N U I
Where N = Native catalytically active enzyme.
U = Reversible unfolded catalytically inactive enzyme.
I = Irreversible inactive enzyme
Enzyme activity is affected by several factors such as temperature, pH, and sometimes even stress. However, the factor which we are testing in this experiment is temperature. Enzyme activity is changed by variation in temperature. As temperature raises the rate of chemical reactions increases because temperature increases the rate of motion of molecules. This leads to more interactions between an enzyme and its substrate. However, if the temperature is too high, enzymes can be denatured and they can no longer bind to a substrate and catalyze reactions.
This study aims to determine the effect of temperature on the amylase catalytic activity on starch at several different temperatures.
Methods:
The solution of 5 cm3 of amylase was were placed into 6 separate test tubes incubated for ten minutes at various temperatures of 0 oC, 20oC, 37oC, 60oC and 100oC. Test tube 1 (0oC) was placed in ice, Test tubes 2, was placed in 20oC water bath, Test tube 3, was placed in a 37oC water bath Test tube 4 was placed in a 60 oC water bath, Test cube was placed in 100 oC water bath and test tube 6 (control ) was left at room temperature 25oC.
The volume of 5cm3 of starch solution (substrate) was added to each amylase solution at the end of the 10 minutes incubation and mixed using clean glass rod.
The starch presence was tested at five minute interval for one hour by withdrawing the starch-amylase mixture from each of the above tubes and placing on a white tile. A drop of iodine solution was added to the starch-amylase mixture and mixed with clean glass rod. The observed colour change was recoded. The test tubes were left in their respective environments as they are being tested. The process was continued until a blue colour was no longer produced.
Results:
Table 1. The reaction rate of amylase expressed by the time of starch disappearance.
This result shows the time it took for the starch to be digested by the amylase as the temperature was increased. The results above support the hypothesis of the experiment, the reaction rate would increase as temperature was increased, until a certain point at which the amylase would become denature and would stop working. The amount of time for the amylase to digest all the starch at 0°C was 4 minutes, 20 oC was 0 minutes, 37 oC was 3 minutes, 60oC was 5 minutes and at both 25oC control no action of amylase as it was mot added to the solution and at 100 oC the blue colour was recoded through out as the enzyme was denatured.
Discussion:
The result of the supported the hypothesis that the reaction rate would increase as temperature was increased, until a certain point at which the amylase would denature and would stop working. Amylase does not become denatured at extreme cold (approx. 0°C) and this is supported by the result of the experiment by which the rate of starch disappearance (being hydrolysed by amylase) was 4 minutes. The low temperatures do not appear to cause damage to enzymes as observed the results of test tube 1 (0oC) in the experiment. The result of the rate of starch hydrolysis at temperature condition of 20oC is not correct and dose not support the hypothesis that reaction rate would increase as temperature increase. This error could have occurred due to incorrect temperature setup or faulty equipment. At temperatures below the optimum the enzyme-substrate reaction is dominant, and a rise in temperature within this range increases the rate of the process. This has heen observed as temperature was increased to 37oC the rate of starch disappearance was reduce to 3 minutes as compared to results at 0oC which was 4 minutes. When temperature was increased to 60 oC the time taken for starch to be hydrolysis was 5 minutes However, at an elevated temperature of approximately 100°C amylase does become denatured. When substances are exposed to high heats the atoms bounce more quickly and hit each other with greater force than they do at lower temperatures. High temperature often causes irreversible damage to the molecular structure of enzymes due to the velocity with which the atoms move about. Denatured amylase no longer catalyzes the hydrolysis of starch into glucose as observed the permanent blue colour indicating presence of starched.
.
In conclusion this experiment further confirmed that enzymes do increase/decrease their rate of reaction due to change in temperature of the environment. Each enzyme has its own individual prime functional temperature, therefore if they are removed from these environments they will not function as well or possibly denature if it the temperature is too high. Although, if an enzyme’s optimal temperature is maintained then the enzymes will catalyze reactions at optimum rates.
Although it appears that the data supports our hypothesis it would be a good idea to repeat the experiment several times to establish a trend, and prove that this set of data was not a coincidence. Also it would be interesting to actually find the point of denaturisation, this would be achieved by performing the experiment again with environmental conditions between 22°C and 37°C, as it appeared as though the denaturisation of the enzyme occurred during this interval
However we were able to establish a general range at which amylase denatures, not all enzymes denature at this range. Some enzymes which have just recently been discovered can thrive in temperatures of 100°C. This newly discovered enzyme could possibly give some information on the early development on life on the earth, as the climate of the primordial earth was much warmer than it is now (Cowen 11). This enzyme was discovered in an organism that thrived in 100°C temperatures near volcanic vents on the sea floor. However amylase has a much lower denaturisation temperature than this newly discovered enzyme; it is important not to make the wide assumption that “all” enzymes denature at the same range as amylase
Ref
Campbell, Neil, J. Reece, and L. Mitchell. 5th Edition. Menlo Park, CA.
Benjamin/Cummings, 1999.
Morgan, Judith. General Laboratory Bio III. Menlo Park, CA. Pearson Custom
Publishing, 2000.
Takai, Noriasu. “Effects of Psychological Stress On the Salivary Cortisol and Amylase
Levels in Healthy Young Adults.” Archives of Oral 49.12 (2004): 963.
Cowen, Robert. “Study of Life’s Origins May Improve Everyday Life Today.”
Science Monitor 87.84 (1995):11.
Cold temperature is similar to what cold blooded animals face in winter every year. The amylase cells which could not handle the cold died. Conversely the boiling presented conditions which amylase was not prepared to deal with. that the enzyme is denatured at high temperatures..
. At temperatures above the optimum, the denaturation reaction is dominant, and a rise in temperature within this range further depletes the system of active native enzyme and hence apparently slows the enzyme-substrate
reaction. Denaturation of a protein involves breaking of chemical bonds within the molecular structure of the protein. Chemical groupings such as SH and OH which, in the native protein are held in tight combination and are not reactive to any great extent with externally added agents, after denaturation become free at the molecular surface and may be detected by chemical means. The catalytic activity, a function of the peculiarly individual structure of the protein molecule, is generally lost. In addition, the denaturation process is characterized by a very large heat of activation, that is, 50,000 calories or more, as would be expected if a number of chemical bonds were broken in the process.