Temperature and Enzyme Activity

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Introduction

In this lab, our main purpose is to determine the relationship between enzyme and temperature. As we know, enzymes are mainly composed by proteins, which is easily degenerated under high temperature. Also, enzymes are highly specific creatures towards temperature. For example, amylase and catalase are both adapted human body temperature, which is about 7-45¢J. There is an apparent optimum temperature for different enzymes, after which the reaction rate decreases rapidly, because of denaturation of the enzyme. However, the optimum temperature of enzymes depends on the way and which enzyme which the activity is measured. Some enzymes are heat resistant and can work at fairly high temperatures. Our goal in this experiment is to find out the enzyme activity at different temperatures.

Method

In this experiment, we will test the effect of temperature on amylase activity. For that the variable is temperature (5¢J, 7¢J, 50¢J, 80¢J), and the main control variable is pH, which will be standardized my a certain buffer(pH=6.). The reactant will be starch, and tester will be IKI (Iodine potassium iodine). Distilled water is used as control groups. The procedures are as follow

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1. Label test tubes and droppers so that we don¡¦t contaminate the experiment by mixing them together.

. Turn on the temperature maintenance machines, and set 1 at 7¢J, another at 50¢J, the other at 80¢J.

. Set up spot plates to test drops of experiment and control groups, and label each spot with minutes preceding one another.

4. Add 1ml of buffer pH=6. to both control and experimental test tubes.

5. Add 1ml of water to the control group.

6. Add 1ml of starch solution to the experimental test tube.

7. Place experimental test tube (E-7) and control test tube (C-7) in the temperature maintenance machine, which is set to 7¢J.

8. Warm test tubes for 5-10 minutes in order to achieve the desired temperature.

. Get the amylase ready in ice, and the dropper filled with amylase.

10. Place 1~ drops of IKI in the spots indicated 7¢J.

11. Add 0.5ml of amylase to both the control and experimental test tubes, and so the reaction starts. Squeeze and release the dropper gently to mix the solution in the test tubes. Take down the time when amylase is added.

1. Start to take samples at 10 second intervals for the first minute. If there are no significant changes within the first minute, then start to record the change every minute. While you do this, put a drop of the control test tube solution on another spot in correspond to the experimental solution.

1. Record the color changes on the data table.

14. Continue to record the color change in between 1 minute interval, and if need additional time points, wash the spot plates out and re-use them.

15. When the experimental color reaches the same color as the control solution (yellow), all the starch has been hydrolyzed. This is the reaction end point. Take this number down carefully.

Results

During the experiment, we acquired results displayed as below, and also seen in figure1.

5¢J 7¢J 50¢J 80¢J

T=reaction end point 15 10 7 0

1/T=Enzyme Activity 0.06666667 0.1000000000 0.0707070 0

Table1

As seen in this table, we obtained the outcome that 7¢J has the highest enzyme activity (lowest reaction time period), 5¢J comes in second, 50¢J comes third, and there is no end point at 80¢J, meaning during the certain time period, the solution wasn¡¦t able to change color completely. The figure shows a curve with a highest point of 0.1, which occurred at 7¢J, and no enzyme activity at 80¢J.

Conclusion

A predicted graph of amylase reaction with temperature change is shown as below

The graph showed a optimum temperature at 40¢J, and drops quite rapidly afterwards. This suggests that amylase works best at temperature slightly below 40¢J. Therefore body temperature, 7¢J, is certainly ideal for amylase to work under, which is also illustrated in our figure1. In figure1, we obtained an optimum at 7¢J; however, there was still enzyme activity at 50¢J, which differed from the predicted graph. This could be caused by several human errors, such as not turning the temperature maintenance machine high enough, or did not leave the experimental and control test tube in the machine long enough, so the temperature of solution was not 50¢J. Moreover, the enzyme value may be miss measured, that we put more than 0.5ml of enzyme into the test tube, increased the enzyme concentration thus showed enzyme activity at 50¢J.

Another interesting phenomenon we acquired during the experiment is that it took us a lot longer to achieve the end points than the other groups. As seen in the table, it took us 10 minutes for 7¢J experiment and 15minutes for 5¢J experiment while normally it should only take 1 to minutes. We reached the conclusion that either the amylase we used was contaminated by other substances or they denatured during the testing. Maybe we did not put them in the ice correctly. Also, one of our teammates forgot to put amylase into some of the solutions. Even though we re-ran the experiment again, it might affect our results. These are the reasons why our experiment are not as accurate as it was predicted.

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