on a microbial protease enzyme. I will observe how and if temperature affects how quickly the
enzyme works on removing the silver salts from photographic film. The protease enzyme breaks
down a layer of gelatine on the film that holds the silver salts to the actual film. I predict that
with the increase or decrease of temperature of the enzyme, its working time will increase or
decrease accordingly, showing a peak temp. and where the enzyme denatures and stops working.
In my experiment I will keep all variables constant such as the temperature of the
enzyme during the test. I will do this by using a water bath which keeps the water, and therefore
enzyme, at a constant temperature. Also, I will use the same size test tubes for each experiment
as this may alter results. And to make sure of no change in the enzyme I will use the same
volume of protease enzyme for each test (3cm). The final variable I will keep constant is the
method used for putting the photographic film into the enzyme. This will be done by the pieces of
film having wire attached to them and then dropped into the enzyme at the same time. To make
sure my results are as acurate as possible I will take 3 tests at a time and average these 3
results, ruling out any mistakes that might have been made. If i come across any anomolous
results I will take them out of my results and average the other two, stopping my graph from
looking irregular.
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The equipment I will be using is as follows: Water bath, Protease enzyme, Photographic
film, Test tubes, Thin wire, 2 Thermometers and a Syringe. (see diagram below)
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To do this investigtion I will set up the equipment as shown on the previous page and
continue as follows. First I will set the water bath to the desired temp. Then I will prepare my
three test tubes by adding 3cm of protease enzyme to each. Next I will place the the test tubes into
the water bath and use the thermometers to tes both the water and enzyme temp. When both of the
thermometer readings correspond I will place the three piece's of photographic film into the three
test tubes all at the same time so that mistakes in timing are not made. As the photographic
film goes into the enzyme another person will start the stopwatch, this is so that the times are
not shorter than they should be. Then I will keep checking the experiments to see if the enzyme
has finished working. When I can see that the enzyme has finished working the stopwatch will be
stopped and the times recorded.
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In the experiments I got a couple of anomolous results. I took these out and averaged the
other two. This gave me a more acurate reading. The Highest temp. I tried was 84C. After 13 mins
nothing had happend, so prove that the enzyme had denatured I lowered the enzyme temp. to 34C
and then put in the photographic film again to check if it just wasnt working. I waited longer
than 136secs ( the time the enzyme took previously at 34C) and saw that still nothing had
happened. From this we can tell that the enzyme had in fact denatured. The lowest temp. I tried
was 0C. After 13mins again nothing had happened so, again, to prove that the enzymes were not
vibrating enough to work I put it in a water bath at 34C. This time after 137secs the film had
cleared. This proved the fact that at 0C the enzyme does not work but was not denatured. The
enzyme was just not warm enough to vibrate enough to clear the film. The results were as I
expected, showing a peak temperature at around 60C and a point at which the enzyme
denatures, which I predicted. So in that respect my results support my prediction. One thing I
did find out though was there was a temp. at which the enzyme did not work but was not
denatured.
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Overall I am pleased with the outcome of the experiment, and my results support my
original prediction. My conclusion is that the protease enzyme vibrates in the test tube. Sat in
the tube by itself this is not visible but if somthing is put in which has a protein holding it the
enzyme will start to break down the protein. As can be seen by the results the enzyme must
vibrate more as it reaches its peak temperature. As it reaches its peak temp. therefore it takes less
time to break down the protein as it has more succesful collisions per second. As the temp. rises
above the peak the process slows down and eventually comes to a stop as the enzyme denatures.
When the protease enzyme is at a very low temperature, it does not vibrate as much or not at
all. This means either less or no sucessful collisions per second. Therefore the protein does not
get borken down but it does not mean the enzyme has denatured, as my results show. To improve
my investigation I could take lots more readings around the peak temp. area to really pin
down the actual peak at which the enzyme works. But as I had limited time this would be
impractical. I could also look at other aspects of the experiment such as, does the concentration
of the enzyme have any effect on the speed of the process. If I was to do more work on this, that
is the aspect I would look at.
