Internal Assessment

Biology

Research to investigate the impact of the cofactor Ca2+ on alpha-amylase thermal

stability

What effect does the presence of Ca2+ (calcium ions) have on the thermal
integrity of alpha-amylase enzyme, as gauged by the rate at which alpha-amylase
hydrolyzes starch at various temperatures (30, 40, 50, 60, 70, 80°C), gauged by
the rate of change in absorbance values (Au/s) of starch in iodine solution,
utilizing a colourimeter (635nm) in 300 seconds?

Jashan Kumar

Personal Code - lch759

Session - May 2024

Aim: The impact of the cofactor Ca2+ on alpha-amylase thermal stability

Research Question: What effect does the presence of Ca2+ (calcium ions) have on the thermal integrity of
alpha-amylase enzyme, as gauged by the rate at which alpha-amylase hydrolyzes starch at various
temperatures (30, 40, 50, 60, 70, 80°C), gauged by the rate of change in absorbance values (Au/s) of starch
in iodine solution, utilizing a colourimeter (635nm) in 300 seconds?

Introduction: Since a child, I have been captivated by the operations of enzymes in our bodies, as well as
the intricacies and versatility of enzymes that aid in the biosynthesis processes of organisms. When we
covered cellular respiration in my biology HL class, I was particularly interested in subject 8.2, which
discussed how acetyl CoA works as a cofactor in the link process. This sparked my interest in learning more
about cofactors and how they work. When investigating numerous sources connected to characteristics
determining the biological efficacy of enzymes, calcium ions were identified as one of the elements
influencing amylase activity. So, as I looked into the matter further in my IA, I became interested. Also, the
practical approach to my chosen course topic piqued my interest, since I enjoy experimenting with cuisine.
Indeed, this raised the question of whether calcium ions may improve amylase's heat tolerance, with possible
consequences for food manufacturing and storage. Answering my key issue required me to create an
experiment in which I pre-heated amylase with various concentrations of calcium ion solutions, fully aware
would be a difficult task and, therefore used complicated experimental approaches in my trials as a manner
of data collecting.

Background Information: In each living organism, there are many biochemical reactions which require
chemical reactions. For example, the process of digestion involves the breaking down of starch into
monosaccharides. These reactions are catalyzed by enzymes, which significantly speed up the reaction, such
as amylase which catalyses the process of the breakdown of starch. With that mentioned, a few enzymes find
that adding a non-protein molecule aids their functionality, especially from a stability viewpoint. These
molecules are called cofactors, and the catalogue of what can be a cofactor extends from ions (e.g. iron/zinc
ions) or vitamins/vitamin-derived molecules (Yokoyama What is an enzyme cofactor?). Essentially, what
cofactors do is they provide chemical groups to the enzymes, contributing to the structure and stability of the
enzyme in a cellular environment (Marchetti The role of cofactors in protein stability and homeostasis:
Focus on human metabolism).
Keeping in mind that the stability of enzymes, which are globular
proteins, can be affected by the temperature and pH of the cellular
environment, I’ve chosen to investigate Ca2+ as cofactors affect the
thermal stability of alpha-amylase, which is an enzyme which requires
calcium ions as cofactors. It has been proven that calcium ions are an
effective co-factor for the alpha-amylase enzyme, mainly contributing
by strengthening the thermal properties of the named enzyme (Morris et
al. Impact of calcium on salivary α-amylase activity, starch paste
apparent viscosity, and thickness perception). Enzymes have an
optimum temperature, and this is the temperature where the enzymatic
activity is greatest, it is different for each enzyme as they are specialised
to function in different cellular environments depending on their
environment’s conditions. The optimum temperature of alpha-amylase is somewhere in the ballpark of 35°C.
which is why I’ve chosen my temperature range which can show how the optimum temperature changes
with the addition of calcium ions and how calcium ions affect the activity of alpha-amylase at higher

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temperatures. My lowest temperature used is 30°C because it acts as a control as it is at room temperature.
My highest temperature utilised is 80°C because alpha-amylase is known to denature at around that
temperature, giving my temperature range of 30-80°C. Furthermore, a way to measure the activity of
alpha-amylase is to measure the rate of change in absorbance values using a colourimeter, as the activity of
the enzyme will cause the colour to shift from blue-black to a whitish hue.

Hypothesis
Null Hypothesis (H0): The existence of Ca2+ doesn’t impact alpha-amylase’s thermal stability.

Alternative Hypothesis (H1): The existence of Ca2+ aids alpha-amylase’s thermal stability.

Scientific Support: Previous studies have shown that alpha-amylase can become more thermally stable in the
presence of calcium ions. According to Gadad et al. (2013), calcium ions help alpha-amylase maintain its
conformational stability, which is essential for the enzyme's thermal stability.

Independent, Dependent, Controlled, and Uncontrolled Variables

Table 1: Independent, dependent, controlled variables along with description(s)
Independent variable Method for varying

Temperature of alpha-amylase & By using a water bath to equalize and vary the temperature of the
calcium chloride solution/distilled reacting solutions (30, 40, 50, 60, 70, 80°C)
water and starch (°C)

Presence of calcium ions By segregating trials into one with Ca2+ and the other with plain,
distilled, water for replacing Ca2+ solution.

Dependent variable Method for measurement

Rate of change in the absorbance Using LoggerPro3 as the data collection application, with Vernier
values of starch in iodine (Au/s) Colorimeter connected to the laptop through Vernier LabQuest
Mini Interface.

Controlled variable Method for Rationale for controlling

controlling

Source of alpha-amylase enzyme
For every trial, use pure Alpha-amylase enzymes from varied
alpha-amylase, origins and batches may have various
obtained from only one rates of reactions and optimal
bottle (to avoid batch temperatures, resulting in an unfair and
discrepancies) inconsistent comparison.

Volume and concentration of
alpha-amylase solution utilized
(enzyme)
For all trials, use Increased alpha-amylase volume
1.0cm3 of 2.0% increases the reaction rate, resulting in
alpha-amylase solution. an inaccurate comparison of findings.
When the concentration of the enzyme
increases, substrates are more likely to
collide with it, increasing the pace of
the reaction and altering the accuracy of
the findings.
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Volume and concentration of starch
solution utilised (substrate)
Volume and concentration of calcium
chloride solution utilised (cofactor)
Volume of iodine
pH level
Time limit for reaction
Colourimeter light wavelength
For all trials, use An increased amount of starch solution
1.0cm3 of 1.0% starch causes the color change to be delayed,
solution. resulting in an inaccurate comparison
of findings. When the concentration of
substrates increases, they are more
likely to collide with the enzyme,
increasing the pace of the reaction and
influencing the accuracy of the
findings.
For all trials, use An increase in the amount of CaCl2
1.0cm3 of CaCl2 solution increases the number of
solutions with a calcium ions, resulting in an inaccurate
concentration of 0.01M comparison of findings. Increased
calcium chloride solution concentration
may alter the reaction pH, therefore
causing temperature to not be the only
factor influencing findings, resulting in
erroneous results.
For all trials, use Increased iodine concentration darkens
0.1cm3 of iodine the color of the solution, resulting in an
erroneous comparison of absorbance
values.
Because the optimal pH pH impacts the activity of
of alpha-amylase is 7.5, alpha-amylase and must be maintained
use water as a buffer. consistently to guarantee that the only
(Cordeiro et al. factors impacting alpha-amylase are the
Production and presence of Ca2+ and temperature.
properties of
alpha-amylase from
thermophilic bacillus
sp..)
On LabQuest Mini, set As time passes, more starch is
the time restriction for hydrolysed by alpha-amylase, causing a
all trials to 300 bigger color shift and influencing the
seconds. absorbance value of the findings.
For all experiments, Because various wavelengths provide
calibrate the varying absorbance values, comparing
colorimeter to 565nm. findings might be erroneous.
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Apparatus and Materials Required: Table 2: Apparatus and materials for the experiment, along with
quantities, uncertainties, and justification
Apparatus and Quantity Uncertainty Justification
Materials
2.0% 2.0% alpha-amylase is a suitable concentration since it is
alpha-amylase 250cm3 ± 0.5 cm3 low enough to permit the recording of results. Excessive
solution concentrations can be harmful.
A concentration of 1.0% starch solution is appropriate
1.0% starch
250cm3 ± 0.5 cm3 since the color shift from blue-black to brown may be seen
solution
within the 300s time range.
High CaCl2 concentrations may cause a pH shift, altering
the R-group linkages of the enzyme and perhaps causing it
to deteriorate. Furthermore, 0.01M was a suitable
0.01M calcium
150cm3 ± 0.5 cm3 concentration to detect any alteration in the enzyme's
chloride solution
structural integrity. (Yadav and Prakash Stabilization of
alpha-amylase, the key enzyme in carbohydrates properties
alterations, at low pH).
To guarantee that the alpha-amylase was at pH 8 in every
Distilled water 150cm3 ± 0.5 cm3
trial and that the total volume was the same across all trials.
The quantity of starch in a solution is determined by the
color shift of iodine (iodine becomes blue-black from
Iodine indicator 1 -
brown in the existence of starch), which is utilised to
quantify the dependent variable.
To calculate the dependent variable, the absorbance value
Colourimeter 1 - of the solution must be measured. (quantitative data
collection).
LabQuest Mini For connecting the colourimeter to the computer.
connected to 1 -
computer
LoggerPro 3 A piece of software that measures the shift in absorbance
1 -
application value of a solution to calculate the dependent variable.
Cuvettes 4 - Insert the solution into a colourimeter.
Since a minute volume, 1.0cm3 is required, utilizing a
5.0cm3 syringes 3 ± 0.05 cm3 5.0cm3 syringe reduces uncertainty when compared to
using a 10cm3 measuring cylinder.
To measure 0.1cm3 of iodine indicator as it is a small
Micropipette 1 ±5 µL
volume
When in the water bath, contain the alpha-amylase, starch
50cm3 beakers 4 ± 1cm3
solution, CaCl2 solution, and distilled water.
20cm3 measuring To measure the volume of the solutions used in the
4 ± 0.1cm3
cylinders experiment.
250cm3 As a vessel to prepare and contain the starch solution, the
3 ±0.1cm3
volumetric flasks calcium chloride solution, and the amylase solution.
To agitate the starch and amylase solutions so that the
Glass stirrer 1 -
molecules are more equally distributed in the solution.

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Risk assessment: Table 3: Risk assessment and precautions taken for the experiment
Concern Hazard Precaution

Safety Handling of the Eye protection in the form of goggles are worn throughout the
alpha-amylase experiment
solution, calcium
chloride solution

Use of water bath When working with high-temperature liquids, use gloves. After
setting up the water bath, do not touch the electrical plugs,
colourimeter, or computer with wet hands. To avoid putting water
on the colourimeter and computer, place them on a different table
from the water bath.

Ethical No animals were harmed as a result of the experiment

Environment Disposal of remaining Flush them into a basin and rinse them with water. Because small
enzymes and amounts were used, no additional dilutions were necessary.
chemicals

Usage of chemicals Only amounts which were sufficient for the experiment were
and enzymes utilized, minimizing the waste of enzymes and materials.

Pre-lab: Before the main experiment, a pre-lab was conducted to produce and test an appropriate method for
this experiment, one that would produce accurate and reliable results without anomalies, therefore errors are
minimized in the actual experimental trials. Furthermore, through the preliminary experiment, it can be
ensured that there will be a change observed by adding calcium. For the pre-lab, the temperatures of 30°C,
50°C, and 70°C were utilized for three trials. I have utilized these temperatures to ensure that:
a) LabQuest mini was functioning optimally for the trials,

b) Ca2+ ions affected the thermal stability of alpha-amylase at higher temperatures,

c) the range and the increments utilized for the temperature were appropriate for the experiment,

d) a colour change is observable with the chosen duration, which is 300 seconds.

The pre-lab results show that calcium ions have an effect on alpha-amylase activity and that this effect can
be replicated across the range of temperatures and time (300 seconds) utilized. Preliminary testing also
suggested that the solution of iodine should be added before the CaCl2 solution. If we skip this step, the
iodine will not diffuse equally across the solution, leading to systematic errors due to erroneous colourimeter
readings. Furthermore, the modifications that were made to the procedure after conducting the pre-lab
included increasing the volume of the iodine indicator as low volumes didn’t display a change. Furthermore,
calcium chloride solution and alpha-amylase solution were mixed before heating to ensure that the heat
didn’t denature the alpha-amylase enzyme.

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Control Experiment: Control experiments are important as they enable us to separate and assess the impact
of particular variables in an experiment. We can assess whether the effects we find are attributable to the
intervention we are investigating or if they are the result of other factors by using a control group or
experiment as a baseline comparison. Without a controlled trial, it might be difficult to tell whether an effect
is attributable to the treatment or intervention under test or to other variables like bias, the placebo effect, or
random fluctuation. We can compare the two results and make sure that any changes shown in the
experimental group are a result of the treatment or intervention under test and not just random chance, bias,
or other external factors by performing a controlled trial alongside the experimental group. Control
experiments can bolster trust in the findings by confirming the accuracy and dependability of the
experiment's findings. Control experiments are therefore essential in scientific research because they enable
us to distinguish between the effects of the treatment and those of other factors, resulting in more precise
and trustworthy results.

Procedure
a. Preparation of solutions

1. Using a balance, measure 5 grams of alpha-amylase powder.

2. Add the powder to a volumetric flask, adding a bit of water and swirling the flask to mix the
alpha-amylase in the water.
3. Once the solution is homogenous, add in the remaining water until the 250cm3 mark.
4. Using a balance, measure 1 gram of starch powder.
5. Add the powder to a volumetric flask, adding a bit of water and swirling the flask to mix the starch in
the water.
6. Once the solution is homogenous, add in the remaining water until the 250cm3 mark.
7. Using a balance, measure 0.365 grams of calcium chloride dihydrate.
8. Add the powder to a volumetric flask, adding a bit of water and swirling the flask to mix the starch in
the water.
9. Once the solution is homogenous, add in the remaining water until the 250cm3 mark.

b. Colourimeter set-up

10. Attach the colourimeter to the LabQuest Mini, which is then connected to the laptop.
11. Launch LoggerPro on the laptop and configure it for data gathering.
12. Set the wavelength of the colorimeter to 635nm.
13. Using LoggerPro, set the data collection duration for each trial to 300 seconds, capturing data every
second.
14. Calibrate the colourimeter by inserting a cuvette loaded with distilled water and waiting for the
absorbance to reach zero.

c. Experiment

15. Using four distinct measuring cylinders, pour 15cm3 of distilled water, the alpha-amylase solution,
the starch solution, and the calcium chloride solution, and then pour each liquid into four separate
50cm3 beakers.
16. Put the 50cm3 beakers containing the alpha-amylase solution, starch solution, calcium chloride
solution, and distilled water in a 30°C water bath and allow them to heat up.
17. Check the temperature of the alpha-amylase solution, starch solution, calcium chloride solution, and
distilled water with a thermometer, taking care to use a separate thermometer for each beaker.
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 Note: Ensure that step 4 is only started after all solutions have reached 30°C.

18. Using a 1.0cm3 syringe, measure and add 1.0cm3 of starch solution into a cuvette.
19. Using a 1.0cm3 syringe, measure and add 0.1cm3 of iodine into the same cuvette.
20. Using a 1.0cm3 syringe, measure and add 1.0cm3 of calcium chloride solution into the same cuvette.
21. Using a 1.0cm3 syringe, measure and add 1.0cm3 of alpha-amylase solution into the same cuvette,
then quickly seal the cuvette's lid and insert it in the colourimeter.

Note: Ensure a different syringe is used in steps 4 to 7 to prevent contamination

22. Press start on LoggerPro to start collecting the data

23. After 300 seconds, record both the initial and final absorbance values as indicated on LoggerPro.
24. Repeat steps 1–10, using distilled water for the calcium chloride solution in step 6.
25. Repeat steps 1-11, at the ensuing temperatures: 40, 50, 60, 70, and 80°C.

Results and Data

Raw Data: Qualitative Data

1. A standard starch solution with iodine added for reference

2. Starch solution with alpha-amylase, iodine, and distilled water in place of calcium chloride solution
3. Starch solution with alpha-amylase, iodine, and calcium chloride solution:

Qualitative data shows the activity of calcium chloride as a cofactor at 50°C, therefore verifying that
calcium chloride assists alpha-amylase in the hydrolysis of starch due to a lesser blue-black colour.

Quantitative Data- Table 4: Raw data as collected from the LoggerPro application
Temperature Ca2+ ions present Ca2+ ions absent
(°C) Trial Initial absorbance Final absorbance Trial Initial absorbance Final absorbance
(± 0.05°C) (Au) (Au) (Au) (Au)
(± 0.0005Au) (± 0.0005Au) (± 0.0005Au) (± 0.0005Au)
30 1 0.875 0.512 1 0.932 0.588
2 0.919 0.547 2 0.883 0.566
3 0.939 0.466 3 0.894 0.493
4 0.865 0.515 4 0.905 0.462

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 5 0.860 0.540 5 0.926 0.511
40 1 0.945 0.297 1 0.889 0.347
2 0.936 0.338 2 0.942 0.375
3 0.949 0.284 3 0.910 0.301
4 0.867 0.341 4 0.875 0.271
5 0.902 0.314 5 0.927 0.295
50 1 0.931 0.454 1 0.855 0.441
2 0.879 0.427 2 0.796 0.403
3 0.913 0.386 3 0.915 0.487
0.888 0.351 4 0.852 0.482
5 0.904 0.409 5 0.927 0.477
60 1 0.885 0.471 1 0.912 0.628
2 0.802 0.465 2 0.973 0.642
3 0.901 0.484 3 0.991 0.673
4 0.885 0.504 4 0.875 0.585
5 0.879 0.449 5 0.924 0.668
70 1 0.880 0.506 1 0.856 0.667
2 0.925 0.519 2 0.737 0.681
3 0.870 0.540 3 0.917 0.686
4 0.859 0.520 4 0.870 0.653
5 0.942 0.565 5 0.937 0.693
80 1 0.944 0.586 1 0.844 0.712
2 0.935 0.604 2 0.919 0.774
3 0.958 0.611 3 0.930 0.785
4 0.873 0.577 4 0.886 0.726
5 0.894 0.532 5 0.891 0.744

Processed Data: Formula for calculating processed data

∆𝐴 = 𝐴𝑖 − 𝐴𝑓
∆𝐴1+∆𝐴2+∆𝐴3+∆𝐴4+∆𝐴5
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 ∆𝐴 = 5
𝑑𝑠
𝑑𝐴
= ∆𝐴/300
Where:
𝐴𝑖= Initial absorbance 𝐴𝑓= Final absorbance ∆𝐴 = Change in absorbance 𝑑𝑠
= Rate of change of
𝑑𝐴
absorbance

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Table 5: Processed data, showing average values obtained in each trial along with standard deviation
Ca2+ present Ca2+ absent
Temperature Average rate of change in Standard Average rate of change in Standard
(°C) absorbance value (Au/s) deviation absorbance value (Au/s) deviation
30 0.0013 0.000193 0.0013 0.000173
40 0.0020 0.000183 0.0020 0.000120
50 0.0017 0.000117 0.0014 0.000103
60 0.0013 0.000125 0.0010 0.000098
70 0.0012 0.000103 0.0006 0.000034
80 0.0011 0.000890 0.0005 0.000033

Result and Analysis

The line graph shows the average change in the
absorption rate as temperature was increased for
both treatments, with and without Ca2+. First off,
both lines are similar from 30°C to 40°C, where
the enzyme reaches its optimum temperature,
thus peak activity. Then, the effect of the
calcium ions as a cofactor starts becoming
pronounced, as at 50°C and beyond, the samples
with calcium ions don’t show as much of a shift
in activity relative to those without calcium ions
as a cofactor present. In higher temperatures,
e.g. 70 & 80°C, the difference becomes more
pronounced as calcium ions do their job as a cofactor to keep the amylase thermally stable, being able to
withstand higher temperatures and not denature as much, almost plateauing after 70°C. Another observation
to be made from the graph is that our standard deviation values decrease as the temperature increases,
signifying more accurate results. Recalling our alternative hypothesis, H1: The addition of calcium ions
strengthens the thermal stability of alpha-amylase, which can be said true using the graph as supporting
evidence to accept the alternative hypothesis.

Statistical Test: Paired t-test (one-tail)

The paired sample t-test is a statistical test that determines if the mean
difference between two sets of observations is zero. A paired sample t-test
involves measuring each subject or entity twice, yielding pairs of
observations. It was employed due to its reliability in deciding if one
variable is better than the other, making it a perfect fit for dependent
samples, with unequal variances, and also with data following a
non-normal distribution. From the test, it is evident that the p-value of
around 0.02 is smaller than our set significance (0.05), thereby offering
sufficient support for rejecting the null hypothesis. It is also worth noting
that the t-stat value obtained is higher than the t-critical value, therefore

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giving us support to accept the alternative hypothesis.

Conclusion: This investigation has displayed and affirmed that the presence of Ca2+ (calcium ions)
positively affects the thermal integrity of alpha-amylase enzyme, as gauged by the rate at which
alpha-amylase hydrolyzes starch at various temperatures (30, 40, 50, 60, 70, 80°C), gauged by the rate of
change in absorbance values (Au/s) of starch in iodine solution, utilizing a colourimeter (635nm) in 300
seconds. This is verified by the qualitative analysis, as it has been observed that the presence of calcium ions
causes a greater colour shift than without using calcium ions in 300 seconds. The quantitative analysis
confirmed and elaborated the qualitative analysis as showing an increase in thermal stability as
alpha-amylase resisted denaturing as the temperatures increased, not showing as much of a decrease as the
group without calcium ions. Furthermore, this conclusion has been verified by graphical interpretation and
also the paired T-test conducted, from which we have rejected the null hypothesis and have accepted the
alternative hypothesis, thereby concluding our investigation.

Evaluation: The evaluation focuses on identifying the experiment's merits and flaws. Starting with the
strengths, it is built around three pillars: the large number of trials, the broad range of the independent
variable, and the favourable outcomes of the statistical tests. Firstly, the large number of trials allowed for
the minimization of the effect of random error on my final result as if one trial doesn’t go well, it doesn’t
make as much of an impact compared to if 1 or 3 trials were utilized for each independent variable. Next, the
broad range of independent variables, with 6 different temperatures. This guaranteed that a definite pattern
could be identified and that random mistakes had no substantial influence on the final data. Lastly, the
reliability of the experimental analysis is cemented as there has been evidence to reject the null hypothesis.
All in all, the strengths show that this experiment produced reliable results. Though it was reliable, the
experiment had a few weaknesses, listed in the table below:

Table 6: Weaknesses from the experiment along with significance and improvement(s)
Weakness Significance Improvement

Independent A broader range of temperatures would allow a Utilize a temperature range

variable range is holistic understanding of how far calcium ions are to above 80°C, probably up to
broad but could be effective in maintaining the thermal integrity of around 120°C.
be more varied alpha-amylase.

Long heating Since I was using a water bath, it took a long time to Heating the solutions without
times heat the solutions, and this may affect the alpha-amylase to ensure that
denaturation of alpha-amylase, therefore leading to the enzyme is in perfect
inaccurate results to an extent. condition before use before
trials.

Freshness of Due to the length of the experiment, the Prepare new solutions every
alpha-amylase alpha-amylase solution may have degraded, even week.
though it was kept in a refrigerator when not in use.

Use of The colour produced by the presence of starch when Use of a spectrophotometer.

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 colourimeter iodine is added (blue-black) might be unsuitable for a
colorimeter. Instead, the use of a spectrophotometer
would be more optimal due to its ability to recognize
a broader spectrum of wavelengths, producing more
accurate absorbance figures.

Lack of Only one colourimeter was present in the school Use more colourimeters to
equipment. laboratory, therefore all the trials had to be done one carry out trials simultaneously.
by one, not simultaneously. Due to this, trials were
carried out within a span of a few weeks, therefore
the fluctuations in conditions might’ve influenced the
results slightly.

Extensions: The significance of this experiment was to prove that calcium ions could help us achieve better
results with alpha-amylase at higher temperatures, due to their nature as a cofactor. However, this
experiment could be made more elaborate through extensions, which would be of greater real-world
significance as we could apply them to industrial applications. One such extension would be to investigate
how different concentrations of calcium chloride impact the thermal stability of alpha-amylase at a certain
temperature (e.g. 80-100°C as these are temperatures where enzymes without cofactor denature) as this
could help determine the optimal concentration of calcium chloride, therefore, minimizing waste of
materials. Another extension to this experiment would be to compare different ions to see which ions work
best as a cofactor, as this would help us determine which ion is the most efficient as a cofactor.

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