Enzyme activity is an enzymatic reaction which is the conversion of one

molecule into another while enzyme kinetics is the study of the chemical
reactions that are catalyzed by enzymes. An experiment was conducted to
study the kinetics of an enzyme by estimating its Michaelis-Menten parameters
and oobserving the effect of pH and temperature and substrate concentration
on the enzyme activity. The enzyme used in this experiment is amylase. A
starch solution was prepared during experiment and the effect of enzyme
activity with pH, temperature and substrate concentration was observer from
data recorded and graph was plotted. The glucose standard curve was plotted
to determine glucose concentration from absorbance value. From the
experiment, the enzyme activity is recorded highest at pH6, while the
temperature and substrate concentration fail to obtain its optimum value
however according to theory optimum temperature for enzyme amylase is at
37C and the optimum activity of the amylase enzyme is proportional to the
concentration of the substrate. The kinetic of the amylase enzyme can be
determine by using Michaelis-Menten equation using line weaver graph to
determine the Vmax and Km.The V max and Km of the amylase enzyme ware
found to be 0.8112 mol/min and 3.1548, respectively. In this experiment, the
data obtain for temperature and substrate concentration contains error and
does not follow the theory. However, all mistakes and error was discussed on
this report.

Table of Contents

NO.
1.0
2.0
3.0
4.0

TITLE
Abstract
Introduction
Objectives
Theory

5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0

Apparatus
Methodology
Result
Calculation
Discussion
Conclusion
Recommendation
Reference/Appendices

PAGES
2
4
5
5
10
11
13
17
19
20
21
21

2.0

INTRODUCTION

Enzymes are protein molecules composed of amino acids and are manufactured by the
living cell and these molecules provide energy for the organism by catalyzing various
biochemical reactions ( Dr. Eby, 2003 ). If enzymes were not present in cells, most of the
chemical reactions would not proceed at measurable rates at the temperatures of living
systems. Each enzyme has at least a single active site which is the location where the
enzyme binds to the substrate. According to Dr. Eby 2003, in this way the substrate is held
rigidly in the most favorable orientation. Within the active site there are various chemical
groups that are involved in the reaction.
Enzyme activity is an enzymatic reaction which is the conversion of one molecule
into another. Enzyme activity involves a chemical reaction which catalyzed at the reactive
sites on the enzyme. Many parameters will affect the rate of this catalytic activity considering
as the complex nature of the enzyme. Enzyme activity can be influenced by pH,
temperature, substrate concentration and other factors. While, Enzyme kinetics is the study
of the chemical reactions that are catalyzed by enzymes. In enzyme kinetics, the reaction
rate is measured and the effects of varying the conditions of the reaction are investigated.
Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this
enzyme, its role in metabolism, how its activity is controlled, and how a drug or an agonist
might inhibit the enzyme.
This experiment is about to investigate the relation of enzyme activity and enzyme
kinetics with changes in enzyme concentration. The enzyme used in this experiment is
amylase. This enzyme usually found in saliva and germinating seeds. Amylase is an enzyme
that catalyzes the hydrolysis which is splitting of a compound like starch by addition of a
water molecule of starch into smaller carbohydrate molecules such as maltose, a molecule
composed of two glucose molecules. There are two categories of amylases which are alpha

and beta and these two differ in the way they attack the bonds of the starch molecules. In
this experiment, Amylase breakdown starch into maltose :

Figure 2.0: Reaction of starch with Amylase to maltose

The hydrolysis of starch can be measured through the use of an enzyme test or
assay. An enzyme assay will test for the simple presence of enzyme activity but can also be
used to measure the reaction rate of an enzyme-catalyzed reaction. The assay can measure
either the appearance of one of the products or the disappearance of one of the substrates
over time.

3.0

OBJECTIVES

The objectives of this experiment are:

To determine the effects of temperature on the enzymatic activity and the changes in

enzyme catalyst reaction.

To study the relationship between substrate concentration and the maximum velocity

of an enzyme.
To study the estimation of Michaelis-Menten parameters, effect of pH and
temperature on the enzyme activity and kinetics of inhibition.

4.0

THEORY

Enzymes are protein molecules composed of small chains of amino acids and are
produce by the living cell. These molecules provide energy for the organism by
catalyzing various biochemical reactions. If enzymes were not present in cells,
most of the chemical reactions would not be able to take place at measurable
rates and at the temperatures of living systems. Each enzyme has at least a
single active site which is the location where the enzyme binds to the substrate.
In this way the substrate is held rigidly in the most favorable orientation. Within
the active site there are various chemical groups that are involved in the
reaction. It is important to remember that enzymatic reactions usually result in
the addition or removal of some molecule or radical such as H2O, -OH, -H, -NH2
( Dr. Eby, 2013 ) .
Each enzyme possesses a pH and a temperature optimum for its activity.
This optimum pH and temperature can be easily determined in the laboratory by
carrying out the reaction in buffers over a wide range of pH or conducting tests
at different temperatures. Enzymes demonstrate a rather high degree of
specificity with respect to their substrates (Hedstrom, 2010). The degree of
specificity varies from enzyme to enzyme: some enzymes carry out a reaction in
only one direction but some will catalyze a reaction in both forward and reverse
directions although usually at greatly different rates for example, hydrogenation
in addition to dehydrogenation. Some enzymes will accept only one or two
specific substrate molecules; others accept whole classes or subclasses of
molecules as substrates. This specificity is the basis for enzyme nomenclature:
according to the kind of reaction performed or, even more specifically, according
to the substrate acted upon.
In enzyme activity, 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. according to James Nishiura (n.d), 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. In general enzyme have a pH optimum. However the optimum
is not the same for each enzyme.
6

The temperature of a system is to some extent a measure of the kinetic

energy of the molecules in the system. Thus the lower the kinetic energy, the
lower the temperature of the system and , likewise, the higher the kinetic
energy, the greater the temperature of the system. Increases in the temperature
of a system results from increases in the kinetic energy of the system(James
Nishiura, n.d). This has several effects on the rates of reactions. Each enzyme
has a temperature range in which a maximal rate of reaction is achieved. This
maximum is known as the temperature optimum of the enzyme. Enzyme activity
increase as the temperature increase and when reaching the optimum
temperature, the enzyme activity will rapidly declines as the further temperature
increase.

Figure 4.1: Effect of temperature on reaction rate (Worthington

Biochemical Coperation, 2015)
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, [S]. 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
7

depend on the [S] (vlab.amrita.edu, 2011). 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 catalysed reaction under
substrate saturation is called the Vmax , Maximum velocity.

Figure 4.2: Graph substrate concentration on the X axis and corresponding

velocity on Y axis (vlab.amrita.edu, 2011)

To measure amylase activity, the disappearance of starch is observed by a

colorimetric assay and spectrophotometric readings. The colorimetric assay that
can be applied is the DNS method, which is widely used to estimate the
concentration of reducing sugar. A reducing sugar in a basic solution is able to
form aldehyde or ketone. This aldehyde group, meanwhile, can convert 3,5dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, which is the reduced
form of DNS. In this reaction, water is used up as a reactant and oxygen gas is

released. The formation of 3-amino-5-nitrosalicylic acid results in a change in the

amount of light absorbed in a spectrophotometer. At wavelength 540 nm, the
absorbance measured is directly proportional to the amount of reducing sugar.
The colour changes that can be expected to occur is from a clear chrome-orange
to a slightly darker shade.
To determine the enzyme kinetics, the Michaelis-Menten equation can be
utilized. According to vlab.amrita.edu, (2011), 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, [S] and Initial velocity of enzyme,
V0 (Figure 2) has the same general shape for most enzymes (it approaches a
rectangular hyperbola). This can be expressed algebraically by the MichaelisMenten 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
9

The necessary terms in this reaction are [S], V0, Vmax, and Km (Michaelis
constant),.All these terms can be measured experimentally. 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 graph is
designated Vmax. Using this maximum velocity and equation 1 and 2. Michaelis
developed a set of mathematical expressions to calculate enzyme activity in
terms of reaction speed from measurable laboratory data (Worthington
Biochemical Coperation, 2015).
The linear representation of the Michaelis-Menten equation can be represented
by the Lineweaver-Burk plot, Eadie-Hofstee plot and Hanes plot. The Km and
vmax are determined from the intercepts at the x- and y-axis and the gradients
of the graphs respectively.

Figure 4.3: Linear represantations of Michealis-Mentan equations by the Lineweaver-Burk

plot (A), Eadie-Hofstee plot (B), and Hanes plot (C).

5.0

MATERIAL & APPARATUS

10

Apparatus

Beaker

Measuring cylinder

Cuvette

Falcon tube rack

Falcon tube

Micropipet and tips

Label sticker

Schott bottle

Vortex mixer

Water bath

Spectrophotometer

Hotplate

Meterial/Reagent

Alpha Amylase enzyme

Starch

pH buffer solution (pH 4-9)

DNSA Reagent

6.0

METHODOLOGY

Part A. Starch solution preparation

1. Four gram of starch powder was measured and 50 mL of cold water was
measured using measuring cylinder.
2. Mixing was done in a 500 mL beaker on a hot plate and using a stirrer
3. 150 mL cold water was added later into beaker until the solution become
homogeneous.

Part B. Effect of pH on the activity and stability of amylase enzyme

1. Five test tube were labelled with pH 5,6,7,8, and 9.

2. 1 mL of 20% starch solution and 1 mL appropriate buffer was added.
3. 2 mL of amylase solution was added into another five test tubes.

11

4. All the test tubes were incubated in a 30C water bath for 10 minutes to
equilibriate the temperature.
5. Amylase solution were added into the test tubes with different pH.
6. The test tubes with different pH were placed again in the water bath for
another 10 minutes for hydrolysis reaction.
7. Four mL DNSA reagents were added into each different pH test tube to
stop the hydrolysis reaction.
8. The test tubes were placed in the boiling water for then minutes and
cooled at room temperature.
9. The absorbance is determined using spectrophotometer at 540 nm.
10.The result were recorded and analyzed by plotted a graph of enzyme
activity level vs pH.

Part C. Effect of temperature on the activity and stability of amylase enzyme

1. A test tube was labelled with 30C,

2. 1 mL of 0.2% starch solution and 1 mL of pH7 were added into the test
tube.
3. Another test tube was prepared to pour in 2 mL of amylase solution.
4. Both of the test tube were places into the water bath at 30C for 5
minutes.
5. Liquid in both test tubes were mixed in the test tube labelled 30C.
6. The test tube was then placed again in the water bath for 10 minutes for
hydrolysis reaction.
7. DNSA reagent was added after the 10 minutes duration to stop the
hydrolysis reaction.
8. The test tube was placed inside a beaker of boiling water for 10 minutes
and cooled at room temperature.
9. The absorbance was determined using spectrophotometer at 540 nm.
10.The procedure are repeated under temperatures 40C, 50C, 60C and
70C.
11.The result were recorded and analyzed by plotting a graph of enzyme
activity level vs temperature.

Part D. Effect of substrate concentration on the activity and stability of amylase

enzyme

1. Starch solutions with concentration of 0.5, 1.5, 2.0, 2.5 and 3.0%(w/v)
were prepared by serial dilution.

12

2. Each test tube were labelled with the different concentrations and filled
with respective starch concentration and 1 mL of pH 7 buffer.
3. Another five test tube were filled with 2 mL of amylase solution.
12.All the test tube were placed in the incubator for 5 minutes (to equilibrate
temperature), mixed with the amylase and incubate again for 10 minutes
for hydrolysis reaction, and was stopped by adding DNSA.Then, the test
tubes were put into the boiling water for 10 minutes and cooled at room
temperature.
13.The absorbance was determined using spectrophotometer at 540 nm.
14.The result were recorded and analyze by plotting a graph of enzyme
activity level vs starch concentration.

Part E. Glucose standard curve preparation

1. Standard solution of glucose at different concentration ranging from 0100mg/L were prepared by serial dilution. For example 25 g of glucose in
250 mL of water are at concentration 0.1 mg/L.
2. 1 mL of each glucose concentration were added in each test tube and
labelled.
3. 1 mL of DNSA reagent was mixed in each test tubes.
4. All the test tube were placed in the boiling water for 10 minutes before
being cooled at room temperature.
5. The absorbance was determined using spectrophotometer at 540 nm.
6. The result were recorded and analyze by plotted standard curve of
absorbance against glucose.

7.0

RESULTS

Glucose standard curve

Table 7.1: Value of absorbance at different glucose concentration
glucose concentration, (mg/L)
200
400
600
800
1000

Absorbance value
0.378
0.668
0.801
1.224
2.052

13

Glucose Standard Curve

2.5
2
f(x) = 0x
R = 0.97

1.5
Absorbance Value

1
0.5
0
0

200

400

600

800

1000 1200

Glucose Concentration, mg/L

Figure 7.1: Graph of glucose standard curve

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

Table 7.2: Value of enzyme activity at different pH
pH

5
6
7
8
9

Absorbance

Glucose

Glucose

Enzyme

reading

concentrati

released

activity, V

(nm)

on, X

(mol)

(mol/min)

2.68
5.17
2.625
2.546
2.35

(g/mL)
0.0015
0.0029
0.0015
0.0014
0.0013

8.2610-6
1.5910-5
8.0910-6
7.8510-6
7.2610-6

8.3310-7
1.5910-6
8.0910-7
7.8510-7
7.2610-7

14

Graph of enzyme activity against pH

Enzyme activity, V (mol/min)

0
0
0
0
0
0
0
0
0
0
4.5

5.5

6.5

7.5

8.5

9.5

pH

Figure 7.2: Graph of enzyme activity against pH

Effect of temperature on the activity and stability of amylase enzyme

Table 7.3: Value of enzyme activity at different temperature
Temperat

Absorbanc

Glucose

Glucose

Enzyme

ure (C)

e reading

concentrati

released

activity, V

(nm)

on, X

(mol)

(mol/min)

2.63
5.32
7.29
8.07

(g/mL)
0.0015
0.0030
0.0041
0.0045

8.3310-6
1.6710-5
2.2810-5
2.5010-5

8.3310-7
1.6710-6
2.2810-6
2.5010-6

30
40
50
60

15

A Graph of Enzyme activity, V (mol/min)against Temperature (C )

0
0
0

Enzyme activity, V (mol/min)

0
0
0
0
25

30

35

40

45

50

55

60

65

Temperature (C)

Figure 7.3: Graph of enzyme activity against temperature

Effect of substrate concentration on the activity of amylase enzyme.

Table 7.4: Value of enzyme activity at different substrate concentration
Substrate

Absorbanc

Glucose

Glucose

Enzyme

concentration

e reading

concentration,

released

activity, V

(%)
0.5
1.5
2.0
2.5
3.0

(nm)
4.06
3.39
3.26
2.66
2.18

X (g/mL)
0.0023
0.0019
0.0018
0.0015
0.0012

,S(mol)
12.27x10-6
10.55x10-6
9.991x10-6
8.326x10-6
6.661x10-6

(mol/min)
12.27x10-7
10.55x10-7
9.991x10-7
8.326x10-7
6.661x10-7

1/V

1/S

8.15105
9.48105
1.00105
1.20105
1.50105

2.00
0.67
0.50
0.40
0.33

16

Graph of enzyme activity against substrate concentration

14
12
10
8
Enzyme activity, V (X10-7 mol/min)

6
4
2
0
0 0.5 1 1.5 2 2.5 3 3.5
Substrate concentration (%)

Figure 7.4: Graph of enzyme activity against substrate concentration

17

Graph of 1/V against 1/S

10
9
8

f(x) = 3.89x + 1.23

R = 0.42

7
6
1/V

5
4
3
2
1
0
0.33 0.53 0.73 0.93 1.13 1.33 1.53 1.73 1.93 2.13
1/S

Figure 7.5: Graph of 1/V against 1/S

8.0

CALCULATION

Determination of glucose concentration, X (g/mL)

The calculation is made based on the result obtained for substrate concentration of 0.5%
1

Glucose Concentration.
From the equation of standard curve:

Y = 0.0018X
where; X = protein concentration and Y = absorbance reading. Therefore, to calculate
protein concentration,

18

X=

Y
0.0018

X=

4.06
0.0018

X=

2255.56 mg
L

1L
1000 mL

1 103
mili

X = 0.0023 g/mL

Determination of glucose released (mol)

Moles of glucose released (mol) =

Concentration of glucose( g /mL)

MW of glucose(g/mol)

MW of glucose = 180.1559 g/mol ; Volume of starch= 1 mL

Moles of glucose released (mol) =

0.0023 g/mL
180.1559 g/mol

1mL

Moles of glucose released (mol) = 12.27x10-6 mol

Determination of enzyme activity, V (mol/min)

Enzyme activity (mol/min) =

mol of glucose released

Hydrolysis reactiontime

Duration of hydrolysis reaction: 10 minutes

Enzyme activity (mol/min) =

12.27 x 106 mol

10 min

Enzyme activity (mol/min) = 12.27x10-7 mol/min

19

Equation for Michaelis-Menten:

V = Vmax [S]
Km + [S]

Double reciprocal;
1
V

Km
V max

1
S

1
+

V max

From graph, the linear equation obtained is:

y = 3.8889x + 1.2327
Finding value of Vmax, maximum enzyme activity:
1
V max

= 1.2327

Vmax = 0.8112 mol/min

Finding value of Km, Michaelis constant

Km
V max

= 3.8889

Km = (3.8889)(0.8112)
Km = 3.1548

9.0

DISCUSSION

In the experiment, the kinetic of enzyme are determined by using the

absorbance value by detecting the amount of glucose present in the each test
tubes based on different pH, temperature and substrate concentration. It is being
measured by using spectrophotometer at wavelength of 540 nm.
20

Based on figure 7.1, the graph shows a glucose standard curve. This graph
is plotted in order to get the equation straight line of glucose standard curve.
From this experiment the equation of the glucose standard curve is y = 0.0018x.
Thus, the enzyme activity can be calculated. The enzyme used is

2mL of

amylase and the hydrolysis reaction occured for 10 minutes at temperature

30C. The DNSA reagent is added in the test tube to make no more hydrolysis
occured. From the graph y=absorbance value, x= glucose concentration. The
glucose standard curve shows a high regression value at 0.89, which indicates
that it is can be consider as accurate.
As for Effect of pH on the activity and stability of amylase enzyme, all
results and calculation was tabulate from table 7.2 above and graph enzyme
activity against pH was plotted on figure 7.2. The enzyme activity is highest at
pH 6. From the graph, it show the enzyme activity increasing from pH 5 to 6 and
decrease after passing pH 6. As state by Chul-Won and Erik (2000). Most
proteins, and therefore enzymes, are active only within a narrow pH range
usually between 5 and 9. Several factors are influenced directly by the pH in
which the reaction takes place. In this case, this experiment shows the optimum
pH for amylase is at pH 6 for optimum enzyme activity. This explanation prove
that from above theory, enzyme have a pH optimum. However the optimum is
not the same for each enzyme.
As for Effect of temperature on the activity and stability of amylase
enzyme, the results and calculation was tabulate from table 7.3 above and graph
enzyme activity against pH was plotted on figure 7.3. From the graph, it shows
the enzyme activity increase as the temperature increase. Based on theory
stated above, as temperature increase the enzyme activity increase but after
reaching optimum temperature the enzyme activity will be declines. In this
experiment, the graph doesnt show any decline where by the optimum
temperature for enzyme amylase cannot be observe. This is probably due to
misconduct experiment or equipment error. Based on research done by
Rudeekulthamrong et. al., 2012, the optimum temperature for amylase is 37
degrees C. However, in this experiment after reaching 37C the enzyme activity
seems increase as the tempereture increase.
Concentration of 0.25 to 3.0% was taken out and enzyme activity at each
substrate concentration was checked to analyze using Michaelis-Menten kinetics.
The Lineweaver- Burke plot showing the Michaelis Menten type kinetics of the
21

amylase enzyme indicating Km values under maximum velocities(Vmax).The

effect of substrate concentration on the activity of amylase enzyme shows that
as substrate concentration increase the enzyme activity also increase until it
reach a stationary point where on that point the enzyme is saturated when the
active sites of all the molecules are occupied most of the time. At the saturation
point, the reaction will not speed up, no matter how much additional substrate is
added. this can be seen from the graph proposed on theory above. However, this
experiment is observe that the enzyme activity decrease as the substrate
concentration. It is because during conducting experiment, perhaps some of
mistakes and measurement error was done and its affect the whole result.
Thus the Lineweaver- Burke plot graph shows incorrect plot comparing to the
theory. However, by drawing a linear line with regression value at 0.41, which indicates
that it is can be consider as inaccurate, the line equation can be obtain which
relate to in finding values for Vmax and Km. The line was draw to indicate the possible
actual the Lineweaver- Burke plot. From the plot, Vmax and Km was found to be 0.8112

mol/min and 3.1548.

10.0 CONCLUSION
As conclusion the enzyme activity of the enzyme are affected by temperature,
pH and the substrate concentration. The optimum activity of the amylase
enzyme is at pH 6, and for temperature optimum temperature cant be found in
this experiment. The optimum activity of the amylase enzyme is proportional to
the concentration of the substrate. However according the theory optimum
temperature and pH for amylase is at 37C and pH 7, respectively.
The kinetic of the amylase enzyme can be determine by using MichaelisMenten equation using line weaver graph to determine the Vmax and Km. The V
max and Km of the amylase enzyme ware found to be 0.8112 mol/min and 3.1548,

respectively.
From the experiment, it showed that the maximum activity of the amylase
enzyme are at pH 6.Thus the relationship between effect of pH and enzyme
activity can be observe and experimental objective achieve. However, the
relation between enzyme activity and 2 parameter of temperature and substrate
concentration fail to be obtain according to theory because error while
22

conducting experiment and incorrect data gain. Thus, this failure affecting the
Michaelis-Menten parameters even it is obtain but it is not as correct as it should be.

11.0 RECOMMENDATION
These are some recommendations that should be done while carrying out this experiment:

Carry out proper absorbance reading to avoid low regression value from the

calibration curve
Maintain correct water bath temperature by not huddling around it as it may cause

inconsistent temperature drop and rise inside it

Reduce waste of raw materials by mixing small amount during preparation.
Every effects of the parameters involved should be handled carefully according to the

laboratory manual and guidelines.

With higher concentration of starch, the reaction can proceed until it reaches

constant level and the Vmax and Km of the reaction can be determined.
Starch analysis method can be used to enhance the reaction process. It can be

accomplished by mixing the starch solution with the buffer.

To measure the starch consumed in the reaction carried out, the starch-iodine assay
can also be used.

12.0 REFERENCES
Dr. Eby Bassiri (2013), ENZYME KINETICS: THEORY. Molecular Biology of Life
Laboratory. Retrieved on 10 October 2015 from
http://www.sas.upenn.edu/LabManuals/biol123/Table_of_Contents_files/8dEnzymeKinetics-Theory.pdf
Chul-Won Park and Erik Zipp (2000). The effect of temberature and pH on
enzyme kinetic. Introduction to Biochemical Engineering. Retrieve on 10
October 2015 from http://www.rpi.edu/dept/chem-eng/BiotechEnviron/Projects00/temph/enzyme.html
Hedstrom Lizbeth (2010) Enzyme Specificity and Selectivity. In: eLS. John Wiley & Sons Ltd,
Chichester. http://www.els.net
James Nishiura (n.d). The effect of pH on enzyme activity. Retrieve on 10 October
2015 from
http://academic.brooklyn.cuny.edu/biology/bio4fv/page/ph_and_.htm
23

Rudeekulthamrong et. al., 2012. Kinetic inhibition of human salivary alphaamylase by a novel cellobiose-containing tetrasaccharide. epartment of
Biochemistry, Phramongkutklao College ofMedicine, Phramongkutklao Hospital,
Bangkok, Thailand.
vlab.amrita.edu, (2011). Effect of Substrate Concentration on Enzyme Kinetics.
Retrieved 10 October 2015, from vlab.amrita.edu/?
sub=3&brch=64&sim=1090&cnt=1
Worthington Biochemical Coperation, 2015. Introduction to Enzymes. Retrieve on
10 October 2015 from http://www.worthingtonbiochem.com/introbiochem/tempeffects.html

13.0 APPENDIX

24

Appendix A: Result of absorbance at different pH

Appendix B: Result of absorbance at different substrate concentration

25

Appendix C: Result of glucose standard curve

Appendix D: Result of absorbance at different temperature

26

Appendixe E: Preparation of buffer solution

Appendix F: Spectrophotometer for measure absorbance value

27

Appendix G: Sample of glucose with amylase solutions

28