What is the effect of chloride ion concentration (0%, 0.4%, 0.8%, 1.2%, 1.6%, and 2.

0%) on starch
hydrolysis by α-amylase measured by a change in absorbance (± 0.001 au) after 200 seconds?

Biology HL | May 2021

Introduction:

Starch is shockingly a component of over a third of the human diet (Starchy foods and carbohydrates, 2021). The
main sources of starch are the cereals such (as rice, barley and maize) and root vegetables (such as potato, carrot
and yam) (Wang, n.d.). Due to its insolubility at room temperature and compact size, starch can be found stored in
chloroplasts in the form of granules (Wang, n.d.).

In class, when we were studying the section regarding enzymes, we were told that the factors that affect enzyme
activity are temperature, pH, and substrate concentration (Allott and Mindorff, 2014). However, upon further
research on my part, due to mere curiosity, I came across some other factors that affect enzyme activity. These
factors were: compartmentalization, feedback inhibition, the presence of Inhibitors, enzyme cofactors, and the
presence of allosteric activators (Enzyme regulation (article).). I gained sufficient knowledge about inhibition and
enzyme cofactors. However, the information that was readily available of allosteric activation was quite limited.
This limitation drove me to investigate the effect of the presence of an allosteric activator on enzyme activity.

For this experiment, I chose the enzyme, α-amylase, and starch as the substrate, as these were readily available.
Through my research regarding allosteric activation, I found that ADP, cations, and anions can function as
activators (D. Lopina, 2017). Keeping this information in mind, I randomly chose Cl - as the allosteric activator for
this investigation. Because this experiment involved the use of starch as the substrate, Iodine was needed as an
indicator for starch. When iodine is added to starch, a dark shade of violet can be observed. This occurs because of
the amylose subunits that make up part of the starch molecule. The iodine molecules are trapped by the coils of
these amylose chains. As a result, linear triiodide ion complexes are formed- causing the mixture to turn into a
remarkably dark shade of violet (Calabrese et al., 2020). When the starch molecules are broken down into a
disaccharide, maltose, by α-amylase, the maltose does not react with the iodine molecules. Due to the absence of
amylose, the solution appears to be a light shade of yellow rather than a dark shade of violet (Calabrese et al.,
2020). Obtaining only qualitative data for an experiment based on enzyme activity would be insufficient. These
types of experiments require numerical/qualitative data of multiple trials for them to be considered as reliable.
Hence, I used the spectrophotometer to measure the initial and final absorbance values. Using these values, I then
calculated the difference between the initial and final absorbance readings for each trial from each concentration,
the mean standard deviation and standard error. Along with my curiosity to learn about the effects of an allosteric
activator on an enzyme’s activity, I was also curious to know if changing the concentration of an allosteric
activator would have an effect on the activity of the enzyme. Due to this, I carried out this experiment with
different concentrations of chloride ions. These concentrations were 0%, 0.4%, 0.8%, 1.2%, 1.6%, and 2.0%. Six
different concentrations were chosen in order to show a proper correlation and a clear trend of the effects of one
variable on another.

Allosteric activation occurs when the binding of a ligand to a site different to the active site enhances the affinity
of the enzyme to bind to the substrate- which in this investigation is the chloride ions, α-amylase and the starch
respectively (Sumit Kumar). The enhanced affinity is due to a “ligand-induced conformational change”. This

PAGE 1
should theoretically increase the rate of hydrolysis (Einav, Mazutis and Phillips, 2017). Allosteric enzymes such as
α-amylase are enzymes that undergo a conformational change when bound to a ligand. These enzymes tend to be
larger and complex in nature. The ligand that binds to the allosteric site (also known as the recognition site) of an
enzyme is known as the effector. When an effector, that is an allosteric activator, is bound to the allosteric site of
the enzyme, the activity of the enzyme should increase (Allosteric Enzymes | Facts, Summary & Definition |
Chemistry Revision, n.d.).

Research question:

What is the effect of chloride ion concentration (0%, 0.4%, 0.8%, 1.2%, 1.6%, and 2.0%) on starch hydrolysis by
α-amylase measured by a change in absorbance (± 0.001 Au) after 200 seconds?

Hypothesis:

After understanding the information present above about allosteric activation, I can now make an educated guess
and say that: as the concentration of sodium chloride (Cl-) increases, there will be a decrease in absorbance values
at a faster rate. This will occur due to the increased rate of starch hydrolysis by α-amylase (Au min-1), which is
caused by the increase in the concentration of the allosteric activator that could potentially catalyze the reaction
between starch and α-amylase even further.

Variables:

Independent variable:

Concentration of sodium chloride (Cl-): 0, 0.4, 0.8, 1.2, 1.6, and 2%.

Uncertainty of the electric balance = ± 0.001 g, uncertainty of the 100 cm3 graduated cylinder = ±0.5 cm3

Example 0.4%: 0.4% can also be written as 0.004

𝑈 0.001 0.5
= + = 0.0025 + 0.005
0.004 0.4 100

0.0025+0.005
𝑈= = 0.00003 = 0.003% or 0.400 ± 0.003%
0.004

PAGE 2
Dependent variable:

Rate of starch hydrolysis which is presented by the change or the difference in the absorbance values from the
initial absorbance to the final absorbance is considered to be the dependent variable. In order to calculate the
change in absorption, the initial and final absorbance values for each trial were obtained from the experiment
conducted through the use of a spectrophotometer (±0.001 Au).

Controlled Reason: Method:

variable:
Temperature of the An increase or decrease in temperature would affect the The starch and α-amylase
solutions involved rate of reaction, as enzymes perform best at the optimum solutions were placed in a
temperature. If the temperature of the enzyme is too high or water bath which was set to
too low, this could lead to denaturation of the said enzyme. 37°C.
(Temperature Effects (Introduction to Enzymes), 2021)
pH An increase or decrease in pH would affect the rate of Distilled water was used in
reaction and may denature the enzyme (Effects of pH this experiment as a buffer
(Introduction to Enzymes), 2021). The optimum pH for the in order to maintain neutral
bacteriological α-amylase used in this experiment is 6.0. conditions for all the
Any pH value above 6.0 will denature the enzyme (Mehta). samples. A pH meter was
used in order to ensure that
this occurred.
Concentration of Changing the concentration of iodine would result in Iodine of the same
iodine inaccurate absorbance values. Adding a higher concentration was used for
concentration of iodine will give the solution a darker color each sample in this
which will give higher absorbance values. Adding a lower experiment.
concentration of iodine will give the solution a lighter color
which will give lower absorbance values. (The Beer-
Lambert Law, n.d.)
Concentration of As the starch is considered to be the substrate, increasing Starch solution with a
starch solution the concentration of the starch solution would increase the concentration of 1% was
rate of reaction. If only a few selected samples have a used in this experiment.
higher concentration of the substrate, the results will be
flawed and will be considered unreliable. (Substrate
Concentration (Introduction to Enzymes), 2021)
Concentration of α- In this experiment, α-amylase is the enzyme used to The α-amylase solution
amylase solution breakdown starch. Increasing the concentration of the α- with a concentration of 2%
amylase solution would increase the rate of reaction. If only

PAGE 3
 a few selected samples have a higher concentration of the was used in this
enzyme, the results will be flawed and will be considered experiment.
unreliable. (Enzyme Concentration (Introduction to
Enzymes), 2021)
Wavelength of light When the cuvette is placed in the spectrophotometer, a In this experiment the
emitted by the beam of light of a certain wavelength is emitted from the wavelength of the light
spectrophotometer spectrophotometer. Once this beam of light passes through emitted by the
the sample, the absorbance value is recorded. If the samples spectrophotometer was
have been hit with beams of light of different wavelengths, 430.4 nm.
the absorption values recorded will be flawed and will be
considered unreliable (Wathall, J.)
Source and age of Age and the source of the enzyme could affect the rate of All the α-amylase that was
α-amylase reaction. If an older enzyme is used, the rate of reaction used in this experiment was
will be lower compared to that of a fresher enzyme. Using taken from the same
α-amylase from different sources could also affect the rate package. The α-amylase
of reaction. If this occurs, the results will be flawed and was obtained from the
will be considered unreliable (Erika Vyskočilová) bacteria Bacillus
amyloliquefaciens.

Method:

Apparatus and materials:

o α-amylase obtained from the bacteria Bacillus amyloliquefaciens

o Potato starch
o Sodium chloride, NaCl
o Iodine stored in a brown lab glass reagent bottle
o Spectrophotometer with an uncertainty of ±0.001 (to find absorbance values)
o Data logger (to collect data) and computer (to view and save results)
o Excess Distilled water stored in distilled water bottles
o Polystyrene diamond shaped weighing boats ×3
o 250 cm3 conical flask with rubber bung (±10) ×3
o 100 cm3 graduated cylinder (±0.5) ×1
o 10 cm3 graduated cylinder (±0.2) ×9
o Stirring rod ×3 and Spatula ×3

PAGE 4
 o Graduated pipette (±0.03) ×8
o Thermometer (±0.01) ×1
o 100 cm3 beaker (±5) ×10
o Cuvette (±0.03) ×10
o Electric balance
o Water bath ×1
o Dropper ×1 Figure 1 shows the apparatus used for this experiment.
o Labels ×6

Procedure:

Preparing the diluted sodium chloride solutions:

Preparation of the 2.00% sodium chloride solution:

1) Place the polystyrene weighing boat on the electric balance and tare its mass.
2) Using a spatula, take out some of the sodium chloride from its respective container and drop the substance
into the weighing boat until the balance reads 2.000 g.
3) Add the 2 g of sodium chloride to the 250 cm3 conical flask.
4) Measure 100 cm3 of distilled water using a graduated cylinder.
5) Empty the contents of the graduated cylinder into the conical flask containing sodium chloride.
6) Mix the two substances well until a homogenous mixture is formed.
- Preparation of the 0.40% sodium chloride solution:
7) Using a pipette, add 2 cm3 of the 2.00% sodium chloride solution to a 10 cm3 graduated cylinder.
8) Add distilled water to the graduated cylinder until the bottom of the meniscus reaches the 10 cm 3 mark.
9) Follow steps 6 and 7 in order to prepare the 0.80%, 1.20% and 1.60% sodium chloride solutions. Use the
table below:
Concentration of the NaCl solution (%) Volume of the 2.00% NaCl added (cm3)
0.00 0 .00
0.40 2.00
0.80 4.00
1.20 6.00
1.60 8.00

Preparation of the 1.00% starch solution:

10) Place a polystyrene weighing boat on the electric balance and tare its mass.
11) Using a spatula, take out some of the starch from its respective container and drop the substance into the
weighing boat until the balance reads 1.000 g.

PAGE 5
 12) Add the 1 g of starch to the 250 cm3 conical flask.
13) Measure 100 cm3 of distilled water using a graduated cylinder.
14) Empty the contents of the graduated cylinder into the conical flask containing starch.
15) Mix the two substances well.

Preparation of the 2.00% α-amylase solution:

16) Place a polystyrene weighing boat on the electric balance and tare its mass.
17) Using a spatula, take out some of the α-amylase from its respective container and drop the substance into
the weighing boat until the balance reads 2.000 g.
18) Add the 2 g of α-amylase to the 250 cm3 conical flask.
19) Measure 100 cm3 of distilled water using a graduated cylinder.
20) Empty the contents of the graduated cylinder into the conical flask containing α-amylase.
21) Mix the two substances well until a homogenous mixture is formed.

To setup the spectrophotometer:

22) Connect the spectrophotometer to a data logger.

23) Place a clean, blank cuvette into the spectrophotometer.
24) On the meter screen, tap on the button “Sensors”.
25) From the drop down box, choose “Calibrate”.
26) After another screen comes up, choose to “Skip warm up” and “Finish calibration”.
27) Tab on the “OK” button to complete the action.
28) Exchange the blank cuvette with a cuvette containing a dark violet solution.
29) Tap on the green arrow to start data collection.
30) After a graph is show on the screen, tap on the red square button to stop data collection.
31) Check the absorption at different wavelengths. The wavelength that gives you the best absorption is to be
used in this experiment.
32) Move the cursor along the graph in order to set the wavelength which is to be used throughout this
experiment (430.4 nm).
33) Go to the meter screen and tap on the “Mode” button.
34) Change the settings and add the following:
Mode: Time Based, Interval: 2 s/sample, Duration: 200 s

Preparing the cuvettes and conducting the experiment:

35) Using a 10 cm3 graduated cylinder, add 1 cm3 of the 0% sodium chloride solution (distilled water), 1 cm3
of the starch solution and 3 drops of iodine into a beaker using a dropper.
36) After mixing the solutions well, pour out the contents of the beaker into a clean cuvette.

PAGE 6
 37) Add 1 cm3 of the starch solution.
38) Fix the cap of the cuvette back on and invert the cuvette about 5 times.
39) Place the cuvette containing the mixture into the spectrophotometer.
40) Immediately, start the data collection.
41) Repeat steps 21-26 for other concentrations of the sodium chloride solution (0.40%, 0.80%, 1.20%, 1.6%,
2.00%).

Calculation and analysis of the results:

42) Organize the data collected into well-structured tables.

43) Using the data collected, calculate the rate of change.
44) Calculate the mean, standard deviation, and standard error of the rate of change for all the categories.
45) Perform the Pearson’s product moment correlation coefficient test.
46) Uses all the data available to form a suitable conclusion for the experiment.

Safety, environmental and ethical considerations:

o Gloves, lab coat, and goggles should be worn at all times while conducting the experiment in order to
prevent any substances from coming in contact with skin or eyes, as this could lead to potential hazard.

o The α-amylase that is being used in this experiment is obtained from a microbial source (Bacillus
amyloliquefaciens) rather than a human source. Hence, there is no ethical consideration to observe.

o Any substance used in this experiment is to be disposed of in an appropriate manner in order to avoid any
environmental hazard.

Results:Qualitative data:

Figure 2 Figure 3 to
shows the the right
cuvette just shows the
before the same cuvette
enzyme as in figure 1
was added. about 5 to 6
Figure 2 shows the cuvettes about 15 minutes after the
minutes after
final absorbance value was recorded. Order of the cuvettes
the enzyme
with respect to NaCl concentration from left to right as
was added.
shown in the figure: 0%, 0.4%, 0.8%, 1.2%, 1.6%, 2.0%

PAGE 7
Quantitative data:

Raw data:

NaCl Concentration: 0%
Absorbance
Value/Au Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial 1.199 1.204 1.224 1.198 1.230

Final 0.997 0.966 0.989 0.991 0.990

NaCl Concentration: 0.40%
Absorbance
Value/Au Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial 1.202 1.201 1.210 1.225 1.205

Final 0.870 0.861 0.853 0.832 0.867

NaCl Concentration: 0.80%
Absorbance
Value/Au Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial 1.218 1.211 1.214 1.210 1.215

Final 0.722 0.715 0.721 0.713 0.731

NaCl Concentration: 1.20%
Absorbance
Value/Au Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial 1.196 1.210 1.204 1.212 1.208

Final 0.521 0.511 0.517 0.515 0.507

NaCl Concentration: 1.60%
Absorbance
Value/Au Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial 1.202 1.184 1.211 1.176 1.224

Final 0.354 0.355 0.340 0.353 0.341

NaCl Concentration: 2.00%
Absorbance
Value/Au Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

Initial 1.248 1.221 1.260 1.185 1.201

Final 0.202 0.213 0.198 0.195 0.206

Table 1 shows the initial and final absorption values after 200 seconds for the different concentrations.

PAGE 8
 Difference between the initial and final absorption values (Au)
NaCl concentration (%) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5
0.00 0.202 0.238 0.235 0.207 0.240
0.40 0.290 0.270 0.254 0.307 0.269
0.80 0.348 0.601 0.361 0.378 0.348
1.20 0.675 0.699 0.687 0.697 0.701
1.60 0.969 0.932 0.923 0.911 0.978
2.00 1.046 1.008 1.062 0.990 0.995
Table 2 shows the difference between the initial and final absorption values for the 5 trials across all
concentrations.

Sample calculations:

* Values are from the results of the experiment conducted with 1.60% of NaCl

1) Calculating the mean of the rate of change of absorption:

("Analysis Of Mean")

0.969 + 0.932 + 0.923 + 0.911 + 0.978

Mean = = 0.943
5

2) Calculating the standard deviation:

(Rajabi)

(0.969−0.943)2 +(0.932−0.943)2 +(0.923−0.943)2 +(0.911−0.943)2 (0.978−0.943)2

σ=√ = 0.025
5

PAGE 9
4) Calculation of the Standard Error:

(Ilola)

Where,

SE = Standard deviation, Ꝺ = Standard deviation, n = Number of trials

𝜎 0.025
SE = = = 0.0112
√𝑛 √5

Processed data:

Statistics for the rate of change of absorbance for the following concentrations:

Concentration
0.00% 0.40% 0.80% 1.20% 1.60% 2.00%
of NaCl
Mean 0.224 0.278 0.407 0.692 0.943 1.020
Standard
Deviation 0.016 0.368 0.098 0.010 0.026 0.029
Standard Error 0.007 0.165 0.044 0.004 0.012 0.013

Table 3 shows the mean, standard deviation, and standard error of the rate of change of absorbance.
1.400
Mean difference between initial and

1.200
final absorbance (Au)

1.000

0.800

0.600

0.400

0.200

0.000
0.00 0.40 0.80 1.20 1.60 2.00
NaCl concentration (%)

Graph 1 shows how the mean difference between initial and final absorbance changes with an increase in
NaCl concentration.

Statistical test:

The Pearson’s product moment correlation coefficient shows the correlation strength between the two variables
(Wathall, J). In this case, the two variables are: the average change in absorbance and the concentration of NaCl.

PAGE 10
The Pearson’s product moment correlation coefficient, r, is given by the following formula:

X X2 Y Y2 XY
0.00 0.00 0.224 0.050 0.000
0.40 0.16 0.278 0.077 0.031
0.80 0.64 0.407 0.166 0.133
1.20 1.44 0.692 0.479 0.574
Here, the variable X is the concentration of NaCl,
1.60 2.56 0.943 0.888 1.422
whereas the variable Y is the average rate of change of 2.00 4.00 1.020 1.041 2.082
absorbance. Ʃ Ʃ Ʃ Ʃ Ʃ
6.000 8.800 3.564 2.701 4.241
6(4.241)−(6.000)(3.564)
𝑟= Table 4 shows the summation values that are
√[6(8.800)−6.0002 ][6(2.701)−3.564 2]
required to find r.

4.062
r= = 0.529
√58.866

1.200
Graph 2 shows the strong positive
1.020
linear correlation between the
1.000 0.943
concentration of NaCl and the
average change in absorbance.
Concentration of NaCl (%)

0.800
0.692
Conclusion:
0.600
My hypothesis was that as
0.407
NaCl concentration increases,
0.400
0.278 the rate of starch hydrolysis
0.224
0.200 will also increase. All of the
data obtained proves that my
0.000 hypothesis was correct. In
0.00 0.50 1.00 1.50 2.00 2.50
graph 1, we can see that the
Average change in absorbance (Au)
average change in absorption
was at the highest when the concentration of NaCl was 2.00% and at the lowest when the concentration of NaCl
was 0.00% (distilled water). The Pearson’s product moment correlation coefficient, r, was calculated to be exactly
0.529. Any r value of a that lies within the range 0.3 < r ≤ 0.7 is said to belong to relationship that has a weak or
moderate positive correlation. Because the calculated r value for the relationship between NaCl concentration and
rate of starch hydrolysis lies within this particular range, this indicates that there is a moderate positive correlation
between the two variables. This can also be seen in graph 2, where the graph has a steep positive slope. Thus,
indicating a strong positive correlation.

PAGE 11
 Evaluation:

Strengths Limitations Extensions

The calibration process of the The fact that only 5 trials were taken This experiment could be conducted
spectrophotometer helped per concentration makes the results of using other ligands such as ADP or
avoid any systematic errors. the experiment somewhat unreliable. other anions and cations.
Six concentrations of NaCl To avoid this, more trials could be This experiment could be conducted
with an interval of 0.4 were conducted. with another allosteric enzyme such
experimented with. This made The starch that is present in the starch as glycogen phosphorylase or
it easy to see the gradual solution, settles to the bottom of the glutamine synthetase.
changes in rate. conical flask. Hence few trials may This same experiment could be
A data logger was used in contain a lower concentration of starch conducted with animal and plant α-
order to automatically record compared to the rest if the starch amylase.
the data at a 2 second interval. solution had not been mixed thoroughly
This helps reduce human before adding to the beaker containing
error. the sodium chloride solution.

PAGE 12
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