Neev Gupta

Enzyme Catalysis Lab Report

The Effect of Substrate Concentration on Catalase Activity

Abstract:

In this experiment, the objective is to determine how changing the concentration of the

substrate(hydrogen peroxide) would affect the rate of catalase activity. The null hypothesis states

that if the substrate concentration is decreased, then the rate of catalase activity will not be

changed. In the experiment, 5 different tubes were filled up with 15 mL of 5 different substrate

concentrations(0%, 0.375%, 0.75%, 1.5%, 3%) and 50 small yeast spheres with catalase were

made. 10 yeast spheres were carefully put into each tube 1 by 1, and after the yeast sphere

touched the bottom of the tube, a timer was started and the timer was stopped as soon as the

sphere reached the top of the liquid. All this data was recorded and the experiment ended up with

10 trials for each concentration. After the experiment, the data was compiled and it was

determined that the null hypothesis was rejected and that decreasing the substrate concentration

led to a decrease in the rate of catalase activity.

Introduction:

In this experiment, we take a look at how changing the concentration of the substrate affects the

results produced by the catalase. Now, enzymes are proteins located in living cells which act as

catalysts in biochemical reactions in living organisms. A catalyst affects the rate of a chemical

reaction by speeding up the chemical reaction or by lowering the temperature or pressure which

is needed to start a chemical reaction. During these reactions, the catalyst is never consumed

which gives it the ability to be reused as many times as it needs to for certain biochemical
reactions. Now in these catalyzed reactions, the substrate is acted on and when it is, the substrate

binds to the active site of the enzyme. Once this occurs, the reaction with the substrate molecules

can be activated which leads to the formation of the products in this biochemical reaction. After

the reaction is completed, the enzyme can be recycled to be used again to break down more

substrate molecules.

In this lab, the enzyme is catalase and the substrate will be hydrogen peroxide. The structure of

catalase includes 4 identical subunits which are tetrahedrally arranged. Now each subunit

contains a heme group and NADPH in the active center of it. Now, catalase is an enzyme which

is really important for aerobic organisms as one of its functions is the decomposition of H2O to

form water and oxygen. The chemical equation for this breakdown is: 2H2O2 → 2H2O +O2

(gas). Since this enzyme helps produce oxygen and water from 2 water molecules, which are

essential for living organisms which are aerobic, it becomes super essential for them. Now in this

reaction, the compound being broken down, is called hydrogen peroxide. Since the enzyme is

breaking this down, it helps prevent the accumulation of hydrogen peroxide in the cells which

are formed by metabolic processes. In this experiment, yeast is used as the source for the

Catalase enzyme. This is due to the fact the yeast is a much easier object to obtain, and is usually

lying around and also due to the fact that it contains a huge amount of catalase, which would

allow it to take place in the chemical reaction. Yeast or Saccharomyces cerevisiae, which is the

scientific name of a microorganism and is single-celled. It is a type of fungus which is commonly

used in baking and brewing due to the fact that the catalase it has inside can help start reactions

with certain ingredients to form air bubbles, enlarging the mixture. For example, to make bread,

baker’s yeast is mixed with the ingredients to make the dough and it helps the dough puff out and
become larger so that it can be used to make bread. Also the ability it has to ferment sugars into

alcohol and carbon dioxide makes it also very helpful to create beer and win. The ability of it to

produce air bubbles or CO2 is essential for this experiment. Since hydrogen peroxide is the

substrate, the catalase enzyme will be dropped on to the hydrogen peroxide, or the substrate.

Since the functions of catalase cause it to interact with hydrogen peroxide and break it down into

water and oxygen bubbles, once the yeast spheres containing the catalase are dropped onto the

hydrogen peroxide, a reaction will start to occur. In this reaction, the catalase will do its job and

eventually start producing air bubbles in the substrate, showcasing its function of breaking down

hydrogen peroxide and also allowing for the rate of fermentation to be measured by seeing how

fast bubbles star to form and how fast the yeast sphere comes to the top of the substrate.

In the experiment, the null hypothesis states, decreasing the concentration of the substrate will

not affect the rate of catalase activity. This means that changing the amount of concentration will

not speed up or slow down the times it takes for the catalase to break down the hydrogen

peroxide. But in reality, decreasing the concentration will cause the rate of catalase activity to

become much longer. This is due to the fact that decreasing substrate concentration gives the

enzyme less room to take action and bind to the substrate meaning that there isn’t enough of the

catalase to break down the hydrogen peroxide fast enough. There are fewer substrate molecules

available to interact with the enzyme’s active site leading to this decrease in the rate of catalase

activity.

This procedure works for this experiment for many reasons. Since the experiment is trying to test

out how substrate concentration affects catalase activity, the substrate has to be changed in order

to give accurate results. This means that you have to find a way to change the concentration of
the hydrogen peroxide. In the procedure, the concentration of the original peroxide is 3% so that

means the procedure has to contain many diluting steps in order to achieve the lower

concentrations. Also since the main function of the catalase enzyme is to break down hydrogen

peroxide. The procedure using Hydrogen Peroxide makes the most sense as that is the only thing

that gets broken down by catalase. Using another substrate would be useless as enzymes only

bind to specific substrates which means that using another substance would cause a scenario

where there is no enzymatic reaction, making us get no results from the experiment. This

procedure can only be used for this specific experiment as in the other ones, the hydrogen

peroxide or substrate is staying the same in each of them as they do not need to be changed since

that would create invalid results. This makes it so that this procedure is the only one where you

have dilute the concentration multiple times.

Procedure:

Start off the experiment by forming the yeast spheres. Mix 2% of the sodium alginate solution

with an equal volume of a 10% yeast suspension. Now with this mixture, put it into a syringe and

slowly let small drips out, into a beaker of calcium chloride which allows the drops to harden and

turn into spheres after 5 minutes. There should be 50 of these spheres and set them aside. Now

get 5 different test tubes, one for each concentration of substance. The first trial will be the

control which is a concentration of 0%. Fill up the test tube with 15 mL of just water and put it

on a test tube holder. Now prepare a stopwatch and a table to record this information. With some

forceps, carefully grab a yeast sphere and then slowly place it into the test tube. After the sphere

hits the water in the tube, then wait for it to reach the bottom of the tube and when it does, start

the timer. After the timer is set, let the bubbles sit there until the reaction starts and the yeast
sphere comes all the way to the top of the liquid. When the sphere reaches the top, the timer

should be stopped and the data should be recorded and the yeast sphere should be taken out and

be gotten rid of. The same test tube is being used for each concentration. Then, repeat this trial

for the 0% concentration, 9 more times, making it a total of ten times, with all the data being

recorded. After the control is done, then the 3% solution has to be tested. Since the 3% is the

stock solution, put 15 mL of it into another test tube and then redo the experiment done before,

but this time with a 3% concentration. This should also be done 10 times with 10 different yeast

spheres with the data being recorded. After this, the 1.5% concentration will have to be made. To

do this, the hydrogen peroxide will have to be diluted in half. To do this, mix 7.5 mL of water

with 7.5 mL of the 3% solution to create 15 mL of a 1.5% solution. Then repeat the experiment

in another test tube 10 separate times with the data being recorded. After this the 0.75%

concentration will have to be made. To do this, we first have to make 7.5 mL of 1.5% solution

and then with that, add it to 7.5 mL of water and then mix them together to create a 0.75%

solution of concentration gradient. Then put it into another test tube and then repeat the

experiment 10 separate times and record all data. For the final trial, we will need to make a

0.375% solution. To do this we will need to dilute the 0.75% solution. So we need to mix 7.5 mL

of water with 7.5 mL of the 0.75% solution creating a 0.375% solution. Then once again, put the

solution into another test tube, and then continue with the experiment, doing it 10 separate times

and then recording the data. After this, the experiment is over and everything has to be cleaned

up.

Variables:

- Independent variable: Substrate concentration

 - Dependent variable: Catalase activity

Control:

- The control in this experiment is the 0% substrate concentration

- Negative control

- This is the control in this experiment as this gives us a base point to measure the other

substrate concentrations. Since there is no substrate, this becomes a negative control as

there is an absence of the component needed to formulate this chemical reaction. This

determines the effect with catalase has on no substrate and helps us validate the other test

results since the other ones should be much faster then the results this produces.

Constants:

- Same type of yeast: Having the same type of yeast is very important for this experiment.

Since there are different types of yeast, they may have different properties and may have

different amounts of catalase in them. This means that if you used different types of

yeast, some of the results you get may be inaccurate as some trials may use more

catalase, increasing another factor which is not being tested.

- Same amount of liquid in each test tube(15 mL): Having the same amount of liquid in

each test tube is important as it makes sure that each concentration is being tested in a fair

environment. The amount of fluid can change the results of an experiment as a yeast

sphere may reach the top of the liquid faster if there is less of it. If all tubes are 15 mL, it

means that each yeast sphere will have to travel 15 mL before the timer stops making a

fair scenario for each sphere and getting an accurate result.

 - Size/amount of the yeast sphere: This also has to be the same for similar reasons as the

type of yeast. Adding more yeast to a sphere means that there is guaranteed to be more

catalase in the sphere which means that once it interacts with the substrate, it will have a

faster activity time as there is more enzyme making the reaction faster. This means that

the size of the yeast sphere and the amount has to be the same so that there is no extra

amount of catalase in the experiments being conducted .

Data and Results:

Figure 1 Showing a data table of the results of the conducted experiment.:

Concentratio Trial Trial Trial Trial Trial Trial Trial Trial Trial Trial Average
n 1 2 3 4 5 6 7 8 9 10 time
time time time time time time time time time time

0% 0 sec 0 sec 0 sec 0 sec 0 sec 0 sec 0 sec 0 sec 0 sec 0 sec 0 sec

3% 10.57 10.76 11.37 10.97 11.12 11.09 10.9 11.39 10.36 10.37 10.89 sec
sec sec sec sec sec sec sec sec sec sec

1.5% 17.36 17.49 17.59 17.89 17.94 18.84 17.06 15.8 17.44 18.23 17.564 sec
sec sec sec sec sec sec sec sec sec sec

0.75% 36.17 38.34 36.82 32.57 37.87 35.12 39.53 33.62 38.49 42.81 37.134 sec
sec sec sec sec sec sec sec sec sec sec

0.375% 119 115 107 117 121 123 126 117 110.6 sec
sec sec 67 sec sec sec 94 sec sec sec sec sec
Figure 1: This figure is showing the data table of the results from the conducted experiment. The

Vertical axis lists the 5 different substrate concentrations which were tested out in the

experiment. The Horizontal axis includes the trial number and the time it took for the yeast

sphere to reach the top of the test tube. It also includes the average time for each substrate
concentration. Each time is in seconds. From the table, as the concentrations went down, the

time it took for the catalase activity to happen increased showing a decreasing trend in catalase

activity.

Figure 2: Graph of Data table above showing Catalase Activity in seconds based on

Substrate Concentration.

Figure 2: This figure shows a graph of the data above. The x-axis includes the 5 different

substrate concentrations tested in the experiments. The y-axis contains the catalase activity

shown in seconds. This because the amount of seconds shows how fast it took for the reaction to

take place and for the yeast sphere to come to the top of the tube. The graph shows that as the

concentration increases, the catalase activity also increases since the number of seconds
decreases, meaning that the reaction is faster and the effect of the catalase is greater. Each point

also includes a standard error bar disregarding 0. When looking at the error bars, you can

determine that everything is significantly different from each other.

Figure 3: Calculations of Standard deviation, Standard error, and the 95% CL range of the

0.375% concentration.

Figure 3: This shows the work used to find the standard deviation, standard error, and the 95%

CL range for the 0.375% substrate concentration. This math was required in order to find the

standard error bars on the graph in Figure 2. This process was done for each substrate

percentage.
Conclusions/discussion:

Claim:

In this experiment, the null hypothesis, decreasing the concentration of the substrate will not

affect the rate of catalase activity, is rejected. The results of the experiment show that the

changing and decreasing the substrate concentration does in fact change the rate of catalase

activity. Specifically, decreasing the substrate concentration, leads to less catalase activity.

Evidence:

The data from the experiment shows that as the percentage of substrate concentration decreased,

it took longer for a reaction to occur and for catalase activity to happen. From the data in Figure

1 and the graph in Figure 2, we can prove this statement. The table shows the average time it

took for the yeast sphere to reach to the top of the liquid during the experiment for 5 different

concentrations of substrate. The control ended up with no reaction as the yeast sphere did not go

up at all. If we look at the results of the 3% concentration, the average time was 10.89 seconds

for the reaction to occur. Now if we look at the 0.375% concentration, the average time was

110.6 seconds. The time of the 0.375% concentration was 99.71 seconds longer than the average

time for the 3% substrate concentration. Since the 3% substrate concentration is much larger than

the 0.375% concentration and the time it took was much fewer, it can be determined that as the

substrate concentration decreases, the amount of time it takes for the reaction to occur is much

longer. Also if you take a look at the graph in Figure 2, you can see that as the concentration of

the substrate increases, the y-axis or the time in seconds decreases, which further proves this
statement. Since the graphs were also statistically analyzed and examined, error bars were drawn

on the graph.Since none of the error bars overlap with each other, this means that each value is

statistically different from each other, meaning that each one is unique, and the huge difference is

real.

Reasoning:

Whenever an enzymatic reaction occurs, the enzyme has to bind to the substrate for the reaction

to start. The substrate is essential for the biochemical reaction to take place. Changing the

substrate concentration is saying that the amount of that substrate is changing. At lower substrate

concentrations, there are fewer substrate molecules which is what the enzyme has to interact

with. This is what happened in the experiment as the concentrations went down which led to less

substrate molecules to bind to which made it much harder for the enzyme to bind to the substrate.

Since the process of binding together became much more difficult, the time it took for the

chemical reaction to occur increased as well. Another thing is the active site occupancy in the

substrate and cell. The catalase has active sites, but when the concentration is lower, the number

of molecules, like said before, is much fewer which results in a reduced probability of successful

collision between the enzyme and substrate molecules. This leads to a slower overall reaction

since collison helps form more product. Now if you look more in depth into the materials of the

experiment, the catalase was represented by yeast spheres and the substrate represented as

hydrogen peroxide. As the hydrogen peroxide concentration was decreased, more water was

added to it diluting the solution. This made it so the number of molecules which the peroxide

usually contained, to become a fewer number as water was being added. Yeast, which contains a

decent amount of catalase, doesn’t have any specific reactions with water, and since the solutions
get more and more water in them, it becomes harder and harder for the enzyme to find the right

part of the substrate to bind with, so that the reaction can occur. This leads to an increase in wait

time before the reaction happens as the substrate concentration decreases.

Design Flaw:

This experiment was definitely not a perfect experiment by any means.One task which was very

tough was perfectly measuring out all of the measurements. Sometimes when trying to get the

right amount of certain liquid, it was not always right. This could be fixed by using items like

pipettes and being more precise with the measurements. Also, another design flaw was the first

procedure as it was not clear enough. This led us to make a mistake and at first make a trial with

only 10 mL of liquid. This made it very hard to take the yeast sphere out of the tube as the

forceps were too big to take them out. This led to us wasting resources and forced us to restart

the experiment. In order to prevent this, the procedure should have been more thought out before

since it would make it easier on the experiment day to complete all the tasks. Another issue due

to the procedure was the amount of hydrogen peroxide. In the original procedure, it was not

taken into account of how much hydrogen peroxide will be given in the experiment. This meant

that we could not make any mistakes since we need 15 mL of liquid for each trial. This led us to

delay our experiment and made it take longer. Overall, a more specific and thought out procedure

would of led to a much better and more valid experiment.

Predict:

To determine if an inhibitor is competitive or noncompetitive can be done in a similar way to the

procedure for this experiment. To determine this you can use the same catalase and hydrogen
peroxide pair for the enzyme and substrate. The initial test will be the same with it measuring the

control. The control will be measuring the enzyme-catalyzed reaction without any inhibitor. This

would be set as a baseline. Now the next test would be wanting to determine the competitive

inhibitor. A competitive inhibitor is when an inhibitor is competing with the substrate for the

active site of the enzyme. But unlike the earlier procedure, this time the substrate concentration

will be staying the same while the concentration of the inhibitor will be different. The inhibitor

would be pre-incubated with the enzyme before the substrate is added. And if the scenario is

competitive inhibition, the concentration would reduce the ability of the enzyme to bind with the

substrate which means that the expected reaction would be a decrease in the reaction rate. Now

an non-competitive inhibitor is when the inhibitor binds to an allosteric site on the enzyme rather

than on the active site. This means that in this scenario the enzyme will still have a reaction with

the substrate, but it will be changed as the enzyme and substrate don’t fit perfectly together any

more as the non-competitive inhibitor creates a different shape. This means that when the

reaction is measured, the reaction rate will also decrease. Increasing the concentration rate of the

non-competitive inhibitor will just lead to a decrease in the reaction but won’t cause it to hit zero

since it is not competing with the substrate. The catalytic activity will decrease just like the

results of the completed procedure.

Follow-Up Questions:

1. If you increase the concentration of an enzyme in the solution, the reaction will be sped

up more and it would have a higher rate of enzymatic activity. If you decrease the

concentration, the opposite effect will happen and the rate of enzymatic activity will
 decrease. This is because as the enzyme concentration decreases, there are fewer enzymes

to complete the chemical reaction. Now if you use the example with hydrogen peroxide

being the substrate and the yeast spheres being the enzyme, the more yeast which is put

in with the tube of peroxide, the more oxygen bubbles it will form and the faster it will

reach the top of the tube. This represents an increase in the catalase activity.

2. Sodium Chloride or salt concentration varies depending on the enzyme and the conditions

of the environment. It can both have positive and negative effects. Since sodium chloride

is composed of sodium and chloride ions, they can form a salt bridge between these ions.

A salt bridge will help maintain the structure of the enzyme and will help stabilize the

active site which helps the process of the chemical reaction and gives a better binding and

catalytic activity. But also on the other hand, if there is too much salt, it can lead the

enzyme to denaturation because it disrupts the hydrogen bonds and the interactions

within the structure of the enzyme, breaking the protein and making it a denatured

protein. This will cause the enzyme to lose its shape which means that it won't be able to

bind with substrate, causing no chemical reaction. This makes sodium chloride a

double-edged sword based on how much of it you use.

3. The effect of temperature also varies on the activity of catalase. In higher temperatures,

as it increases, the enzymes and substrates collide more frequently and the enzyme active

increases due to the increased kinetic energy produced by the heat. But if it gets too

warm, the structure of the enzyme gets disturbed since the weak hydrogen bonds are

being disrupted. This will lead to denaturation making the enzyme useless. This

obviously has a negative effect on catalase activity. At lower temps, kinetic energy is

reduced which means that there is slower molecular motion. This means the rate of
 catalase activity decreases. Since the lower temps also reduce the molecular motion, it

becomes harder for the enzyme and substrate to bind together, decreasing the activity

rate. The most optimal temperature for the best enzymatic activity with catalase is 40

degrees celsius since at that temperature, the kinetic energy is high and the temperature

isn’t high enough to denature and ruin the protein.

4. Both acidic and basic pH levels lead to a decrease in catalase activity. In higher pH

conditions, or more basic pH, the excess of OH- ions lead to the deprotonation of certain

amino acid side chains. This means that the structure of the active site is being changed

which makes it so that the enzyme and substrate won’t bind perfectly. The change in

ionization disrupts the environment and makes it unfavorable for substrate binding which

leads to a decrease in enzyme activity. In lower pH or more acidic conditions, excess of

H+ ions does the same thing as the OH- ions. This once again changes the structure, and

the ionization disrupts the environment of the active site again which makes it

unfavorable for binding. This also leads to a decrease in the enzymatic activity. Since

both too acidic and too basic levels do not suit the environment for catalase and decrease

activity, the best range of pH would be 6-8 with 7 being the most optimal. The water

levels in 6-8 are more balanced and don’t favor acidic or basic water, making it so that

the environment inside the active site is better and so that the catalase activity becomes

better as well.

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