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The Effect of Temperature on the Activity of Pectinase

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The Effect of Temperature on the Activity of Pectinase



Plan
====

Aim: - To investigate how temperature affects the rate of reaction of
pectinase on its substrate apple fruit pulp.



Introduction: -
===============

Enzymes are proteins that catalyze reactions in biological systems by
reducing the activation energy needed for any given reaction. They do
this by temporary binding to one or more reactants of the reaction
they catalyze. By doing so, they lower the amount of activation energy
needed and thus speed up the reaction. The activation energy is the
amount of energy needed for molecules to react when they collide. The
fact that molecules need to collide in order to react is known as the
collision theory.

[IMAGE]

Most enzymes are simple globular proteins, and the tertiary structure
of the enzyme protein is well developed. Enzymes are specific which
means a particular enzyme only works on one substance known as its
substrate. The precise folding of the polypeptide chains ensures that
each globular protein has its own 3D shape or confirmation. Substrates
bind with an enzyme at a specific spot on the enzyme called the active
site. The combination of an enzyme with its substrate is known as the
enzyme-substrate complex. This formation is explained by two theories.
The Lock and Key hypothesis suggests that the substrate fits into the
active site like a "lock and key" where the substrate is the lock and
the enzyme is the key. The Induced Fit theory suggests that the shape
of the active site changes shape when the substrate molecule attaches
to it.

Pectins are large polysaccharide molecules, made up (mainly) of chains
of several hundred-galacturonic acid residues, and they are found in
plant cell walls. Pectin is able to form jellies (e.g. jam) but in
fruit juices and other liquids it is undesirable. Pectinases are
enzymes that break down complex pectin molecules to shorter molecules
of galacturonic acid, as pectinase causes the disintegration of the
cell wall, and so allowing the cell sap (juice) to flow out. This is
why the biggest industrial use for pectinases is in the extraction and
clarification of fruit juices; as some fruit juices e.g. apple juice
may have bits of apple in it. This is where pectinase is used to
catalyze the breakdown of the solid apple bits into apple juice, thus
clarifying the apple juice. Pectinases are obtainable from fungi, such
as species of Aspergillus and Penicillium, and also from bacterial
organisms.

[IMAGE]

Temperature has a complex effect on enzyme activity. The "optimum
temperature" of an enzyme is the temperature in which the enzyme
functions most efficient. An increase in temperature below the optimum
results in an increase in the kinetic energy of enzymes. This leads to
higher efficiency of enzyme-substrate complex formation; therefore a
higher rate of reaction. It differs in each enzyme, depending on its
nature and structure. At extreme temperatures above the optimum, the
increased kinetic energy disrupts the bonds holding the active site;
the enzyme is unstable and the shape of its active site changes. This
means that the enzyme is less efficient and successful at
enzyme-substrate complex formation. The enzyme is said to be
denatured; it has lost its ability to catalyze reactions.

[IMAGE]

Effect of temperature on rate of reaction



Hypothesis: -
=============

As temperature of pectinase rises, the rate of reaction will rise till
40°C. As it rises above 40°C the rate of reaction falls.

The rate of reaction will increase as the temperature of pectinase
increases because kinetic energy of pectinase increases, thus it moves
faster and it is more likely to collide with apple fruit pulp. This
results in a greater frequency of collisions. This will result in the
rate of reaction increasing. Pectinase works best within the range
(18°C - 40°C). Rises in temperature above 40°C will cause the rate of
reaction to fall because pectinase will denature. The shape of the
active site of pectinase will have changed and will lack the ability
of degrading the pectin in apple fruit pulp to apple juice. The higher
the temperature above 40°C, the lower the rate of reaction.

Below is the predicted graph for the temperature of pectinase against
the rate of reaction:

[IMAGE]


Materials: -

Equipment used

Quantity

Volume in Cm3 (if applicable)

Diluted fungal pectinase 1%

1

50

Filter funnel

2

Filter paper

10

Test tubes

10

Test tube rack

2

Cut-off Syringe

Apple fruit pulp

1

5

Water bath

1

60

Pipette

1

Scalpel spoon

1

5

Glass rod for mixing

1

Stopwatch

1

Measuring cylinder

1

Permanent marker

2

5

Aluminum foil

1

Method: -

1. I will label each of the following temperatures on 2 test tubes:
25°C, 30°C, 35°C, 40°C, and 45°C.

2. In each test tube, I will pipette 3cm³ of pectinase and syringe
6cm³ of fruit pulp.

3. I will stir the mixtures in each test tube thoroughly with the
glass rod, then I shall place the test tubes in a test tube rack.

4. I will put the 2 test tubes labeled 25°C in the water bath at 25°C
for 15-20 minutes. Then I will cover the remaining test tubes with
aluminum foil until needed.

5. While waiting for the test tubes to reach the desired temperature,
I shall set up the 2 5cm³-measuring cylinders and place the filter
funnels on top with the filter paper folded in them. I will place the
stopwatch in front of it for time recording.

6. I will then filter the solutions of the 2 test tubes into the
5cm³-measuring cylinders, and start stopwatch immediately.

7. I will record the volume of the juice collected in each measuring
cylinder at 1-minute intervals for 10 minutes.

8. Finally, I will repeat steps 4-7 with the following temperatures:
30°C, 35°C, 40°C, and 45°C.

The constant factors in this experiment are the concentrations of the
enzyme & substrate, their volumes and the pH at which the reaction
takes place in. For maintaining pH I will not use pH buffer solutions
but I will use only one apple as it will have a specific pH and it
will be constant throughout the experiment. I will be using fungal
pectinase at 1% concentration, which will be prepared by mixing
49.5cm³ of distilled water to 0.5cm³ of the 100% concentrated fungal
pectinase. I have decided to use the temperatures 25°C, 30°C, 35°C,
40°C, and 45°C because in my preliminary work I found out that these
temperatures are adequate for showing how the rate of reaction is
affected by changes in temperature. I will use a permanent marker to
label the beakers and test tubes to avoid confusion. I will use a
water bath to maintain the temperature of each test tube throughout
the experiment, and a thermometer to check the temperature of the
water bath before placing any test tube in it. I will use 3cm³ of
pectinase and 6cm³ of apple fruit pulp in one test tube. This is
because my preliminary experiments showed that using 1cm³ of pectinase
with 5cm³ of fruit pulp would take too long to show an increase in
volume over 1-minute time intervals. Using 2cm³ of pectinase with 8cm³
of apple fruit pulp made up a 10cm³ solution or mixture, which took
too long to change temperature when placed in a water bath. Measuring
the volume of juice at 1-minute intervals is appropriate as in my
preliminary work I found out that measuring the volume at 5-minute
intervals would take too long, and at 30 second intervals the
measurements barely showed any variations in volumes. When mixing
pectinase with apple juice in the beakers, I will use a glass rod to
assure that they have been mixed together properly. For precise volume
measurement of pectinase (enzyme) I will use a 5cm³ pipette and for
the apple fruit pulp (substrate) I will use a cut-off syringe. I
intend to use scalpel spoons for aid in collection of fruit pulp
within a syringe. I will use a 5cm³-measuring cylinder because, as I
found out in my preliminary experiments, it gives the most precise
volume measurements at 1-minute intervals. I will use a stopwatch to
attain accurate time recordings for my experiment. Finally, for
accurate results I will carry out this experiment twice for each
temperature, and average the results afterwards.

Safety: -

The enzyme pectinase may cause sensitization when inhaling its fumes /
vapor, so to ensure that a very minimum amount is inhaled I will wear
a mask. Most of the materials I will use are of low risk but I will
wear safety glasses and wash my skin if anything spills on me. I will
take extreme precautions when using glassware and I will be very
careful not to break any of it. I will take caution not to put my hand
in the water bath, as it may be extremely hot. I will wipe any enzyme
spills and wash the area with water if a spillage occurs. The product
(fruit juice) is not suitable for consumption, as it will contain the
enzyme within it.



Analysis
========

From the results obtained it is clear that for each temperature, the
volume of juice rises after each minute. The amount of juice collected
after 10 minutes for each temperature must be greater than 3cm³
because the enzyme alone takes up 3cm³ of the mixture. This is because
enzymes, like chemical catalysts, are not used up in the reaction they
catalyze and can therefore be collected after product formation. Below
is a table showing the volume of juice extracted after 10 minutes for
each temperature i.e. the total volume minus the amount of enzyme
3cm³.

Temperature (°C)

Volume (cm³)

25

1.5

30

1.6

35

1.8

40

2.7

45

1.5

The volume of juice produced after 10 minutes rises as the temperature
rises, because an increase in temperature would lead to an increase in
kinetic energy of pectinase. This means that it is more efficient in
enzyme-substrate complex formation. Thus, the amount of juice produced
is directly proportional to temperature; the higher the temperature,
the higher the amount of juice produced.

From the graph of total volume against temperature we can see that the
highest volume of juice extracted was at 40°C. The volume produced at
25°C was 4.5cm³, and then it rises with temperature until it reaches
its maximum at 40°C. This happens because at low temperatures the
pectinase has less energy, so it will collide with the substrate
(fruit pulp) less often, meaning the frequency of collisions is low,
and the rate of reaction is low. At higher temperatures, the rate of
reaction is higher because the enzyme has higher kinetic energy so it
will collide more often with the substrate. After 40°C the enzyme
denatures because it becomes unstable and the shape of its active site
changes. Therefore, the rate of reaction falls; hence less juice is
produced.

After conducting the experiment I found the following anomalous
results:

* The total amount of juice produced after 10 minutes for the third
test at 30°C and the fourth and fifth tests at 35°C are lower than
3cm³, which -in theory- is not possible because the amount of
enzyme used is 3cm³, so the total amount of juice after 10 minutes
must be above 3cm³. This does not affect the analysis as the
readings were ignored when taking averages.

* On the graph of total volume vs. temperature, the total volume for
30°C was 0.6cm³ below the curve and the total volume for 35°C was
0.7cm³ below the curve. This was taken into account when I drew
the line of best fit by using error bars for these temperatures.



Evaluation
==========

The results obtained for each temperature show a very slight variation
e.g. at 30°C the total volume of juice was 1.6 whereas at 35°C it was
1.8; a 0.2cm³ difference. This may be due to the following: the mixing
time for each solution at preparation was different as -in the method-
it was not specified for how long the mixtures were to be stirred. The
method also states that the test tubes containing the mixtures are to
be placed in the water bath for 15-20 minutes. Some mixtures may have
been placed for 15 minutes and some for 20 minutes.

An apparent anomaly occurred for the results of the third test at 30°C
and the fourth and fifth tests at 35°C. The total amount of juice
produced after 10 minutes was lower than 3cm³, which -in theory- is
not possible because the amount of enzyme used is 3cm³, so the total
amount of juice after 10 minutes must be above 3cm³. This may have
occurred because the solutions weren't mixed long enough while being
prepared. This does not affect the analysis as the readings were
ignored when taking averages. The other anomalous result was on the
graph of total volume vs. temperature, where the total volume for 30°C
was 0.6cm³ below the curve and the total volume for 35°C was 0.7cm³
below the curve. This was taken into account when I drew the line of
best fit by using error bars for these temperatures.

For each temperature, I have a minimum of 3 results that aided in
achieving a better average. At 35°C I felt that there was a need for
repetition as the total volume of juice for the first three sets of
results was less than the total volume at 30°C. This contradicts the
theory about the effect of temperature on enzyme activity: An increase
in temperature below the optimum would result in an increase in the
kinetic energy of enzymes. This would lead to higher efficiency of
enzyme-substrate complex formation; therefore a higher rate of
reaction. Because the third test at 30°C was anomalous, I repeated the
experiment at that temperature twice as the more the experiment is
repeated, the more precise the average.

The results obtained in my experiment can support my hypothesis that
as the as temperature of pectinase rises, the rate of reaction will
rise till 40°C. As it rises above 40°C the rate of reaction falls. The
amount of juice extracted may have varied by an insignificant amount,
but as the temperature increased the amount of juice produced
increased till 40°C, after which it fell.

The only limitations I encountered while conducting my experiment were
that different apples were used to prepare the substrate. Although
they were of the same type, different apples tend to have different pH
values. Also, after removing the mixtures from the water bath they
were filtered and then the volume was taken at 60-second time
intervals, but the temperature may have slightly dropped 5 minutes
after removal. If I were to do the experiment again I would try even
more temperatures because this would enable me to find the optimum
temperature or range, and I would also obtain more results for each
temperature to achieve high precision in my averages. A different
substrate e.g. banana fruit pulp, and/or different concentrations of
pectinase could be used to see how this would affect the rate of
reaction and the amount of juice extracted.

How to Cite this Page

MLA Citation:
"The Effect of Temperature on the Activity of Pectinase." 123HelpMe.com. 17 Apr 2014
    <http://www.123HelpMe.com/view.asp?id=122363>.




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