Investigation of the Effect of Temperature on the Rate of a Lipase Reaction


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Investigation of the Effect of Temperature on the Rate of a Lipase Reaction

I am going to look at the effect of temperature on the rate of a
Lipase Reaction, and to look at this I will be changing the
temperature variable. In order to measure this, I will be doing the
following practical:

1) Add 5cm³ of milk and 1cm³ of Lipase solution at different
temperatures.

2) Then I will measure the reaction every 30 seconds, from 30 seconds
to 2 minutes with a pH probe (as lipase breaks down into fatty acids)
to test the strength of the acid produced. This will show how well the
enzyme has worked and a pattern should emerge in my results relating
the temperature to the strength of the acid. The stronger the acid,
the better the enzyme has worked. This will be shown in my results as
the more under a pH of 7 the final pH is, the better the enzyme has
worked during the reaction at a certain temperature. For example, a
final pH of 6.1 and a final pH of 7.3 shows that the first pH is
stronger and therefore more acid has been produced as the lipase have
broken down into fatty acids.

I will be using a range of temperatures at which to add the milk and
lipase. This should ensure that I get a fair result as I am using a
range of high and low temperatures from which to compare. I will get
readings at 20, 30, 40, 50 and 60°C from which a trend should show. I
shall repeat the experiment 3 times, and then take average results to
ensure the most reliable results possible.

Temperature (°C)

Lipase Amount (cm³)

Milk Amount (cm³)

20

1

5

30

1

5

40

1

5

50

1

5

60

1

5

I will use a pH probe to test the pH of the acid produced.

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This should
reduce the chance of human error as it is computerised and therefore
more accurate.

To ensure a fair test, all variables (the amount of lipase and milk
used every time) will stay the same, except for the temperature at
which the reaction takes place at, which I will be controlling. I will
ensure all of these factors are the same as it would be unfair to
change the dependant variables and would give me inaccurate and
unreliable evidence on which to base a conclusion. I will also ensure
that the milk is always the same pH before use and I will do this by
using the same milk every time, ensuring it has been stored and
treated the same.

To change the temperature variable, I will be using a water-bath and
will start at the lowest temperature value (20°C). I have chosen
values between 20 and 60°C as this will clearly show me a range of
results and therefore make my results more accurate. All measurements
collected will be as accurate and reliable as possible because I will
strive to conduct a fair test, keeping all variables except the
temperature the same. I will also check the temperature of my water
bath constantly with a thermometer to make sure it is the correct
temperature.

To do my experiment I will need the following equipment:

* pH probe

* 5cm³ lipase

* 25cm³ milk

* Teat pipette

* Test tube rack

* Test tubes

* Water bath

I have chosen this equipment because I feel it is the most suitable
for the experiment and that combined with careful variable control,
they will produce the best results.

I predict that the lipase will have the optimum reaction rate at
around 37°C, as this is human body temperature and therefore the
reaction would usually take place at around this temperature. I also
predict that at around 45°C, the reaction will slow down as the heat
will begin to denature the enzyme.

As acid is being produced, there are general safety considerations to
consider and goggles will be worn at all times when acid is present.
Also, basic classroom rules will be followed, such as no running, to
again ensure the complete safety of all present.

My reason for introducing steps to produce reliable evidence is
simple: to be able to write an accurate and concise conclusion, all
evident provided has to be reliable and accurate. Human error is
unavoidable, but steps can be taken to reduce this as much as
possible. I have chosen a pH probe as this is simply more accurate
than humans and can do a more precise reading as it is computerised. I
have also chosen now to use a temperature probe when checking the
temperature of my water bath, as this is computerised and therefore
would be expected to be more reliable than using a thermometer. This
would also be quicker, as a thermometer would have to adapt from the
rooms temperature before being able to reliably use it, whereas a
probe would be immediately able to take an accurate temperature
reading. Scientifically, I have based my prediction on my previous
knowledge of digestive enzymes, which states that all enzymes have an
optimum temperature at which to work. Too low, or too high, enzymes
will not work or become denatured. This is also why I chose a range of
temperatures. As human body temperature is around 37°C, I would expect
that the lipase would work better around this, although as it is
produced in the pancreas the temperature may vary.

My Results:

Results from my first attempt:

Temperature (°C)

Milk pH

pH after 30 Seconds

pH after 1 Minute

pH after 2 Minutes

20

7.0

7.17

7.12

7.06

30

7.0

7.06

7.00

6.81

40

7.0

7.03

6.91

6.59

50

7.0

6.86

6.75

6.68

60

7.0

6.75

6.67

6.51

Results from my second attempt:

Temperature (°C)

Milk pH

pH after 30 Seconds

pH after 1 Minute

pH after 2 Minutes

20

7.0

7.44

7.43

7.40

30

7.0

7.34

7.32

7.29

40

7.0

7.19

7.15

7.05

50

7.0

7.11

7.05

6.90

60

7.0

6.93

6.83

6.73

Results from my third attempt:

Temperature (°C)

Milk pH

pH after 30 Seconds

pH after 1 Minute

pH after 2 Minutes

20

7.0

7.30

7.21

7.14

30

7.0

7.15

7.05

6.95

40

7.0

7.02

6.97

6.88

50

7.0

6.98

6.86

6.76

60

7.0

6.75

6.66

6.54

I will now collect averages of all my results and then will plot these
results onto a graph. A trend should show. I will round my averages up
so that they will be easier to read and more clear when plotted.

Table of Averages

20°C

30°C

40°C

50°C

60°C

Average pH after 30 Seconds

7.33

7.18

7.08

6.98

6.81

Average pH after 1 Minute

7.25

7.12

7.03

6.89

6.72

Average pH after 2 Minutes

7.20

7.0

6.84

6.78

6.59

[IMAGE]Graph to show how the pH of a Lipase & Milk reaction is
affected by temperature using how acidic the pH is as a guide

My graph shows me that Lipase worked better at 60°C. After only 30
seconds it had reduced the pH to just above 6, much more quickly than
at the other temperatures. It had a final pH of just below 6.6,
showing it to be the most acidic of all of the reactions. The lowest
acidic reading was when I used a water bath of 20°C and therefore it
would be safe to assume that lipase does not work well at lower
temperatures.

This contradicts the prediction I earlier made. I expected that the
optimum temperature would be around 37°C, andtherefore expected the
best results when I was using a water bath of 30°C and 40°C. This was
clearly not the case and to have made a prediction more scientifically
supported, I could have done research into the Pancreas, finding
information about temperature and conditions. I would then have used
this information in order to predict the results. If I were to do this
experiment again, this is what I would do as I would have had more
idea scientifically about what to expect.

I found I had no anomalous results and I believe this to be because I
followed a strict procedure and was thoroughly conscientious at all
times. I controlled variables accurately and checked and double
checked readings. I used the same stopwatch throughout when timing and
used all the same equipment, such as pH probes and temperature probes.
I constantly monitored the temperature of my water baths, ensuring
that they were always the exact and same temperature throughout each
experiment.

To provide an even firmer conclusion, if I were to perform this
experiment again I would do a number of things. I would firstly use
more temperatures, for example not just 20, 30, 40, 50 & 60°C but 20,
25, 50, 35, 40, 45, 50, 55, 60°C. This would provide more information
as more results are being obtained on which to base a conclusion. I
would also research the conditions of the pancreas more thoroughly so
that I could predict scientifically what would happen using better
knowledge.


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