Abstract
Temperature is one of the great determinants while conducting reaction experiments. An increase in temperature causes an increase in the rate of reaction of chemicals during experiments. This may be attributed to the increase in molecular motion due to high temperatures. The increased rates of reactions are also caused by the increased kinetic energy of the molecules. Therefore substrates would collide more frequently with enzyme active sites causing the reaction rates to soar. However, the rate of reactions will increase with rising in temperature to a given limit after which any further rise in temperature will cause a permanent denature of enzymes, thus a sudden decline in the reaction rates.
This experiment focuses on the effect of temperature on the activity of the Amylase enzyme. It was observed that the amylase activity increases with rising in temperature but declines with further increase in temperature which was contrary to the expected results.The experiment was carried out in different temperature conditions to determine the influence of temperature on each reaction. The experiment was undertaken in different water baths of different temperatures while observing the color change from blue-black to yellow-amber. The results of the experiment indicated an increase in reaction rates with rising in temperature but to a specific temperature beyond which the enzyme activity declined.
Introduction
Amylase is an essential enzyme in the human body since it aids in the digestion of starch. Starch comprises two polysaccharides, amylase and Amylopectin. It is a protein in its nature and like other enzymes, it catalyzes reactions without being consumed in such reactions (Campbell, 1999). Moreover, enzyme reaction rates are influenced by different factors such as temperatures, pH, and even stress in the human body. According to Takai (2004), stress in the human body regulates the release of amylase enzyme hence determining the reaction rates.
The focus of this experiment was on the effect of temperature on the activity of amylase as a catalyst. A rise in temperature results in increase in motion of molecules as they possess more kinetic energy hence their interaction with substrates is increased. In this experiment, starch was used as a substrate with a mixture of glucose in it. However, higher temperatures could also denature enzymes and thus cause a decline in the reaction rates as they are not able to play their significant role as catalysts.
Through the process of hydrolysis, the amylase enzyme breaks down starch into smaller and less complex glucose chains. The reaction produces a complex mixture that is composed mainly of maltose and glucose. This mixture will fail to react to iodine to produce a blue-black color.
Hypothesis and Predictions
The experiment was conducted to investigate the impact of temperature on the activity of the amylase enzyme. The research question was if the temperature rise resulted in a linear increase in the reaction rate. Therefore the hypothesis of this research targeted the activity of amylase at different temperatures and was stated as follows: the higher the temperature the faster the rate of reactions. The expectation of the experiment was a steady rise in the rate of reaction with an increase in the temperature during the experiment. The prediction of this experiment was that an increase in the temperature of water would shorten the reaction time.
Materials and Methods
Four standard-sized test tubes marked one to four were used for this experiment. A volume of 5ml 1% starch solution was added to these test tubes using a calibrated pipette. Add 4ml D1 water and 1ml of 6.8 buffer to every tube.
Different conditions were provided for each test tube by placing them in water baths of different temperatures. The first test tube was placed in a water bath of 80C, second test tube in a water bath of 37C, the third test tube in a test-tube rack which was at room temperature of about 22C while the last test tube in crushed ice was about 4C. In addition a test tube of starch and another of amylase were placed in each of the above conditions and each was observed for 10 minutes.
Several rows of test plates were filled with one or two drops of I2KI per compartment, while test tubes 1 and 1A were mixed while time was recorded. Add 1 to 2 drops of the mixture to the I2KI on the test plate and observe the color change (Campbell, Reece & Mitchell, 1999).
Subsequent mixture drops were added to other new wells of I KI at intervals of 30 seconds until the blue color was filled with a yellow-amber color being observed in each well. This is an indication that starch is fully digested. The experiment was stopped for each well when there was no color change within the first 10 minutes. The following were the results as recorded in the figure below:
Figure1.0.
The above steps were repeated for the other three test tubes while recording the changes and time for those changes. For more advanced results, the test tube with crushed ice was transferred into the water bath of 37C and waited for an average of 2 minutes. Test the contents and record the results at intervals of 10 seconds.
Results
According to the hypothesis and predictions of this experiment which were based on the fact that the higher the temperature the quicker the reaction rates and the higher the temperatures of water baths the shorter the reaction period. While using temperature as an independent variable on the X-axis and time of starch disappearance as a dependent variable on the Y-axis, the following results were recorded:
According to the table shown above, in tube 1 there was no change even after 5 minutes of the experiment. In the second tube, the color changed from blue to yellow-amber after 1.22 minutes. The third tube recorded the shortest time during the experiment as the blue color disappeared after 0.69 minutes only. The last tube which was placed in crushed ice recorded 2.26 minutes before the color change was observed (Campbell, Reece & Mitchell, 1999).
Discussion
The results of this experiment were contrary to the hypothesis and expected results. It was noted that an increase in temperature resulted in increased reaction rates but was not linear as expected before. The water bath of 80C recorded no change even after 5 minutes of the experiment. Both higher and lower temperatures slowed down the reaction resulting in longer time before the change in color was observed. Higher temperatures denatured the enzymes resulting in slow rate of reaction. This was mainly because the experiment went beyond the 10-minutes limit given. It also shows that starch failed to be broken down by the amylase.
From the experiment it was observed that the amylase enzyme was denatured between temperatures 37C and 22C thus more experiments were needed to establish the exact temperature when they were denatured. However, when the test tube placed in crushed ice was transferred to the water bath of 37C, the reaction was observed to increase. This was because the low temperature did not alter the structure of enzymes but only made them inactive. Therefore when the environment was altered, the motion of the enzymes was increased and thus the rate of reaction was increased (Takai, 2004).
According to the observation of the experiment, the period of reaction at 22C was longer compared to the period taken by the reaction while placed at the water bath of 37C. This could have been caused by uncontrolled room temperature and the non-standard behavior of the enzyme in different conditions.
References
Campbell, N., Reece, J. & Mitchell, L. (1999). Biology.5th Ed. Menlo Park, CA: Benjamin/Cummings.
Dutta, T., Malabendu, J., Priti, R. P., &Tanmay, B. (2006). The Effect of Temperature, pH, and Salt on Amylase in Heliodiaptomus viduus. Turk J Zool. 30.187-195.
Takai, G. (2004). Effects of Psychological Stress on the Salivary Cortisol and Amylase Levels in Healthy Young Adults. Archives of Oral Biology, 49(12), p. 963.