What is the effect of changing pH (5, 6, 7, 8, 9) on the reaction rate, as measured by

disaccharide production (mM/min), of amylase?

MAHMAL

BIOLOGY HL
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Personal Engagement
During the first year of IB Biology, one of my favorite units was enzymes, but I would
have liked to do more experimental procedures on how changing the pH affects an enzyme’s
ability to catalyze reactions. Specifically, the enzyme amylase intrigued me the most because it is
part of saliva, whose pH routinely changes based on the acidic or alkaline food we eat and drink
like lemon or soda. It made me wonder: if pH is changing, to what extent does it actually
interfere with amylase’s ability to break down starch?

Research Question
What is the effect of changing pH (5, 6, 7, 8, 9) on the reaction rate, as measured by
disaccharide production (mM/min), of amylase?

Introduction
Biology Background Information
Enzymes are proteins that act as biological catalysts to speed up the rate of chemical
reactions such as digestion, photosynthesis and cellular respiration by reducing the activation
energy. Specifically, the activation energy is reduced as the enzyme is able to position the
substrate, the substance the enzyme acts on, in a specific way, allowing for more collision and
less kinetic energy to “break the bonds” (bthoole, 2018). In order to function properly, there is
enzyme-substrate specificity: each specific enzyme has a active site, a binding site that has
complementary shape and chemical properties that allow it to attach to a specific substrate
(Eed, 2013, p. 48).
The enzyme then is able to help break down the substrate into smaller product subunits as
shown by the equation below.
𝐸 + 𝑆 (𝑒𝑛𝑧𝑦𝑚𝑒 & 𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒) → 𝐸𝑆(𝑒𝑛𝑧𝑦𝑚𝑒-𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒 𝑐𝑜𝑚𝑝𝑙𝑒𝑥) → 𝑃 (𝑝𝑟𝑜𝑑𝑢𝑐𝑡)
Enzyme Chosen
The enzyme amylase is predominantly found in human saliva, liquid that is secreted by
salivary glands to aid in the process of starch breakdown for digestion and “determining liking
and preference for food,” and pancreas although they are coded by different genes (Peyrot des
Gachons & Breslin, 2016). However, the specific amylase I have chosen is the salivary kind that
breaks down starch, which is composed of amylose and amylopectin, through hydrolysis as the
enzyme breaks the alpha 1,4 glyosidic bonds and produces the smaller disaccharide subunits of
maltose (Peyrot des Gachons & Breslin, 2016).

Figure 1: The hydrolysis of starch by amylase (Klimek-Ochab, 2016)

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Additionally, if this experiment was done manually, amylase can be inexpensively

bought or sourced from a person’s saliva.
Factors That Affect Enzyme Activity
However, the rate of enzyme activity can be affected by three main factors: substrate
concentration, temperature and pH. The focus of the experiment, pH, affects reaction rate as it
can change the active site of enzyme, decreasing the amount of substrate that can be “converted”
into product. Moreover, extreme pH that are too alkaline or acidic can affect the hydrogen and
peptide bonds that hold protein structures together as the amino acids’ charges change; this
causes the enzyme to unfold and denature as the bonds are unable to keep the secondary and
tertiary structure of the protein intact tightly (Sky-Peck & Thuvasethakul, 1977, p. 309) (Morris,
n.d.). From previous investigations in which researchers used common enzyme assays with pH
buffer solutions, it can be seen salivary amylase’s activity works at its optimum, or fastest, rate at
a pH of 7. Moreover, the reaction was “active in the pH range of 4.5 to 10,” but rapidly
decreased in any pH level that was below or above the optimum while retaining only “40 to 60
percent of its activity at pH of 2 and 11 (Sky-Peck & Thuvasethakul, 1977, p. 311).
Procedure
In this experiment, I will be determining how changing pH, done by changing the
parameters on the simulation, will affect enzyme activity, which will be determined by the rate at
which amylase convert starch into disaccharides. The rate requires a calculation that will be
derived from the slope of the disaccharides produced graph on the simulation.

Importance
As stated before, enzymes are commonly found in animals and humans and contribute to
much of how the food we eat is digested, a very important bodily process as it provides nutrients
for growth, metabolism and cell repair. Moreover, it has been increasingly important in
biotechnology as amylase is routinely used in the preparation of fermented food and
pharmaceutical industries. Therefore, it is imperative we investigate pH, a common variable
factor that fluctuates due to simple reasons like air pollution, affects the reaction rate of enzymes.

Hypothesis
Working Hypothesis: As pH increases, there will be a significant effect on disaccharide
production and therefore, the reaction rate of amylase. Additionally, the optimum rate for
amylase where disaccharide production is the greatest will occur at the pH of 7.
Scientific Rationale: The experiment discussed in Factors That Affect Enzyme Activity as
well as scientific literature show that salivary amylase has optimal enzymatic activity at a
pH of 7 as it is the common pH of human saliva (Sky-Peck & Thuvasethakul, 1977, p.
309)

Null Hypothesis: As pH changes, the disaccharide production will remain stagnant and
therefore, there is no effect on the reaction rate of amylase.
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Variables
Units Rational
I chose these pH values as my levels because a lot of the
scholarly literature’s experiments I found online for other
enzymes made sure to cover a wide range of values that
included acidic to alkaline. I also wanted to make sure that I
didn’t repeat myself when the amylase is denatured by the
extremes of the pH scale, so I did not include 0-4 and 10-14
Independent Level of pH n/a
because looking at previous literature, they produced
Variable (5, 6, 7, 8, 9) (±1 pH)
similar values of minimal reaction rate (close to 0). In
addition, when picking my levels, I wanted to have 2 that
were more acidic that the predicted optimum of 7 and 2
more alkaline. pH was controlled using a parameter in the
simulation

Rate of
Dependent
disaccharide mM/min
Variable
production

Control
Units Why? How?
Variable
Temperature also affects enzyme
Temperature was set at 25℃ during
activity as raising temperature can
Celsius the entire data collection process by
Temperature increase reaction rate, but extremely
(±1 ℃ ) using the slider on the simulation
high temperatures can denature
enzymes, which would affect my data
Time for The more time an enzyme has, the
Minutes Collection was only 20 minutes
collection of more substrate it can break down,
(±2 min)
data which could alter rate
An enzyme can’t catalyze different Used only starch, the substrate for
Type of
n/a substrates as there is enzyme-substrate amylase
substrate
specificity
Rate of enzyme activity is affected by
Only 50 mM of substrate was added
Substrate Millimolar substrate concentration, so
for all trials
concentration (±1 mM) adding/subtracting concentrate will
alter the rate
A different enzyme may have different
Type of Used only amylase
n/a optimal pH which can skew the results
enzyme
on how pH affects activity

The more enzyme there, the faster Simulation doesn’t specifically state
Amount of
n/a substrate is broken down, skewing rate amount of enzyme, but it is assumed
enzyme
and data of how pH affects rate it is same amount each trial
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Safety, Environmental & Ethical Considerations

As I used a simulation, I took proper care of my laptop and shut it down if it was
overheating or not functioning properly. There were no ethical or environmental considerations
as I did not use any live organisms.

Materials
• Laptop with adequate Internet connection
o Data collected (disaccharide production) had uncertainty of ±30 mM

Methods
1. Basic Preparation of Simulation
a. Open the simulation with the following link:
https://sites.google.com/site/biologydarkow/enzyme-diversity-enzymes-products-
and-substrates
b. Click on the “Enzyme Behavior over Time” tab making sure the screen looks as
below

Figure 2: Enzyme Behavior over Time tab selected

c. Set initial starch to 50 mM using the slider, ignoring the other substrates as they
are not pertinent to this investigation
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Figure 3: The constant variable of amount of starch is set

d. Select “Add Amylase”

Figure 4: The constant variable of type/amount of enzyme is set

e. Set temperature at 25ºC

Figure 5: The constant variable of temperature is set

2. Data Collection
a. Using the “pH” slider, set the pH at 5.

Figure 6: The independent variable is changed with this parameter

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b. Hit “run” to begin data collection

Figure 7: The run button is used to collect the unprocessed data for the investigation

c. Repeat step 2b. 4 more times for a total of 5 trials for the pH
d. Repeat steps 1c.-2b., moving the pH 1 increment towards the right each time for a
total of 5 different pH values tested (5, 6, 7, 8, 9 pH)

Collected Data

Figure 8: Disaccharides Production For Amylase Over 20 Minutes With Various pH levels.
Data was collected (± 30 mM) by setting the specific parameters and hitting “run” 1 sample trial
for each pH level (5, 6, 7, 8, 9) is shown. The runs represent pH as followed: run 1 is 5, run 2 is
6, run 3 is 7, run 4 is 8, and run 5 is 9. The dependent variable is calculated by taking the slope of
the linear line from minutes 2-7.

Observations
• Each trial had a linear reaction rate in production for at least 6 minutes before becoming
flatter
o The 6 minutes in the simulation went quicker (enzyme is catalyzing reaction with
starch at optimum speed), but the last 14 minutes were slower in production
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• Each trial for each pH level returned approximately the same number of disaccharides
produced at the end of data collection
• For 2 of the trials, the simulation glitched and produced no line.
o These trials were not included in the data collection process
• No smell/visuals could be seen due to virtual nature of the simulation.

Sample Calculation For Rate of Disaccharide Production For pH 5

∆ 𝐷𝑖𝑠𝑎𝑐𝑐ℎ𝑎𝑟𝑖𝑑𝑒𝑠 𝑃𝑟𝑜𝑑𝑢𝑐𝑒𝑑
𝑅𝑎𝑡𝑒 =
∆ 𝑀𝑖𝑛𝑢𝑡𝑒𝑠

150 𝑚𝑀 − 60 𝑚𝑀
𝑅𝑎𝑡𝑒 = = 𝟏𝟖 𝒎𝑴/𝒎𝒊𝒏
7 𝑚𝑖𝑛 − 2 𝑚𝑖𝑛

Rate of Disaccharide Production (mM/min)

Trial pH level

5 6 7 (control) 8 9

1 18.00 23.73 29.63 22.68 22.70

2 17.82 22.67 26.37 24.12 19.62

3 17.10 23.50 27.80 22.76 22.25

4 16.10 23.27 28.20 21.95 21.42

5 17.62 24.30 27.22 21.49 20.87

Table 1: Rate of Disaccharide Production For Various pH Levels. All the data collected
using the equation above has been formatted into the data table.
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Rate of Disaccharide Production (mM/min)

pH level
5 6 7 8 9
AVG 17.33 23.49 27.84 22.60 20.97
STD DEV 0.76 0.60 1.21 1.00 1.18
T-TEST 0.0000 0.0004 0.0001 0.0000
T-TEST (8 vs 9) 0.0670
T-TEST (6 vs 8) 0.1329
T-TEST (5 vs 6) 0.0000

Table 2: Average Rate of Disaccharide Production For Various pH Levels. An average and
standard deviation was performed for each pH level. A t-test using pH 7 as a control was
performed as 7 is considered the pH of pure water. Additional t-tests were performed between
pH 8 & 9, pH 6 & 8 and pH 5 & 6. All calculations were performed using Microsoft Excel.

The Effect of Various pH Levels on the Average Rate of

Disaccharide Production
35.00
Average Rate of Disaccharide Production (mM/min)

30.00

25.00

20.00

15.00

10.00

5.00

0.00
pH 5 pH 6 pH 7 (control) pH 8 pH 9
Level of pH

Figure 9: The Effect of Various pH Levels on the Average Rate of Disaccharide

Production. The average rate and standard deviation from Table 2 are graphed
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Conclusion
According to the collected data, changing the pH has a significant effect on the reaction
rate as measured by the rate of disaccharide production of amylase. In Figure 9, it can be clearly
seen that the pH of 7 creates the fastest rate and any pH that diverges from 7 constitutes a
decrease in rate. Overall, there is a bell shape that shows that as the pH increases to 7, the
reaction rate increases and when pH decreases from 7, reaction rate decreases. This observation
is further supported by data in Table 2 where the average for pH 5 is 17.33 ± 0.76 mM/min,
p=0.0000; 23.49 ± 0.60 mM/min, p=0.0004 for pH 6, 27.84 ± 1.21 mM/min for pH 7; 22.60 ±
1.00 mM/min, p=0.0001 for pH 8; and 20.97 ± 1.18, p=0.0000 for pH 9. Through this, it is
evident that the average rate of disaccharide production of amylase for each level of pH and
standard deviation barely overlap when compared to the control. Additionally, the p-values are
all under 0.05 which shows that the rate of disaccharide production for each pH level is
significantly different than the control of pH 7 (which is both the pH of human saliva and
distilled water).
Since pH 7 was used as a control, the other pH levels were statistically different such that
their rates were less (see Table 2) and the general distribution of the bars in Figure 9, it can also
be concluded that the optimum rate for salivary amylase in terms of disaccharide production
occurs when pH is 7.
However, looking even further, not each pH level was significantly different from each
other. In Figure 9, the bars for pH 6 & 8 and pH 8 & 9 are similar with overlapping standard
deviations. Additional t-tests performed (p=0.1329 for pH 6 & 8 and p=0.0670 for pH 8 & 9)
show that these rates were not statistically different and rate did not change significantly.
However, when pH became more acidic from 6 to 5, there was a decrease in rate as the average
and standard deviation did not overlap in Figure 9 and were statistically different (p=0.0000).
Overall, it can be said that a continuous increase and decrease in pH from the control does not
necessarily create a linear relationship as the increase/decrease does not always change the rate
of disaccharide production.

Evaluation
Scientific Comparisons
When considering the claim for the collected data, it is evident that overall, changing the
pH has a significant effect on the reaction rate even though there is not a linear relationship. As
described before, when compared to the control of pH 7, there is statistically different average
for each pH. The claim is supported by the fact that increasing or decreasing the pH from an
enzyme’s ideal environment “affects ionic and hydrogen bonds which are important to enzyme
shape” and therefore, decreasing rate of reaction (Eed, 2013, p. 50). Additionally, it has been
proven that the enzyme was still function from pH 4.5 to 10, but there is not “a relationship that
illustrates how much enzymatic activity will decrease” when removed from optimum (Sky-Peck
& Thuvasethakul, 1977, p. 313). In other words, there is only a general negative correlation. The
facts put forward by both studies are also illustrated by Figure 9 where introducing salivary
amylase into different pHs created staggering heights in the bars that are clearly less than pH 7,
showing that reaction rate has decreased.
In the conclusion, pH 7 was shown to be the ideal environment for salivary amylase as
Table 2 shows that its average rate was the highest. As discussed in the Introduction, this specific
amylase thrives in a range pH 6.8-7.0 as it allows the enzyme to retain its active site for breaking
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down starch in the human mouth (Peyrot des Gachons & Breslin, 2016). Once again, the value
from this scholarly source is illustrated by Figure 9 where the rate of disaccharide production at
pH 7 is the tallest bar, showing that optimum reaction rate has been reached.
Strengths
The fact that the data correlates with previous scientific literature also shows that my
experiment was extremely precise as the standard deviation/error were low and small compared
to the average (see Table 2) and the p-values show that there is high confidence (nearly 100% for
pH 5,6,8,9) that the reaction rate is different from the control. Even when additional t-tests were
performed, there was somewhat of a high confidence that the rate of disaccharide production for
pH 8 & 9 (99.3%) and pH 6 & 8 (87%) were not significantly different or that rate for pH 5 & 6
(100%) was different. Moreover, the low standard deviation can be credited to the minimal
outliers in Table 1 (most rates only differentiated by less than 1 mM/min) and slopes calculated
from the raw data in the simulation. Finally, the overall trend in the data was extremely clear as it
supported my hypothesis that as pH increases, there will be a significant effect in the rate of
disaccharide production and the optimum rate will be at pH 7.
When looking at the experimental procedure as a whole, the simulation itself has its
strengths. Prominently, it allowed many variables such as temperature, which cannot be easily
controlled in the real world due to factors like central climate control, to be set a certain
temperature like 25ºC. This can be considered a strength as it allows for a more precise focus on
how the independent variable affects the dependent without external factors skewing the data.

Weaknesses & Improvements

In terms of measuring tools, although the measure values were above the uncertainty, the
error rate was somewhat high. For example, when measuring disaccharide production throughout
the 20 minutes in Figure 8, the range of the final extent produced was approximately 400-420
mM for each trial. This leaves an error rate of 7.5% which is somewhat high and could have led
to high or lower rate of production, and skewed reliability of data. In order to fix the issue
presented by high error rate, the experiment can be improved by finding a simulation (real
measuring tools if done in person) that included tools with smaller uncertainties, allowing for
more precise measurements of disaccharide production.
Secondly, the simulation itself can be considered a weakness as it is run by computer
software rather than real world experience, meaning that the results found in raw data like Figure
8 are mainly based on algorithms. This would illustrate why the data collected was nearly perfect
and did not fully illustrate the systematic/random errors present in any experiment. Simulations
are unable to capture disparities such as the fact that materials taken from a biological source
such as salivary amylase is different in different individuals. Therefore, in the real world, the
optimum rate does not only occur at pH 7, but a range from 6.2-7.6 (Morris, n.d.). This weakness
cannot be improved unless a more precise simulation that takes into account the real world can
be used or the experiment is done in the real world.
Finally, the most prominent weakness in the experiment would be the systematic error of
calculating the rate of enzyme activity with the slope formula. The simulation did allow for
precise measurements of disaccharides produced at different time intervals, but it was difficult to
pinpoint the exact same times (2 & 7 minutes) for each trial as the simulation’s x-axis for time
went up by intervals of 2. This most likely caused a systematic error in the calculation of rate,
possibly skewing data collected. The error could be improved by choosing times that are
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intervals of 2 (ex. 2 and 8 minutes) or finding a simulation that uses computer processes to
calculate slope.
Overall, even though the weaknesses found in the experimental procedure cannot be
fixed manually through the chosen simulation, the data could be stronger with even lower
variation on standard deviation if more trials for each level were conducted in Table 1.
Additionally, the flaws found in calculating rate and the original simulation could be improved
by finding a new simulation that takes into account disparities and provides a way to calculate
rate through computer software.

Further Investigation
An extension that can be done to this lab as a next step would be to investigate how the
age of a person affects rate of disaccharide production. In the current experiment, pH level is
investigated as the independent variable, but it fails to account for how age impacts the
efficiency of amylase.
Research Question
What is the effect of changing age of person from which amylase is sourced from on the
reaction rate, as measured by disaccharide production (mM/min), of amylase?
Independent & Dependent Variable
The independent variable would be the age of the person from whom amylase is sourced.
There could be 10 different levels that included age groups varying from birth to age 100. This
would create a diverse experiment as it would allow for an investigation of which age group has
the most effective saliva, and therefore amylase. In this experiment, the dependent variable
would be rate of disaccharide production like the preexisting experiment. The dependent variable
should be kept the same as it is crucial to see which age range effects enzymatic activity the
most.
Control Variables
Some of the control variables include the time allowed for each reaction, amount/type of
starch, amount of salivary amylase and environmental conditions (temperature and pH). The
basic demographics (gender, race, medical conditions, etc.) should also be kept constant in order
to accurately claim that age solely effects reaction rate.
Importance
This extension is important to investigate as it is already known that as a person gets
older, their salivary glands are less productive and therefore, produce less saliva and the salivary
enzymes found in the secreted liquid (Morris, n.d.). It would be interesting to see if protein
structure changes with age and if this change affects how effective amylase is at breaking down
starch into smaller subunits.
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References

bthoole. (2018). Enzymes lower activation energy. Writing in Biology.

https://bcrc.bio.umass.edu/courses/fall2018/biol/biol312section1/content/enzymes-lower-

activation-energy

Eed, J. (2013). Factors affecting enzyme activity. Essai, 10(19), 48-51.

https://dc.cod.edu/cgi/viewcontent.cgi?article=1411&context=essai

Klimek-Ochab, M. (2016). Figure 8 [Illustration]. ResearchGate.

https://www.researchgate.net/figure/Enzymatic-starch-hydrolysis-Glucose-product-of-

starch-hydrolysis-is-a-substrate-for-the_fig5_321759280

Morris, H. (n.d.). Human salivary alpha amylase: The reason why foods taste sweet. University

of Virginia. Retrieved May 11, 2021, from

https://collab.its.virginia.edu/access/content/group/f85bed6c-45d2-4b18-b868-

6a2353586804/2/Ch22_Morris_H_Alpha_Amylase_(Human_Salivary)-_-/index.html

Peyrot des Gachons, C., & Breslin, P. A. S. (2016). Salivary Amylase: Digestion and Metabolic

Syndrome. Current Diabetes Reports, 16(10). https://doi.org/10.1007/s11892-016-0794-7

Sky-Peck, H. H., & Thuvasethakul, P. (1977). Human pancreatic alpha-amylase. II. Effects of

pH, substrate and ions on the activity of the enzyme. Annals of Clinical & Laboratory

Science, 7(4), 310-316. http://www.annclinlabsci.org/content/7/4/310.full.pdf+html