The Hydrolyzation of Starch
Abstract
There are thousands of different types of enzymes with each enzyme with its own optimal temperature . Optimal temperature is where the enzyme works at its best. If enzymes kept at the wrong temperature the enzyme will start losing its shape(denatured). Fungal amylase and bacterial amylase were examined to determine their optimal temperature. It was hypothesized that fungal amylase’s optimal temperature was going to be 55 degrees Celsius with a time of 6 minutes. It was also hypothesized that bacterial amylase’s optimal temperature was going to be 85 degrees Celsius with a time of 8 minutes. Fungal amylase and bacteria amylase were each mixed with starch at different temperatures and were left there for two minutes. This was done for 10 minutes for each amylase. The optimal temperature for bacterial amylase was at 85 degrees Celsius at the 4-minute mark because this is when the starch is first fully hydrolyzed. The optimal temperature for fungal amylase was between 0 and 25 degrees Celsius at the 0-minute mark because this is when the starch is fully hydrolyzed. The data obtained in this lab is inconclusive because using the iodine test is not an accurate way to determine if the starch is still present or not. While the lab was being done more than two drops of fungal amylase and starch which affected the results. These findings have no significant impact in determining the optimal temperature because of the critical errors made.
Introduction
Enzymes are catalyst that speed up a chemical reaction using low activation energy. At the molecular level, enzymes catalyze biochemical reactions by accelerating the conversion of substrates into products in a buried pocket within the active site of the enzyme (Verma & Mitchell-Koch ,2017). It was determined that enzymes specificity and catalytic power came from the inflexible fit of the right substrate onto the preformed enzyme surface, the way a key fits a lock also known as the lock and key model (Ringe & Petsko, 2008). Optimal temperature is where the enzyme works at its best. If enzymes are kept at the wrong temperature the enzyme will start losing its shape(denatured). The factors that could affect the proper function of an enzyme are pH, substrate concentration, salt concentration, the presence of inhibitors, activators and cofactors (Alberte et al.,2012). Any of these factors found in the enzyme could have denatured (changed in shape) the enzyme preventing substrate binding.
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Without enzymes most in life will not be useful in life. Enzymes has improved the lives of people. Fungal amylases are used for hydrolyzing carbohydrate, protein and other constitutes of soybeans, wheat into peptides, amino acid, sugars and other low molecular weight compounds (Naidu &Saranraj, 2013). The application of an amylase in industrial reactions depends on its unique characteristics, such as its action pattern, substrate specificity, major reaction products, optimal temperature, and optimal pH12 (Saranraj & Naidu, 2013). Amylases are enzymes which hydrolyze starch molecules to give diverse products including dextrin and progressively smaller polymers composed of glucose units (Naidu &Saranraj, 2013). When starch and fungal amylase was mixed the optimal temperature was 55 degrees Celsius with a time of 6 minutes because at this temperature the starch was fully hydrolyzed. When starch and bacterial amylase was the optimal temperature was 85 degrees Celsius with a time of 8 minutes because at this temperature the starch was fully hydrolyzed.
To determine the optimal temperature of the fungal amylase and the bacterial amylase this experiment was done. The temperatures used to determine the optimal temperature were 0 degrees Celsius, 25 degrees Celsius, 55 degrees Celsius and 85 degrees Celsius. As the optimal temperature was being tested, the experiment provided the opportunity to examine the effect of temperature on the ability of amylase to break down starch to maltose (Alberte et al., 2012). The iodine test was used to determine the optimal temperature. The iodine test turns yellow to blue-black in the presence of starch.
Methods
Before the experiment is done the proper procedures of cleaning up has to be reviewed. Placed a napkin under the spot plates and wrote the temperatures 0 degrees Celsius, 25 degrees Celsius, 55 degrees Celsius, and 85 degrees Celsius and wrote on the side the time trials of 0, 2, 3, 4, 6, 8, 10 minutes. 4 test tubes were labeled FA and 4 other test tubes was labeled S 4 test tubes labeled FA are obtained then fill each up with 1 mL of fungal amylase. When the four test tubes are filled with fungal amylase, then another 4 test tubes labeled S was each filled up with 5 mL of 1.5% of starch solution. The 8 test tubes were placed to the temperature they belonged. The 0 degrees Celsius was placed in a beaker filled with ice, the 25 degrees Celsius was placed in water while 55 degrees Celsius and 85 degrees Celsius were placed in their respective temperature. All 8 test tubes were allowed 5 minutes to adjust to their temperature. Two iodine drops were added to O minutes row then the starch and amylase tubes were mixed. After being mixed drop two drops to O minutes. Set the timer for 2 minutes. When the 2 minutes is reached add two iodine drops and two drops of starch-amylase is also added. This process is repeated until the 10 minutes are reached. The data of which temperature the starch was fully hydrolyzed is recorded. The same procedure is taken for the bacterial amylase. The data is also recorded for this amylase.
Results
The Fungal Amylase
0
25
55
85
Group 1
3
4
4
5
Group 2
4
4
4
5
Group 3
4
4
4
5
Group 4
3
2
3
5
Group 5
1
1
4
5
Group 6
4
4
4
5
0 minutes Mean ± SD
3.166666667
3.166666667
3.833333333
5
Group 1
1
2
2
5
Group 2
2
2
2
5
Group 3
2
2
3
5
Group 4
3
2
3
5
Group 5
2
2
3
5
Group 6
3
3
3
5
2 minutes Mean ± SD
2.705128205
2.705128205
3.294871795
5
Group 1
1
2
2
5
Group 2
2
2
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
3
5
Group 6
3
3
3
5
4 minutes Mean ± SD
2.333333333
2.5
2.666666667
5
Group 1
1
2
2
5
Group 2
3
2
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
2
5
Group 6
3
3
2
5
6 minutes Mean ± SD
2.5
2.5
2.333333333
5
Group 1
1
2
2
5
Group 2
3
2
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
2
5
Group 6
3
3
2
5
8 minutes Mean ± SD
2.5
2.5
2.333333333
5
Group 1
1
2
2
5
Group 2
3
3
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
2
5
Group 6
2
2
2
5
10 minutes Mean ± SD
2.333333333
2.5
2.333333333
5
OPTIMAL TEMP.
0-25
TIME TO 100% HYDROLYSIS AT OPTIMAL TEMP.
0
From the results the mean was taken. The highest color change according to iodine test was 2 which is medium yellow.
The bacterial amylase mean
0
25
55
85
Group 1
Group 2
4
4
4
5
Group 3
5
5
5
5
Group 4
5
5
5
5
Group 5
5
5
5
5
Group 6
4
4
5
5
0 minutes Mean ± SD
4.6
4.6
4.8
5
Group 1
Group 2
3
3
3
4
Group 3
4
3
4
3
Group 4
3
3
2
3
Group 5
3
2
4
2
Group 6
3
3
3
3
2 minutes Mean ± SD
3.2
2.8
3.2
3
Group 1
Group 2
3
2
3
2
Group 3
4
4
4
2
Group 4
4
3
2
2
Group 5
4
4
4
3
Group 6
3
3
3
2
4 minutes Mean ± SD
3.6
3.2
3.2
2.2
Group 1
Group 2
3
3
3
2
Group 3
4
4
4
2
Group 4
3
3
2
1
Group 5
3
4
4
2
Group 6
3
3
4
1
6 minutes Mean ± SD
3.2
3.4
3.4
1.6
Group 1
Group 2
3
3
3
2
Group 3
4
4
3
1
Group 4
4
4
3
1
Group 5
4
4
4
2
Group 6
3
3
4
1
8 minutes Mean ± SD
3.6
3.6
3.4
1.4
Group 1
Group 2
3
3
3
1
Group 3
4
4
3
1
Group 4
5
3
3
1
Group 5
3
4
4
2
Group 6
3
3
5
1
10 mintues Mean ± SD
3.6
3.4
3.6
1.2
OPTIMAL TEMP.
85
TIME TO 100% HYDROLYSIS AT OPTIMAL TEMP.
10
From the results the mean was taken. It turned yellow at the optimal temperature of 85 at minute 10.
The fungal amylase standard deviation
0
25
55
85
Group 1
3
4
4
5
Group 2
4
4
4
5
Group 3
4
4
4
5
Group 4
3
2
3
5
Group 5
1
1
4
5
Group 6
4
4
4
5
0 minutes Mean ± SD
1.169045194
1.329160136
0.40824829
0
Group 1
1
2
2
5
Group 2
2
2
2
5
Group 3
2
2
3
5
Group 4
3
2
3
5
Group 5
2
2
3
5
Group 6
3
3
3
5
2 minutes Mean ± SD
0.752772653
0.40824829
0.516397779
0
Group 1
1
2
2
5
Group 2
2
2
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
3
5
Group 6
3
3
3
5
4 minutes Mean ± SD
0.816496581
0.547722558
0.516397779
0
Group 1
1
2
2
5
Group 2
3
2
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
2
5
Group 6
3
3
2
5
6 minutes Mean ± SD
0.836660027
0.547722558
0.516397779
0
Group 1
1
2
2
5
Group 2
3
2
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
2
5
Group 6
3
3
2
5
8 minutes Mean ± SD
0.836660027
0.547722558
0.516397779
0
Group 1
1
2
2
5
Group 2
3
3
3
5
Group 3
3
3
2
5
Group 4
3
2
3
5
Group 5
2
3
2
5
Group 6
2
2
2
5
10 minutes Mean ± SD
0.816496581
0.547722558
0.516397779
0
OPTIMAL TEMP.
0-25
TIME TO 100% HYDROLYSIS AT OPTIMAL TEMP.
0
From table 1 the standard deviation was taken from. Fungal amylase had an optimal temperature between 0 and 25 according to group 5.
The bacterial amylase
0
25
55
85
Group 1
Group 2
4
4
4
5
Group 3
5
5
5
5
Group 4
5
5
5
5
Group 5
5
5
5
5
Group 6
4
4
5
5
0 minutes Mean ± SD
0.547722558
0.547722558
0.447213595
0
Group 1
Group 2
3
3
3
4
Group 3
4
3
4
3
Group 4
3
3
2
3
Group 5
3
2
4
2
Group 6
3
3
3
3
2 minutes Mean ± SD
0.447213595
0.447213595
0.836660027
0.707106781
Group 1
Group 2
3
2
3
2
Group 3
4
4
4
2
Group 4
4
3
2
2
Group 5
4
4
4
3
Group 6
3
3
3
2
4 minutes Mean ± SD
0.547722558
0.836660027
0.836660027
0.447213595
Group 1
Group 2
3
3
3
2
Group 3
4
4
4
2
Group 4
3
3
2
1
Group 5
3
4
4
2
Group 6
3
3
4
1
6 minutes Mean ± SD
0.447213595
0.547722558
0.894427191
0.547722558
Group 1
Group 2
3
3
3
2
Group 3
4
4
3
1
Group 4
4
4
3
1
Group 5
4
4
4
2
Group 6
3
3
4
1
8 minutes Mean ± SD
0.547722558
0.547722558
0.547722558
0.547722558
Group 1
Group 2
3
3
3
1
Group 3
4
4
3
1
Group 4
5
3
3
1
Group 5
3
4
4
2
Group 6
3
3
5
1
10 mintues Mean ± SD
0.894427191
0.547722558
0.894427191
0.447213595
OPTIMAL TEMP.
85
TIME TO 100% HYDROLYSIS AT OPTIMAL TEMP.
10
From Table 2 the standard deviation was taken from. The optimal temperature was 85.
The fungal amylase
According to group 2, the optimal temperature of the fungal amylase was 55 degrees Celsius. Also according to this group, the presence of starch was fully there in 85 degrees Celsius.
The bacterial amylase
According to group 2, the optimal temperature was between 0 and 55 degrees Celsius. Also according to group 2 the presence of starch was in 85 degrees Celsius.
Discussion
According to group 5, the fungal amylase optimal temperature was between 0 to 25 degrees Celsius at minute 0. In table 4 the average color was middle yellow which proves that an error was made. More than two drops of iodine were added. According to table 3, the bacterial amylase optimal temperature was 85 degrees Celsius at the 10th minute.
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It can be concluded from my graphs and tables that the hypothesis was accepted because if starch and amylase are not at their optimal temperature, the starch will not be hydrolyzed. The results showed that if the temperature had a blue-black color then the amylase did not break down starch. In other words, it meant that the starch was fully present. If the temperature had a yellow, then the amylase broke down the starch. In other words, the starch was fully hydrolyzed. The results also accepted the hypothesis that if the enzymes are not in their optimal temperature then the enzyme will have a different shape because the enzyme was denatured At low temperatures decrease the low movement of molecules, resulting in less contact between enzymes and substrates, which slows down the frequency and the rate of reaction and ultimately diminishes product formation (Alberte et al.,2012). Amylase catabolizes starch polymers (a storage polysaccharide) into smaller subunits (monomers = saccharides) including maltriose, maltose and short oligosaccharides comprised of 2-20 monosaccharide units (Alberte et al., 2012). Proteases and amylases, which are produced by the body to break down protein and starch are also used commercially to bake bread, biscuits and crackers (Alberte et al.,2012). Filamentous fungi have been used for the industrial production of a wide varietyof native products, such as antibiotic (e.g., penicillin andcephalosporin), organic acid (Citric and acetic acid) andcommercial enzymes (e.g., protease, catalase, amylase) (Saranraj & Stella, 2013).
The teaching assistant committed an error he forgot to say the chart that explained the colors at which the starch is fully hydrolyzed or when starch is present. He said it after the data was taken which could have caused the data to be misinterpreted. The test tubes were not cleaned enough after the first lab use. This data does not account for human error because many students added more than two drops of amylase-starch and iodine. The data obtained in this lab is inconclusive because using the iodine test is not an accurate way to determine if the starch is still present or not. While the lab was being done more than two drops of fungal amylase and starch which affected the results. These findings have no significant impact in determining the optimal temperature because of the critical errors made. What could be done to improve the experiment is improve communication between the testers and the teaching assistant. The test tubes could have been new so none of the residue left behind could have affected the experiment. The drops of starch, iodine and amylase could have been added more accurately. For future testing of the breakdown of starch more temperatures should be added so that the results could be accurate. The teaching assistant could have done one to compare it to the ones’ the students did.
References
Alberte J., Pitzer T., Calero K. (2012).General Biology Lab Manual / Second Edition. Florida International University: The McGraw Hill Companies.
Ringe D., Petsko G. A. (2008). How Enzymes Work. Science 320: pp. 1428.
Saranraj P., Naidu. (2013) A Bacterial Amylase A Review
Saranraj P., Stella D. (2013) A Fungal Amylase Review
Velma R., Mitchell-Koch K. (2017) In Silico Studies of Small Molecule Interactions with Enzymes Reveal Aspects of Catalytic Function. Wichita State University
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