The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
Sophia Hawks
Lab Section: BSC2010L-U14
Panther ID: 6125674
Lab Partners: Sergio Lopez, Kailynn Santos, Gian Zaldivar
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
Abstract
In almost every living system, an organism has several enzymes for specific functions. Enzymes are accountable for regulating biological reactions within the organism and aid in an organism’s function. Amylase, an enzyme that is found in both animals and plants, has such a function that assists in the breakdown of protein and starch. In this report, an experiment was conducted and evaluated to identify the optimal temperatures for the fungal Aspergillus oryzae bacterial Bacillus lichenoformis at different temperatures. To determine the starch catalysis, an Iodine test was used to help distinguish the breakdown of starch in a color spectrum from dark brown to bright yellow, from the least to the most broken-down starch. This helped to show the amount of the breakdown reaction between the starch and the enzyme. With a predicted optimal temperature of twenty-five Celsius, there was an expectation that this would be the optimal temperature. However, once the data was collected and evaluated, it was shown that both the fungal and bacterial amylases had an optimal temperature of about fifty-five Celsius. In other temperatures, the color spectrum was darker because of either denaturation or slow movement of amylase. This experiment helped show the significance of the difference of temperatures and how it can affect greatly the function of the enzyme.
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
Introduction
Enzymes are essential in maintaining life and happen in all forms of life. With over three thousand identified, there are many specific enzymes that aid in cell function. (Total Health 2009). By regulating the functions of many cellular reactions, enzymes are crucial in reducing the activation energy, which is the sum of energy needed to make cellular responses to occur. Without enzymes, the functionality of the cell will be hindered and result in death of the organism. Enzymes, however, also need to function properly by having their optimal conditions met. Optimal conditions, which can be affected by factors such as pH and temperature of the surrounding environment, assist in the enzymes to perform their maximum proficiency (Donaldson 2000). If these optimal conditions are not met, the enzyme could either denature because of high temperatures or have its reactions slowed down due to being in an environment with cooler temperatures.
Another significance of the enzyme is that the active site establishes the specificity of the enzyme. There are two models for the active site: the “lock and key model” and the “induced fit model.” In the “lock and key model,” the substrate, considered as the key, gets placed into the enzyme’s active site and stimulates multiple chemical reactions. The shape of the substrate must be accurate, compared to the “induced fit model.” (Alberte et al. 2012). In the “induced fit
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
model,” the enzyme has more of a flexibility to change to an appropriate shape to improve its placement of another type of substrate near the active site (Alberte et al. 2012).
A necessity in the human diet, starch is a storage polysaccharide made up of glucose. Found in many crops such as corn, wheat, and so on, starch is collected and expended in its original form or through modification of enzymes such as glucoamylase, pullulanase, amylase, and so on. (Baroroh et al. 2017). One of these enzymes, amylase, catabolizes the polymers within starch into monomers such as maltose to create a food source and to store energy ( Alberte et al. 2012). Amylase, which is also found in almost all organisms, catabolizes starch by hydrolyzing it into glucose and other monomers that could be used by several types of bacteria for nutrition (Haase et al. 2015).
In this experiment, samples of amylases from Aspergillus oryzae and Bacillus lichenoformis were taken to observe the efficiency of the two amylases in different temperature environments. To monitor this visually, an Iodine test has been conducted to observe the presence of starch and its catalysis. After monitoring and identifying the breakdown of starch, the results recorded will show the breakdown of starch of both amylases at an optimal temperature of fifty-five degrees Celsius and slow-down of the breakdown at lower temperatures and denaturation at higher temperatures.
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
Method
Napkins were placed underneath a pair of spot plates and were labeled, “Time,” including zero, two, four, six, eight, and ten minutes horizontally and labeled, “Temperature,” including zero, twenty-five, fifty-five, and eighty-five degrees Celsius vertically. Four test tubes were labeled with a temperature of zero, twenty-five, fifty-five, and eighty-five degrees Celsius, the group number, and labels, “B,” or “F” to identify the amylases from Bacillus lichenoformis and Aspergillus oryzae. Four more test tubes were obtained and labeled with the given temperatures, the source of amylase labeled, “B” or, “F,” the group number, and labeled as, “S” to identify as the starch solution. Five mL of 1.5% of starch solution was placed in the test tubes that were labled, “S.”
One mL of amylase was placed into the remaining test tubes that had no presence of starch. If the group was appointed to do bacterial amylase, the amylase must be placed into the test tubes selected for bacterial amylase. If the group was chosen to do fungal amylase, the amylase then must be placed into the test tubes selected for bacterial amylase. Four test tubes containing starch and four test tubes containing amylase must be placed into their assigned temperatures. The test tubes must then stay in their respective temperatures for five minutes.
Add three drops of iodine into the first row of the spot plate labeled zero minutes. Once five minutes has passed, a few drops of the starch solution from
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
the distinctive temperatures must be placed on the first row without moving the test tubes and using different pipettes for each temperature. This is done to prevent different temperatures of starch from mixing with each other, which may create a variation of incorrect data. The starch solution of each temperature must be combined with the amylase those that has the same temperature. The timer must then be set for two minutes.
Add three drops of iodine. Then, the transfer of the iodine and starch solution must be placed in three drops in the row labeled two minutes. The timer must be set up once more for two minutes and the process is repeated for each row until reaching the ten-minute row in the spot plate. A color spectrum chart is given to differentiate the amount of starch within each spot, ranging from bright yellow with the least amount of starch to dark brown with the most amount of starch present.
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
Results
(Figure 1.1): Photograph of Bacillus lichenoformis spot plate. This shows that the optimal temperature for this amylase from the bacteria is around fifty-five Celsius. This row shows the least amount of starch present on the spot plate based on the Iodine Test.
Temp
Time (min) 0 25 55 85 Aspergillus oryzae
0 4.6 4.4 4.6 5
2 3 2.6 3.2 4.8
4 3 2.6 3.2 4.6
6 3.4 2.6 3.2 4.8
8 3.4 3 3 4.8
10 3.2 2.4 3.2 4.8
(Data Table 1): Class average of starch present in Aspergillus oryzae based on the color spectrum from Iodine test. The table shows the optimal temperature is around twenty-five degree Celsius.
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
(Figure 1.2): Visual representation of class average of starch present over time of the amylase of Aspergillus oryzae-starch mixture in different temperature environments. The graph shows the optimal temperature is around twenty-five degree Celsius.
Temp
Time (min) 0 25 55 85 Bacillus lichenoformis
0 4.6 4.6 4.8 5
2 3.2 2.8 3.2 3
4 3.6 3.2 3.2 2.2
6 3.2 3.4 3.4 1.6
8 3.6 3.6 3.4 1.4
10 3.6 3.4 3.6 1.2
(Data Table 2): Class average of starch present in Bacillus lichenoformis based on color spectrum from Iodine test. The table shows the optimal temperature is around eighty-five degrees Celsius.
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
(Figure 1.3): Linear graph displaying average class data of recorded changes at different temperature environments. The graph shows that the optimal temperature for the amylase of Bacillus lichenoformis is around eighty-five Celsius.
Discussion
Based on the results presented, the optimal temperature for the amylase of Aspergillus oryzae and Bacillus lichenoformis were not what was expected. With the amylase of Aspergillus oryzae, the graph shows what seems to be the optimal temperature of around twenty-five degrees Celsius, however some linear representations at different temperatures follow a similar pattern. There may be because of a possibility of extraneous variables playing a role, such as not mixing the amylase-starch solution correctly, incorrect data evaluation, and other possibilities. Because of these errors, the class data may be inaccurate and can affect the whole experiment.
The results presented by the amylase of Bacillus lichenoformis also shows the optimal temperature that was not expected. Based on the class data, the optimal temperature for the amylase is around eighty-five degrees Celsius. However, the linear graph shows that most of
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
the amylase-starch mixtures at different temperatures had a sudden “dip,” around four minutes into the main portion of the experiment. A possible scenario of why this occurred in the graph is because of poor timing from some groups, inaccurate data collection, and so on, which become part of the many extraneous variables involved in the whole experiment.
Even though there were multiple errors in the experiment that could have created inaccurate data representation, there was still some sort of correlation between temperature and amylase function. It is now applicable to reject the null hypothesis that states that there is no correlation between temperature and amylase function, however we cannot accept the alternative hypothesis at this time because of insufficient data from both the experiment and outside factors that may affect the experiment.
To reduce any errors that may affect the data of the experiment, several precautions must be implemented. For instance, the surrounding environment around the experiment must be controlled, such as the temperature of the room. Another way to reduce unnecessary errors is by implementing the method of mixing the amylase-starch mixture part of the experimental procedure to establish more accurate data of the observable starch catalysis in the spot plates. An additional solution for the reduction of errors in the experiment is for there to be more room to conduct it because cross-contamination could have easily occurred with the amylase-starch mixture of different temperatures. If applied, this may reduce the amount of error in the data because of less contamination compared to the current set-up of the experiment.
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
Based on the results of the experiment, it is a possibility that there are many more organisms, especially bacterial organisms, that are not yet found because of the adaptability of being able to survive at high temperatures. An example of this is the optimal temperature of the amylase of Bacillus lichenoformis, which is eighty-five degrees Celsius. Most organisms, especially humans, are not able to survive in such temperatures normally, bringing up the possibility that there are much more unknown organisms such as prokaryotes, bacteria, and other simple organisms that is not found yet. However, even though most organisms cannot live in high temperatures due to the denaturation of enzymes, several organisms such as gut bacteria within the stomach and intestines are able to survive around a low pH level (Alberte et al. 2012).
With the knowledge of knowing that several organisms are able to live at extreme temperatures because of the functionality of their enzymes, it may be possible that in the distant future, humans may be to acquire the enzyme’s proficiency and apply it to themselves and find it useful to harsh environments in either extremely hot temperatures or areas that would not normally be possible for humans to live and function normally.
Bibliography
Alberte, Jose, Thomas Pitzer and Kristy Calero. “Enzymes.” Alberte, Jose. General Biology 1 000 Lab Manual. McGraw-Hill Education, 2012. 49-61.
Baroroh U, Yusuf M, Rachman SD, et al. (December 2017). The Importance of Surface-Binding 00000Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of 00000View. Enzyme Research.:1-11. doi:10.1155/2017/4086845.
Donaldson, D. (2000). Enzymes: What are they and what do they do? The Journal of the Royal 00000Society for the Promotion of Health, 120(3), 148-149. Retrieved from
The Outcome of Temperature on the Breakdown of Starch using Amylase on Aspergillus oryzae and Bacillus lichenoformis
http://ezproxy.fiu.edu/login?url=https://search-proquest-com.ezproxy.fiu.edu/docview/72411931?accountid=10901
Haase E, Feng X, Pan J, et al. (Jul 2015). Dynamics of the Streptococcus gordonii Transcriptome 00000in Response to Medium, Salivary α-Amylase, and Starch.Appl. Environ. Microbiol. 81 (16) 000005363-5374; DOI: 10.1128/AEM.01221-15
Overview. (2009, Mar). Total Health, 30, 39-46. Retrieved
http://ezproxy.fiu.edu/login?url=https://search.proquest.com/docview/210156750?accountid=10901