Leonarda Matic

Mrs. Hawkins
Biology IB DP
16.2.2023.

Enzyme Lab Report: How do varying temperatures affect lipase activity?

Background Information:

Enzymes are globular proteins that catalyze the rate of biochemical reactions, by lowering the energy
necessary for activation (“Energy, Enzymes, and Catalysis Problem Set”). Enzymes bind to substrates at
the active site, where the reaction is catalyzed and finally the product is released. Enzymes are necessary
for muscle, liver function, digestion, and metabolism of a cell, and are overall necessary for a functioning
body (“Lipase Tests”). Enzyme activity depends on the environment's temperature, pH, and substrate
concentration (“2.5 Enzymes”).

Different pH will change the charge of an enzyme, affecting its solubility and shape. Any change in shape
can prevent the substrate from binding to the active site. Each enzyme has an optimum pH where the
activity is highest. Each enzyme has unique pH optimums.

Substrate concentration increases the rate of reaction. Unlike with pH and temperature, once the
maximum rate is achieved, the substrate concentration will remain constant, as there can not be a bigger
number than the maximum of active sites available. At an optimum concentration of substrate molecules,
all active sites are open and ready for reaction, creating maximum efficiency (“Enzyme Activity |
BioNinja”).

When a liquid is heated the particles gain kinetic energy. The substrate and enzyme move at higher
speeds, increasing the chances of collision between the substrate and active site. Therefore, the rate of
enzyme activity is increased and thrives best at optimum temperature. However, higher temperatures
threaten the hydrogen bonds and stability within an enzyme. The enzyme and active site are vulnerable to
change in shape and drastic loss in activity, also known as denaturation. Too low temperatures also do not
support enough thermal energy for activation and reaction to occur (Allott and Mindorff) When
temperatures are cold the molecules are moving slower, and causes less rate of substrate and enzyme
collision (Dr. Lisa Bartee and Brook).
In the body, there are several types of lipase: hormone-sensitive lipase, lipoprotein lipase, pancreatic and
hepatic lipase. For example, the average pH in the pancreas is 8.3, while in the liver it is 7.0, meaning that
hepatic lipase will function in different optimum conditions than pancreatic lipase (Itoyama et al.)
(Buddies).

Lipase is a digestive enzyme made in the stomach, pancreas, and in salivary glands. Lipase catalyzes the
hydrolysis of triglycerides into glycerol and three fatty acids throughout the body. Fats must be digested
to be absorbed easily through the membrane of the gut and be soluble enough for blood transport
(“Investigating Effect of Temperature on Lipase Activity) Lipase digests different forms of triglyceride,
one of them being dietary triglycerides found in milk (Yasaman Pirahanchi and Sharma).

Lactose, Lipase, and Phenolphthalein are all acidic. For this experiment, sodium bicarbonate was added to
the milk to create an alkaline solution, imitating the pancreas that secrete sodium bicarbonate to neutralize
the acid in the stomach and protect the duodenum (Bartel). This transformation creates an environment
with a pH similar to the one of the duodenum, small intestine, where lipase is typically found.
Phenolphthalein is an indicator that is colorless in acidic solutions and pink when found in alkaline
solutions with a pH of around 10 (“Investigating Effects of Temperature on Enzymes”). The formation of
ions impacts the color of the solution. Once the lipase is mixed with the milk, it will break down the
lipids, exposing the fatty acids and thus neutralizing the pH. As the pH level decreases, the
phenolphthalein turns clear. This experiment will investigate the effect of varying temperatures on the
breakdown of lactose by lipase (Britannica).

Independent variable Temperature (15°C-75°C by 10 degrees increments)

Dependent variable Time taken for phenolphthalein to turn colorless (± 0.5 sec)

Control variables Room temperature

Starting enzyme concentration

Starting pH concentration

Volume and the surface area of pH

Substrate concentration

Materials:
1. Marker pen for
 2. Test tube rack
3. Ice cubes (6-8 pieces)
3
4. Measuring cylinder 10 𝑐𝑚
3
5. 1 beaker 100 𝑐𝑚 for milk solution
−3 3
6. Sodium bicarbonate solution 0. 05 𝑚𝑜𝑙 𝑑𝑚 (7 𝑐𝑚 per temperature being assessed)
7. 40 ml lipase
8. 20 ml phenolphthalein
3
9. 1 beaker 100 𝑐𝑚 for sodium bicarbonate solution
3
10. 2 beakers 250 𝑐𝑚 to act as a water bath for temperatures
3
11. Full-fat milk 5 𝑐𝑚 for temperature being assessed
12. Thermometer
13. Test tube
14. Glass rod
2
15. Syringe 2 𝑐𝑚
16. Stopwatch

Procedure:

1. Fill the test tube with 5 ml milk, 7 ml sodium bicarbonate, and 5 drops of phen.
2. Fill the second test tube with 1 ml lipase
3. Turn on the hot plate, and place the beaker on the hot plate for temperatures above 25
4. For temperatures below 25, add ice cubes to the beaker instead of heat
5. In the beaker, add water and both test tubes
6. Place a thermometer in the test tube with milk, and let the liquid reach desired temperature
7. When the liquid reaches a specific temperature, pour lipase into the milk test tube, and start
stopwatch
8. While the liquid is changing color, maintain the temperature of solution
9. Once the color changes from pink to clear, stop the timer and remove the tubes from solution
10. Record the information and repeat steps from 1-9 for each range
a. Each temperature will have 5 different trials

Risk Assessment:
Sodium bicarbonate of higher concentrations can be irritant, so it is important to use low concentrations
and wear eyeglasses, to prevent sodium from accidentally splashing in eyes. Phenolphthalein contains
ethanol which is flammable. Avoid using or placing Phenolphathelin near the Bensen burner. Lipase is an
acid that can burn through the retina and blind the eye, it is necessary to avoid lipase from contact with
the eyes and wash hands well after use. There are no ethical issues with this lab report.

Figure 1.

Temperature: (C°) Qualitative data:

15 ● Light at top light pink almost pinkish

● Darker pink on bottom
● 1st: slowly changing to lighter shade
● 3rd: light pink while others changing
● 2nd trial completed transformation first

25 ● Not enough tubes for lipase at first, had to wash out

● 1st: started turning light pink first
● 2nd: turned pink, ombre, slower
● 5th: color difference more drastic= not mixed well
○ Changed white first
● Only stirring in the beginning

35 ● Immediately started changing

● Change milkfish almost immediately, only little pink on the bottom
● All turned back to milkish
● Only one tube remained slightly pinkish

45 ● Milkish at the top immediately

● Still efficient
● Little pink on the bottom

55 ● 4th: started reaction the quickest

● Slow change rate, colors more blended (ombre)
● Still - 2 + 4 = palest pink shade compared to others (1,3,5)
● Long time to change
● Tested pH of different tubes= altering results
● Lightest - pH 8
● Darkest - pH 9

65 ● No change
● Slight change in color, all together, no ombre
● Stayed same color

75 ● Started changing slightly, remained dark pink

● Phenolphthalein did not turn colorless
 ● No significant change
● Remained darker color than 65

Figure 2.

Tempera Time taken for phenolphthalein to turn colorless ± Average Standard Average
ture (°C) 0.5sec deviation rate of
lactose
breakdown
(1/t)

Trial 1 Trial 2 Trial 3 Trial 4 Trial 5

648 11.3137 0.0015
15 656 710 904 710 640

25 480 395 480 480 390 393 3.5355 0.0025

35 72 111 100 80 120 76 5.6569 0.0132

45 573 585 583 580 590 582 6.3008 0.0017

55 3600+ 3600+ 3600+ 3600+ 3600+ 3600 0.0000 0.0000

65 3600+ 3600+ 3600+ 3600+ 3600+ 3600 0.0000 0.0000

75 3600+ 3600+ 3600+ 3600+ 3600+ 3600 0.0000 0.0000

Figure 3. Figure 4. Figure 5.

Figure 6.

Figure 7.
Figure 8.

Figure 9.
Analysis:

Lipase digested lactose fastest at a temperature of 35°C, with an average time of 76 seconds. Lactose at
55°, 65°, and 75° did not show a significant color change, suggesting that the temperature was too high
and denatured the enzyme, making it unable to complete the reaction, creating a reaction rate of 0. This
spike in rate is supported by the weak hydrogen bonds between the acid and R-group, which are easily
broken in environments other than optimum (Allott and Mindorff). As seen in figure 5, the color
gradually changed from pink to clear, working from top to bottom, suggesting that lipase is lighter than
the milk solution and therefore lipolysis occurs in the higher region. This was more noticeable in test
tubes where the milk and lipase were not mixed as well. The ombre formed by the colors indicates a
gradual change in pH as the limited amount of enzymes is continuously creating the reaction or
catalyzing. As seen in figure 9 the temperatures approach the optimum and the time taken for
phenolphthalein to turn colorless decreases. The difference between the breakdown rate for 35°C and the
rest of the temperature is significantly bigger, demonstrating the sensitivity and dependency of lipase on
body temperature. This sensitivity of lipase is supported by a study that determined only 65% of lipase
activity at 35°C was retained at a temperature of 45°C (Hiol et al.). The color went from a Barbie pink
shade to a clear, or the original milk color (figure 4). The several anomalies in each trial explain the large
error bars in figure 8 In this figure, the standard deviation is high, but the results are significant and most
likely not due to chance because the error bars do not overlap. Figure 7 was created without the anomalies
to further analyze the data and consistency within trials. Figures 9 and 2 show that at temperatures above
55°C, it took lipase more than 3600 seconds, or an hour, for color to change as noticed in. Data recorded
for 55-75°C was not included in Figures 7 and 8, because the raw data is the same, and does not offer
insight into the interpretation of graphs. However, the data recorded for 55-75 suggests denaturation
Figure 10.

occurs, and the enzyme is no

longer of correct shape and can not
go through with reactions.
Vladimir N. Krukovsky and B.L.
Herrington conducted a similar
experiment at Cornell University,
testing the activation of milk lipase
by temperature changes. Their
results in figure 10 show a similar
trendline to the one in figure 6,
strengthening the validity of findings in this investigation. However, their experiment was conducted
several times with more variables controlled. Their optimum was at 30°C, unlike the optimum in this
study, 35°C, weakening the accuracy of the data (Krukovsky and Herrington). The R-squared calculation
has a strong positive correlation between the variables of 0.915. While calculating r2, the values for
55-75°C were removed because they are the same value, and if included they alter the trendline and r2.
The strong correlation demonstrates that the relationship between the rate of reaction curve has biological
reasoning and is most likely not by chance. The biological reasoning is an optimum of 35°C when the rate
of reaction is fastest, and a decrease in the rate at temperatures above or below.

Conclusion:

The optimum temperature for lipase activity is 35°, at which lipase will break down at the fastest rate.
Any temperature above or below the optimum will decrease the rate of reaction, and above 55°C will lead
to the denaturation of the enzyme.

Evaluation:

This lab included seven values for independent variables. It was decided to go drastically above body
temperatures to try and denature the enzyme. The data with the anomalies included has large standard
deviations, and such inconsistencies could weaken the precision of the results. The average is not a true
reflection of the trials. This is a result of a random error with altered lipase concentrations in test tubes.
Temperatures 15-45°C were completed on one day, and 55-75°C on another. A trial run of 55°C was done
on the first day and gave different results than the one recorded on the second day. The results recorded
suggest denaturation occurs at 55°C, but the trial done the day before showed a change in phenolphthalein
but at a slower rate. It is suspected that data of 55°C are the result of random error, but results for 65 and
75°C are due to denaturation, as supported by the National Library of Medicine, which claims lipase starts
to denature at temperatures above 45°C (Rajakumara et al.). For those three temperatures, each trial
started changing color but stopped at different seconds and did not further continue the change in color.
This is seen in Figures 1 and 2, and could be the result of a systematic error with the sedimentation of the
solution. The sodium bicarbonate was mixed with water but was not mixed well prior to adding lipase,
causing the powder to sink and creating an uneven concentration throughout the solution. It is possible the
lipase powder fell out of the solution. This unequal spreading will be different in each test tube, therefore
changing the color at different rates for the same independent variable, which can be noticed in figures 3
and 1.These figures further show a pH test was done with two trials of temperature 55°C to discover a
significant difference, the lightest color had a pH of 8, while the darkest had a pH of 9. The result leads to
ambiguity and weak validity on what happened above optimum temperature and how much time is
necessary for phenolphthalein to change color at 55°C. However, the results do support the theory of
denaturation, that above the maximum temperature at which lipase can function, the rate will remain
consistent because the reaction will not happen. Overall, this experiment has been recreated by other
scientists, Krukovsky, Herrington, Hiol et al, showing the findings from temperatures ranging from 15° to
45° are significant and valid.

The raw data and the average time taken for lipase to break down lactose were rounded to the nearest
whole number, including an uncertainty of 0.5 seconds. It was rounded because more precise results
would not benefit the findings of this experiment. The standard deviation and rate of reaction were
recorded to four decimal places because if they were rounded the same as raw data, there would be no
significance. The uncertainty of measuring equipment is representative of the anomalies within trials, as
noticed in figure 4, specific test tubes changed color slower than average per trial. It also decreased the
ability to control variables such as enzyme and substrate concentration. While the uncertainties impact the
results, the error bars do not overlap and therefore the data is significant.

The data can be further statistically explored by creating a t-test to test if the difference in results between
the control and experimental groups is significant or due to chance. A separate experiment should test the
effect of pH and substrate concentration on the activity of lipase. It can be expanded by connecting it to
real life: what happens to the glycerol and fatty acids after they have been absorbed by the digestive tract
("Investigating Effect of Temperature on the Activity of Lipase")? Bile salts are found in the gallbladder
and are necessary for the digestion of fats. How do bile salts affect the rate of reaction (Young)? What
happens to the body when lipase does not function properly or breaks down lactose at the necessary rate?
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