Optimizing Amylase Activity: Temperature Effects on Bacterial and Fungal Amylase

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Abstract

Enzymes play a crucial role in speeding the reaction by lowering its activation energy to produce a precise biochemical reaction. This lab’s purpose is to find the optimal temperature of bacterial and fungal amylase. Amylase is an enzyme found in saliva and pancreas that help break down sugar. In this experiment, four test tubes that contained the amylase and sugar were used. By testing various temperature water baths that ranged from 0 to 85 degrees Celsius for each one, the optimal temperature of the amylase was determined.

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Numerous test tubes with the different amylase and sugar solution in the plates were tested to see what their optimal temperature was.

This was observed by leaving a mix of amylase and sucrose in the different temperatures for 2-minute intervals, then mix the iodine and observe if the sugar was broken down. The color in the dish indicated how much enzyme activity was present. If the sugar had broken down, the liquid would be yellow, since it is the color of iodine and there would be no sucrose mixed in.

If the color was brown or black it meant that there was still sugar in the solution, therefore indicating that it was not the optimal temperature of the enzyme. After analyzing the results, it was concluded that the optimal temperature of the amylases were about 55 degrees Celsius (Alberte, Pitzer, Calero, 2012)

Introduction

Enzymes are catalysts that speed up chemical reactions within cells by lowering their activation energy. Without enzymes, life would not be possible because such reactions would occur too slowly to keep one alive.

Most of the time they are found in the form of a protein and they bind to reactant molecules for the reaction to take place more efficiently (Clark, 2018). For this to occur, the substrate must bind to the active site of the enzyme, creating an enzyme-substrate complex.

There are two main theories on how this occurs, the lock and key model and the induced fit model. The lock and key model explains that the substrate and enzyme both have complementary unique geometric shapes that align perfectly for each reaction. In the induced fit model, the substrate does not fit exactly in the enzyme, so the substrate produces changes that aligns them both. The enzyme substrate complex ends up having a unique shape and size and therefore a unique function as well. This complex needs very precise environments, also called optimal conditions, for it to function properly. These conditions include temperature and pH level (Cooper, 2000).

An extreme change in the optimal temperature or pH can cause an enzyme to denature, or change shape, and therefore not be suited for the substrate. This prevents the needed reaction from occurring. In humans, certain health conditions can affect the efficiency of the enzymes. A minor fever can affect the body’s temperature, causing it to denture. It is essential to return the temperature back to its normal range to restore the enzyme. More serious illnesses like pancreatitis, cystic fibrosis, or pancreatic cancer reduce the quantity of enzymes in your body, therefore preventing the body from fully digesting all the food and obtain the necessary nutrition to be healthy (Whitcomb, Lowe, 2007).

Scientists have found a way to treat some of these conditions and make sure the body gets the nutrients it needs by making dietary enzyme supplements. One crucial enzyme supplement is amylase. Amylases are enzymes that break down starch into smaller molecules. If the body is not able to break down starch, it will not be able to make the ATP needed, causing fatigue and muscle weakness. (Roland, 2019)

Amylases can also be found in plants, animals, and microorganisms such as bacteria and fungi. Since microbial amylase is so easily available, it has been favored by industries over any other source (Mishra, Behera, 2008). They are utilized in a wide range of industries such as food, detergent, and pharmaceutical. They have numerous uses, including aid in maltose and high fructose syrup production, and the improvement of cleaning effects in detergents (Vidyalakshmi, Paranthaman, Indhumathi, 2009).

Knowing enzymes’ optimal conditions can allow one to acquire the best results when utilizing them. In this experiment, fungal, specifically aspergillusoryzae, and bacterial amylase were put in a range of temperatures to determine their optimal conditions. This was done by bathing a test tube with a mixture of amylase and sucrose in four different temperatures that ranged from 0 to 85 degrees Celsius in 2-minute intervals and adding it to iodine to indicate whether the sugar had broken down. Amylase is usually found in temperatures around 37 degrees Celsius, therefore it was predicted that their optimal temperature would be between 25 degrees and 55 degrees Celsius (Mishra, Behera, 2008).

In this experiment time and temperature were two variables that were being controlled, therefore referred to as independent variables. The dependent variable was the color of the iodine after the mixture of amylase and sucrose was heated. The color varied from light yellow, which meant the sugar was broken down, to black, indicating that the sugar was not broken down. To ensure the experiment was done correctly, a negative control group was used. This was the sucrose by itself at zero minutes in which no response was expected since it did not have any amylase in it, therefore could not have been broken down.

Methods

To test the optimal temperature of the amylases, first, two spot plates were placed on two napkins that were labeled bacterial and fungal. On the top four rows, the temperatures were labeled 0, 25, 55, and 85 degrees Celsius. The columns were labeled in minutes, starting with 0 and adding two minutes per column until ten minutes were reached. Next, sixteen test tubes were labeled, eight were for 5mL 1.5% starch solution, four for 1mL bacterial amylase, and four for 1mL fungal amylase.

They were then put into their corresponding station depending on the temperature. For 0 degrees Celsius, the tubes were put in a beaker filled with crushed ice and for 25 degrees Celsius, the tubes were placed in a beaker that contained room-temperature water. Lastly, for 55 and 85 degrees, the tubes were place in individual water baths for each temperature. For the first five minutes they sat in their respective beaker to equilibrate. After the five minutes, about three drops of iodine were added in each spot for time zero of the spot plate, and three drops of the starch was added to the matching temperature with a pipette without taking the amylase tube out of their beakers to avoid temperature change.

Then, the starch was added to the amylase test tube and mixed gently with the pipette by absorbing it and releasing it back inside the test tube. The timer was set for two-minute intervals. After each interval, three drops of iodine were added to the corresponding spot and three drops of the amylase and starch solution were added to the iodine at their corresponding temperatures. They were each mixed with different toothpicks to avoid any contamination. After this occurs, the color of the mixture should indicate the optimal temperature for both the bacterial and fungal amylase. (Alberte, Pitzer, Calero, 2012)

Data

In this image, most enzyme activity is seen in the yellow color at 55 degrees Celsius. The Dark brown and black colors at 0, 25, and 85 degrees Celsius indicate little to no enzyme activity.

Data Table 1: Class Average and Standard Deviation of the Effect of Temperature on Bacterial Amylase
0 °C		25°C		55°C		85°C
Time (Minutes)	Average	Standard Deviation	Average	Standard Deviation	Average	Standard Deviation	Average	Standard Deviation
0	4.67	0.52	4.50	0.55	4.67	0.52	5.00	0.00
2	4.33	0.41	3.00	0.75	3.33	0.82	4.67	0.52
4	3.83	0.00	3.83	0.84	2.67	0.82	4.67	0.52
6	4.00	0.55	3.50	1.17	2.33	0.84	4.67	0.52
8	4.00	0.98	3.17	1.38	2.50	0.75	4.67	0.52
10	4.17	0.98	3.50	1.38	2.17	0.75	4.33	0.52

After numbering the colors that indicate the bacterial amylase activity, the results were shared and organized into a table which showed the mean of each temperature at every time period as well as the standard deviation. The lowest numbers are at 55 degrees Celsius, followed by 25, 0, and lastly 85.

Class Average of the Effect of Temperature on Bacterial Amylase

The colors were numbered one through five. One being the lightest color and five the darkest. In this graph, the temperatures have their assigned colors and the lower the line is, the lighter the color in the spot plate.

In this spot plate, little to no enzyme activity is shown given the dark colors on the spot plate. This indicates an error in the experiment.

Data Table 2: Class Average Standard Deviation of the Effect of Temperature on Fungal Amylase
0 °C		25°C		55°C		85°C
Time (Minutes)	Average	Standard Deviation	Average	Standard Deviation	Average	Standard Deviation	Average	Standard Deviation
0	5.00	0.00	4.50	0.84	4.67	0.52	5.00	0.00
2	4.33	0.82	4.17	0.41	3.83	0.75	5.00	0.00
4	4.50	0.84	4.50	0.55	4.00	0.63	5.00	0.00
6	4.50	0.84	4.50	0.55	3.83	0.75	5.00	0.00
8	4.33	0.82	4.33	0.82	3.83	0.75	5.00	0.00
10	4.33	0.82	4.17	0.75	4.00	1.10	5.00	0.00

In this table, the class the mean and standard deviation are shown. Overall, the lightest color was at 55 degrees Celsius and the darkest at 85.

Class Average of the Effect of Temperature on Fungal Amylase

The graph shows the enzyme activity, indicated by the color in the y axis—the higher the number, the darker the color—as the time passed shown in the x axis.

Results

In the bacterial amylase group experiment, the 25 degrees Celsius and 85 degrees Celsius had the darkest colors, being black, as well as the spots at time zero. The third darkest color was dark brown at 0 degrees Celsius, followed by light brown at 55 degrees from times two and four, and yellow from times six to ten. For fungal amylase, 0 degrees Celsius had the lightest color, being dark brown, and the rest were black with the exception of time zero at 85 degrees Celsius and time four at 55 degrees Celsius.

The colors were numbered from one to five, one being the lightest, or yellow; and five being the darkest, or black. Given this information, the data tables were created by taking the mean of the class trials as well as the standard deviation. The information was then put into a graph which showed color on the y-axis and time on the x-axis. Each temperature tested had a different shade assigned to it, demonstrating each amylase activity occurred throughout the ten-minute time period. In the bacterial amylase graph, greater changes are observed than in the fungal amylase graph. In the bacterial amylase graph the greatest numbers, indicating the darkest colors, occurred when the temperature was 85 degrees Celsius.

The lowest overall numbers, which meant the lightest colors, occurred at 55 degrees Celsius. In the fungal amylase graph, the highest number occurred when the temperature was 85 degrees. Unlike for bacteria, this color remains constant throughout all of the time intervals. For 0, 25, and 55 degrees Celsius, the colors are very similar throughout and indicate a dark shade of brown, but 55 degrees Celsius is the lightest shade, which can be seen as it is the lowest line in the graph.

Discussion

In this experiment, the effect of temperature on bacterial and fungal amylase was tested. Amylase is an enzyme that breaks down sucrose into smaller molecules. In humans, it is found in the saliva and pancreas; but it is also found in bacteria and fungus.

The experiment included having a mix of the amylase in sucrose and adding it to iodine to observe its activity under certain temperatures. Under the ideal conditions, the enzyme would break down the sugar and when mixed with iodine, the color would be a light yellow. If there was still sugar present, the color would be brown or black since that is the color of sucrose and iodine mixed together (Clark, 2018). In the bacterial spot plate picture, the optimal temperature is clearly seen to be 55 degrees Celsius since it has the lightest color, meaning more enzyme activity. The least amount of activity is seen at 25 and 85 degrees Celsius given the dark brown and black colors. This meant that at those temperatures the enzyme had denatured and therefore could not perform its function.

The fungal amylase plate does not show an optimal condition for the amylase, this is due to errors in the experiment. The class data was gathered and shown in the graphs. This is important because repetition in an experiment leads to more accurate results. The bacterial amylase graph shows that the optimal condition for this enzyme is about 55 degrees Celsius since it ends up having the lightest color at the ten-minute mark. On the other hand, the fungal amylase graph seemed to have darker colors throughout the experiment, meaning little to no activity occurred with any of the temperatures. Given that, the lightest overall color still matched with 55 degrees Celsius, suggesting that if the experiment had been done correctly, that would have been the optimal temperature. In both the bacterial and fungal amylase tests it was shown that the ideal temperature was around 55 degrees Celsius, although the results were not an identical match with the hypothesis.

This was a result of errors in the experiment. Factors that could have affected this could have been the temperature of the room, not having the test tube with the enzyme and sucrose mix under the exact temperature water bath, contaminated test tubes or pipettes, not mixing the exact amount of drops of the solution with the iodine, not leaving the enzyme and sucrose mix in their corresponding water baths for the required two-minute period, among others. Errors are part of every experiment, but it is important to minimize them as much as possible to acquire the most accurate results.

This can be achieved by being more careful and paying more attention to the surroundings and small details of the experiment, making sure everything is clean, controlling as much of the outside environment as possible, as well as being overly informed on the procedures and the expected results based on previous research.

References

Alberte J., Pitzer T., Calero K. (2012). General Biology Lab Manual / Second Edition. Florida International University: The McGraw Hill Companies.
Clark, Mary Ann (2018) “Enzymes - Biology 2e.” OpenStax, openstax.org/books/biology-2e/pages/6-5-enzymes.
Cooper, G. M. (2000). The Central Role of Enzymes as Biological Catalysts. NCBI https://ncbi.nlm.nih.gov/books/nbk9921
Mishra, S., & Behera, N. (2008). Amylase activity of a starch degrading bacteria isolated from soil receiving kitchen wastes. African Journal of Biotechnology, 7(18).
Roland, James. (2019) “Why Are Enzymes Important?” Healthline, Healthline Media, www.healthline.com/health/why-are-enzymes-important. replace
Vidyalakshmi, R., Paranthaman, R., & Indhumathi, J. (2009). Amylase production on submerged fermentation by Bacillus spp. World Journal of Chemistry, 4(1), 89-91.
Whitcomb, D. C., & Lowe, M. E. (2007). Human pancreatic digestive enzymes. Digestive diseases and sciences, 52(1), 1-17.