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The Effect of Temperature on Enzyme Activity
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Hire a WriterThis lab was about the examination of how the activity of enzyme amylase is affected by the variations in temperature. Moreover, it determined the optimal temperatures for the fungal and bacterial amylases. It was hypothesized that the active site denatures, preventing or reducing substrate binding at temperatures that are greater than the optimal. As a consequence, the formation of product is either completely arrested or reduced. The experimental procedure involved placing a napkin/ paper under the spot plates and across the top write temperature (00C, 250C, 650C, 850C) as well as on the Time’s side write (0, 2, 4, 6, 8, and 10 minutes). After allowing the test tubes to equilibrate, the data were recorded in various tables. Fungal and bacterial amylases were presented in tables 1 and 2. Also, tables 3 and 4 were used to show the behavior of time and temperature of bacterial and fungal amylase. Finally, the colors of these substances were presented in table 5 and 6. Additionally, the experimental set up were also presented in figures. Each of the time and temperature had different mean ± SD values. For both fungal and bacterial amylases, it was evident that they work best when the temperature is close to optimal. In summary, the hypothesis was correct.
Introduction
In this lab, functions of the enzymes were determined. Furthermore, the lab helped in understanding as well as providing the knowledge of predicting how the enzyme activity is affected by the temperature. Also, the lab was quite significant in making it easy to understand how to examine and collect qualitative data. It was evident that several factors affect the enzymatic activity. In particular, these factors include the concentration of substrate, pH, the presence of activators, inhibitors, cofactors, as well as the concentration of salt. For this reason, this lab tried to answer two questions. One, the variation of the effect of temperature on the enzyme amylase activity, and two, the optimal temperature for the amylase from the fungal and bacterial sources. In practice, the rate at which the enzyme molecules and substrate collide is affected by temperature. For this reason, it was hypothesized that the active site denatures, preventing or reducing substrate binding at temperatures that are greater than the optimal. As a consequence, the formation of product is either wholly arrested or reduced.
The enzyme is simply a substance acting as a catalyst in the living organisms. It functions by regulating the rate at which the reactions of chemicals proceed without the alteration of itself in the process. The chemical reactions are the processes of biology that occur within the living organism. They are mostly regulated by the help of enzymes (Cadenas & Sies, 1985). Practically, several of these reactions fail to take place at a perceptible rate without enzymes. All of the aspects of cell metabolism are catalyzed by enzymes. In particular, some of these cell metabolism include the food digestion, in which large molecules of nutrients such as carbohydrates, fats, and proteins are broken down into smaller ones. Other cell metabolism aspects are the transformation and conservation of the chemical energy, as well as the cellular macromolecules construction from smaller precursors. Several human diseases are inherited such as phenylketonuria and albinism (Smith, 1977). In most cases, these diseases are caused by a particular enzyme’s deficiency.
Also, there are valuable medical and industrial applications for enzymes. For example, the leavening of bread, beer brewing, fermenting of wine, and curdling of cheese have been practiced in the past, but not until the nineteenth century were such reactions observed to result from the enzymes catalytic activity (Turner & Vulfson, 2000). Since then, increasing importance in the processes of an industry that involves organic chemical reactions has been assumed by the enzymes (Tobi, 2013). Moreover, there are other uses of enzymes in medicine. Specifically, it is used to promote the healing of the wound, kill disease-causing microorganisms, and diagnose a variety of diseases in hospitals. In the earlier times, people thought of enzymes to be proteins. However, it has been possible to demonstrate the catalytic ability of nucleic acids knowns as RNAs or ribozymes, thus helping to refute the axiom (Chang & Wetmore, 1986).
Typically, a large molecule of protein enzyme is composed of either one or more chains of amino acids called polypeptide (Lomthong, Lertwattanasakul & Kitpreechavanich, 2016). The sequence of amino acids plays a significant role in determining the characteristics folding patterns of the structure of proteins, which is vital in the specificity of the enzyme. Subjecting enzymes to changes such as the fluctuations in the structure of the protein, pH, or temperature may make it denature (lose its integrity), as well as its enzymatic ability. In some cases, it is possible to reverse the denaturation. Moreover, there is always an additional chemical component called cofactor that is bound to some enzymes. A cofactor acts as a direct participant in the event of catalysis, and thus it is necessary for the activity of enzymes. Either an inorganic metal ion or a coenzyme can serve as a cofactor (Herzog, Ou & Whitley, 2013). Normally, a cofactor is either loosely or tightly bound to the enzyme. A prosthetic group is a cofactor that is tightly connected to the enzyme.
Methods
In the first place, a napkin/ paper was placed under the spot plates and across the top write temperature (00C, 250C, 650C, 850C) as well as on the Time's side write (0, 2, 4, 6, 8, and 10 minutes). Afterward, four test tubes were obtained and labeled with different temperatures. At this point, 5 milliliters of 1.5% solution of starch was added into each of the test tubes. Again, 1 milliliter of amylase was added into each of the test tubes that had no starch. All the 4 test tubes that contain amylase and the 4 test tubes containing starch were then placed into their respective temperatures. For example, 00C was placed into the ice bath. Also, the 250C was put into the 250C water bath. At the same time, 650C and the 850C were also placed into the 650C and the 850C respectively.
These tubes were then allowed to equilibrate for about 5 minutes in their respective temperatures. Subsequently, about 2 to 3 drops of the iodine were added to each of the tubes at the row of 0 minutes. At the end of the process of equilibrium, a few drops of the starch solution were transferred from each treatment of temperature to the first row of the plate of spot that corresponds to the time 0 minutes. Within each of the temperatures treatment, the starch solution was poured into the tube that contains amylase. A timer was set for 2 minutes at the amylase addition’s moment. About 2 to 3 drops of iodine was added to each well at the row of 2 minutes. Remember the procedure was repeated before the transfer of a mixture of each starch amylase to the spot plates. The correct transfer pipettes were used after 2 minutes for each temperature for removing a few drops of the mixture of starch amylase from each of the tubes. Additionally, 2 to 3 drops of the mixture were then placed in the second row on the spot plate under the corresponding temperature. Finally, the color change was noted and observation tabulated.
Results
Table 1: Predictions of the Fungal Amylase
Temperature (0C)
Expected Results
Reasoning
0
Will not work.
Because the temperature is too cold.
25
Work best.
The temperature is too cold.
65
Will work best.
It is close to the optimal temperature.
85
Will not work.
The temperature is too hot. It denatures.
Table 2: Predictions of the Bacterial Amylase
Temperature (0C)
Expected Results
Reasoning
0
Will not work.
The temperature is too cold.
25
Will not work.
The temperature is still too cold.
65
Will work best.
It is close to the optimal temperature.
85
Will just work a little but not as that of the temperature of 65 0C.
The bacterial amylase gets too hot thus making it not to be as efficient as the one in optimal temperature.
Figure 1: Fungal Amylase Results
Table 3: Behavior of Time and Temperature of Fungal Amylase
Time (min)
Temperature (0C)
Group 1
5.00
5.00
5.00
5.00
Group 2
5.00
5.00
5.00
5.00
Group 3
2.00
4.00
4.00
5.00
Group 4
3.00
5.00
5.00
5.00
0 minutes, Mean ± SD
4.00 ± 1.41
4.80 ± 0.45
4.70 ± 0.45
5.00 ± 0.00
Group 1
5.00
5.00
4.50
5.00
Group 2
5.00
5.00
4.50
5.00
Group 3
2.00
4.00
3.00
5.00
Group 4
4.00
5.00
5.00
5.00
Group 5
5.00
4.50
4.00
5.00
2 minutes, Mean ± SD
4.20 ± 1.30
4.70 ± 0.45
4.20 ± 0.76
5.00 ± 0.00
Group 1
5.00
5.00
4.50
5.00
Group 2
5.00
5.00
4.50
5.00
Group 3
2.00
5.00
4.00
5.00
Group 4
5.00
5.00
5.00
5.00
Group 5
5.00
5.00
4.00
5.00
4 minutes, Mean ± SD
4.40 ± 1.34
5.00 ± 0.00
4.40 ± 0.42
5.00 ± 0.00
Group 1
5.00
5.00
4.50
5.00
Group 2
5.00
5.00
5.00
5.00
Group 3
2.00
5.00
4.00
5.00
Group 4
4.50
5.00
4.50
5.00
Group 5
5.00
5.00
3.50
5.00
6 minutes, Mean ± SD
4.30 ± 1.30
5.00 ± 0.00
4.30 ± 0.57
5.00 ± 0.00
Group 1
5.00
5.00
4.50
5.00
Group 2
5.00
5.00
5.00
5.00
Group 3
2.00
5.00
3.00
5.00
Group 4
4.00
5.00
4.00
5.00
Group 5
5.00
5.00
3.50
5.00
8 minutes, Mean ± SD
4.20 ± 1.30
5.00 ± 0.00
4.00 ± 0.79
5.00 ± 0.00
Group 1
5.00
5.00
4.50
5.00
Group 2
5.00
5.00
5.00
5.00
Group 3
2.00
5.00
3.00
5.00
Group 4
4.00
5.00
4.00
5.00
Group 5
5.00
5.00
3.50
5.00
10 minutes, Mean ± SD
4.20 ± 1.30
5.00 ± 0.00
4.00 ± 0.79
5.00 ± 0.00
Figure 2: Bacterial Amylase Results
Table 4: Behavior of Time and Temperature of Bacterial Amylase
Time (min)
Temperature (0C)
Group 1
5.00
5.00
5.00
5.00
Group 2
5.00
5.00
5.00
5.00
Group 3
5.00
5.00
5.00
5.00
Group 4
5.00
5.00
5.00
5.00
Group 5
5.00
5.00
5.00
5.00
0 minutes, Mean ± SD
4.80 ± 0.45
5.00 ± 0.00
5.00 ± 0.00
5.00 ± 0.00
Group 1
5.00
5.00
4.50
3.00
Group 2
5.00
5.00
4.50
4.00
Group 3
5.00
5.00
4.00
3.00
Group 4
3.00
4.00
4.00
3.00
Group 5
5.00
5.00
3.00
3.50
2 minutes, Mean ± SD
4.60 ± 0.89
4.80 ± 0.45
4.00 ± 0.61
3.300 ± 0.45
Group 1
5.00
5.00
4.00
2.50
Group 2
5.00
5.00
4.00
4.50
Group 3
5.00
5.00
3.00
4.00
Group 4
3.00
5.00
4.00
3.00
Group 5
5.00
5.00
3.00
4.00
4 minutes, Mean ± SD
4.60 ± 0.89
5.00 ± 0.00
3.60 ± 0.55
3.60 ± 0.82
Group 1
5.00
4.50
3.00
2.00
Group 2
5.00
5.00
3.50
4.00
Group 3
5.00
5.00
3.00
4.00
Group 4
4.50
5.00
3.00
2.00
Group 5
5.00
5.00
2.00
4.00
6 minutes, Mean ± SD
4.80 ± 0.45
5.00 ± 0.00
3.00 ± 0.61
3.20 ± 1.10
Group 1
5.00
4.50
3.00
2.00
Group 2
5.00
5.00
3.50
3.50
Group 3
5.00
5.00
3.00
4.00
Group 4
4.00
5.00
3.00
2.00
Group 5
5.00
5.00
3.00
4.00
8 minutes, Mean ± SD
4.80 ± 0.45
4.90 ± 0.22
2.90 ± 0.55
3.10 ± 1.02
Group 1
5.00
4.00
2.50
1.50
Group 2
5.00
5.00
5.00
3.00
Group 3
2.00
5.00
3.00
4.00
Group 4
4.00
5.00
2.00
2.00
Group 5
5.00
5.00
1.00
3.00
10 minutes, Mean ± SD
4.80 ± 0.45
4.80 ± 0.45
2.30 ± 0.84
2.70 ± 0.97
Table 5: Bacteria Amylase
Temperature
0 0C
25 0C
65 0C
85 0C
Time (mins)
Color
#
Color
#
Color
#
Color
#
0
Brown
4
Brown/ black
5
Blue/ black
5
Blue/ black
5
2
Blue/ black
5
Blue/ black
5
Dark orange
3
Brownish orange
3.5
4
Blue/ black
5
Blue/ black
5
Dark orange
3
Brown
4
6
Blue/ black
5
Blue/ black
5
Orange
2
Brown
4
8
Blue/ black
5
Blue/ black
5
Orange
2
Brown
4
10
Blue/ black
5
Blue/ black
5
Yellow
1
Dark orange
3
Table 6: Fungal Amylase
Temperature
0 0C
25 0C
65 0C
85 0C
Time (mins)
Color
#
Color
#
Color
#
Color
#
0
Brown
5
Blue/ black
5
Dark brown
4.5
Blue/ black
5
2
Blue/ black
5
Dark brown
4.5
Brown
4
Blue/ black
5
4
Blue/ black
5
Blue/ black
5
Brown
4
Blue/ black
5
6
Blue/ black
5
Blue/ black
5
Brownish Orange
3.5
Blue/ black
5
8
Blue/ black
5
Blue/ black
5
Brownish Orange
3.5
Blue/ black
5
10
Blue/ black
5
Blue/ black
5
Brownish orange
3.5
Blue/ black
5
Discussion
The first hypothesis was supported in the sense that the active site denatures, preventing or reducing substrate binding at temperatures that are greater than the optimal. As a consequence, the formation of product is either completely arrested or reduced. As can be seen in Tables 1 and 2, the enzymes work best at a temperature of 65 0C where it is close to the optimal. Cold temperatures such as 0 0C make the enzymes not to work. In the same way, the enzymes also fail to work at high temperatures (85 0C) due to hotness. At this temperatures, the enzyme denatures. Practically, the rate at which molecules of enzyme and substrate collide is affected by temperature. Evidently, the movement of molecules is decreased by low temperatures such as 0 0C. As a result, there is less contact between the substrate and enzymes which results in reduced rate and frequency of reaction. Ultimately, the formation of product is diminished.
Based on the scientific, Ho and Ha, a hypothesis was also formulated that there will not be any correlation between different treatments and time for the fungal amylase activity (Ho). On the other hand, there will be a correlation between different treatment and time for fungal amylase activity (Ha). For Ho, it is evident that either low or high temperatures make the enzymes not to wok hence creating no possibility of a correlation between the different treatments. In Ho, the correlation is brought about by the temperature that is close to optimal (65 0C). Remember the optimal temperature is about 70 0C. Even though the temperatures effects outside the range of optimal on substrate catalysis is the same, there is a difference in the mechanism through which the activity of the enzyme is reduced.
The values of temperatures are also evident to vary with time for the fungal and bacterial amylases as shown in Table 3 and 4 respectively. For all the two substances, the mean ± SD for all the values are also different. In table 1 for example, the mean ± SD for all the four tubes are 4.00 ± 1.41, 4.80 ± 0.45, 4.70 ± 0.45, and 5.00 ± 0.00 respectively. At this point, it can be said that various factors affect the enzymatic activity. Specifically, these factors are the concentration of the substrate, pH, and the presences of the activators, cofactors and inhibitors, and the concentration of salt. These factors might have also contributed to some deviations in the experimental and the expected values of the lab. However, the use of functional apparatuses as well as being more careful in the lab can help in enhancing the accuracy of the values obtained.
Table 5 and 6 also shows various colors of bacterial and fungal amylase respectively. In general, the two substances appear to be producing different colors at various temperatures. There is also a variation of these colors with time. Such variations are also presented in figure 1 and 2. Here, the variation in temperature is seen to have effects on the bacterial and fungal amylase results. In conclusion, the hypothesis was supported in this lab. Also, the primary objectives were met.
References
Cadenas, E., & Sies, H. (1985). Oxidative stress: Excited oxygen species and enzyme activity. Advances in Enzyme Regulation, 23, 217-237. http://dx.doi.org/10.1016/0065-2571 (85)90049-4
Chang, C., & Wetmore, J. (1986). Effects of Water Stress on Starch and Its Metabolizing Enzyme Activities in Cotton Leaves. Starch - Stärke, 38(9), 311-313. http://dx.doi.org/10.1002/star.19860380907
Herzog, T., Ou, L., & Whitley, C. (2013). Increased substrate concentration boosts enzyme activity levels of fluorometric α-l-iduronidase enzyme activity assay. Molecular Genetics and Metabolism, 108(2), S48. http://dx.doi.org/10.1016/j.ymgme.2012.11.111
Lomthong, T., Lertwattanasakul, N., & Kitpreechavanich, V. (2016). Production of raw starch degrading enzyme by the thermophilic filamentous bacterium LaceyellasacchariLP175 and its application for ethanol production from dried cassava chips. Starch - Stärke, 68(11-12), 1264-1274. http://dx.doi.org/10.1002/star.201600018
Smith, C. (1977). Temperature and the regulation of the activity of some mitochondrial enzyme systems in ecto- and endo-thermic animals. Journal of Thermal Biology, 2(4), 215-221. http://dx.doi.org/10.1016/0306-4565 (77)90034-1
Tobi, D. (2013). Large-scale analysis of the dynamics of enzymes. Proteins: Structure, Function, and Bioinformatics, 81(11), 1910-1918. http://dx.doi.org/10.1002/prot.24335
Turner, N., & Vulfson, E. (2000). At what temperature can enzymes maintain their catalytic activity? Enzyme and Microbial Technology, 27(1-2), 108-113. http://dx.doi.org/10.1016/s0141-0229 (00)00184-8
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