A. Deconstructing the question: How can a washing powder enzyme be used to remove stains most efficiently?

What Is An Enzyme?

Enzymes are vital biological molecules that are responsible for catalysing numerous different chemical reactions that occur
within cells. Metabolism and digestion are types of chemical reactions that are catalysed by enzymes and are absolutely
necessary for life. Other reactions that enzymes catalyse include the breaking down of larger molecules into smaller pieces as
well as the binding of two molecules. Enzymes however are highly selective, meaning that they only catalyse a specific
chemical reaction at a time.

Enzymes work with molecules that are known as substrates, which bind to a specific part of an enzyme which is known as the
active site. The current theory, the induced fit model, that is believed by most scientists is that although the active site present
in the enzyme is complementary to the substrate, the enzyme and substrate don't fit perfectly together and instead alter their
shape to bind together. It is for this reason that enzymes are highly selective as only specific substrates can bind to an enzyme.
As a result of the substrates binding to the enzymes, a new product is formed and is separated from the enzymes, which then
proceeds to catalyse other reactions.
As enzymes can significantly speed up chemical reactions, they are regularly used by different industries which include food
and pharmaceutical in order to control and catalyse reactions to efficiently produce a final product. Enzymes are also
prominent in the detergent industry as they can catalyse reactions that break down other molecules which allows for
detergents to efficiently remove stains present on clothes and fabric.

Factors That Affect Enzyme Activity

Factor Effect on enzyme activity

Temperature Enzyme activity generally increases as the temperature increases and decreases at colder
temperatures. However, enzymes at extremely high temperatures denature as their active site is
changed, rendering the enzyme ineffective. All enzymes have a specific optimum temperature range
where they work optimally.

In the context of enzymes used in detergents, temperature is one of the key factors that can be used to
determine the most effective enzyme for removing stains. Specifically, enzymes that are active in lower
temperatures will be more effective as stains can then be removed at lower temperatures which as a
result saves energy.

pH levels All enzymes are affected by changes in the pH, which is a measure of the alkalinity or acidity of a solution.
Similar to temperature, all enzymes have a specific pH value in which they work optimally. Completely
wrong pH values can also lead to the denaturation of enzymes. Most enzymes have an optimum pH value
that is close to neutral as extremely high or low pH values denatures most enzymes although some
enzymes do function best in highly acidic or alkaline conditions.

Similar to temperature, pH levels are another factor that can be taken into consideration when selecting
an enzyme to be used in a laundry detergent. The pH of most laundry detergents normally ranges from 7-
12 as acid can often damage the fibre present in clothes. As a result, an enzyme that is active within this
pH range will be more effective than other enzymes that are active at a different pH range.

Enzyme Increasing the enzyme concentration will generally increase enzyme activity as long as substrates
concentration remain.

Similar to enzyme concentration, increasing the concentration of substrates will also increase the rate
Substrate of reaction. Once all the enzymes are bound to the substrates, an increase of substrate concentration at
concentration that point will have no effect on the rate of reaction as all the enzymes will already be working at their
maximum potential.
 Specifically these enzymes catalyses
proteolysis, which is the breakdown of proteins
into smaller polypeptides or single amino acids.
This breakdown is done through the splitting of
peptide bonds with the process of hydrolysis
(reaction where water breaks down bonds).
As protease enzymes are responsible for
breaking down proteins, they are effective in
removing protein related stains which could
include blood and other protein containing foods
such as egg.
pH level
Another factor that can affect the activity of protease
enzymes is the pH value. Most protease enzymes work
optimally in neutral conditions around a pH value of 8 and are
active around the pH range of 6-11. However, pepsin works
optimally around a pH level of 2. This is due to pepsin being
involved in the breakdown of proteins in the highly acidic
stomach.

Lipase Lipase enzymes are also found in the human Temperature

body and are responsible for the breakdown of
lipids (fats). The three main types of lipase
enzymes are gastric, pharyngeal and hepatic
lipase.
Lipase are enzymes that are responsible for
catalysing hydrolysis of lipids. Hydrolysis is the
chemical breakdown of a compound due to
reaction with water.
As a result, lipase enzymes are effective in the
removal of fat related stains.
Similar to protease, lipase enzymes also work optimally
around the temperature of 40°C. This is due to the
temperature of the human body, where these enzymes are
found, which is around 37°C. Lipase enzymes are also active
around temperatures that are as low as 20°C and as high as
60°C.
pH level
Likewise to protease, lipase enzymes usually have an
optimum pH level of 8 and are active within the pH range of 7-
11. Gastric lipase, which is found in the stomach has an
optimum pH value of 3 and is active within the range 3-6.

Amylase Amylase enzymes are commonly found in the Temperature

saliva of humans and are responsible for
catalysing the process of hydrolysis and
ultimately converting starch into sugar. The
three main types of amylase enzymes include
alpha, beta and gamma amylase.
As a result, amylase enzymes are found in some
washing detergents as they are effective in
removing starch related stains e.g. chocolate.
Identical to protease and lipase, amylase enzymes work
optimally at a temperature of around 40°C and can be active
at temperatures as low as 20°C and as high as 60°C.
pH level
Amylase enzymes commonly have an optimum pH of around
6 and can be active in a pH range of 5-8. Gamma amylase,
similar to pepsin and gastric lipase, works optimally in acidic
conditions and has an optimum pH level of 3.

Choosing An Enzyme And A Factor

Protease was chosen as the enzyme that will be investigated as it is most commonly found in washing detergents. This would
later help in analysing the results as there are an abundance of studies present on this enzyme that can be used as a benchmark
when comparing the results. As most washing detergents have a pH of around 6-12, due to highly acidic conditions causing the
fibre present on fabric, it was decided that pepsin will not be used and instead any other type of protease that is not acidic will
be investigated. The temperature was chosen as the factor because the main advantage of using enzymes in detergents is the
fact that unlike other detergents, they can be effective at lower temperatures, thus saving energy. As a result, the temperature
was favoured over other factors as it is the most important one in the context of stain removal.

Process That Could Be Used To Determine The Effect Of Temperature On Protease Activity
A spectrophotometer can be used to measure the effect of temperature on protease activity. Specifically, a
spectrophotometer is an instrument that can be used to measure how much a chemical substance absorbs light by measuring
the intensity of light as a beam of light passes through sample solution. In this context, the absorbance of the protease solution
can then be measured beforehand. Furthermore, a textile swatch can be embedded with a protein related stain such as blood
or egg yolk and placed inside the protease solution. After a specific amount of time, the absorbance of the protease solution
can then be measured again and compared to the initial absorbance of the solution, which would then effectively showcase
how much of the stain has been degraded by the protease. The effect of temperature on protease activity can also be
measured using this process by simply altering the temperatures of the protease solution. Therefore, the effect of
temperature on protease activity in removing a stain can be determined using a spectrophotometer.

Potential Risk Factors Present

One of the major risk factors that is involved in this process is the potential exposure to harmful UV-radiation. To prevent this
risk, safety glasses must be used and the lid of the spectrophotometer must not be opened during the measuring of the
absorbance. Another potential risk factor is the potential exposure to harmful acidic substances. This risk can be minimised
through the use of lab coats and safety glasses which would prevent the acid from making direct contact to skin and eyes.
B. Design
Enzyme selected: Protease
Factor to investigate: Temperature
Inquiry Question: How does temperature affect the activity of protease in removing stains?
Types of Variables
Dependent
The activity of protease in the
removal of stains.
This will be measured through the
spectrophotometer which can
measure and provide a quantifiable
colour measurement of the stained
fabric.
Materials
• 250 ml Beaker
• Spectrophotometer
• Protease
• White denim fabric
• Egg yolk
• Different temperatures of water (20°C, 40°C and 60°C)
• Magnetic stirring plate
Independent Constant
Temperature pH
This will be changed by The pH value of the protease solution will be kept
using different constant as wrong pH values can significantly affect
temperatures of water in protease activity and in extreme cases may even lead to
which the protease will its denaturation.
be dissolved.
The pH will be kept constant through conducting the
experiment using water (7 pH). Additionally, in order to
confirm that the pH is kept constant, the pH value of the
solution will be measured using a pH meter.
Enzyme concentration
The concentration of the enzyme used is another factor
that will be kept constant as it can also affect the activity
of the enzyme. Specifically, higher concentrations of
enzymes results in an increase in the speed of the
reaction. Therefore, in order for the results to be valid, the
concentration of the protease has to be kept at a constant
amount.
This can be done by using the same concentration of
protease which can be measured using a weighing scale
or measuring solution.
Type of stain
The type of stain will also be kept constant as different
enzymes are more effective in removing different kinds of
stains. Protease in particular is most effective in
removing protein related stains which include blood and
egg stains.
Therefore, in this investigation the type of stain will be
kept the same.
The amount of water
The amount of water used will also be kept the same as it
could potentially affect protease activity in removing the
stain.
Step Method Justification

1 Stain a white denim fabric with egg yolk.
White denim fabric is used as the fabric as it one of the stronger
fabrics which would prevent the fibres present on the fabric from
degrading due to the protease solution. Egg yolk is used to stain the
chosen fabric as it has the most amount of protein. Thus, protease
activity in removing an egg yolk stain can then be
effectively measured.

2 Pour 200 mL of 60°C water in a 250 ml
beaker and mix 0.1 g of protease with it.
60°C is used as one of the temperatures as protease is known to be
active around this temperature. 200 mL is chosen as the amount of
water as other experiments that were found online had also used this
amount. Similarly, the amount of protease is also reasonable as other
similar experiments found online had also used this amount.

3 Measure the absorbance of the enzyme The absorbance of the protease solution is measured before the
solution. stained fabric is placed inside it as this value can then be compared
with the absorbance of the solution after the stained fabric is placed
inside. This would effectively showcase the amount of stain that the
protease has degraded.

4 Add the stained white denim fabric to the A magnetic stirring plate is used to simulate the action of a washing
beaker and place it on a magnetic stirring machine. The time chosen as 5 minutes is reasonable as it only takes
plate for 5 minutes. around 30 minutes for protease to completely break down the stain.

5 Once the 5 minutes are over, measure the The absorbance is measured again as this value can then be
absorbance of the protease solution once compared to the initial absorbance of the protease solution. The
again using a spectrophotometer. difference between the two values can then effectively showcase
the amount of protein that the protease has degraded.

6 Repeat the same experiment with different
temperatures (20°C, 40°C and 80°C ).
In order to determine the effect of temperature on the protease
activity, the same procedure has to be repeated with different
temperatures. This would effectively showcase how different
temperatures lead to differences in the amount of protein that
protease can break down. The temperatures chosen are 20°C, 40°C
and 80°C. This is because protease is found to be active at
temperatures around 20°C and is known to work optimally at
temperatures around 40°C. 80°C is also used as a temperature as
protease like most other enzymes is known to denature at that
temperature. Thus, including 80°C as one of the temperatures can
be beneficial in the analysis of results as well as the evaluation of the
procedure. .

7 Conduct three trials of the procedure The procedure is replicated three times in order to increase the
reliability of the results. Conducting three trials will prevent the risk
of an outlier affecting the results thus increase the precision of the
procedure.

Potential Safety/Ethical Risks Present In The Procedure

Potential Safety/Ethical Risk How To Prevent The Risk

Chemical solutions potentially spilling in This risk can be easily prevented with the use of protective eyeglasses.
eyes

Exposure to UV-radiation emitted from This risk can also be minimised through the use of protective eyewear. Another
spectrophotometer preventive measure that can be taken to reduce this risk is to not open the
spectrophotometer lid at times when the absorbance is being measured.