Substrate concentration affecting enzyme activity

William Wang
Date Submitted
Class: Grade 10 Biology

Timeline
Date Task

Friday, October 1st @12:00pm First Draft: hypothesis and variables

Wednesday, October 6th @10:00am Final Draft

First Draft and Final Copy of Criterion B Summative must be uploaded to MB.

Write-up
Research question
How does the concentration of lipids in milk, ranging from 0.03g/ml, 0.02g/ml, 0.01g/ml, 0g/ml affect
the time it takes for the Phenolphthalein indicator to turn white?
Background research

Enzymes
An enzyme is a group of proteins that acts as a catalyst in chemical reactions in organisms. It is important
as enzymes could speed up the chemical reactions. (Classnotes)
The chemical reactions that enzymes are involved in require the breaking down and/or forming of a
bond. The substrate which is any substance that enzymes will bind to is going to join enzymes at the
active site. The enzyme changes the shape slightly and forming the enzyme-substrate complex. The
enzymes break a molecule down and form a new molecule. Once the reaction is complete it is left with
enzyme products complex. Last, the enzyme releases the products from their active site, Enzymes help
large substances to break into smaller substances such as digestion.
(Classnotes)

Lipids
Lipid is same as fats. The type of lipids in milk is called triglycerides. The main function of triglycerides is
to store energy in our bodies. Whereas carbohydrates storage is used up in one day, lipid storage can
help sustain needs of energy for several weeks therefore, it is important in humans as it helps to sustain
and store the energy. For example, if a person eats excessive amounts of food, lipids help to store energy
in the form of fat molecules in the body to use later. In humans, triglycerides are stored in the specialized
cell called an adipose cell. Within these cells, triglycerides aggregate in the cytoplasm to form a large fat
globule that makes up the majority of the volume of the cell.(Berg , Triacylglycerols are highly
concentrated energy stores) When we eat fats, we can break them down by lipases which is one of the
 types of enzymes. For example, triglycerides are broken by hydrolysis into smaller units of fatty acids and
glycerol. This process is called lipolysis and it happens in the cytoplasm. (OpenStax Anatomy and
physiology I). Therefore, Digestive enzymes such as lipase break down lipids in the diet into fatty acids
and glycerol. Lipase enzymes are produced in your pancreas and small intestine.

Concentration of lipids
The amount of substance in the liquid is called ‘concentration’.In other words, it is a measure of how
much solute(a substance that is being dissolved) is dissolved in a solvent(dissolving medium)(classnote).
A more concentrated solution would have more solute compared to solvent. Therefore, for this
experiment lipid in the milk is going to be the solute and water being the solvent.

Sodium carbonate
Sodium carbonate is a sodium salt of carbonic acid. For this experiment, Sodium carbonate is going to be
used to make the milk in an alkaline solution. Milk has the ph of 6.5 to 6.7 which is an acidic solution and
needs to change to an alkaline solution as lipase optimum ph is 8.0 and sodium carbonate is going to
increase its ph. Lipases are more active and stable in alkaline media. Alkaline solution means that ph is
over 7 which makes it easier for phenolphthalein indicators turning colorless as when the pH drops
below 8.3 phenolphthalein goes colorless. (Bora and Bora Optimization of extracellular thermophilic
highly alkaline lipase from thermophilic Bacillus sp isolated from Hotspring of Arunachal Pradesh,
India)We must make it into an alkaline solution, which turns colorless in only alkaline solution showing
the process of fat in milk is broken down to form fatty acids, which is our main topic in this experiment.

Phenolphthalein
​Phenolphthalein is an indicator that is pink in alkaline solutions of pH10. When the pH drops below pH
8.3 phenolphthalein goes colorless. In this experiment, an alkaline solution of milk, lipase, and
phenolphthalein will change from pink to colorless as the fat in milk is broken down to form fatty acids
reducing the pH to below 8.3. (Alkalinity - Whitman College).

Hypothesis
If the concentration of the lipid in milk increases, then the rate of the enzyme activity will also
increase causing the time it takes for the Phenolphthalein indicator to turn white to decrease. This
prediction can be justified with the already existent scientific evidence that we learned in class. When the
substrates are bound with an enzyme to form an enzyme-substrate complex, the reaction occurs where
some enzymes help large substances to break apart into smaller molecules while others help smaller
chemicals to join together to make the larger product, therefore more substrate molecules will be colliding
with enzyme molecules which means that more products will be formed(khan academy, Enzymes and the
active site (article)). Also, as there is a high concentration of substrate more enzyme molecules can bind
with the substrate at the active site, speeding up the reaction.

Variables (IV, DV, CV)

Independent Variable Unit Describe how you will change

your independent variable

Lipid(fat) concentration in milk I will be using 4 different The selection of listed

independent variables which is
lipid concentration in milk
ranging from 0.03g/ml, 0.02g/ml,
0.01g/ml, and 0g/ml. The unit is
going to be g/ml as we are
measuring with different milk
concentrations. I will be putting
all same amounts of milk for
every experiment which is going
to be 5ml cube per trial.
concentrations was based on
grams that were near the
average of the fat content by
weight in regular milk which is
3.25%. (BSc Milk 101: Nutrition
Facts and health effects). For my
experiment “Seoul milk” contains
about 1000ml per container
which will be based on. If we
convert into grams per 1ml
(percentage/100) it turns out to
be 0.0325 g/ml. I will be
changing the different types of
milk that satisfy each listed
concentration of lipid: 0.03g/ml-
whole milk, 0.02g/ml-
reduced-fat milk, 0.01g/ml-
low-fat milk, 0g/ml- non-fat
milk(Types of milk explained:
Whole milk, 2 percent, skim and
more: Gonnaneedmilk).

Dependent variable Unit Describe how you will change

your dependent variable

Time measured for the rate of
reaction
As we are measuring the time
until the reaction turns colorless
for the Phenpletian indicator, I
will be using seconds for this
experiment.
The dependent variable of this
experiment is the time it takes
for the Phenolphthalein indicator
to turn white. I will be measuring
the time it takes for the two
substances: 1. the
Phenolphthalein indicator 2. the
mixture of three liquids; milk,
lipase, and sodium carbonate to

Control variables
The volume and percentage of
lipase solution
The temperature of all the
substances used during the
experiment
The volume of Phenolphthalein
indicator
The volume and the
concentration of sodium
carbonate solution
Impact on Data
It is important to use the same
percentage of lipase solution
because if the lipase solution
increases, then the speed of
reaction will also increase.This
will have a huge impact on the
data because our experiment is
about changing the substrate
concentration not the enzyme
concentration.
Temperature is a significant
procedure to keep it consistent
during the experiment. It may
impact the data by as the
temperature increases, the rate
of reaction will speed up and
extreme temperatures can cause
denaturing of enzymes.
The volume of the
Phenolphthalein indicator,
should be kept all the same for
this experiment. It could impact
the data as the volume of
indicators increases, the faster
the indicator will turn colorless.
The volume and the
concentration of sodium
carbonate solution, which is an
fully turn colorless. I will be using
a stopwatch in this experiment
and I will start the timer as soon
as two substances: 1. the
Phenolphthalein indicator and 2.
the mixture
of three liquids; milk, lipase, and
sodium carbonate are both in the
flask from the measuring
cylinder. Then I will stop the
timer as the color pink in alkaline
solutions in the Phenolphthalein
indicator turn colorless as it
breaks fat in milk to form fatty
acids and glycerol.(Alkalinity -
Whitman College)
Method of control
I will make sure to keep the
percentage of lipase solution as
5%.
I will make sure to keep the
experiment lab at 24 degrees
using an air conditioner in the
lab. I will start the experiment
after 20 minutes so that all the
temperatures could be settled.
I will be keeping the volume of
the Phenolphthalein indicator
the same by always dropping 5
drops per trial by using a spoiler.
I will be keeping the volume and
the concentration of sodium
carbonate solution the same by
 alkali that makes the using the spoiler and measuring
Phenolphthalein indicator turn cylinder to always measure
pink(Wannabemadsci and 7cm^3 of sodium carbonate per
Instructables Magic trial with a concentration of 0.05.
color-changing water), will be
kept the same in the experiment.
As sodium carbonate help to
increase pH solution, if the
volume and the concentration of
sodium carbonate solution
increases, the time it takes for
enzymes to break down the
substrate will also increase which
will have an impact on my data.

Materials/Equipment list
1. Safety materials: Goggles, lab coat, gloves, and mask
2. 15ml for each different concentration of ‘Seoul Milk’, (15ml of whole milk, 15ml of reduced-fat milk,
15ml of low-fat milk, 15ml of non-fat milk)
3. Total 4ml of Phenopheltain indicator (1drop=0.25ml)
4. Total 40ml of Sodium carbonate (The concentration of 0.05)
5. Total 20ml of Lipase (The concentration of 5%)
6. Three measuring cylinders(25ml)
7. Erlenmeyer flask
8. A stopwatch
9. Four Pipettes
10. Six beakers (50ml)
11. Tape
Safety Considerations
Safety materials; goggles, lab coat, gloves, and mask
During the experiment, it is significant to prevent dangerous substances like lipase and sodium carbonate
which is skin and eye irritant. The inhalation of sodium carbonate could affect the respiratory systems in
our body such as coughing and shortness of breath. The experiment also contains strong lipase solutions
which might sting your eyes when accidents occur, therefore, wearing goggles would help to avoid this kind
of situation.

Standing up for the whole experiment

Make sure to always stand up during the whole experiment in case of accident occurrences like chemical
spills and glass breakage. Even after the occurrences of chemical spills and glass breakage, make sure to
always call a supervisor for help. After the experiment is done, make sure to clean out all the salt solutions
in the beakers and cylinders in the order guided by the supervisor.

Method/Procedure
1. Using tape, stick it to all two beakers and label it by writing down two solutions which are sodium
carbonate, lipase as you could get confused later

2. Pour a total of 84ml of sodium carbonate and 24ml of lipase into the beaker where we taped in step
1.

3. Using tape, stick it to all 4 beakers that contain different concentrations of lipid 0, 0.01, 0.02, 0.03.

4. Pour a total of 15ml of whole milk in the beaker where it says 0.03g/ml, 15ml of reduced-fat milk in
the beaker where it says 0.02g/ml, 15ml of low-fat milk in the beaker where it says 0.01g/ml, 15ml
of non-fat milk in the beaker where it says 0g/ml.

5. Using tape, stick it to all three measuring cylinders and label them with Lipse solution, milk, and
sodium carbonate.
6.
The The volume of The volume of The volume of The drops of
concentration of milk(cm^3) sodium lipase(cm^3) Phenopheltain
lipid in carbonate(cm^3) indicator (cm^3)
milk(g/ml)

0.03 5 7 2 5

0.02 5 7 2 5

0.01 5 7 2 5

0 5 7 2 5

7. Using the table above, start with 0.03g/ml of the concentration of lipid in milk and use the pipette
to move the whole milk into the first measuring cylinder by the volume of 5cm cubed.

8. Move the Sodium carbonate into the second measuring cylinder by the volume of 7cm cubed.

9. Move the Lipase into the third measuring cylinder by the volume of 2cm cubed. (Make sure to use
all three different pipettes in order to prevent the mixture of solutions during separation)

10. Put all the solutions in the three different measuring cylinders into the Erlenmeyer flask in the
order of 1. Lipase solution, 2. Milk with different fat concentrations, 3. Sodium carbonate
concentration.

11. Lastly, drop exactly 7 drops of Phenpheltain indicator using the pipette. (Make sure to use the new
pipette at the reaction that could happen inside the pipette.

12. Then start the timer as soon as all the liquids are in the flask.

13. Stop the timer as the color pink in alkaline solutions in the Phenpletain indicator turns colorless as
it breaks fat in milk to form fatty acids and glycerol.
 14. Follow steps 7-13 three times per concentration of lipids in milk in order to get three trials. Make
sure to clear out the flask and the measuring cylinders after each trial.

15. Follow step 14 for the other values of the concentration of lipid in milk(0.03g/ml: whole milk,
0.02g/ml: reduced-fat milk, 0.01g/ml: low-fat milk, 0g/ml: non-fat milk) and record the data. Make
sure to do three trials per value and record the data as soon as you stop the timer.

Works Cited

Alkalinity - Whitman College.

www.whitman.edu/chemistry/edusolns_software/AlkalinityBackground.pdf.

Berg, Jeremy M. “Triacylglycerols Are Highly Concentrated Energy Stores.” Biochemistry. 5th Edition.,
U.S. National Library of Medicine, 1 Jan. 1970, www.ncbi.nlm.nih.gov/books/NBK22369/.

Bora, Limpon, and Minakshi Bora. “Optimization of Extracellular Thermophilic Highly Alkaline Lipase
from Thermophilic Bacillus Sp Isolated from Hotspring of Arunachal Pradesh, India.” Brazilian
Journal of Microbiology : [Publication of the Brazilian Society for Microbiology], Sociedade
Brasileira De Microbiologia, Jan. 2012, www.ncbi.nlm.nih.gov/pmc/articles/PMC3768969/.

BSc, Atli Arnarson. “Milk 101: Nutrition Facts and Health Effects.” Healthline, Healthline Media, 25
Mar. 2019,
www.healthline.com/nutrition/foods/milk#:~:text=Whole%20milk%20straight%20from%20the,lo
w%2Dfat%20milk%201%25.

Department of Biotechnology - Bapuji Institute of ...

www.bietdvg.edu/media/department/BT/data/learning-materials/18BT53_Enzyme_Technology
_Biotransformation_pdf.pdf.

“Enzymes and the Active Site (Article).” Khan Academy, Khan Academy,
www.khanacademy.org/science/ap-biology/cellular-energetics/enzyme-structure-and-catalysis/
a/enzymes-and-the-active-site.
Kozlowski, Rosann. “Why Does Phenolphthalein Change Color?” Sciencing, 16 Feb. 2020,
sciencing.com/phenolphthalein-change-color-5271431.html.

“Lipase.” Encyclopædia Britannica, Encyclopædia Britannica, Inc.,

www.britannica.com/science/lipase.

OpenStax, Lumen Learning &. “Anatomy and Physiology II.” Lipid Metabolism | Anatomy and
Physiology II, courses.lumenlearning.com/suny-ap2/chapter/lipid-metabolism/.

“Types of Milk Explained: Whole Milk, 2 Percent, Skim and More: Gonnaneedmilk.” Milk, 16 Apr. 2021,
gonnaneedmilk.com/articles/types-of-milk-explained/.