ACTIVITY NO.

5
ENZYME KINETICS OF α -AMYLASE
Cecilia, Jasmin S. (1), Chan, Janelle Mae A. (2), So, Shannen Louise T. (3)
CHEM 43 LB1A
Ms. Fatsy Cruz

ABSTRACT
Enzymes, most of which are proteins, play an important role as they increase the otherwise slow reaction
rates of biochemical reactions. This is possible when the substrate binds to the active site of an enzyme and
turns into its respective product, as is the case of the enzyme α -amylase used in this activity, which catalyzes
the hydrolysis of starch into glucose. In order to determine the effects of incubation time, temperature, pH,
and substrate concentration on the activity of α -amylase acquired from human saliva, a starch standard curve
was first prepared. This was used in determining starch concentrations from the absorbances of iodine-starch
complexes obtained at 620 nm using a UV-Vis spectrophotometer, as the time was varied from 0, 3, 5, 7, to
10 minutes; the temperature from 10, 40, 60, to 80 ° C; ​the pH from 2.0, 4.0, 6.7, 7.4, to 9.0; and finally, the
substrate concentration from 0.10, 0.08, 0.05, 0.02, to 0.01%. Plots of starch concentration versus each of the
variables were created, except for the variable of substrate concentration, for which a 1/V versus 1/[S] plot
was made and the K​M and V​max values were calculated through the Lineweaver-Burk equation. The standard
curve obtained, with an R​2 value of 0.9307, had an equation of y = 95.432x + 1.2697. The following trends
regarding the four factors were observed: the longer the incubation time, the less the substrate concentration;
the plot for incubation temperature had a graph with an upward concavity; the plot for pH had a relative
minimum at around 7.05; and the greater the initial substrate concentration, the faster the reaction velocity.
Based from the Lineweaver-Burk plot obtained, a K​M value of 0.011 and a V​max value of 5.27*10​-4​. These were
calculated from the linear equation y = 21.212x + 1897.6, which had an R​2 ​value of 0.9456. In conclusion,
optimal enzyme activity occurs at 10 minutes of incubation time, 40 ° C incubation temperature, a pH value of
around 7.05, and a relatively greater initial substrate concentration.

KEYWORDS:​ enzyme kinetics, α -amylase, iodine-starch complex, factors on enzyme activity, Lineweaver-Burk
equation, UV-Vis spectrophotometry

INTRODUCTION One approach to enzyme kinetics is the

In biological systems, an enzyme acts as a catalyst by
lowering the activation energy of the reaction, and may be
able to accelerate the reaction by up to 10​17 than how they
would normally proceed. It participates in the reaction
without being permanently changed nor appearing as final
products.​[1, 2] ​Enzymes are also highly selective and would
only react with one specific reactant called a substrate. This
selectivity allows for them to distinguish between different
substances even up to their optical isomers.​[2]
Michaelis-Menten Model wherein it relates the velocity of
the reaction to the substrate concentration where the
​ inds reversibly with the enzyme ​E to form the
substrate ​S b
enzyme-substrate complex ​ES, ​which then reacts to form
the product and the free enzyme irreversibly.​[5] The model
can be shown as:

Enzymes have active sites that the target substrates Figure 1. ​Formation and Breakdown of the ES Complex
would bind to. These active sites are composed of amino Source: ​http://2016.igem.org/Team:Freiburg/Delivery
acids that would form a cleft or a pocket where the
substrate stays during the reaction. The cleft would provide This relationship can also be represented
a better environment for the reaction to proceed – by mathematically using ​Equation 1.
excluding water, for example.​ [3]

Enzyme kinetics is the study of the rate at which a

reaction catalyzed by enzymes proceeds. The reaction rate
is measured and various factors – incubation time,
temperature, pH, and concentration of the substrate – are
Equation 1.​ Michaelis-Menten Equation​[5]
observed with regards to their effects. By studying the
kinetics of an enzyme, its catalytic mechanism and Where V is the reaction rate, V​max is the maximum
​
enzymatic activity may also be analyzed. [4] velocity of the catalyzed reaction, [​S]​ is the substrate
concentration and K​M is the concentration of the substrate
at half the maximum rate. [5,​ 6] V​max and K​M​, under defined
conditions, can be used to characterize the enzyme, thus
​
are also called kinetic parameters. [2]
In order to represent the data more accurately and
easier, a double reciprocal plot of the Michaelis-Menten
equation was derived. The Lineweaver-Burk plot linearizes
the data making it more precise and would, therefore, give
a more accurate calculation of the V​max and subsequently,
​ 8] The linear equation used to determine the
K​M​. [7,
parameters can be shown as:
Equation 2. ​Lineweaver-Burk Equation
Source:https://pharmafactz.com/medicinal-chemistry-understandi
ng-enzyme-kinetics/
Extracted from human saliva, α -amylase was used as
the enzyme in this experiment. The α -amylase breaks
down starch chains, forming two or three glucose units.​[9]
The starch concentration after the addition of α -amylase –
and consequently, the enzymatic activity – can be
measured through colorimetry using a UV-Visible
Spectrophotometer. Iodine, through I​2 ​in KI, is added to form
an iodine-starch complex with the starch, appearing dark
blue/violet, and because of its colorimetric properties, the
less the starch concentration is, the lighter the color would
be. The intensity of the color can be quantified by
measuring the absorbance at 620nm. ​ [10, 11]
In this experiment, the researchers aimed to determine
the effects of various factors that affect enzyme kinetics, to
construct a standard curve for the determination of product
concentration, and to determine the values of kinetic
parameters, K​M​ and V​max​.
EXPERIMENTAL
Preparation of Starch Standard Curve.​ From 0.2%
starch solution, 2 mL of the following starch concentrations
were prepared in 0.1 M phosphate buffer (pH 6.7): 0%
(blank solution), 0.10%, 0.08%, 0.05%, 0.02%, 0.01%, and
0.005%. An mL of each starch concentration was
transferred to separate test tubes, and 20 ​µL of KI, along
with 5 mL of water, was added to each solution​. The
absorbance of each solution was measured immediately at
620 nm, and the starch concentration vs absorbance was
plotted, generating a standard curve.
Effect of Incubation Time on Enzyme Activity​. A 10 mL
of 0.1% starch solution was prepared using 0.2% starch
solution and phosphate buffer pH 6.7. A saliva solution was
made by mixing 28.5 mL phosphate buffer and 1.5 mL
human saliva. In a 20 mL test tube, 9 mL of the previously
stated starch solution was mixed with 1 mL of the prepared
saliva solution. An mL of this starch-saliva solution was
then transferred into 5 separate test tubes, which were
previously labelled t = 0, t = 3, t = 5, t = 7, and t = 10 to
indicate the number of minutes the solution was incubated
before 100 ​µL of HCl was added​. In the test tube labelled
t=0, 100 µL of HCl was added immediately. For the test
tube labelled t=3, the solution was incubated at room
temperature for 3 minutes before 100 ​µL of HCl was added.
This incubation and addition of 100 µL of concentrated HCl
was repeated, but at varying incubation times, for test tubes
labelled t = 5, 7, and 10 minutes. After all test tubes were
incubated at their respective times, and 100 µL of HCl were
added, 20 µL of I​2 in
​ KI solution and 5 mL of distilled water
was added to each test tube. Their absorbances were read
at 620 nm, and the starch concentration vs incubation time
was then graphed.
Effect of Temperature on Enzyme Activity​. Using 0.2%
starch solution and phosphate buffer pH 6.7, 20 mL of 0.1%
starch solution was prepared. In four separate 10 mL test
tubes, 4.5 mL of this starch solution and 0.5 mL of 0.9%
NaCl were mixed together in each of the test tubes. These
test tubes were then incubated in water baths of varying
temperatures of 10°C, 40°C, 60°C, and 80°C respectively,
for 5 minutes. Without removing these test tubes from their
water baths, 100 µL of the previously prepared saliva
solution was added to each, before being further incubated
for 5 minutes more. An mL of each aliquot was then put into
separate test tubes, before 100 µL of concentrated HCl
was added to each aliquot. Just before reading the
absorbances of each aliquot at 620 nm, 20 µL of I​2 in ​ KI
solution and 5 mL of distilled water was added to all the test
tubes. The starch concentration vs temperature was then
graphed.
Effect of pH on Enzyme Activity.​ In 5 separate test
tubes, 2.50 mL of 0.2% starch solution, 0.5 mL of 0.9%
NaCl solution, and 2.0 mL of 0.1 M phosphate buffer with
varying pH were mixed, making the solutions have the
following pH: 2.0, 4.0, 6.7, 7.4, 9.0. To each test tube, 100
µL of the saliva solution was added. They were then
incubated for 5 minutes. From each test tube, 1 mL aliquot
of each solution was put into separate test tubes, and 100
µL of concentrated HCl was added to each. Before reading
their absorbance at 620 nm, 20 µL of I​2 in
​ KI solution and 5
mL of distilled water was added to all test tubes. The starch
concentration vs pH was then graphed.
Effect of Substrate Concentration on Enzyme Activity.​
Using 0.2% starch solution and 0.1 M phosphate buffer (pH
6.7), a 4.5 mL starch solution with the following starch
concentrations were prepared in separate test tubes:
0.10%, 0.08%, 0.05%, 0.02%, and 0.01%. To each test
tube, 0.5 mL saliva solution was added before incubating
each for 5 minutes. After 5 minutes of incubation, 1 mL of
each solution was placed in separate test tubes, and 100
µL of concentrated HCl was added. The absorbance of
each test tube was then measured at 620 nm after adding
20 µL of I​2 in KI solution and 5 mL of distilled water to each
test tube. The reciprocals of average reaction velocity and
substrate concentration were then graphed, and the Km
and Vmax were determined.
Table 2.​ Absorbances at Varying Incubation Times
RESULTS
A. Preparation of Starch Standard Curve Time Absorbance Concentration
Different starch concentrations were prepared for the (minutes)
standard curve. To get the diluted version as shown in
Table 1, each starch concentration was multiplied by 1 mL 0 0.462 -0.016927
and divided by 6.02 mL by the M​1​V​1 = M​2​V​2 formula. From
3 0.240 -0.02158
this table, the absorbance, with a range of 1.348 to 2.859,
was seen to increase as the concentration increased. 5 0.237 -0.02164

Table 1. Absorbances of Varying Iodine-Starch Complex 7 0.231 -0.02177

Concentrations
Diluted Starch 10 0.223 -0.02194
Starch solution
Concentration Absorbance
(%)
(%)

0.10 0.0166113 2.859

0.08 0.01328904 2.363

0.05 0.00830565 2.382

0.02 0.00332226 1.495

0.01 0.00166113 1.372

Figure 3.​ Starch Concentration vs. Incubation Time
0.005 0.00083056 1.348
C. Effect of Temperature on Enzyme Activity
The concentrations, as presented in Table 3, were
The plot, as shown in Figure 2, obtained by having the obtained by inputting the absorbance values as the
concentration values as the x-values and the absorbances y-values and solving for x.
as the y-values resulted in Equation 3, with an R​2 value of
0.9307. Table 3.​ Absorbances at Varying Temperatures
Temperature Absorbance Concentration
y = 95.432x + 1.2697 ( ° C)
Equation 3.​ Standard Curve
10 0.401 0.01821

40 0.621 0.0136

60 0.298 0.02036

80 N/A N/A

A trend observed from this data was that the starch

concentration was lowest at 40 ° C, while the
concentrations at 10 and 60 ° C were higher, yielding Figure
Figure 2. ​Standard Curve 4, which had an upward concavity.

B. Effect of Incubation Time on Enzyme Activity

The concentrations, as presented in Table 2, were
obtained by solving for x using Equation 3. A trend, as
reflected in Figure 3, where the substrate concentration
decreases as the incubation time increases, was observed.

Figure 4. ​Starch Concentration vs Temperature

 The starch solution with 80 ° C incubation temperature Table 5.​ Absorbances at Varying Substrate Concentrations
produced a suspension of precipitates, as shown in Figure Average
5. Therefore, no absorbance value was obtained for this Starch Initial Reaction
sample. Absorbance Final [S] Velocity
solution [S]
( Δ [S]/ Δ t)

0.10% 0.160 0.07352 -0.011628 0.0170315

941 2 2

0.08% 0.170 0.05882 -0.011523 0.0140693

353 4 8

Figure 5. ​Starch Sample with 80 ° C Incubation 0.05% 0.130 0.03676 -0.011942 0.0097414
Temperature 471 5 5

0.02% 0.039 0.01470 -0.012215 0.0053841

D. Effect of pH on Enzyme Activity 588 7

The concentrations, as shown in Table 4, were obtained 0.01% 0.104 0.00735 -0.012896 0.0040498
from Equation 3 as the calculated x-values. 294 1 1

Table 4.​ Absorbances at Varying pH

Upon plotting the 1/[S] values as the x-values and the 1/V
pH Absorbance Concentration values as the y-values, a linear graph, with Equation 4 and
an R​2​ value of 0.9456, was obtained, as shown in Figure 7.
2.0 0.734 -0.01123

4.0 0.876 -0.008251

6.7 0.259 -0.02118

7.4 0.253 -0.02131

9.0 0.608 -0.01387

The plot obtained from these data, as shown in Figure 6 Figure 7. ​Lineweaver-Burk Plot
had a relative minimum value in between pH 6.7 and 7.4,
indicating that starch concentration is lowest in between y = 21.212x + 1897.6
these two points. Equation 4.​ Lineweaver-Burk Equation

From this linear graph, the V​max​ value was 5.27*10​-4​ and the
K​M​ value was 0.011.

Figure 6. ​Starch Concentration vs pH
E. Effect of Substrate Concentration on Enzyme Activity
The initial concentration of substrate, [S], was obtained
by first taking dilution into account. As a sample calculation,
0.10% was multiplied by 4.5 mL aliquot and divided by the
final volume, 6.12 mL. The final concentration of [S] was
obtained from Equation 3. The average reaction velocity
was obtained by subtracting the final [S] from the initial [S]
and divided by the total incubation time, 5 minutes, as
presented in Table 5.
DISCUSSION
Enzymes are biological catalysts responsible for
accelerating biochemical reactions that would otherwise
take a very long time. Enzymes lower the activation energy
of the reaction by providing an easier path for the
substrates to react on, making the formation of products
faster. They have what is called an active site, or cleft,
where the substrate binds and sits through the reaction.
The clefts will then position the substrates in the right
orientation or wherein the bond breaking and bond forming
happen more obligingly to form the transition state. They
can also control the Each enzyme only acts on a certain
specific substrate and this is due to the characteristic of the
active site which is given by the amino acids making it up -
their acidity/basicity, 3-D orientation, hydrophilic/
hydrophobic tendencies, and more, altering the shape,
​
size, and charge of the cleft, as shown in Figure 8. [12]
 by α -helices. The highly conserved amino acid residues,
which are involved in catalysis and substrate binding, are
found in loops of the “C-termini of β − strands ” in the
aforementioned domain. The B-domain protrudes between
the β -sheet #3 and the α -helix #3, and ranges 44 to 133
amino acid residues. It also plays a role in substrate or Ca​2+
Figure 8. ​Induced Fit Model​[12] binding. Calcium binding to the amylase, whose structure is
shown in Figure 10, is important to the amylase as it may
Starch is a polymer of glucose linked through the C​1 affect its activity and stability. The domain C is folded into
oxygen in a “glycosidic bonds”. This bond is stable at high an antiparallel β -barrel and has no known function.
pH, but hydrolyzes at low pH. A latent aldehyde group may
be found at the end of this polymeric chain, and is called
the reducing end. In starch, two kinds of glucose polymers
are present: amylose, a linear polymer consisting of around
6000 glucose units, and amylopectin, which consists of
significantly less glucose units. It may be noted that
amylopectin is soluble in water, but amylose and the starch
itself is insoluble in cold water, both of whose structures are Figure 10​. Three Dimensional Structures of α -amylases [15]
​
​
shown in Figure 9. [13]
In these sites, the confirmed active sites of glycosyl
hydrolases are made of multiple binding sites, and formed
between domain A and B so domain B may participate in
substrate binding. They also contain many amino acid
residues which form hydrogen bonds to the substrate,
either directly or via water molecules. These substrate
binding sites are commonly aligned with aromatic residues,
making hydrophobic stacking interactions with the sugar
Figure 9​. Amylose and Amylopectin [14a]
​ rings. The glutamic acid residue is said to be the proton
donor, while the first conserved aspartic acid is the
Amylases are enzymes capable of digesting the nucleophile. Below is the reaction of α -amylase and starch.
glycosidic linkages of starch. In humans, they may be found [13]
​ There are two types
in both saliva and in the pancreas. [14]
of amylase: endoamylase and exoamylase. Both types are
capable of hydrolysing starch, and are classified by their
manner of attacking the glycosidic bond. Endoamylases
can cleave α ,1-4 glycosidic bonds found in the inner part of
both amylose and amylopectin chain. One popular example
of an endoamylase is α - amylase. Exo-amylases, on the
other hand, either exclusively cleave α , 1-4 glycosidic
bonds, or cleave both α ,1-4 and α , 1-6 glycosidic bonds.
Figure 11​. Reaction of α - amylase and starch​[16]
The β − amylase is a good example of the former
​
exo-amylase. [13] Unlike typical organic and inorganic catalysts, enzymes
have an unusual behavior with regards to substrate
As mentioned before, α -amylase is an endoamylase concentration, as illustrated in Figure 12.
that may cleave the α ,1-4 glycosidic bonds of the amylose
or amylopectin chain. The end products of this amylase are
oligosaccharides with varying lengths. This amylase is
often divided into two types, saccharifying α -amylase and
liquefying α -amylase, depending on the degree of
hydrolysis of the substrate. Saccharifying α -amylases
hydrolyze 50 to 60%, while liquefying α -amylases only
​
cleave about 30-40% of the linkages of starch. [13]

The α -amylase is a starch hydrolase composed of
several amino acid sequences. [14]​ They are monomeric,
calcium-containing enzymes, with a single polypeptide
chain folded into three domains. The most conserved
domain consists of eight parallel β − strands that are folded
in a symmetrical manner, and arranged in a barrel encircled
Figure 12. ​Effect of Substrate Concentration on the Initial
Velocity of an Enzyme-Catalyzed Reaction
Source:
https://www.khanacademy.org/science/biology/energy-and-enzym
es/enzyme-regulation/a/basics-of-enzyme-kinetics-graphs
At low substrate concentrations, the rate of the reaction
increases with increasing substrate concentration.
However, the rate increases less at relatively higher
substrate concentrations, up to a certain point where the
rate becomes constant (V​max​). In 1913, Michaelis and
Menten derived a mathematical equation to describe this
hyperbolic plot by first proposing the enzyme action where
enzymes, E, and substrate molecules, S, bind to each other
to form an ES complex, as shown in Figure 1. ​Initial rates
are measured so that the concentration of the product, P,
and therefore the rate constant, k​-2​, are both negligible. By
assuming this as well as the steady-state condition of the
ES complex, where its concentration is held constant,
Equation 2 was derived.​[17]
From Equation 2, it was determined that the Michaelis
constant, K​m​, is equal to the substrate concentration that
yields an initial velocity that is half of V​max​. Another useful
constant is the turnover number, defined as the amount of
substrate that is transformed to products in a certain time
period, as shown in Equation 5.​[18]
Equation 5. ​Turnover number, k​cat
Source:
http://wajimypi12.dragon.ru.net/829234/615002/gynebuz_enzyme
-turnover-number-example_ymapihocaz/xygob.asp
Taking the reciprocal of the Michaelis-Menten equation
yields Equation 2, otherwise known as the Lineweaver-Burk
equation. V​max was calculated as the reciprocal of the
y-intercept and the K​M as the product of the slope and V​max​.
Since Equation 2 yields a linear graph and the intercepts
1/v​0 and -1/K​m​, the variables v​o and
​ K​m are
​ measured more
accurately compared to the hyperbolic Michaelis-Menten
graph. Generally, the variables K​m​, V​max​, and k​cat give
information about enzyme activity. A high K​m requires
​ a
greater amount of substrate to saturate the enzyme, which
also means that the enzyme has a low affinity for the
substrate. Hence, low enzyme activity is observed at
relatively low substrate concentrations. Moreover, a high K​m
also means that a greater substrate concentration is
required to reach V​max​, which means that at low substrate
concentrations, maximal catalytic efficiency (V​max​) cannot
be achieved. On the other hand, a high k​cat value​ denotes
higher enzyme activity.​[19]
In order to determine the concentration of the starch left
uncatalyzed, iodine through I​2 in ​ KI was added. Iodine
reacted with starch to form a blue-black complex without
being affected by the glucose present as product of the
reaction. Since it is colorimetric, the absorbance of the
solutions were measured at 620 nm using a UV-Visible
Spectrophotometer and theoretically, as the enzyme
activity increases, the less intense the color will be. Due to
different factors and possible errors, there were deviations
from the expected results. [20] In the following investigations
for the four factors, HCl was added to stop enzyme activity
because HCl can hydrolyze the enzyme and break it apart,
causing it to lose its function.
One factor that may affect enzyme activity is incubation
time, that generally follows a linear trend and is directly
proportional to enzyme activity. Enzyme activity is
reversible, and the formation of products occurs while little
to no products have formed. As the reaction continues, and
a significant amount of product has been formed, the rate
of back reaction becomes more significant, and the rate of
formation of product slows down. Should the incubation
time be too great, the enzyme activity may be measured as
falsely low. Thus, as a general rule, incubation must be
long enough for a moderate amount of product to form and
for error in timing to be insignificant, but not too long that
​
there is a noticeable levelling off of the curve. [19]
Generally, enzyme activity increases over time. This is
because the longer an enzyme is incubated with its
substrate, the more product is formed. However, this rate of
formation of product is not always linear. Should the
substrate be used up during the incubation time, the
formation of product will decrease, regardless of
concentration of substrate.​[19]
Another factor that influences enzyme activity is
temperature. At low temperatures, the reaction rate
increases with increasing temperature. However, enzyme
activity decreases when the temperature, which differs for
each enzyme, is high enough to denature the enzyme itself.
Enzymes are also affected by pH and each has an
optimum pH at which they are most active, mostly
​ 24]
corresponding to the pH of their natural environment. [20,
Above or below the optimum pH, the reaction rate of the
reaction will more or less drop rapidly. This is due to
denaturation and ensuing inactivation caused by the effect
of pH on the state of ionization of the functional groups of
the enzymes and substrates resulting to an inefficacy of
enzyme-substrate complex formation.​ [2, 20]
The last factor that influences enzyme activity is
substrate concentration, which is directly proportional to
enzyme activity. Theoretically, in the absence of enzymes,
the rate of reaction would increase linearly with substrate
​
concentration, as proven by the equation below. [22]
Equation 6​. Reaction Rate in the Absence of Enzyme​[22]
In the presence of enzymes, reaction rates are still
directly proportional to substrate concentration. However,
there is now the limitation of the number of active sites said
enzyme has. At low enzyme concentrations (or high
substrate concentrations), all active sites may be occupied
by substrates, and no increased amount of substrate
concentration can increase the reaction rate any further.
Thus, it may be said that there is a maximum reaction rate ,
Vmax, when all the active sites of the enzyme are
occupied. While substrate concentration may increase the
reaction rate, the presence of enzymes greatly speeds up
the reaction. However, as enzymes become saturated, the
reaction rate may level off. This maximum reaction rate is
dependent on the total enzyme concentration, E, and the
catalytic constant of the enzyme, k​cat​, which is the
frequency ​at which an enzyme-substrate complex is
converted to product. The velocity at which active sites can
become saturated, K, is also taken into account. Thus,
according to the Michaelis-Menten equation (see equation
​
below): [22]
​
Equation 7​. Michaelis-Menten Equation [22]
Thus, enzyme activity may be both increased and
inhibited by enzyme concentration, and increases as
substrate concentration increases. However, the reaction
rate may reach a point wherein it no longer increases as
the enzyme has reached its saturation point.​[23]
It must be noted that in this experiment, the results for
substrate concentration versus incubation time is
influenced by at least two factors: the standard equation
gotten in part A, and the incubation time of 5 minutes,
which may not have been accurately followed. Because of
this, the data may be more prone to errors than the other
tested factors.
CONCLUSIONS
Starch is a polymer of glucose, that may be cleaved or
digested by α -amylase, producing one or two units of
glucose. The α -amylase is an enzyme that catalyzes the
digestion of glucose, and is found in the saliva and
pancreas of humans. There are several factors that may
affect the enzyme activity in various ways. Incubation time
is directly proportional to enzyme activity up to a certain
time. There is a certain temperature that yields the highest
enzyme activity, which is not too high that the enzyme may
be denatured, but not too low that the enzyme may not
have enough energy to react. There is also an optimum pH
which may yield the highest enzyme activity, which
generally corresponds to the pH of their natural
environment. Lastly, the enzyme activity may be affected
by substrate concentration, which is generally directly
proportional to one another, but is limited by the enzyme
concentration. This relationship between enzyme and
substrate concentration may be depicted in the derivation
of the Michaelis-Menten equation called the
Lineweaver-Burk equation.
The researchers recommend immediately measuring
the absorbance of the samples after addition of I​2 in KI
because the color of the complex fades quickly. The
variables to be studied must also be strictly followed. For
example, an incubation time of 5 minutes must be strictly
followed because some enzymes react within seconds.
Inhibitors may also be introduced to the starch solution to
further investigate the Lineweaver-Burk equation.
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CERTIFICATION OF CONTRIBUTION
I hereby certify that I have given substantial contribution
to this report:
_________________________
Jasmin S. Cecilia
_________________________
Janelle Mae A. Chan
_________________________
Shannen Louise T. So