The purpose of this experiment was to determine the optimal temperature for both bacterial enzyme amylase, known as Bacillus licheniformis (B. licheniformis), and fungal enzyme amylase, known as Aspergillus oryzae (A. oryzae). During this experiment, both enzymes were exposed to 4 different temperatures (0 ᵒC, 25 ᵒC, 55 ᵒC, and 85 ᵒC) for specific time intervals. The time intervals were 0, 2, 4, 6, 8, and 10 minutes each. Because we are conduction an experiment, the 0 ᵒC was designated as our control group, and therefore not directly altered. Every group in this lab experiment was assigned two spot plates, one for bacterial amylase and the other for fungal amylase, 16 test tubes, disposable plastic pipettes, 1.5% starch solution, as well as hot-water and cold-water baths. Another reason for this experiment was to determine what would happen when the enzymes were exposed to temperatures higher than their optimal temperature ranges. This experiment did show that the optimal temperatures for both fungal and bacterial amylase were 55ᵒC. This is somewhat surprising because bacterial amylase should have a high optimal temperature than its fungal counterpart. Although this experiments results should have indicated explicitly that the optimal temperature for fungal amylase was 55ᵒC, the results were skewed due to possible contamination from previous lab section(s).
Within the human body and the world at large, enzymes play an incredibly important role. Whether its in the breaking down of food to sustain life or even in the processing of chemical waste, enzymes are very important and widely used nowadays. An enzyme is a biochemical catalyst that helps speed up a reaction by lowering the activation energy required for the reaction to occur (Clark, Choi, & Douglas, 2018). Enzymes are so important that in extreme cases the absence of certain enzymes a can cause illness or even death (Alberte et al., 2012). In the world at large, however, the lack of enzymes would be catastrophic. Because these biochemical catalysts allow for chemical reactions to occur even when enough activation energy is not available, without them chemical reaction would take far longer than our daily lives would permit or those reactions might not even occur in the first place (Alberte et al., 2012). In particular, bacterial Amylase, a common enzyme, is frequently used in the production of many foods and sugars due to it being a fast growing and very productive enzyme.
Enzymes are made of proteins which are formed by large chains of amino acids weakly bonded together (Urry et al.,2017). Enzymes have a specific spot on them in which a substrate, or reactant, binds and can be broken down or changed by the enzyme (Alberte et al., 2012). This specific spot is referred to as the active site. After binding to the active site, the enzyme forms an enzyme-substrate complex. After this, the substrate can now be altered with the desired chemical reaction (Alberte et al., 2012). Because of what they are made of, enzymes are particularly sensitive to both pH and temperature (Urry et al., 2017). When proteins, and in this case specifically enzymes, are exposed to temperatures above their optimal temperature they denature. Denaturing is the process by which the active site on the enzyme is altered due mostly to the high temperature disrupting the weak bonds within the structure of the enzyme itself (Urry et al., 2017). Enzymes can exist with wide varieties in optimal pH and optimal environment temperatures, some even exist with extremely low optimal temperature, as well as high optimal pH (Duarte et al., 2018). Research has shown that the optimal temperature for enzymes are not intrinsic properties of the enzymes themselves, rather optimal temperature is based on the enzyme’s conditions (Almeida & Marana, 2019).
For this experiment, two specific enzymes will be used to break down starch and test their effectiveness with increasing temperature and prolonged exposure. The two specific enzymes are from a bacterial source and a fungal source. The bacterial amylase sample was taken from a bacteria known as Bacillus licheniformis. The fungal amylase sample was taken from a fungus known as Aspergillus oryzae. The purpose of this experiment was to test the enzymatic activity of both Fungal and Bacterial Amylase in response to rising temperature, and how well they would hydrolyze starch directly following exposure.
Upon the instructions given by the lab instructor (TA), each group was given two spot plates (one marked fungal and the other marked bacterial), 16 test tubes, and 1.5% solution starch. Each test tube was marked with an “F” for Fungal amylase or a “B’ for bacterial amylase, as to not confuse the test tubes. The TA assigned each of the 8 total groups either fungal or bacterial amylase to experiment with. Next, one person within each group would fill 4 test tubes with 5 mL of 1.5% starch solution, and 4 test tubes with Fungal amylase. While this was happening, another group assigned the bacterial amylase would prepare 4 test tubes with the bacterial amylase, as well as 4 test tubes with 1.5% starch solution. Before the lab started the TA had prepared two hot-water baths, one at 55°C and the other at 85°C, as well as a cold-water bath set for 0°C. For the 25°C part of this experiment, the test tube was left in a bowl of room temperature water. Once each group was ready, the 4 test tubes containing the amylase as well as the 4 test tubes containing the starch solution were exposed to one of the given temperatures while the TA started a timer for 2 minutes. Once the timer had sounded, the test tubes were removed from the temperature exposers and a few drops of both the starch and the amylase were added to their respective spot plates in the 0 minutes row (top row of both plates respectively). Next, a drop of Iodine solution was added to each well to check for presence of starch. Each group was careful to use separate transfer pipettes for each temperature expose to limit the chances of error. This procedure was repeated for 2, 4, 6, 8, and 10 minutes, making sure that the process stayed the same for each temperature exposure and within the 2 minutes time frame. After all wells were filled with amylase-starch-iodine mixtures, data was recorded, specifically about what color each well was and what time and temperature corresponded to that well.
It is clear from the results that both bacterial amylase Bacillus licheniformis (B. licheniformis) and fungal amylase Aspergillus oryzae (A. oryzae) were affected by being exposed to differing temperatures. Our experimentally determined optimal temperatures for both bacterial amylase Bacillus licheniformis (B. licheniformis) and fungal amylase Aspergillus oryzae (A. oryzae) are both 55ᵒC. The optimal temperature for bacterial amylase was determined by close inspection of the data tables as well as Figure 2.3 in the results section. As can be clearly seen on the bacterial spot plate, under the 55ᵒC column from 2 minutes of exposure to 10 minutes exposure, the wells get increasingly lighter and more yellow which indicates that the amylase enzyme is catabolizing and hydrolyzing the starch present. Somewhat surprisingly, the optimally determined temperature for bacterial amylase is 55ᵒC, when it should have been higher. Bacterial amylase does not hydrolyze starch efficiently at temperatures below 40ᵒC, which is again evident in the data as well as in Figure 2.3. Although the optimal temperature for fungal amylase should be 55 ᵒC, this experiment provided skewed results. From Figure 1.3, we can see that only the enzyme in the first well of 55ᵒC column indicated enzyme hydrolyzation and enzyme activity, but the others in that same column did not. This can be caused by several things, but most notable it could be caused by improper cleaning of the spot plate from the previous lab section. As mention previously, due to the biomaterial composition of enzymes, they are highly sensitive to both pH and temperature (Urry et al., 2017). Another major explanation for faulty results with the fungal amylase would be that the temperature used for the hot-water bath was higher that indicated. The spot plate (Figure 1.3) suggests that the enzyme in 55ᵒC column is either fully denatured or just in a slowed-down state, which would only happen if the enzyme was exposed to a temperature above its optimal temperature range (Divakaran et al, 2011).
- Alberte, J., Pitzer, T., Calero, K. (2012) Enzymes. General Biology I Lab Manual Second Edition (49-62). Florida: McGraw-Hill Education LLC.
- Clark, M. A., Douglas, M., & Choi, J. (2018, March 28). Chapter 6.5 – Enzymes (Biology 2E). Retrieved from https://openstax.org/books/biology-2e/pages/6-5-enzymes.
- Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., & Reece, J. B. (2017). An Introduction to Metabolism. In Campbell Biology in Focus (2nd ed., pp. 122–138). Boston: Pearson.
- Almeida, V. M., & Marana, S. R. (2019). Optimum temperature may be a misleading parameter inenzyme characterization and application. PLoS ONE, 14(2), 1–8. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212977
- Divakaran, D., Chandran, A., & Pratap Chandran, R. (2011). Comparative study on production of a-Amylase from Bacillus licheniformis strains. Brazilian journal of microbiology: [publication of the
- Brazilian Society for Microbiology], 42(4), 1397–1404. doi:10.1590/S1517-838220110004000022
- Duarte, A. W. F., Dos Santos, J. A., Vianna, M. V., Vieira, J. M. F., Mallagutti, V. H., Inforsato, F. J., …
- Durães Sette, L. (2018). Cold-adapted enzymes produced by fungi from terrestrial and marine
- Antarctic environments. Critical Reviews In Biotechnology, 38(4), 600–619. https://doi-org.ezproxy.fiu.edu/10.1080/07388551.2017.1379468