The Outcome Of Breaking Down Starch With Bacterial And Fungal Amylase PDF

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The Outcome Of Breaking Down Starch With Bacterial And Fungal Amylase

The Outcome of Breaking Down Starch With Bacterial And Fungal Amylase in Temperatures: 0°,25°,55°,85° Emily Andrade PID: 5874172 Lab Partners: Leidy Felipe Drew Blumenthal Nathalie Medina Florida International University BSC-1010L U25

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The Outcome Of Breaking Down Starch With Bacterial And Fungal Amylase

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Abstract: Enzymes are biological catalysts that need to have a low activation energy for a chemical reaction to happen. Enzymes can be found in humans salvia, which is when the breakdown of starch happens. Enzymes are found anywhere, even in foods or home products that are used to clean . Products like detergent, syrup, dairy, avocados, and bananas amongst many others contain enzymes. Starch polymers is being broken down when the monomer becomes even smaller during Fungal and Bacterial amylase. The reaction that was asked to be recorded was how the fungal and bacterial amylase reacted towards the breakdown of starch to maltose in multiple temperatures. The independent variable in this experiment were the temperatures which varied from 0°, 25°, 55°, and 85° Celsius of both Aspergillus oryzae and Bacillus Licheniformis. The optimal temperature was decided after when the colors on the spot plate were observed and compared to a starch hydrolysis color-coding scheme provided for us in the lab manual (Alberte, 56). This happens to be the dependent variable in this experiment. When enzymes are exposed to these types of temperature changes they are not able to function correctly because enzymes need to be maintained at a certain temperature so it could function at its optimal temperature. If temperatures keep increasing and decreasing and not kept at a optimal temperature it would disrupt the activity the enzyme are producing. Introduction: In France, 1833 a chemist Anselme Payen discovered the first enzyme. But before that Gottlieb Sigismund Kirchoff in 1812 was already investigating the procedure enzymes undergo. (Gurung, Ray, Bose, Rai, 2013) Enzymes are one of the most important catalyst. Catalysts are like enzymes they work by reducing the amount of energy required for a chemical reaction to

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take place. Enzymes are known to be complex proteins produced by all living organisms, they can speed up chemical reactions in the cell. The type of reactions that cells can undergo are cellular respiration, photosynthesis, DNA replication, Digestion and making new proteins. Emil Fischer is the man that came up with the “lock and key” model for the relationship between enzymes and their substrates. (Copeland, 2000). The lock and key model proposes that the active site, which is the “lock”, is not rigid so a substrate combines with binding groups. That changes the shape of the enzyme but only because the enzyme took in the substrate. (Martin, 1992) The “lock” is the fluid wrapping itself around a key of the correct type to make it fit closer. The change of shape in the enzyme is what reduces the activation energy of the reaction. (Martin, 1992) Amylase is an enzyme that catalyzes the breakdown of starch into sugars. This enzyme can be found in the human body: salvia and the pancreas. The importance of enzymes in a humans everyday life is important, it can be affected if there is an absence of an enzyme. Every enzyme in our body has a different role and if one of these enzymes like pepsin, trypsin, lipase, amylase, are absent then this can cause serious illnesses or even death. (Selisteanu,Tebbani, Roman, Petre, Georgeanu, 2014) Homeostasis is maintained when functions work properly. Enzymes function better in their own environment, which is the optimum temperature. (Alcaíno, Cifuentes, Baeza, 2015). Enzyme activity is impacted by various of factors like as pH, temperature, substrate, enzyme cofactors, inhibitors and activators (Engel, 1981; Raven, 2011). After the optimum temperature is reached the reaction eventually begins to slow down and stop. Making a solution more acidic or alkaline will make the reaction slow down. Enzymes during digestion are used to working faster in unusual pH conditions. Enzymes work better when there is a plethora of substrates available, as the substrate increases so does the enzymes activity.

The Outcome Of Breaking Down Starch With Bacterial And Fungal Amylase

(Alberte, 49) When the enzyme has a higher than the optimal temperature, the enzyme denatures the active site changing the shape of the whole enzyme. At lower temperatures than the optimal temperature the whole enzyme ends up being slow because there is no hydrogen bonds to be broken apart from the interactions. (Alberte, 51) In order to examine the effect that temperature has on enzymatic activity it will be presented as starch amylase since it is a type of enzyme it will be studied in various temperatures. Optimal temperature is determined when hydrolysis happens, the reason why is because that makes starch break down. The optimal temperature for Asperigillus or Fungal amylase and the Bacillus Licheniformis or Bacterial amylase are between 20° and 85°. Fungal’s optimal temperature ranges between 20°and 40°, and Bacterial’s optimal temperature is around 70°-95°. If these enzymes are exposed to other temperatures they will denature and kill themselves. Methods Place two paper towels and under each spot plate, label the top of the paper towels “Temperature” with the values of 0°, 25°,55°,85° Celsius. Towards the side of towel write the times on them which were 0, 2,4,6,8, 10 minutes. Grab 8 test tubes, grab enough tape to label each of them. 4 of them label them with the group number given, the temperatures, and enzyme source, either fungal or bacterial. Then repeat this step but what is done differently this time is that you include the letter S, which stands for starch. Separate them after, make sure there is no confusion between these 8 test tubes. After the labelling you have to grab the plastic pipette pump and a glass pipette so the starch and amylase would be able to be placed in the tubes. Since amylase and starch are placed separately split the group in half and put the required amount of either amylase or starch in the

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The Outcome Of Breaking Down Starch With Bacterial And Fungal Amylase

tubes. 5ml of starch should be placed in all four tubes that have the “S” on it, and 1ml inside the tubes that are labeled “F.” After you have to place the test tubes in their designated bath stations. After you let the tubes equilibrate to the temperature for 5 minutes. The group member who stays at the table is responsible for adding iodine on each hole on the spot plate. When the 5 mins are over you are required to stay at that station because you have to make sure precipitation isn’t created so you go ahead and move the test tube in the bath while the time is running. You could change designated stations between your group just have the communication because people might get confused and end up at the wrong station. Rotation is desired depending on the group you are working with. As the minute go by you are supposed to go back to the table with the tube and you are supposed to add 2-3 drops of the substance in the designated hole. It does not matter which goes first either the amylase or starch but it is the same process. To mix the mixture on the spot plate you use a toothpick, you use a different one every time you have to mix just to be safe. You have to collect date as the experiment goes on. After the experiment is done you clean the test tubes out, clean your area and make it look spotless. Make sure pictures are taken and results are written down. Results: The bacterial amylase optimal temperature should have been 85° Celsius. The result shown is way different then what we imagined it to be. In both Bacterial and Fungal the optimal temperature turned out to be 0° Celsius. In the experiment two errors happened, one of them dealt with out Lab TA giving us contaminated amylase. Mario did not notice that the amylase was contaminated but the lab kept going, it was already too late to start the process all

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over again. The second error was that when we were waiting for those 5 minutes to give the substance an equilibrium my lab partner, Leidy Felipe, and I switched tubes by mistake. The 25° was place in the 85°Celsisus bath which could have triggered our results to turn out to be completely wrong. Bacterial Amylase Average

Table 1.

Minutes Temperature

0

2

4

6

8

10

(°C)

Total Average

0°

2

2

2

2.1

1.2

2.3

1.9333

25°

2.3

1.8

2

2.3

2.3

1.2

1.9833

55°

1.8

1.8

2

2

2.2

2.5

2.05

Table 1. Shows the class average for the bacterial amylase in different temperatures. The class average is found by combining the temperature and minutes together. No starch represents when the lowest number has a lighter color than the other substances in each hole in the spot plates. In this case Bacterial amylase’s optimal temperature is at 0° Celsius. Graph 1. STARCH CONCENTRATION

Bacterial Amylase Average 5 4 3

0°

2

25°

1

55°

0

85° 0

2

4

6

Minutes

8

10

Total Average

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The Outcome Of Breaking Down Starch With Bacterial And Fungal Amylase

Graph 1. The line graph demonstrates the bacterial amylase average. You could tell that there was some kind of error in this experiment because the 0 minutes all groups are supposed to start at the same rate but that did not occur. Fungal Amylase Average

Table 2.

Minutes Temperature (°C)

0

2

4

6

8

10

Total Average

0°

1.7

2.6

2.5

2.6

2.6

2.6

2.4333

25°

2.3

2.8

2.6

3

3

2.8

2.75

55°

2.2

2.8

2.6

2.6

2.6

2.8

2.6

85°

3.6

3.6

3.8

3.5

3.8

3.8

3.6833

Table 2. Shows the class average for the bacterial amylase in different temperatures. The class average is found by combining the temperature and minutes together. No starch represents when the lowest number has a lighter color than the other substances in each hole in the spot plates. In this case Fungal amylase’s optimal temperature is at 0° Celsius.

Fungal Amylase Average Starch CONCENTRATION

Graph 2. 4 3.5 3 2.5 2 1.5 1 0.5 0

0°

25° 55° 85° 0

2

4

6

Minutes

8

10

Total Average

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Graph 2. The line graph demonstrates the bacterial amylase average. You could tell that there was some kind of error in this experiment because the 0 minutes all groups are supposed to start at the same rate but that did not occur.

Figure 1 Figure 1. Shows the iodine test for the Fungal amylase. Starch is seen when the mixture turns a blueish/blackish color. Iodine was placed to determine the optimal temperature, that will indicate if the starch was hydrolyzed into maltose.

Figure 2 Figure 2. Shows the iodine test for the Bacterial amylase. Starch is seen when the mixture turns a blueish/blackish color. Iodine was placed to determine the optimal temperature, that will indicate if the starch was hydrolyzed into maltose.

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Discussions After evaluating the experiments results, Table 1 and 2 has enough evidence to support the hypothesis and predictions when the temperature is optimal for an enzyme it will denature the function. The optimal temperature of fungal amylase indicates in my lab that the Asperigillus Oryzea was at 0° Celsius. Enzymes function better in their own environment. (Alcaíno, Cifuentes, Baeza, 2015). When the enzymes aren’t in their optimal temperature they can denature. The amylase was fully broken down at at 0° Celsius, the optimum temperature for Fungal amylase is estimated to be around 40° Celsius. The denaturing occurred at 55° and 85° Celsius. The optimal temperature for bacterial amylase was as well at 0° Celsius. Bacterial amylase optimal temperature is at 70° Celsius. Bacteria prokaryotes are active throughout the hot areas. The amylase was broken down fully at 0° Celsius, and denatured at 55° and 85° Celsius. The contamination affected both amylase’s and it made the experiment work back words. I reject my null hypothesis because both my hypothesis of fungal and/or bacterial amylase hydrolyzed 100% during the optimal temperature which was 0° Celsius. I support my hypothesis because the temperature for the enzyme to react is its optimal temperature therefore it does not matter. At 0° for both fungal and bacterial temperatures were extremely cold which made the the process slow, but it in this case it made the amylase react fast. The formation of maltose includes the breakdown of starch. The formation of maltose is when a polysaccharide makes a glucose molecule to maltose which is considered a monosaccharide. (Diaz, McDurmon, and Rushton, 2008) A chemical reaction happens when the iodine starts getting a darker color. The colors go from yellow to dark blue and a dark blue. The reason this happens is because there is no reaction occurring between them so the enzymes end up forming a enzyme substrate complex. (Diaz,

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McDurmon, and Rushton, 2008) Optimal temperature did occur on both fungal and bacterial amylase and is seen through starch hydrolysis. In any laboratory setting, there are numerous factors of error. Errors happen naturally in experiments, you just have to avoid them to get the most accurate results. One of the errors that can happen is when the experiment has possible delay in time when adding the starch or the iodine. You have to work as quickly as possible when you are working with such sensitive elements. Another error could be at the bath stations where it is required to fully leave the test tubes in the water and you mistakenly remove it before time can cause error. The room temperature might set an imbalance to the substance inside the test tube. After taking the test tubes out it is required to combine the iodine with the specific enzyme, there could be error just because there wasn’t enough stirring. Adding too many drops or too few drops to each spot on the spot plate can cause error as well. The results were completely flipped, but an optimal temperature was seen as well as the denaturing state of the enzyme. In Figure 1 and 2, a picture of the visual results were added. It is rare to see how the color scheme on the lab manual is correct when stating when the optimal temperature occurred. It is also seen how the group had many errors, some of the spot plates holes were almost overflowing meaning there was too much of either the bacterial or fungal amylase. There was no type of organization due to the fact that there were a lot of toothpicks around the experiment. Having everything neat and in order is very important for these kinds of experiment. Organization is needed just because working with these kinds of enzymes can mess up the results.

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Bibliography Alberte, J. el al. (2012). Enzymes. In: General Biology 1 Lab Manuel 2nd Edition., pp49-61. New York: Mc-Graw Hill. Alcaíno Gorman, J., Cifuentes Guzmán, V., & Baeza Cancino, M. (2015). Physiological adaptations of yeasts living in cold environments and their potential applications. Chandrasekaran, M. (2016). Enzymes in food and beverage proccesing (1st ed.) CRC Press Taylor & Francis group. Copeland, A. R. (2000). The development of mechanistic enzymology. Enzymes (Second ed., pp.

4) JOHN WILEY & SONS, INC.

Dias, Michael, Grant McDurmon, and Gregory T. Rushton. “Enzyme inquiry :a coupled inquiry lesson explores the catalytic activity of amylase on starch.” The Science teacher 75.6 (2008): 60. Student resources In Context. Web. 17 2016. Engel, Paul C. “Enzyme kinetics The steady- state Approach.” 2nd ed. New York: Chapman and Hal, 1981. 7-11, 16,89. Print.

Gurung, N., Ray, S., Bose, S., & Rai, V. (2013). 2013 Raven. el al. “Enzyme: Biological Catalyst.” Biology 9th edition. Ed. Lisa Brudflodt and Rose M. Koos. New York: Mc-Grawhill, 2011. 118-199. Print.

Rowland, M. (1992). Lock and key model of enzyme activity. Biology (First Edition ed., pp. 108) Thomas Nelson and Sons Ltd.

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Selişteanu, D., Tebbani, S., Roman, M., Petre, E., & Georgeanu, V. (2014). Microbial production of enzymes: Nonlinear state and kinetic reaction rates estimation. Biochemical Engineering Journal, 91, 23-36.

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