The Effects of Sodium Chloride Concentration (0.2%, 0.6%, 1.0%, 3.0%, 5.

0%) on α -Amylase
activity and starch breakdown as measured by the Relative Absorbance of glucose and
Benedict‟s Reagent
Research question:
How does increasing the sodium chloride concentration (0.2%, 0.6%, 1.0%, 3.0%, 5.0%) affect
the enzyme activity of α –amylase, as measured by the relative absorbance of glucose and
Benedict‟s Reagent using a colorimeter?

Personal Engagement:
Sodium chloride, also known as salt, is a common ingredient found in many of the foods
eaten in the society. While it is an important part of diets of societies all around the world, a high
intake of sodium chloride is often associated with elevated blood pressure (Rasheed et al. 2016).
High blood pressure is then seen as major risk factor of cardiovascular diseases such as heart
diseases and strokes which can lead to death (Rasheed et al. 2016). Thus, having a high level of
sodium chloride in blood due to consuming more sodium chloride filled foods, drinking too little,
sweating excessively, or vomiting is not necessarily good. Additionally, having a higher
concentration of sodium chloride in the human body causes the enzymes working to be exposed
to more sodium chloride. This raises the interesting question of how this affects the enzymes
needed for processes such as digestion of foods. One particular enzyme that plays a big role in
digestion and is the one that will be investigated happens to A-amylase which is found in saliva
and in the pancreas. A-amylase causes the hydrolysis of 1,4 bonds in starch into maltose and
maltotriose (Allott & Mindorff, 2014). They are then broken down into glucose. This rate at
which amylase functions can be affected by the salt concentration which would mean that the
amount of salt we consume and our choices in diet can affect our body‟s functioning.

While, it is evident that our choices in lifestyles and diet can influence our bodies and our
health, it is not something that people often think about and many people continue to choose an
inactive and unhealthy diet despite the consequences. For example, in this study about salt intake
and hypertension in the province of Shandong, China, it is found that the average amount of
sodium consumed is around 5,745 mg and around 23.4 % are thought to have hypertension (Bi et
al. 2014). Hypertension is a leading factor of cardiovascular disease which accounts for around
40% of the deaths in China (Bi et al. 2014). Additionally, unhealthy diets can lead to childhood
obesity which is a big problem in developed and developing nations around the world (Sahoo et
al. 2014). Obesity can then lead other health problems such as Type 2 Diabetes and Coronary
Heart Disease (Sahoo et al. 2014).

I have always been very interested in nutrition and how it affects our lives. So this
investigation is an excellent opportunity to observe the effects of consuming more salt and higher
salt concentrations on the enzyme activity of amylase which is essential for starch digestion.

Introduction:
Enzymes are biological catalysts that speed up reactions in living organisms and have
many industrial applications such as the food, paper and textile industries (Sundarram & Murthy
2014). One particular type of enzyme is amylase which is needed in the hydrolysis of starch and
can be found in plants, animals and other microorganisms (Sundarram & Murthy 2014). In
humans, amylase is found in many parts of the digestive system such as the mouth and the
pancreas (Allott & Mindorff, 2014). There are three types of amylases, α-amylase, β-amylase,
and γ-amylase. The amylase that was used for this investigation is α-amylase which acts upon
the substrate starch, a polysaccharide made of two types of polymers, amylase and amylopectin.
Amylose is a straight chain of glucose units linked by α-1, 4-glycosidic linkage while
amylopectin is branched chains of glucose units containing an α-1, 6 glycosidic bonds every 15-
45 glucose units where the branching occurs (Sundarram & Murthy, 2014). Α-amylase catalyses
the hydrolysis of α-1, 4-glycosidic linkages in starch to make maltose and maltotriose
(Sundarram & Murthy, 2014). While 1,6 bonds of amylopectin, also called dextrin, cannot be
broken down (Allott & Mindorff, 2014). Later, in the human digestion process, other enzymes
called maltase, maltotriase, and dextrinase are used to break maltose, maltotriose and dextrin into
glucose (Allott & Mindorff, 2014).

The rate at which amylase breaks down starch can be affected by factors such as
temperature, pH, substrate concentration, enzyme concentration, sodium chloride concentration
and other enzyme inhibitors (Campbell et al., 2008). As the temperature increases up to a certain
point, the rate of enzyme activity also increases since more kinetic energy is given to the
particles and collisions between the starch and the active sites on the amylase happen more
frequently (Allott & Mindorff, 2014). As a result, starch is broken down more rapidly. For
human salivary alpha-amylase, the ideal temperature at which enzyme activity is optimized is 37
°C (Rudeekulthamrong & Kaulpiboon, 2012). As the temperature exceeds 37 °C, enzyme
activity drops quickly and the high temperature can eventually lead to the denaturing of the
amylase enzyme when the enzymes bonds break and structure changes. This is a permanent
change that takes away the ability of enzymes to catalyze reactions, which slows down and
eventually stops enzyme activity as more amylase denature. Similarly, a pH level that is too
high/ alkaline or too low/acidic can also denature enzymes and stop amylase enzyme activity.
For amylase, the optimal pH, at which the enzyme works best at, happens to be 7
(Rudeekulthamrong & Kaulpiboon, 2012).

Enzyme concentration and substrate concentration are some other factors that affect the
rate of enzyme activity. As the concentration of starch increase, so does the rate of reaction,
since there are more active sites available for the starch to bind to. However, as the concentration
continues to increase, gradually the rate of reaction levels off since the active sites will become
saturated and unavailable for other starch particles wanting to bind to it (Allott & Mindorff,
2014). Similarly, as the concentration of amylase increases, there will be more active sites
available to catalyze the reaction which will speed up the process. Hence, the rate of reaction will
increase.
As a result, these variables, temperature, amylase concentration and starch concentration
will act as the control variables of this investigation.
Finally, enzyme inhibitors, activators and sodium chloride concentration can also affect
amylase enzyme activity. Some metal cations such as Ca2+, Li+, and Mg2+ are activators as they
increase amylase enzyme activity, while heavy metals such as Fe2+, Cu2+and Zn2+ are inhibitors
as they decrease enzyme activity (Mageswari et al. 2012). Additionally, sodium chloride can also
affect enzyme activity to some degree. This is an ingredient that is common in food and is part of
human diet. In humans, sodium chloride makes up around 0.4% of our mass and the salt
concentration of blood is around 0.9 %. Sodium chloride at low concentrations does not really
affect amylase activity, but sodium chloride at higher concentrations, such as 2M and 5M, does
slow down the reaction of amylase activity by a bit (Dutta et al. 2006). This is because changes
in salinity can disrupt its bonds and the 3D structure of the enzyme (Sinha & Khare 2014).
Additionally, it can cause a disturbance in the water structure nearby, affect its intermolecular
hydrogen bonds and affect solubility, binding and stability (Sinha & Khare 2014). All of this
contributes to the denaturation of the enzyme as the active sites change shape and the substrate is
no longer able to bind (Allott & Mindorff, 2014).

Hypothesis:
If the concentration of sodium chloride increases, the rate enzyme activity will gradually slow
down because high amounts of sodium chloride disrupts the bonds and structure of the active
site. This then leads to denaturation and the starch is no longer able to bind to some of the active
sites. Enzyme activity will gradually slow down as more and more enzymes become denatured
and finally stop.

Variables:
Type of Variable Explanation
Variable
Independent Sodium Sodium chloride concentration is one of the factors that affect
chloride enzyme activity. If the concentration is low there is little to no
concentration effect on amylase activity. However, if the NaCl concentration is
(0.2%, 0.6%, too high, it can affect the bonds of the enzyme and its active sites.
1.0%, 3.0%, The changes in the 3D structure can then cause denaturation as
5.0%) starch is no longer able to properly bind to the active sites of
amylase. These specific concentrations of sodium chloride were
chosen because they are around the sodium chloride concentration
in blood which is 0.9%. Unfortunately, 0.9% solution could not be
made since it requires 0.45g of sodium chloride for 50ml of
solution and the scale only displays one decimal place. As a result,
0.45g cannot be precisely measured so 1.0% was chosen instead.
Other concentrations chosen were bigger and smaller than 1.0% to
see the effects of very little and excessive amount of sodium
chloride on a-amylase activity.
Dependent Relative Benedict‟s reagent is an indicator that turns yellow when glucose, a
absorbance product of the hydrolysis of starch, is present. Depending on the
amount of glucose present, the colour of the solution can be light
yellow to dark yellow/ brown. A colorimeter can be used to shine
light of wavelength 470nm (blue light) through a small sample
containing amylase, salt and starch solutions and Benedict‟s
Reagent to determine the relative absorbance. The higher the
number, the more glucose that is present. This gives an indication
of the enzyme activity of amylase.
Control Amylase This concentration must be constant throughout the experiment to
Concentration stop amylase concentration from affecting the amount of glucose
(1.0%) present and the relative absorbance. If there is too little amylase,
the rate of enzyme activity will be slower as there are fewer active
sites to catalyze the reaction. As the enzyme concentration
increases, there will be more active sites available to catalyze the
hydrolysis of starch which results in a higher rate of enzyme
activity. However, as amylase concentration continues to increase
 even further with the starch concentration remains the same, the
increase in enzyme activity with become smaller until it becomes
constant. This is because there will be too many active sites
available and not enough substrate to attach to them. Hence, the
rate of enzyme activity becomes constant. In this experiment, the
amylase concentration chosen was 1.0% which was not too high or
too low.
Starch Another variable to keep constant throughout the trials is substrate
Concentration concentration. If the starch concentration is too low then there will
(1.0%) not be enough substrate available to bind to all the active sites
which would result in a less glucose produced and a lower relative
absorbance (Allott & Mindorff, 2014). However, if the
concentration increases and the enzyme concentration remains the
same, the relative absorbance will also be higher. Eventually,
enzyme activity will slow down since the active sites will become
saturated and unavailable for other substrates to bind (Allott &
Mindorff, 2014). The concentration of substrate chosen for all trials
is 1.0%, the same of the amylase concentration, as it is not an
overly high or low concentration.
Temperature The optimal temperature for amylase is 37°C which is the
of hot bath temperature the hot bath will be kept at (Rudeekulthamrong
(37°C) & Kaulpiboon, 2012). If the temperature is over 37°C, the amylase
will start to denature due to its bonds breaking and the structure
and active sites changing (Allott & Mindorff, 2014). When amylase
becomes denatured, it is no longer able to catalyze reactions which
will eventually slow down enzyme activity. If the temperature is
below 37°C, there is less kinetic energy which means less collision
between active sites and starch and a slower rate of reaction (Allott
& Mindorff, 2014). The temperature of the hot bath must be kept
constant so that temperature is not the factor that caused
fluctuations in relative absorbance.
Time The amount of time that the amylase solution is allowed to be
(25 minutes in reacting with the sodium chloride and starch solution and the
the test tube amount of time spent in the hot bath are also factors that need to be
and 2 minutes kept constant. The longer the amylase solution and the starch and
in the hot sodium chloride solutions are sitting there mixed together affects
bath) the more glucose is produced which leads to a higher absorbance
shown on the colorimeter. Thus, the time chosen must be kept
constant. The hydrolysis of starch by amylase is not a particularly
fast reaction so 25 minutes was the time chosen for each trial.
Finally, the solution of amylase, starch and sodium chloride was
also put into the hot plate for 2 minutes following the addition of
Benedict‟s reagent to allow it time to react to the glucose and
change colour. As a result, each trial took 27 minutes which
allowed enough time for the hydrolysis of starch and provided
similar results for all the trials of one condition.
Materials:
 1.25 g of A-Amylase powder  1 Stirring rod
 1600 ml of Water  1 Hot plate
 1.25 g of Starch  1 thermometer (±0.5 °C)
 5.0 g of Sodium chloride  5 Test tubes
 100 ml Graduated cylinder (±0.5 ml)  250 ml Benedict
 250 ml volumetric flask  Vernier Colorimeter (±0.001)
 1 volumetric flask cap  6 Colorimeter tubes
 five 50ml beakers  1 timer
 one 250 ml beaker  1 marker
 10 ml graduated cylinder (±0.1 ml)  1 roll of tape
 1 scale (±0.1g)

Safety:
 Wear goggles, gloves, and an lab apron for protection
o Benedict may cause irritations when in contact with skin or eyes (ChemLab 2014)
o A-Amylase can also cause irritations when exposed to skin or eyes (AquaPhoenix
2014)
 Benedict contains copper sulfate, Sodium Carbonate, Potassium Ferrocyanide, Potassium
Thiocyanate, Sodium Citrate and water, so it should be disposed in the caustic waste
container. (ChemLab 2014)
 A-Amylase should be kept away from heat as it can be explosive at high temperatures
and should be stored in an air-tight container (AquaPhoenix 2014)
 Amylase and sodium chloride-starch solutions can also be disposed of in the caustic
waste containers
 Be cautious when working with hotplates, unplug and turn off hot plate when it is not in
use to avoid getting burned and injured
 Be careful with the glassware (beakers, flasks etc) to avoid breaking them and getting
injured

Methodology/Procedure:
Preparation of starch solution:
1. Measure out 2.5 g of starch using the scale.
2. Measure out 250 ml of water using the 100 ml graduated cylinder and pour the 250 ml of
water into a 250 ml beaker.
3. Pour the 2.5 g of starch into the 250 ml beaker containing the water and place on top of
the hotplate.
4. Plug in the hotplate and set it to around 25°C using the thermometer to check the
temperature.
5. Mix the solution using the stirring rod until the starch has fully dissolved creating a 1.0 %
starch solution. Then remove the beaker from the hot plate.
6. Using a 100 ml graduated cylinder, measure out 50 ml of the 1.0% starch solution and
place inside a 50 ml beaker.
7. Repeat step 6 four more times to obtain five 50 ml beakers containing 50ml of starch
solution each.
Preparation of the 0.2 %, 0.4%, 1.0%, 3.0%, and 5.0% sodium chloride solution
8. Measure out 0.1g of sodium chloride using the scale.
9. Place the 0.1 g of sodium chloride into one of the five beakers containing 50ml of 1.0%
starch solution.
10. Mix the solution using a stirring rod until the sodium chloride has dissolved to create a
solution with 0.2 % sodium chloride and 1.0% starch.
11. Using tape and a marker, label this 50 ml beaker 0.2% sodium chloride and 1.0% starch.
12. Repeat steps 8-11 using 0.3g, 0.5g, 1.5g, and 2.5g of sodium chloride instead of 0.1g to
create the 0.6%, 1.0%, 3.0%, and 5.0% sodium chloride solutions.
Preparation of Amylase solution:
13. Measure 1.25 g of a-amylase powder using a scale.
14. Measure 125 ml of water using a 100 ml graduated cylinder and pour it into a 250ml
beaker.
15. Pour the amylase into the water and mix with a mixing rod until the amylase dissolves
creating a 1.0% amylase solution.
16. Pour the 1.0% amylase solution into a 250ml volumetric flask and seal it with a cap.
17. Store inside a refrigerator.
Conducting the Experiment:
18. Measure out 5.0 ml of the solution containing 0.2 % sodium chloride and 1.0% starch
using a 10 ml graduated cylinder and pour this into a test tube.
19. Place the test tube in a test tube stand.
20. Repeat step 18-19 four more times using 4 other test tubes.
21. Remove the amylase solution from the fridge and heat it to 37°C using a hot plate and
thermometer.
22. Measure 5.0 ml of amylase solution using a 10 ml graduated cylinder.
23. Add the amylase solution to one of the test tubes containing the 5 ml of sodium chloride
and starch solution and set the timer for 25 minutes.
24. Repeat steps 22-23 four more times by adding the 5.0 ml of amylase into each of the
other four prepared test tubes containing the 0.2 % sodium chloride and starch solution.
During the 25 minutes:
25. Fill a 2000 ml beaker with 1000ml of water and heat up the water using a hot plate to
35°C (use thermometer to check the temperature)
26. Set up the colorimeter and gather 6 colorimeter tubes.
27. Fill one of the colorimeter tubes with distilled water.
28. Set the wavelength of light on the colorimeter to 470nm (blue light).
29. Place the tube containing distilled water inside the colorimeter and calibrate.
After the 25 minutes:
30. Place 10 drops of Benedict‟s reagent into each of the 5 test tubes.
31. Place the 5 test tubes containing Benedict‟s reagent inside the 2000ml beaker of water
sitting on the hot plate for 2 minutes.
32. Remove test tubes from the beaker and place them back in the test tube rack.
33. Fill each of the 5 colorimeter tubes with solution from a different test tube.
34. Place each colorimeter tube into the colorimeter one at a time to find the relative
absorbance and record the data in the raw data table.
35. Repeat steps 18-34 using the 1.0% starch solutions containing 0.6%, 1.0%, 3.0% and
5.0% sodium chloride instead.
Data Collection and Processing:
Raw Data Table:
Table 1: Quantitative raw data of the relative absorbance (±0.001) when 1.0% amylase solution is
combined with1.0% starch solution containing 0.2%, 0.6%, 1.0%, 3.0%, and 5.0% sodium chloride
(±0.2%) after 25 minutes at room temperature in a test tube and 2 minutes in a hot bath
Sodium Relative absorbance (±0.001)
Chloride
Concentration Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
(±0.2%)
0.2% 1.101 1.143 1.171 1.127 1.109
0.6% 1.261 1.224 1.277 1.195 1.284
1.0% 1.146 1.110 1.215 1.122 1.130
3.0% 1.059 0.983 1.063 0.943 0.995
5.0% 1.021 0.949 1.034 0.877 0.972
Note: The uncertainty for relative absorbance is based on the lowest increment on the colorimeter, 0.001.
The uncertainty for sodium chloride concentration is calculated using the smallest increment on the digital
scale, 0.1g, and half the smallest increment on the 100 ml graduated cylinder, 0.5ml. First the percentage
error is calculated for both mass and volume separately by dividing the total uncertainty of each piece of
equipment by the specific amount measured out. Then these two percentages are added to find relative
uncertainty. Finally absolute uncertainty is found by multiplying the relative uncertainty and the
conditions. An actual calculation will be provided in the section below entitled “Sample Calculation”.

Qualitative Data Table:

Table 2: Qualitative observations of the 1.0% amylase solution and the 1.0% starch solution containing
0.2%, 0.6%, 1.0%, 3.0%, and 5.0% sodium chloride (±0.2%) before, during and after 25 minutes at room
temperature in a test tube
NaCl Before During After
Concentration
(±0.2%)
0.2% Sodium chloride: -liquid After the addition of Benedict:
-White -transparent -colour is now pale blue
-Small granular shape -some of the -a bit of undissolved amylase
-solid amylase sunk remains
-no smell to the bottom After the hot bath at 37°C:
Amylase: of the test -test tube is warm
-white tube -colour of the solution become light
-solid -uncoloured yellowish brown
-powder -transparent
0.6% Starch: After the addition of Benedict:
- white -colour is pale blue
-solid After the hot bath at 37°C::
-powder -test tube is warm
1% Amylase solution: -colour of the solution become
-liquid brown
-low viscosity - more opaque
1.0% -slightly white colour After the addition of Benedict:
-transparent -colour is now pale blue
-a bit of amylase powder After the hot bath at 37°C:
remained at the bottom of the -test tube is warm
solution -colour of the solution become light
 Benedict: brown
-blue -a bit opaque
3.0% -transparent After the addition of Benedict:
-liquid -colour is now medium blue
1% starch solutions of After the hot bath at 37°C:
varying salt concentrations: -test tube is warm
Characteristics in common: -colour of the solution become light
-liquid yellow
-low viscosity -transparent
5.0% -transparent After the addition of Benedict:
Other characteristics: -colour is now pale blue
0.2% NaCl: uncoloured After the hot bath at 37°C:
0.6% NaCl: uncoloured -test tube is warm
1.0% NaCl: slightly white -colour of the solution become very
3.0 NaCl: slightly white light yellow
5.0 NaCl: more white than -transparent
3.0%

Sample Calculations:
The mean is useful as it provides the average of the 5 trials of one independent variable condition
and helps to cover up the effects of outliers. Additionally, the means calculated are the points
are plotted on the graph. Standard deviation is needed as it will be used to create the error bars on
the graph. A bigger standard deviation correlates with bigger error bars. Thus it is useful when
analyzing the reliability of the results and seeing if there is a strong trend.

 Mean absorbance when1.0% amylase solution is exposed to a solution containing 1.0%

starch and 0.2% sodium chloride for all 5 trials

Mean =

Mean = = 1.130 (±0.001)

 Standard deviation of the absorbance when 1.0% amylase solution is exposed to a

solution containing 1.0% starch and 0.2% sodium chloride for all 5 trials
̅
σ=√ =√

σ=√ =

Uncertainty of 1.0% NaCl concentration:

Relative uncertainty= 20% + 1%
Percentage error (g) = = = 20% =21%
Percentage error (ml) = = = 1% =0.21

Absolute uncertainty = NaCl concentration × relative uncertainty = 1.0% × 0.21 = 0.2%

Processed Data:
Table 3: Processed data of the relative absorbance (±0.001) when 1.0% amylase solution is exposed to
1.0% starch solution with a sodium chloride concentration of 0.2%, 0.6%, 1.0%, 3.0% and 5.0% (±0.2 %)
for 25 minutes at room temperature and 2 minutes in a hot bath of 37°C
Sodium Chloride Relative Absorbance
Concentration (±0.2 %)
Mean of 5 trials (±0.001) Standard deviation (SD)
0.2% 1.130 0.025
0.6% 1.248 0.034
1.0% 1.145 0.037
3.0% 1.009 0.046
5.0% 0.971 0.056
Note: The method of calculating uncertainties is shown in “Sample Calculations”

Graph:
Figure 1: The relative absorbance (±0.001) when 1.0% amylase solution is exposed to 1.0%
starch solution with 0.2%, 0.6%, 1.0%, 3.0%, 5.0% sodium chloride (±0.2%) for 25 minutes at
room temperature and 2 minutes in a hot bath at 37°C
The effects of NaCl concentration (±0. 2%) on the relative absorbance (±0.001)
Relative absorbance (±0.001)

1.400
1.300 y = -4.842x + 1.196
R² = 0.763
1.200
1.100
1.000
0.900
0.800
0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0%

sodium chloride concentration (±0.2%)

Conclusion and Evaluation:

According to the data, it appears that as the salt concentration increases, there is a
decrease in relative absorbance. This relationship is mostly linear and is a somewhat strong
correlation for the conditions chosen. However it should be noted that the difference in mean
absorbance gradually becomes smaller as the concentration of sodium chloride rises. For
example between sodium chloride concentrations of 1.0% and 3.0%, the absorbance decreased
by around 0.005. While on the other hand, the decrease between 3.0% and 5.0% sodium chloride
concentration was only 0.001. Despite the fact that they both involve an increase in sodium
chloride concentration by 2.0%, the difference between 1.0% and 3.0% is far greater.
Furthermore, the fact that the errors bars at 3.0% and 5.0% were mostly overlapping and the
difference between the mean absorbance of these concentrations was only 0.001, which is the
uncertainty of the colorimeter, can indicate that the decrease in absorbance becomes very small
and absorbance will remain relatively constant after 3.0% NaCl. Additionally of the 5 sodium
chloride conditions, it was 0.6% sodium chloride that provided the highest rate of enzyme
activity instead of 0.2% which is the smallest concentration and should have theoretically
corresponded with the highest rate of enzyme activity. This could be because certain enzymes
are more structurally adapted to perform under specific saline environments (Sinha & Khare
2014). In this cause, the a-amylase used may be more suited to work under 0.6% salt
concentration than higher or slower salt concentrations. All of this may be suggesting that if the
experiment were to be continued with higher and lower salt concentrations, the trend line may be
of a similar shape to a shifted pH/ temperature graph with an optimum NaCl concentration and
sharp decline on both sides that slowly becomes less steep the farther away from the optimum.
This is a very possible option given the fact that the R2 value of the linear trend line is 0.763
which shows that the data collected does not fit very strongly with the linear line drawn.
Something else to realize is that the slope of the trend line drawn is not very steep at -4.8 which
indicates that NaCl concentration does not play a huge role in amylase activity. This seems so
mean that consuming a bit more sodium chloride would not really effect the digestion of starch
in the body to a large extent. However if the it were to be over 3.0% sodium chloride, there
would be a notable decrease in amylase activity when compared to the lower concentrations.
Overall my hypothesis was correct as I believed that enzyme activity, as measured by the
relative absorbance, will decrease as sodium chloride concentration increases. This is shown by
observing the trend line of the graph which has a negative slope of 4.8 as the sodium chloride
concentration increases. This is probably due to high sodium chloride concentrations affecting
the structure of the amylase and slowly diminishing its ability to cause the hydrolysis of starch
into maltose, maltotriose and eventually glucose. However, due to the 5 sodium concentrations
chosen, it is impossible to see if the enzyme activity will eventually stop if the concentration is
too high. This can only be observed if the experiment continued into higher sodium chloride
concentrations.
The results from this investigation are relatively reliable as similar results were obtained
in other published papers over the years. Firstly, in the experiments published by Dutta et al.,
they witnessed similar results of amylase activity decreasing as sodium chloride concentration
decreases (2006). Moreover, their results had a downward trend, but did not appear to be
perfectly linear (Dutta et al. 2006). For example, on their graph, there appeared to be 5%
difference in relative activity between the sample containing 3.0M and 4.0M of sodium chloride
and a 20% difference in the trial containing 4.0M and 5.0M after the same 24 hours (Dutta et al.
2006). This is similar to my results which had a R2 value of 0.763 when a linear line of best fit is
drawn. This helps to show that the affect of salt concentration on amylase activity is not a
perfectly linear trend.
Additionally, other published papers found similar results when analyzing the effects of
NaCl on amylase. In the journal entry by Oliveira et al., it mentions that NaCl at a concentration
of 20 mM had little to no effect on the activity of a-amylase in plants, however at a higher
concentration of 100 mM, there is a significant decrease in activity (2013). This is shown by
their graph that shows that at 0 mM of NaCl, the rate of amylase activity is about 21 mMol/mg
protein and it only experienced a slight decrease to around 19 mMol/ mg protein when NaCl
concentration is increased to 20 mMol (Oliveira et al. 2013). However, at 100 mM of NaCl, the
amylase activity decreased to 6 mMol/mg protein which only around 29% of its original activity
(Oliveira et al. 2013). This is similar to the results I collected since at relatively low
concentrations of 0.2%, 0.6%, 1.0% NaCl, the enzyme activity was similar with relative
absorbance of 1.130, 1.248 and 1.145 respectively. On the other hand, when the NaCl
concentration increases drastically to 3.0%, there was a decrease of around 12% from the
absorbance at 1.0% NaCl. Thus, it appears that at low concentrations of sodium chloride, salt
has little affect on amylase activity however that changes if the sodium chloride concentration is
too high.
Moreover, the effects of increased amounts sodium chloride on amylase are also true for
other types of enzymes. For example, a published lab that conducted a similar experiment using
nuclease found similar results of decreased activity when NaCl concentrations increased. In this
journal, the data was collected at 3 different time intervals, 1.5, 3.5 and 7.0 hours. For all these
time intervals the general shapes of the graph were very similar to each other and somewhat
similar to my graph. For the graphs of nuclease activity in response to different NaCl
concentrations, it appears that the optimum sodium concentration is around 1.0%. Nuclease
activity decreases as NaCl concentration increases or decreases from that point. Similarly on my
graph, the salt concentration that achieves the absorbance is 0.6% and any value above or below
this concentration results in lower amylase activity. In all these cases, it seems if more data was
to be collected using other salt concentrations, the resulting graph may actually look like a
shifted parabola.
While my results were relatively reliable, there are still some outliers in the raw data table
that impacted the mean absorbance and the R2 value of the graph. For example, one of the trials
for 5.0% sodium chloride got an absorbance of 0.877 which significantly lower than all other
absorbance of 5.0% which were in 0.900s and 1.000s. This causes a bigger standard deviation
and error bars. Some of the variations in absorbance which led to bigger error bars were due to
the fact that the experiment was carried out over multiple days which caused the amylase
solution to slowly denature and decrease its ability to catalyze reactions. This is due to the fact
that the experiment had to be conducted during regular class hours so there were limitations on
how many trials can be done each day. As this was the case, variations in the quality of the
amylase solution, which was created on the first day, and the temperature of the room affected
the data collected.
Additionally it was difficult to keep the temperature of the amylase solution and the hot
bath consistent throughout the lab given the equipment available. The limitations due to the
equipment available contributed to the relatively high uncertainty of the independent variable,
the sodium chloride concentration. The scale used only displayed one decimal place while the
100 ml graduated cylinder had an uncertainty of 0.5 ml. This had a pretty big effect in this
investigation since the quantity of solution required for each sodium chloride concentration was
relatively small at only 50 ml. This resulted in a high percentage error for both mass and volume
which led to a high relative uncertainty and high absolute uncertainty. When the absolute
uncertainties of all five conditions were rounded to one decimal place, it was ±0.2%. This is a
particular big uncertainty considering the fact that the percentages chosen were rather small and
one of the conditions happened to be 0.2%. This diminished the accuracy and precision of the
data. This high certainty could be decreased with the use of more precise equipment such as a
scale that shows 2 decimal places and the use of pipettes instead of 100 ml graduated cylinders.
These equipments would be more accurate and would have led to a smaller uncertainty for the
independent variable.
 On another note, the colorimeter can display 3 decimal places and has an uncertainty of
±0.001. This allows it to provide a more precise measurement. However, the number on the
colorimeter never stops at exactly one number so it is difficult to find the “exact” number that
represents the absorbance. Since this was the case for all the data collected, there is quite a bit of
uncertainty in the data making it not as precise as it should be.
Strengths and Weaknesses:

Limitations Explanation
Denaturation The 1.0% amylase solution was made on the first day of the lab, but the actual
of enzyme over trials were carried out over multiple days due to the scheduling of classes. As a
time result, the age of the amylase solution is not constant for all the trials. The
trials conducted with the fresher amylase solution can be more effective in the
hydrolysis of starch compared to the trials conducted later on. This causes the
time that the trial was conducted to affect the absorbance in addition to the
NaCl concentration. However, the general trend of the 5 conditions should still
be relatively similar as less effective amylase will affect all of the conditions.
Although, this may contribute to a higher standard deviation and bigger error
bar.
Difficult to Temperature is another factor that can affect amylase activity (Allott &
keep the Mindorff, 2014). While most of the amylase hydrolysis is taking place happens
temperature of when it is sitting in the test tube at room temperature, temperature can still play
the hot bath at a role in the form of the hot bath and the refrigerator. While the hot bath was
exactly 37°C supposed to be set to 37°C, this was very hard to keep consistent given the
for 2 minutes equipment available (thermometer) for 2 whole minutes. As a result, there
were slight fluctuations in temperature that could have sped up amylase
activity, denatured some enzymes due to high temperatures, and helped speed
up the change in colour. This can make the solution a darker colour which
results in a higher absorbance.
Inconsistency The 1.0% amylase solution was supposed to be left to react with the starch and
in the timing NaCl solutions for 25 minutes and then placed in the hot bath (after the
addition of Benedict‟s reagent) for exactly 2 minutes. However, due to the fact
that 5 reactions in 5 different test tubes were taking place at the same time, it
was really difficult to keep the exact timing for every single one of them. The
slight differences in timing may have allowed some test tubes more time for
the hydrolysis of starch and some less time. This can then affect the amount of
glucose present and the colour of the solution after the addition of Benedict‟s
reagent and the 2 minutes in the hot bath. This can then affect the relative
absorbance shown on the colorimeter.
Improvements and Extensions:

Improvements Explanation
Conduct One way to make the denaturation of amylase over time a smaller source of
experiment in error in this lab would be to conduct the experiment all in one day. This results
one day in less time between the various trials and less time for the amylase to
denature. As a result, the data would be more similar and the error bars would
be smaller.
One possible improvement to help keep the temperature of the hot bath
Use more constant at 37°C for 2 minutes is to use more precise equipment than a
precise thermometer and hot plate. One piece of equipment that can help with this is
equipment Precision‟s Digital Circulating Water Baths which provides ±0.05°C
(Precision™ uniformity at 37°C and comes with the stainless steel cap. This is far more
Digital precise at keeping a constant temperature and it has a smaller uncertainty
Circulating compared to the thermometer which has an uncertainty of ±0.5°C. It would
Water Baths) thus be more capable of keeping a constant temperature for 2 minutes than a
thermometer and hot plate.
Leave a 10 A 10 minute gap between the start of a reaction in one test tube to another
minute gap would cause the time allowed for each reaction to be more similar to one
between start another. This is because there would be a designated time, to remove the test
of the reaction tube from the hot bath, pour it into the small colorimeter tube and place it in
in one test tube the colorimeter to find the relative absorbance, that is unique to each test tube.
to the next As a result, only one test tube will be the focus at a time and the data that
collected will be more accurate since there is no rush to complete all the steps
for 5 test tubes of solution at once. The rush to accomplish all the steps leading
to the collection of data is what led to inconsistencies in the first place, as some
test tubes were allowed more time than others to continue the hydrolysis of
starch. Additionally, the amount of time needed to gather the relative
absorbance, will be similar if working with one test tube at a time. So the
amount of time given for amylase to break down starch will also be similar as
they will all get the 25 minutes mentioned in the procedure as well as the
amount of time it takes to complete the processes of removing the test tube
from the hot bath, pouring its contents into a colorimeter tube and placing it
into the colorimeter to find the relative absorbance.
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