The aim of this laboratory experiment was to determine the optimal temperature for bacterial and fungal, Aspergillus oryzae amylases. Furthermore, it will assess the influence of temperature on amylase’s ability to degrade starch maltose. Collect four test tubes for each amylase. The test tubes were then labeled with the temperatures they corresponded to. In half of each test tube, 5ml of the 1.5 percent starch was applied. Following that, each test tube is put in the corresponding temperatures. To observe the starch hydrolysis mechanism, an Iodine test was performed. For fungal amylase and bacterial amylase, two spot plates were created. Labeling of the spot plates was done with time and temperature as dependent and independent variables respectively. During the process of equilibrating the tubes, 2 drops of Iodine solution were added into the initial rows of the time on each of the spot plates. A few drops of solutions in the tubes were transferred using a pipette to the Iodine solution. This is a process that was carried out at each of the time intervals up to the completion of the experiment. Results of the experiment were then tabulated in figure 1 and figure 2. It was observed that there was no change of color for the majority of the experiments conducted at the temperatures of 85°C and 0°C. The color change was observed at 65°C between 4 and 6 minutes. It can be concluded from the results of the experiment that the optimal temperatures for bacterial and fungal amylases are comparatively 65°C. Therefore, there is a relationship between the metabolic rates of enzyme and its temperature.
Introduction
The aim of carrying out this laboratory experiment was to determine the optimal temperatures for both bacterial and fungal amylases. Amylases are both proteins and enzymes. Proteins are essential in the human body due to the fact that they perform various functions such as storage, regulation, motion, enzyme catalysis, support, defense, and transport. On the other hand, enzymes functions as a biological catalyst. As a result, enzymes increase the rates of chemical reactions that are essential to live. This is done through the reduction of the activation energy of the enzymes. Activation energy refers to the amount of vital energy needed for the process of chemical reactions to happen (Peter, Zakhartsev & Westerhoff, 2006). Activation energy can be reduced by enzymes through the substrate binding. The active site of enzymes has unique shapes that only suit to a particular substrate. This is a model normally referred to as the “lock and key”. Characteristics, charge, and shape of the substrate molecules and enzymes are determined by the specificity of the active site results of the enzyme (Alberte, Pitzer, & Calero, 2012). The shape of the enzyme is transformed to increase the process of chemical reactions after its binding to the enzyme’s active site, commonly known as the “induced fit” model. Nevertheless, there are other factors that can change the shape of the enzyme. These factors constitute salt concentration, substrate concentration, pH level, temperature as well as the presence of cofactors, activators, and inhibitors. Carbohydrates such as sugars and starches are catabolized by amylases (Olivieri et al, 2011). Hydrolysis is a process that helps in the breaking down of the different molecules of carbohydrates. Larger carbohydrate molecules are broken down into smaller molecules through hydrolysis reactions. This involves gaining of a water molecule. Energy is usually released to perform the work after breaking down the larger molecules through hydrolysis reactions. There are certain optimal conditions that help enzymes to carry out their work effectively. For instance, the optimal working conditions for enzymes are normally within a very small range. The Very high temperature will result to the denaturing of the majority of the enzymes hence thwarting substrate binding. In addition, very low temperatures also contribute to low reactions of the enzymes. Nevertheless, the majority of the enzymes have a high tolerance of temperature. Accelerated results will be produced by a warmer temperature in case the temperature is linked to the amylase activity. For instance, perhaps there will be very low temperature at 0°C hence leading to slow chemical reactions. However, there will be high chemical reactions at 65°C and 85°C thus leading to denaturing of the amylase enzyme. This is normally dependent on the hydrogen bonding of the enzymes of a particular organization. The strength of the hydrogen bonding usually determines the shape of the enzymes (Hunter, Jin, & Kelly, 2011). Stronger bonds enable numerous optimal conditions while the weak bonds contribute to low range of the normal conditions. The optimal temperature will be achieved at 25°C.
Methods
Performance of an Iodine test will help to observe the breakdown of starch in fungal amylase and bacterial amylase. Iodine test assists individual to observe the starch hydrolysis process when it changes color from black to yellow. The first step was to set the paper towels below the spot plates with adequate room to facilitate labeling. The next step was to label the left and top side as time and temperature respectively. The various temperatures that would be tested include 0°C, 25°C, 65°C, and 85°C. On the other hand, time will be recorded after an interval of 2 minutes. The labeling of time will be done on the right side as 0, 2, 4, 6, 8, and 10 minutes. Four test tubes were collected and labeled all the bacterial enzymes as “B” where each of the tubes has distinct temperature. Another four test tubes will be collected for the fungal enzymes where they were marked “F”. During this period, a starch solution “S” will also be written for the four test tubes. 5ml of the 1.5% solution of starch was added to all the test tubes that were marked “S”. Thereafter, 1ml of amylase was added into the other test tubes and put each of them into the corresponding temperatures. Allow it to heat for 5 minutes then transfer the solution of starch to the spot plates. During this process, add 2 drops of Iodine solution to the 0 minute row in the spot plate. After equilibration has occurred, add some drops of the starch solution from each of the temperatures to the 0 minute row. The test tubes were not removed from the water bath during the transfer process. Each of the pipettes was labeled with the corresponding temperature for each of the time interval. Results should then be recorded at the end of the laboratory experiment.
Results
Temperature (°C)
0
25
65
85
Time (min)
Color
#
Color
#
Color
#
Color
#
0
5
5
5
5
2
4
4
4
5
4
4
3.5
3
5
6
3.5
3.5
2.5
5
8
3.5
3.5
2
5
10
3.5
3.5
2
5
Fig1: Bacterial Amylase
Temperature (°C)
0
25
65
85
Time (min)
Color
#
Color
#
Color
#
Color
#
0
5
5
5
5
2
5
5
4.5
5
4
5
5
4
5
6
5
5
4
5
8
5
5
4
4
10
5
5
4
–
Fig 2: Fungal Amylase
Fig. 1.1: Starch Catalysis in the Bacterial Amylase
Temperature (°C)
Color (#)
Time (minutes)
Figure 2.2: Starch Catalysis in Fungal Amylase
Temperature (°C) Catalysis in Fungal Amylase Over Time at Varying Temperatures
)
Color (#)
Time (minutes)
According to figure 1, testing of the effects of the starch catalysis in the bacterial amylase after a particular period at various temperatures was carried out. It was observed that the color was a #5 at 0 minute and 0°C. The starch hydrolysis scale indicates that a #1 and a #5 represent smallest and highest amount of starch respectively. The color was a #4 at 2 and 4 minutes while it was a #3.5 at 6, 8, and 10 minutes respectively at the same 0°C. At 85°C, the color remained a #5 at 0, 2, 4, 6, 8, and 10 minutes.
In the second figure, testing of the effects of starch catalysis in the fungal amylase for a period of time was done. It was established that the color was a #5 at 0°C, 25°C and 85°C from 0 minutes to 10 minutes. Nevertheless, the color was a #4 at 55°C for 4 minutes to 10 minutes. The results were represented in the graphs of figure 1 and figure 2.
Discussion
After carrying out the laboratory experiment and examining the results from the graphs and the table, it was concluded that the null hypothesis was disregarded. According to the tabulated information, it demonstrates the association between metabolic rate and temperatures of the enzyme. It was observed that there is a reduction in the reaction of the enzyme at temperatures that are beneath the optimal level. Nevertheless, denaturing of the enzyme occurs at temperatures that are above the optimal level. For instance, according to the graphical representation of the fungal amylase effects at different temperatures of 0°C and 85°C, it indicated that there was no any change of color through the entire laboratory experiment. As a result, there was no start that was broken down at these temperatures. Nevertheless, it was observed that the starch hydrolysis reaction began very swiftly at 0°C and 85°C due to the fact that it illustrated the same results (Arvanitis & Mylonakis, 2015). Half of the laboratory experiment did not indicate any change of color. The color change began to emerge after a period of 4 minutes at temperatures of 25°C and 65°C. It was also observed that there was swift starch hydrolysis at 65°C after a period of 6 minutes due to the fact that the color also changed. Therefore, based on the results obtained from the experiment, a conclusion can be made that the optimal temperatures of fungal amylase and bacterial amylase is comparatively near 65°C. Nevertheless, there might be other errors that might have affected the end results of the laboratory experiments. It was not easy to find the color change at 85°C after 8 and 10 minutes for the fungal amylase. The results might have been very different in case all the information was gathered in the experiment.
It was also observed that there was change of color in the bacterial amylase after 8 minutes at a temperature level of 85°C. In case denaturing of the enzyme could have occurred then it might not have functioned effectively. This might be as a result of the errors incurred during the experiment. Determining whether the experiment was carried out appropriately is essential due to the fact that it helps to get the most precise results. Despite the fact that the experiment was carried out as per the stipulated procedures, there are certain improvements that need to be made. For instance, it is important to assign different individuals a particular temperature for the fungal amylase and bacterial amylase. This is vital due to the fact that it will help make sure that the measurements are taken appropriately as per the time intervals outlined. In addition, it would be appropriate to use higher temperatures so as to observe the effects of higher temperatures on the enzymes. This would be vital in determining the behaviors of enzymes in various environmental conditions. The experiment achieved its objectives since it was able to establish the effects of temperature on amylase activity. The challenges experienced during the experiment should be addressed so as to ensure that future experiments achieve high level of accuracy (Raven et al, 2014).
References
Alberte J., Pitzer T., Calero K. (2012). Exercise Four Enzymes. In: General Biology I Lab Manual, pp 49-53. The McGraw Hill Companies.
Arvanitis, M., & Mylonakis, E. (2015). Fungal-bacterial interactions and their relevance in health. Cellular Microbiology, 17(10), 1442-1446. doi:10.1111/cmi.12493
Hunter, A. J., Jin, B., & Kelly, J. M. (2011). Independent duplications of α-amylase in different strains of Aspergillus oryzae. Fungal Genetics and Biology, 48(4), 438-444. doi:10.1016/j.fgb.2011.01.006
Olivieri, C., Marota, I., Rollo, F., & Luciani, S. (2011). Tracking Plant, Fungal, and Bacterial DNA in Honey Specimens*. Journal of Forensic Sciences, 57(1), 222-227. doi:10.1111/j.1556-4029.2011.01964.x
Peter R., Zakhartsev M., & Westerhoff H.V. (2006). Temperature compensation through systems biology. FEBS Journal. 274 (4): 940-950.
Raven P., Johnson G. B., Mason K. A., Losos J. B., Singer S. S. (2014). The Chemical Building Blocks of Life. In: Biology, pp 44-53. New York: The McGraw Hill Companies.
