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Nimisha Sharma

Effect of the substrate concentration on enzyme catalysed reaction

Aim: To investigate how the concentration of the substrate hydrogen

peroxide affects the rate of the enzyme catalysed reaction, by measuring the
time it takes different concentrations of hydrogen peroxide solution to
produce 35 cm3 of oxygen gas?

Enzymes are large globular protein molecules folded to form a three
dimensional globular structure, found in all living organisms.The biological
processes that occur within all living organisms are chemical reactions, and
most are regulated by enzymes. They are used to speed up specific reactions
in the cells. Without enzymes, many of these reactions would not take place
at anobservable rate.
Hydrogen peroxide (H2O2) is a by-product of metabolism and is made in all
living cells. It is poisonous and must be removed as soon as it is produced in
the cell. Cells make the catalase to remove hydrogen peroxide.
Catalase is an enzyme found in living cells. It is found in food such potato,
carrot, liver, but also in yeast. Catalase is used to speed up the
decomposition of hydrogen peroxide into oxygen and water shown in the
equations given below.

Hydrogen Peroxide -----------------------------Water + Oxygen



2H2O + O2(g)

Enzymes bind with chemical reactants called substrates. There may be one
or more substrates for each type of enzyme, depending on the particular

chemical reaction.They are specific in their action.For an enzyme to catalyze

a reaction, a substrate has to be present and to bind with the active site as a
result of collisions due to random movement.

Catalase is able to speed up the decomposition of hydrogen peroxide

because the shape of it s active site matches the shape of the hydrogen
peroxide molecule. Enzymes speed up chemical reactions because they
lower the activation energy needed to activate the reacting molecule. Factors
that affect the rate of reactions are temperature, pH, substrate concentration
and enzyme concentration.
In this experiment, I will be examining the effect of substrate concentration
(hydrogen peroxide) at the rate of an enzyme catalysed reaction, in this case
catalase found in yeast.

I believe that the increase in the concentration of the substrate
concentration will increase the rate of the reaction.If the concentration of
substrate increase, there will be more frequent collisions between the
substrate molecules and the enzyme active site, so the rate of reaction
increases. The rate of the reaction increases because there are more
substrate molecules added and it collides and bind with the available active
sites on catalase.This results in more no. of reactions so the amount of
oxygen released also happens quickly. But it will only increase to a certain
point, after which the rate of reaction will reach to its optimum level or
plateau and show no further increase by further increasing the concentration
of the substrate concentration.This is because the substrate molecules
exceed in the no. and there will be no active site available to collide and bind

At a low substrate concentration there are many active sites that are not
occupied. This means that the reaction rate will be low.

How to control them

Time it takes for 35 cm3 of oxygen

gas to be released in the gas
syringe in seconds. We can

calculate the rate of enzyme

reaction for this.


Concentration of the hydrogen

peroxide: 20%, 16%, 12%, 8%, 4%
and 0%.The volume of hydrogen
peroxide will be measured using
graduated cylinder. The different
concentrations of the solution are
prepared by adding right amount of tap
water in it.

Volume of hydrogen peroxide

solution- 4 cm3 using a 5cm3
graduated cylinder.
Concentration of the yeast
solution-The concentration of
the suspension is prepared for all
the samples ensuring the
constant concentration.
Volume of the yeast
suspension- 2cm3 using a 5 cm3
Type of yeast- Fresh Dry yeast
is used of the same


Room temperature and pressure

-Room temperature and pressure are
not measured but will be the same for
all the trials as all trials carried at the
same time in the same room.


Yeast catalase suspension (pre-prepared)

Hydrogen Peroxide solution (pre-prepared) with the concentrations: 0%,
4%, 8%, 12%, 16% and 20%
Tap Water
Gas Syringe with the rubber tubing and the bung
7Test Tubes
Test Tube Rack
Pipette filler
Syringe (5cm3)
Graduated cylinder (5 cm3)
Stop Watch

Risk Assessment

Wear safety goggles and protectiveclothing from hydrogen peroxide

while making the concentrations and also during the experiment.

Wash hands with liquid hand wash after using hydrogen peroxide.


Prepare the yeast suspension 1 hour before the experiment by adding dry
yeast with lukewarm water. It will be used as an enzyme in this
experiment for all trials ensuring that the enzyme concentration is
constant in all trials.The amount of yeast suspension will be measured
using the 5 cm3 syringe.

Prepare the different concentrations of hydrogen peroxide solutions by

using tap water with the hydrogen peroxide in an accurate amount.Use
pipette to fill hydrogen peroxide solution in the graduated cylinder. The
volume of hydrogen peroxide will be measured using graduated cylinder.

Label 6 test tubes according to the concentrations of the hydrogen

peroxide. E.g. 0%, 4%, 8%, 12%, 16%, 20%. In each test tube, using a
5cm3 graduating cylinder put the different concentrated solutions of the
hydrogen peroxide.

Make a table to note the time it would take each concentration of the
hydrogen substrate to produce 35 cm3 of oxygen in the gas syringe.

It is very important to accurately measure the amounts of Hydrogen

Peroxide, Yeast and water to ensure the accuracy of the results.
In order to find out how the concentration of hydrogen peroxide affects the
rate of reaction, first set up the apparatus below.
1. Measure and add about of 2cm3 of yeast suspension to an another test
2. Measure about 4 cm3 of hydrogen peroxide solution of concentration
20% and pour itin the test tube containing the yeast and immediately
place the gas syringe bung on the end of the test tube, at the same
time start the stopwatch.
3. Bubbles should start to rise up in the tube and the gas syringe will
move outwards, as shortlyas the gas syringe passes the 25 cm3 mark
stop the stopwatch and note the elapsed time down to the nearest
1/10th of a second.
The 0% concentration of hydrogen peroxide solution is performed as
a control solution to show that at 0% concentration no reaction
takes place.
4. Repeat the experiment with hydrogen peroxide concentrations of 16%,
12%, 10%, 8%, 4% and 0%.