An Investigation into factors affecting the rate of breakdown of sucrose by the enzyme sucrase

        Therefore, I predict that the rate of breakdown will increase with temperature, up to about 50°C (Jones), when it becomes denatured the rate of breakdown will gradually slow down until the enzyme no longer works at all.

This is a graph of what I predict will happen:

In my experiment, I have to make sure that I keep the following 3 variables constant:

1. Enzyme concentration
2. Substrate concentration
3. pH

I must keep these variables constant because I know that they affect the rate of breakdown. However, it should be easy to control these factors, because I just need to keep the concentration of the enzymes and substrate the same by just using the same concentration of solution, and I can keep the pH the same by the use of a buffer.        

I am planning on performing the experiment 3 times, and then taking an average of my results. I plan on doing this in order to make the experiment more accurate and make up for human errors.

Apparatus: Water bath, test tubes, test tube rack, stopclock,                  

     Clinistix, 10% sucrose solution, 5% sucrase solution,      

           Thermometer, buffer.

Method: This is the experiment I intend to carry out in order to obtain my results:

1. Take 18 test tubes. Fill 9 test tubes with a solution containing sucrose concentration, and fill the other 9 test tubes with some sucrase, and place them in a test tube rack.
2. Put 2 test tubes in the water bath, 1 containing sucrose, 1 containing sucrase, of volume 10cm³, having set the water bath to a temperature of 20 degrees Celsius.
3. Wait for the solutions to acclimatise.
4. Once the solutions have acclimatised, mix the 2 test tubes together, so that you have 1 test tube containing 10% sucrose solution, 5% sucrase solution, and immediately switch on the stopclock.
5. Place a Clinistix strip, a buffer, and a thermometer in the test tube.
6. Study the colour of the Clinistix. As soon as its indicator patch turns from pink to blue, stop the clock, and record the time in seconds. This will happen because Clinistix indicates the presence of glucose, so once the sucrase has broken down the sucrose, glucose should be present and the Clinistix will indicate this. This will give us a time for the reaction.
7. Repeat the same experiment with the 16 remaining test tubes, setting the water baths to the following 8 temperatures for the 8 pairs of test tubes remaining:

30, 35, 37, 40, 43, 47, 55, 65.

I have chosen these temperatures concentrated around 40° because I expect this to be about the optimum temperature so this will enable me to accurately decide on the optimum temperature.

8. Perform this whole experiment 3 times, and take an average of all      

of your results.

This experiment will give a set of results showing the different times it takes for the breakdown of sucrose at different temperatures. This should be an indication of the differing rates of breakdown of sucrose by the enzyme sucrase. These results could then be presented as a graph, showing the rate of breakdown.

Obtaining Evidence

In my plan, I stated that I would record the time taken for the reaction with the following temperatures (ºC):

30, 35, 37, 40, 43, 47, 55, 65.

I also said that I would use a buffer solution in the experiment to keep the pH constant. However, I was not able to do these two things.                                                                    

        For a start, I was not able to use the temperatures I planned to use, because the water baths were set to values of every 10ºC, so it was difficult to change the temperature of the water baths to values of temperature such as 37 and 43ºC. Therefore, I modified my plan and recorded the time taken for the reaction between sucrose and sucrase, using the following temperatures (ºC):

10, 20, 30, 35, 40, 45, 50, 60.                                                                      

I also said that I would repeat the experiment 3 times, but I only repeated it twice because I did not have enough time.

        

I was not able to use a buffer solution because there was not one available for my use. Therefore I was not able to use any, as I said I would in my plan, and I had no way of ensuring that the pH was always constant.

Here is a table showing my results:

When I refer to time on my results table, I mean the time the sucrase took to break down the sucrose.

Rate of reaction is calculated by 1 divided by the time taken for the reaction. I have multiplied these values that I got for rate of reaction, by 1000, so that the figures I get are more workable.

Analysis

I have decided that the best way of presenting my results is by constructing a line graph, with a line of best fit. The line graph showing my results is on a separate sheet of paper.

My graph shows that the rate of reaction increases if you increase the temperature, until you get to 40ºC, when the sucrose breaks down the sucrase best. Then, the rate of reaction decreases as you raise the temperature.

I think that my work shows that the rate of breakdown of sucrose by the enzyme sucrase increases quite rapidly until 40ºC, the optimum temperature. This is the temperature that the enzyme works best at. After the optimum temperature has been reached, the enzyme reacts less well and the rate of breakdown decreases with increasing temperature. The enzyme denatures, and this happens at around 60ºC.

I predicted in my planning stage that the rate of breakdown will increase with temperature, up to about 50°C (Jones), when it becomes denatured and the rate of breakdown gradually slows down until the enzyme no longer works at all. I also mentioned that before the optimum temperature is reached, every 10ºC rise in temperature, doubles the rate of reaction. This is the Q10 theory. I came to my prediction having referred to the collision theory:

“If the temperature at which a reaction is taking place is increased, then the rate of reaction will also increase. This is because as the temperature is raised, the kinetic energy of the particles increases. This means that the particles will move around faster and will therefore collide more often. Therefore the number of collisions taking place per second between particles with the necessary collision energy will increase so the rate of reaction will increase.” (M Reynish)

However, I think my prediction was slightly wrong. I predicted the basic outlines of the outcome correctly: I said that the rate of reaction would increase up to a certain point, and that then the rate would decrease as the enzyme gets denatured, which is right. However, I said that the optimum temperature of the enzyme is 50ºC, but my results show that it is 40ºC. And I predicted that the Q10 theory would apply, however, it does not. If you study the graph, you can see that every 10ºC rise in temperature does NOT double the rate of reaction. This is almost the case in a few cases, but it is not exactly right all the time and sometimes it does not work at all.

 

Q10 calculation: The following calculations show how the Q10 theory does not work for my results.

1) 10ºC to 20ºC = 10ºC rise in temperature = double rate of reaction?

   10ºC= 4.9 (rate of reaction)

   20ºC= 10.6 (rate of reaction)

   4.9 x 2= 9.8

   9.8 is not equal to 10.6, therefore Q10 does not work.

2) 20ºC= 10.6

   30ºC= 15.4

   10.6 x 2= 21.2

   21.2 is not equal to 15.4, therefore Q10 does not work.

Evaluation

        I think that the experiment went quite well. The aim of the experiment was to investigate the effect of temperature on the rate of reaction. I have found that the rate of reaction increases until it reaches the optimum temperature, 40ºC, and after that the rate of reaction decreases as the enzyme is denatured. Although this is not exactly what I predicted, the optimum temperature is usually said to be 40ºC in most biology text books. However, I feel that a few of my readings were very strange and my graph shows that the Q10 theory does not work for my results. Therefore there are quite simply flaws in the experimental method and the accuracy of it.

        To start with, I do no think the experiment was very accurate. I think the only reason for this is that I was not able to set the water baths to precise temperatures. The temperature in the water baths were difficult to keep constant, and I feel that combined with human error, the temperatures that the reaction took place at were quite often not the same temperatures that I thought the reaction took place at. So in this way, the experiment was not accurate enough. If I could do the experiment again, I would definitely try and use some water baths which can have more accurate and precise temperatures set to them.

        

I definitely think that the experiment was not reliable. If you look at the results table, you can clearly see that there is a large difference between most of the results, between the 2 times that I performed the experiment. I think a lot of this may be due to human error, but I think one has to criticise the experiment for this unreliability.

        

I also think that the experiment was not suitable either. There are many reasons for this. For a start, when noting when the reaction was complete, I looked at the colour of the Clinistix. If it was blue, the reaction was complete. However, this was not a very good method because the change in colour of the clinistix, from red to blue, is a gradual process, so it was difficult to tell to what degree of blueness I should note the time of the reaction at. Therefore, at different temperatures, I may have accidentally assumed the reaction over, when the Clinistix had got to a different degree of blueness, so my results would not be very reliable. If I could do the experiment again, I would try and use an indicator which is more specific, so that there is no doubt when the reaction is fully finished.

Another problem was that there was no way of ensuring that the pH during the experiment was constant. This may have made the experiment even more inaccurate, because the pH was different at different times at different times. If I could do the experiment again, I would use a buffer solution to keep the pH constant.

        Overall, I think the results of the experiment are quite good, considering the number of flaws there were in the method. However, I think it would be very interesting to do the experiment again, adapting it in the ways that I have mentioned, because I feel that I may manage to get results that agree with the Q10 theory, and which are more reliable.