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A Comparison of the Effect of Temperature on Immobilized Lactase and Non-Immobilized Lactase

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A Comparison of the Effect of Temperature on Immobilized Lactase and Non-Immobilized Lactase

Aim:

To investigate the effect of temperature on the activity of
non-immobilized lactase and the effect of temperature on the activity
of immobilized lactase.

Introduction

Enzymes are substances that act as catalysts; they are biological
catalysts, which increase the rate of chemical reactions. Most enzymes
are large globular protein molecules with complex 3D shapes.

Enzymes have a specific site on their surface, known as the active
site, where the substrate, the substance which the enzyme combines
with, combines at. The shape of the active site is complementary to
the shape of the substrate molecule, to form an enzyme-substrate
complex. This process is called the ‘lock and key’ hypothesis, as the
enzymes are specific which means that each enzyme only combines with
one type of substrate.

[IMAGE]

It has now also been suggested that the active site is able to change
shape in order to combine with the substrate molecule. This process is
known as induced fit hypothesis.

Enzymes catalyze a reaction by lowering the activation energy.
Activation energy is the minimum amount of energy required for the
molecules to react together in a chemical reaction. By reducing the
activation energy, the barrier to a reaction occurring is lower,
therefore the reaction requires less energy to occur therefore it
takes place at a faster rate.

[IMAGE]

Effect of temperature on activity of enzymes

As enzymes are proteins, changes in temperature can cause changes in
the bonding and shape of an enzyme which can cause an affect on the
activity of the enzyme.

A rise in temperature increases activity of the enzyme, as the higher
the temperature the more kinetic energy the enzyme and the substrate
molecules have. Which means that more enzyme-substrate complexes are
likely to form. The increase in movement of the molecules enables a
higher chance of the substrate molecules colliding with the enzymes
molecules, with sufficient energy, to overcome activation energy, and
therefore combining at the active site.

However very high temperatures can also affect the stability of the
molecules, because if the temperature is increased too high the
molecules will gain too much kinetic energy, therefore the huge
increase in collisions could break or disrupt the chemical bonding of
both the substrate and enzyme molecules. This can cause the molecules
to change shape, and as enzymes are specific, a change in shape of the
active site, means that it is unable to combine with its complementary
substrate molecule. The enzyme is therefore inactive so has become
denatured and cannot be re-used.

Once an enzyme has become denatured, as it is not able to form
enzyme-substrate complexes, the rate of reaction would decrease as the
activation energy has not been lowered.

So the overall rate of activity of the enzyme increases as the
temperature increases, but only up to the optimum temperature, at
which the enzyme is most active, temperatures above that cause
denaturing, and the higher the temperature above the optimum, the
higher the extent to which the enzyme has become denatured.

[IMAGE]

Immobilized enzyme

Enzymes that are used in industrial processes are often immobilized
which improve their stability.

Isolated enzymes can be immobilized by attaching to an insoluble
support material.

This involves attaching the enzyme to an inert surface such as plastic
beads and then bringing the surface into contact with a solution of
the substrate.

Immobilised enzymes have some advantages over non-immobilised enzymes:

· As the enzyme in held in position by the support material, the
enzyme is more stable and more likely to withstand higher temperatures
or pH, as its ability to change shape is decreased.

· The immobilized enzymes can be re-used which means that the running
costs are reduced

· It may be possible for them to used continuously, because of the
fact that they are more stable and can re-used

· May have increased activity in some cases

Background information on Lactase

[IMAGE]The enzyme I am using in my investigation is lactase; its
substrate is lactose, contained in milk. Lactase hydrolyses lactose
into glucose and galactose.

Lactase has an optimum temperature of about 48 °C for its activity and
an optimum pH of 6.5.

I have chosen to use lactase in my investigation because immobilised
lactase is used in industry.

Alginate solution is the insoluble material the lactase molecules will
be held in, in my investigation.

In humans lactose is normally hydrolysed into glucose and galactose,
but some people do not produce lactase, so cannot digest lactose, this
condition is known as being lactose intolerant. Therefore lactose-free
milk is manufactured by passing milk through a column of immobilised
lactase enzyme.

Hypothesis

I am investigating how changes in temperature will affect the activity
of non-immobilized lactase compared with immobilized lactase, when the
concentration and PH of enzymes and substrate are constant. This is
important because an increase in concentration of the lactase would
increase the rate of reaction, as there are more active sites
available to combine with the substrate. An increase in concentration
of the substrate would also increase the rate of reaction, as more
substrate complexes are able to form at a quicker rate.

A change in pH also affects the rate of reaction because a too high pH
can disrupt the chemical bonding within the molecules, therefore
changing the shape of the active site and substrate.

I predict that the higher the temperature, the higher the activity for
both types of enzymes, therefore more glucose would be produced. This
is because increasing the temperature causes the molecules to gain
more kinetic energy, therefore are more likely to have more successful
collisions, so more substrate-complexes are able to form. So the rate
of reaction increases, producing more glucose.

Non-immobilized Lactase has an optimum temperature of 48 °C, which
means at this temperature the rate of reaction is fastest, so the most
amount of glucose would be produced. At temperatures above 48°C, I
would expect the amount of glucose produced to decrease as temperature
increases, as the enzyme would begin to become denatured. Because the
temperatures would be too high, causing the chemical bonds, including;
hydrogen, ionic bonding and disulphide bridges, to break within the
lactase molecules, resulting in the active sites to change shape,
therefore substrate-enzyme molecules are less likely to form. So the
rate of reaction would decrease producing less glucose.

Whereas I predict that immobilized lactase will begin to denature at a
higher temperature, as it is more stable, held in the porous material,
it takes more energy to break the chemical bonds within the lactase
molecule. Therefore the optimum temperature, would higher than the
optimum for non-immobilized lactase. Therefore I would predict the
graph results to look similar to this:

[IMAGE]

The blue dotted line represents the predicted activity of
non-immobilized lactase, and the red, immobilized lactase. As you can
see, the immobilized enzyme is active at temperatures above, the
optimum temperature for non-immobilized enzyme.

Preliminary experiment

For my trial experiment, I am going to measure the amount of glucose
produced, when immobilized/non-immobilized lactase is added to its
substrate, milk. I will do this for two temperatures, one high
temperature, 60 °C and a low temperature, 20 °C. This will give me a
rough idea of whether the basis of my hypothesis is correct.

Methods

Firstly I would test the affect of temperature on immobilized lactase:

Materials:

2cm3 lactase (Lactozym)

8cm3 of 2% sodium alginate solution

100cm3 2% calcium chloride

Semi-quantitative glucose strips (Diabur 5000)(max 3%)

5cm3 plastic syringes (precision +/- 0.05cm3 )

100cm3 beaker

Test tubes and rack

Glass rod

Plastic tea strainer

Distilled water

Pasteurized milk (semi-skimmed)

Stop watch (+/- 0.05sec)

Water baths at 20°C and 60°C (thermostatically controlled)(precision
+/- 0.5°C)

Thermometer (precision +/- 0.5°C)

Method 1:

· Mix the sodium alginate solution with the enzyme solution in a
beaker, and transfer into a syringe.

· Add this mixture this mixture drop-wise into the calcium chloride
solution. Alginate beads will form in the beaker, leave for 15 to 20
mins for the beads to harden.

· Strain off the beads using the tea strainer and rinse with distilled
water.

· Transfer the beads into a test tube

· Place the test tube containing the beads and a test tube containing
2cm3 of milk into a water bath of 20°C and wait until they have
reached the temperature of the water bath.

· Then pour the milk in the test tube into the test tube with the
beads and start the stopwatch immediately.

· After 5mins test the solution with a glucose strip and record the
amount of glucose present.

Repeat this for 60°C

Note: the alginate beads re-used after been rinsed with distilled
water

Note: thermometer is placed in a beaker containing the test tubes,
throughout the whole experiment, and the temperature is recorded
before and after each experiment.

Now I will test the effect of temperature on non-immobilized lactase:

Materials:

2cm lactase (Lactozym)

Semi-quantities glucose strips (Diabur 5000)(max.5%)

Test tubes and rack

Pasteurized milk (semi-skimmed)

5cm3 plastic syringes (precision +/- 0.5cm3 )

Distilled water

Stop watch (precision +/- 0.05sec)

Water baths set at 20°C and 60°C (thermostatically
controlled)(precision +/- 0.5°C)

Thermometer (precision +/- 0.5°C)

Method 2:

· Put both the test tube with the lactase and test tube with 2cm3 milk
into the 20°C water bath until they have reached the temperature of
the water bath

· Then mix them and start the stop watch immediately

· After 5mins test for glucose and record the amount glucose present.
Repeat for 60°C

Temperature (°C)

Concentration of glucose produced for non-immobilized lactase (%)

Concentration of glucose produced for immobilized lactase (%)

20.0

0.25

0.25

60.0

0.00

2.00

Results:

The results support my hypothesis, as no glucose is produced for
non-immobilised lactase, because 60°C is above the optimum temperature
so the enzyme has become denatured. Whereas there is some glucose
produced for immobilized lactase at 60°C, this also supports my
hypothesis, that immobilised enzymes can withstand higher temperatures
as the enzyme is more stable.

However this information is not enough to form a conclusion, as we can
not tell from these results how high the temperature can increase
until the immobilized lactase is denatured, nor can we tell at what
temperature the non-immobilized enzyme started to become denatured. So
therefore I have not fully proves my hypothesis to be correct.

Modifications to the preliminary

· Carry out the experiment for a larger range of different
temperatures

· Repeat each experiment twice, so the results retrieved and more
reliable

· Carry out a control experiment for each temperature, which shows
that the substrate, milk alone, when mixed with distilled water not
lactase, cannot produce any glucose at any temperature.

· Place a thermometer in a beaker containing the test tubes, in the
water bath, throughout the whole experiment, and record temperature is
recorded before and after each experiment, in order to make sure the
temperature for each experiment is constant.


Independent variables
---------------------

· The variable in this investigation is the temperature, which will be
tested across the range , 20 °C, 35 °C, 50°C, 65°C, 80°C for each
temperature the temperature will stay constant as the water baths I am
using are thermostatically controlled and precise to 0.5°C. the
temperature will be recorded before and after each experiment.

· Each mixture will be kept in the water baths for 5mins once they
have reached the temperature of the water bath, therefore time is kept
constant

· The volume of enzyme, both immobilized and non-immobilized will be
2cm3 and the volume of semi-skimmed milk (substrate) will be also 2cm3

· The concentration of the substrate and enzyme will also have to be
constant, as a change in concentration affects the rate of reaction;
the milk that is used will always be semi-skimmed and the
concentration of the lactase is _ where 2cm3 is always used.

· The pH of the enzyme and substrate must also be kept constant. The
form of lactase I am using is Lactozym, which already contains a
buffer solution of pH6.5and the ph of the semi-skimmed milk is_. As
glucose is the product, which is not acidic so the pH will not.

· I am going to do the experiments for the same temperature at the
same time, so the comparisons between the results are accurate. E.g.
for 20 °C, I will carry out the experiments for both types of enzymes
at the same time, so the temperature is exactly the same. I would
repeat this twice for this temperature.


Actual experiments
------------------

I am going to repeat both methods 1 and 2 for all 5 temperatures
mentioned above. Then repeat them until I have at least two results
the same.

I will also set up a control experiment for each temperature

Control method:

· Stand a test tube with 2cm3 distilled water and a test tube with 2cm3
lactase in the water bath

· When they have reached the temperature of the water bath, mix them
and time for 5mins

· After the 5mins are up test for glucose



Risk assessment
===============

· Chemical hazards:

Calcium chloride solution 100cm3 - irritating to eyes, also irritating
the skin and respiratory system. Dangerous wit water: can cause water
to boil

Enzyme (lactozym) 2cm3 - All enzymes may produce allergic reactions.
Concentrated solutions can cause asthma and irritate eyes, nose and
skin

Sodium alginate 8cm3 2%

Precautions

Wear eye glasses at all times during the investigation. Wash hands
before and after handling the chemicals. If chemicals came in contact
with eyes flood with running water for 10 mins and seek medical help.
If come in contact with skim wash immediately wit water.

· Experimental hazards

Glass ware – be careful when handling

Water baths – when kept at high temperatures do not let hot water in
contact with skin

Electrical – do not allow electrical appliances in contact with water
and chemicals

· Biological hazards

There are no biological or ethical implications



Results for actual experiments:

Non-Immobilised lactase:

Amount of enzyme and substrate (cm3)

Temp. of water bath

(°C)

Conc. of glucose produced (%)

Expt. 1

Expt. 2

Expt. 3

Expt 4

2cm3 lactase

2cm3 milk

20.0°C

0.10

0.00

0.25

0.10

¾

35.0C

0.25

0.25

1.00

¾

50.0°C

1.00

2.00

2.00

¾

65.0°C

1.00

0.25

0.10

0.25

¾

80.0°C

0.00

0.10

0.00

Immobilized lactase

Amount of enzyme and substrate (cm3)

Temp. of water bath

(°C)


Conc. of glucose produced (%)

Expt. 1

Expt. 2

Expt. 3

2cm3 lactase

2cm3 milk

20.0°C

0.00

0.00

0.00

¾

35.0C

0.25

0.25

0.25

¾

50.0°C

2.00

2.00

2.00

¾

65.0°C

2.00

3.00

3.00

¾

80.0°C

1.00

2.00

1.00

Plotting conc. of glucose produced against temperature can produce
line graphs of the above results. I have plotted graphs only using the
values for conc., of glucose produced that have been recorded twice at
least.

Analysis

The graph shows the relationship between the conc. of glucose produced
and the temperature at which the enzyme and substrate were kept, for
both immobilized and non-immobilized lactase.

The graphs show that as the temperature increases from 20°C to 50°C,
both immobilized and non-immobilized lactase activity increases, this
is shown by the increase in conc. of glucose produced. For both
immobilized and non-immobilized lactase, as the temp increases form 20°C
to 50°C the conc. of glucose produced increases from 0.00% to 2.00%,
which is an increase of 2%.

Therefore this is evidence to show that the rate of reaction when
using immobilized lactase and non-immobilized lactase, increase at the
same rate, this is shown by slopes of both graphs from 20°C to 50°C.
The gradient of both slopes between 20°C and 35°C is 0.02, and the
gradient for the slope between 35°C and 50°C is 0.12. This is not
entirely what I expected, as I expected the slope of the to 35°C to be
less steeper when immobilised lactase is used, as it may take slightly
longer for the heat energy to penetrate through the alginate beads.
Whereas non-immobilised lactase is in immediate contact with the
substrate so therefore may be affected by temperature more quickly
than immobilised lactase.

However the fact that the rate of reaction when immobilized lactase is
used, increases at the same rate to the rate of reaction when
non-immobilized lactase is used could prove that immobilization does
not affect the activity of the enzyme

The increase in conc. of glucose produced is because as the
temperature increases, the lactase and lactose molecules are able to
gain more kinetic energy therefore have more successful collisions.
More collisions mean that it is more likely that more lactose
molecules are able to combine at the active site of the lactase
molecules, forming more enzyme-substrate complexes. The rate of
reaction and therefore the conc. of glucose produced increases,
supporting my hypothesis.

However as the temperature increases above 50°C the concentration of
glucose produced decreases when the non-immobilized enzyme is used.
When the temp is 65°C, the conc. of glucose is 0.25% this is a
dramatic decrease of 1.75% form the conc. of glucose at 50°C. Whereas
when immobilized lactase is used, as the temperature increases form 50°C
to 65°C, the conc. of glucose increases by 1%.

The decrease in conc. of glucose produced when using non-immobilized
lactase is due to the fact that as the temperature increases above 50°C,
the temp is too high for the enzyme to work as the molecules have
gained far too much kinetic energy. Therefore there is an increase in
high-energy collisions, which may be high enough to break hydrogen an
ionic bonding, which make up the tertiary structure of the enzyme.
Thus causing the shape of the active sites to change, and so are
unable to form enzyme –substrate complexes. As the number of
enzyme-complexes formed decrease, the rate of reaction also increases,
and less glucose is produced. The lactase is becoming denatured, this
is proves my hypothesis

At 65°C the conc. of glucose is still increasing when the immobilized
lactase is used, indicating

that the enzyme is still active at high temperatures. The lactase is
supported by an insoluble material the lactase is more stable, this is
why the activity is unaffected by a higher temperature, which also
supports my hypothesis.

At 80°C, the activity of non-immobilized enzyme is zero as no glucose
is produced, which means that the enzyme is now completely denatured.
All the lactase molecules have been distorted so no enzyme-substrate
complexes are able to form.

At 80°C, the immobilized lactase is still active, but the activity has
decreased, as the conc., of glucose decreases form 3% to 1%, as the
temp decreases form 65°C to 80°C. Even when immobilized lactase is
used, extremely high temperatures are still able to break the chemical
bonds within the lactase enzyme. At 80°C, the heat energy is so high
that it can penetrate more strongly through to the enzyme, and
therefore are able to break the chemical bonds in enzyme, so are is
unable to work.

Overall it is clear that my results support my hypotheses. At 50°C the
non-immobilized lactase is most active, therefore 50°Caccording to my
results is the optimum temperature for non-immobilized lactase, which
is very close to the actual optimum temperature, I found out through
research, which is 48°C.

The optimum temperature for immobilized lactase, according to my
results is 65 °C, which does support my hypothesis that immobilized
lactase is still active at higher temperatures than for
non-immobilized lactase.

Evaluation

Accuracy of equipment used:

Equipment

Precision

Max/ min Percentage error

Glucose test strips

5 conc: 0%, 0.2%. 0.25%, 1%, 2%, 3%

Plastic syringes (5cm3)

+/- 0.05cm3

0.05/2 x 100 = 2.5% (max)

0.05/8 x 100 = 0.6% (min)

Stop watch (mins/secs)

+/- 0.05sec

0.05/300 x 100 = 0.02%

Water baths (°C)

+/- 0.5°C

0.5/20 = 2.5%

0.5/80 = 0.6%

Thermometer (°C)

+/- 0.5°C

0.5/20 = 2.5%

0.5/80 = 0.6%

Overall my results support my hypothesis. My results are reliable as
possible, as I have shown above that the % errors of the equipment
used are very low, except for the glucose test strips, which I will
discuss later. Also I repeated each experiment until I obtained the
same result twice. I plotted graphs using the values that have been
recorded at least twice. The shape of the graph shows the effect of
temperature on immobilized lactase compared with non-immobilized
lactase.

I expected the slope of immobilized lactase up to 35°C to be slightly
less steep than the slope of non-immobilized lactase. I predicted that
the effect of temperature on immobilized lactase would be delayed, as
it is more stable. Therefore not only would the immobilized lactase
have a higher optimum temperature than non-immobilized lactase, but it
would also start to work at a slightly higher temperature, therefore
would begin to speed up the reaction at a slightly slower rate. This
is not clear on my graph because the glucose test strips I used only
detect 5 concentrations 0.0%, 0.1%, 0.25%, 1.0%, 2.0%, 3.0%. They do
not provide a very accurate result especially at high glucose
concentrations. As it is not possible to determine the exact
concentration produced at any point. For example at 20°C, the exact
conc. of glucose produced using the immobilized lactase, can be
anywhere between 0.00% and 0.25%. Therefore it is only possible to
determine the general trend of the affect of temperature on the
activity of both enzymes from these results A more reliable way of
measuring the conc. of glucose produced would be to use a glucose
strip of concentrations of higher precision or perhaps an electronic
devise to measure the glucose concentration such as a colorimeter.

Another limitation would be the fact that this experiment was carried
out for only 5 different temperatures, the exact trend of activity is
not predictable, for example, we cannot determine the exact optimum
temperatures or temperatures at which the enzyme is complete
denatured. Also there are not enough results to highlight any
anomalies. The interval between each temperature investigated is 15°C.
A more accurate way of determining trends in activity of the enzymes
as the temperature increases is by carrying out experiments at more
temperatures with smaller intervals, e.g., 30°C, 35°C, 40°C, 45°C,
50°C, 55°C, 60°C, 65°C, 70°C, 75°C, this would produce a graph, more
similar to a curve.

Other limitations could have occurred during carrying out the
experiments. For example when heating the immobilized enzyme in a
water bath, it not possible to make sure the beads had reached the
right temperature, as it is possible that the beads took longer to
reach a certain temperature than the substrate. Perhaps a more
accurate equipment of measuring temperature, which can measure
temperatures in solids, would produce more accurate results. Also the
size of the beads may make a difference of the affect of the enzyme,
as smaller beads would work more affectively as they have a larger
surface area, for more reliable results, the size of the beads would
have to be kept constant.

A more accurate way of carrying out this investigation would be to use
benedicts reagent in order to test for glucose and use a colorimeter,
which would accurately measure the intensity of the colour, therefore
conc. of glucose produced.

To extend this investigation further, I could investigate whether the
time the enzyme is subjected to a certain temperature affects the rate
of denaturation, rather than just the change in temperature. So
therefore I would not just vary the temperature I would vary the time
the enzyme is subjected to those temperatures.

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"A Comparison of the Effect of Temperature on Immobilized Lactase and Non-Immobilized Lactase." 123HelpMe.com. 16 Apr 2014
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