Investigation into how temperature affects the enzyme: Amylase.

The temperature will be the manipulated independent variable as this is what I will be changing. The dependant variable will be the iodine solution turning brown indicating when the amylase has successfully broken down the starch, this being the rate of reaction.

Preliminary Work

Before I did my investigation, I did some preliminary work. I investigated how PH affected the rate at which starch and the enzyme amylase reacted.

I used a water bath, some test tubes, amylase mixed with water in the ratio 1:4

(1cm cubed of saliva mixed with 4cm³ of water), 3 lots of 5cm³ starch solution, each at different PHs: PH 3, PH 7 & PH 13 & some Iodine. I predicted that the enzymes would work best at PH 7 because PH 7 is the PH of the enzyme amylase. I started with PH 3 & I took a sample every 15 seconds. The Iodine mixed with the starch & saliva did not turn from black to brown until the 21st sample, which took 315 seconds. This result was what I expected because amylase are used to working at

PH 7. This happened again with the PH 13 solution. The Iodine did not turn from black to brown until the 26th sample, which took 390 seconds (table of results at the end). But as expected, the PH 7 solution got to work right away and the Iodine turned from black to brown on the 6th sample, which took 90 seconds.

PH 3

PH 13

PH 7

From my results I noticed some errors in my working which affected my results. These errors will help me with my investigation, as I will know not to make the same mistakes.

I will set up and perform my investigation in the same way as my preliminary work e.g. same equipment (excluding waterbaths).

As predicted, my preliminary work showed that PH 7 reacted the quickest with the amylase.

Equipment List

• Test tubes
• 10cm³ measuring cylinder
• Dimple Tray
• Pipettes (plastic & glass for health reasons)
• Test tube rack
• Timer
• Iodine Solution

Method

I will start by measuring out 1cm³ of amylase & mixing it with 4cm³ of filtered water in the 10cm³ measuring cylinder, I will do this 5 times. Next I will measure out, in a separate measuring cylinder, 5 lots of 5cm³ of starch solution & place both the amylase and the starch solutions in separate water baths set at 20ºC, 30ºC, 40ºC & 60ºC and also in some ice (0ºC). These temperatures are sensible to use because they are in between inactivation temperature (0ºC) and denaturing temperature (60ºC). Next, using a pipette, I will put 2 drops of Iodine into each dimple on the dimple trays. I will leave the solutions in the water baths/ice for 5 minutes. After this I will begin the testing. I will take one test tube of starch and one test tube of amylase from one of the water baths/the ice and mix them together in one separate test tube, I will immediately start the timer & take my first sample at 0 seconds. I will keep on taking samples every 15 seconds until the starch and amylase mixture turns the iodine brown. This will indicate that a reaction will have taken place. I will do this with each of my 5 temperatures. Until I have a table of results. If I get anomalies (recordings that don’t follow the pattern) I will do that particular experiment again, but on the whole I aim to do each experiment 3 times.

Risk Assessment

I must wear safety glasses & gloves to prevent contamination (just in case I come into contact with other peoples saliva). I must be careful not to drop any test tubes/glass pipettes as I may cut myself on the glass or my saliva may splash onto someone. I must be careful not to burn myself on the hot water in the water baths. It is important to label my test tubes so I won’t accidentally use someone else’s saliva instead of mine, to do this I will use a black labelling pen. After finishing the experiment, I will put all of the contaminated equipment into a bucket filled with bleach so they can be fully sterilised. It is important to wear an apron to prevent saliva from splashing onto my clothes, and also to prevent iodine from splashing onto my clothes. It is important I use the iodine with great care because it can stain the skin.

Results & Results Table

Conclusion

Looking at my results table I can see that (as I predicted) the 32ºC & the 39ºC temperatures reacted the quickest. And also (as I predicted) the 64ºC temperature was denatured. The other temperatures (22ºC and 2ºC) still worked but not as quickly. I did however, have a few anomalies: In the 2nd experiment on both 32ºC and 39ºC, the results do not follow the pattern & take twice as long to react. There could be many reasons why this had occurred, e.g. I used too much Iodine or too little/weak starch/saliva mix. These two anomalies pulled my average up by quite a considerable amount. The results show, as I predicted, that the enzyme efficiency decreases with the extremes of temperature. This is because enzymes have a 3D structure with an active site. Even the slightest change in the shape of the active site can decrease enzyme efficiency because enzymes are specific catalysts – they only catalyse and speed up one reaction. The enzyme works by the lock and key method: the substrate and enzyme are specific and fit together for a short time (like a key in a lock) before the enzyme releases a new substrate. The enzyme does not get used up in the experiment. I think my results are quite accurate and give me the general idea of what the optimum temperature is (around 40ºc), around what point the enzyme becomes de-natured (about 60ºc) and at what point the enzyme becomes inactive (0ºc). All my results support my prediction except that I expected the enzyme to be de-natured completely at 60ºc.

Evaluation

I tried to keep everything at a constant while doing my experiment, and I tried to measure everything accurately. There were, however, a few things that caused me problems e.g. at the start of each experiment I needed to shake up the solution as soon as I mixed the two substances together this meant that I could not take an accurate reading at 0 seconds, although I do not think this affected my results greatly. I did not have time to do a repeat so I took other peoples results and this was obviously not as accurate as it would have been had I done the experiment with my own saliva each time as people have different strengths of saliva. Therefore I was not controlling this variable and, had I had time I would have remedied this by repeating the experiment another 2 times to find an accurate average. However this was not possible but despite the anomalous second set of results my results and graph did show the basic idea that in extremes of temperature the enzyme is less efficient. My results are not very reliable as the saliva strength was not controlled as it should have been (I should have repeated with my own saliva) but they are accurate - I did measure everything out carefully with precision and timed to .0 of a second.