The lab conducted focused on examining the effects of temperature on the ability of fungal and bacterial amylase to breakdown starch to maltose, and determine the temperature at which these two amylases work best, which is known as optimal temperature.
The experimental part of the lab consisted in setting up the utensils that were going to be used during the actual experiment. During this section test tubes were labeled, and spot plates were placed in temperature/ time table created. For the second section of the experiment, iodine was placed in each row of the spot plates for each temperatures, and the solutions in the test tubes( bacterial , fungal amylase and starch mixture) were added to those same spots were iodine was added, depending on the time and the temperature corresponding to each amylase. The optimal temperature was deducted by observing the color change in the spot plates and comparing them with a color-coding scheme for starch hydrolysis.
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Conclusions for this task were reached by analyzing the data collected by each group, which suggests that a change in temperature disturbs the activity of enzyme amylase. When exposed to low and high temperatures, these enzymes were not able to function properly, therefore, reducing or eliminating their ability to breakdown certain compounds, especially starch. Enzymes need maintain at a certain temperature to be able to function at its optimal.
Introduction:
Enzymes are complex proteins produced by all living organisms with the function of enhancing chemical reactions through a process known as catalysis. During this process, the substrates, which are the molecules that will undergo the reaction, binds to the active site of the enzyme to form different molecules called products. Each active site on the enzyme is unique, permitting only substrates that match the shape of the active site to bind to the enzyme in a process known as lock and key model, however, active sites are able to adjust their shape to permit the binding with a substrate through the induced fit model, which moves entire protein domains (Raven et al., 2008; Ringe & Petsko, 2008; Whitehurst & Van Oort, 2009).
Catalysts, like enzymes, work by reducing the amount of energy required for a chemical reaction to take place by linking two substrates in the correct orientation or by accentuating chemical bonds of a substrate, which reduces the energy difference between reactants and transition state. Enzymes are not consumed or changed during the reaction and they do not alter the equilibrium of the reactions they catalyze (Garcia et al, 2004; Raven et al., 2008; Whitehurst & Van Oort, 2009; Alberte et al., 2012).
The activity of enzymes is affected by multiple factors, including 1) pH (ranges from pH 6 to 8), 2) temperature (Rate of reaction increases with temperature, but only up to a point called optimum temperature. A change in temperature, either below or above the optimum, causes the active site to denature, decreasing or preventing substrate binding. When exposed to low temperatures enzymes are not flexible enough to permit induce fit, and in high temperatures enzymes are too weak to maintain their shape.), 3) substrate concentration (If amount of enzyme is preserved constant and substrate concentration is gradually increased, the reaction velocity will increase until it reaches a maximum. After this point increasing substrate concentration will not increase the rate of reaction), 4) allosteric inhibitors and activators (Inhibitors are substances that bind to an enzyme and decreases its activity, and they can occur in two ways; competitive inhibitors and noncompetitive inhibitors. Effectors that enhance enzyme activity are referred to as allosteric activators, which bid to allosteric sites to keep an enzyme in its active configuration), and 4) cofactors (Many enzymes required the presence of other compounds, called cofactors, which during the catalytic activity, A cofactor can be a coenzyme, a prosthetic group or a metal ion activator (Harisha, 2006; Raven et al., 2008; Whitehurst & Van Oort, 2009).
Enzymes have a wide spectrum of functions in the bodies of living organisms; they are present from signal transduction to generation of muscle contraction. The also break starch molecules, forming smaller fragments of maltose, which can be easily absorbed by mammals. And it is the ability of enzymes to breakdown starch and the effect of temperature during this process that will be analyzed in the lab (Whitehurst & Van Oort, 2009; Alberte et al., 2012), expecting that the results collected confirm that temperature does have an effect in bacterial and fungal amylase activity.
Methods:
The experiment should be performed once per group, using fungal (Apergillus oryzae) and bacterial amylase. Starch catalysis will be monitored by using Iodine test, which turns from yellow to blue-black in the presence of starch.
Experimental Setup
Place a paper under the spot plates and label the top side with temperature values 0,40,60,95 °C, and the side with the times 0,2,4,6,8,10 min. Obtain 4 test tubes and label each with a different temperature, enzyme source, either bacterial or fungal and group number. Repeat previous step, but this time include the letter S, which stands for Starch solution. Finally add 5ml of 1.5% starch solution into each of the test tubes labeled S.
Effect of temperature in amylase activity
Add 1ml of amylase into each of the test tubes that do not contain starch, and place the 8 test tubes (4 containing starch and 4 containing amylase) into their respective temperatures, allowing all test tubes to equilibrate for 5 minutes. Add 2-3 drops of iodine to the first row of the spot plate corresponding to o minutes. After 5 minutes has passed and test tubes are equilibrated, transfer a few drops of starch solution from each temperature to the row where you added the iodine. Pour the starch solution into the tube containing amylase without taking it put of bath, and set the timer for two minutes.
Add 2-3 drops of iodine to the second row, and after 2 minutes has passes, transfer a few drops of the starch-amylase mixture from each tube to the 2 minutes row using the pipette correspondent to each temperature. After each additional 2 min, add 2-3 drops of iodine and a few drops from starch amylase mixture. At the end of 10 min, note the temperature and the time at which 100% hydrolysis occurred. Repeat the procedure using the other amylase type, and using the color-coding scheme convert results into numerical values.
Results:
Temp (°C)
0
40
60
95
Time (min)
0
5
5
5
5
2
4.333333
3.166667
3
5
4
4.166667
3.083333
2.833333
5
6
4
3
2.75
5
8
4
3
2.683333
5
10
3.833333
3
2.75
5
Table 1: Class Average for Bacterial Amylase activity
After all groups performed the experiment, a class data for bacterial amylase was collected. The average of the data was calculated and presented in Table 1, showing color changes for each temperature.
Temp (°C)
0
40
60
95
Time (min)
0
5
5
5
5
2
3.333333
2.666667
3.166667
5
4
3.333333
2.666667
3.083333
5
6
3.333333
2.666667
2.833333
5
8
3.333333
2.416667
2.833333
5
10
3.333333
2.416667
2.833333
5
Table 2: Class Average data for Fungal Amylase activity
After all groups performed the experiment, a class data for fungal amylase was collected. The average of the data was calculated and presented in Table 2, showing color changes for each temperature.
Graph 1: Class Average for Bacterial Amylase activity Graphical Representation
Results from Table 1 exposed in a graph, showing that all groups’ optimal temperature for Bacterial amylase is 60°C
Graph 2: Class Average Data for Fungal Amylase activity Graphical Representation
Results from Table 2 were exposed in a graph, showing that all groups’ optimal temperature for Bacterial is 40°C
Figure 1: Color coding-scheme for starch breakdown
Starch hydrolysis color coding scheme is used to determine the optimal temperature for each amylase during starch breakdown
Figure 2: Bacterial amylase activity spot plate
Group number 1 spot plate during bacterial amylase experiment showing the amylase reaction during each temperature
Figure 3: Fungal amylase activity spot plate
Group number 1 spot plate for fungal amylase experiment showing starch breakdown during each temperature
Graph 3: Bacterial Amylase Activity graphical representation
Bacterial amylase activity data taken from table 1 showing that optimal temperature for this kind of amylase according to group 1 is 60°C
Temp (°C)
0
40
60
95
Color
#
Color
#
Color
#
Color
Time (min)
0
blue/black
5
blue/black
5
blue/black
5
blue/black
2
blue/black
4
med brown
3.5
light brown
3
blue/black
4
blue/black
4
light brown
3
light brown
3
blue/black
6
med brown
3.5
light brown
3
dark yellow
2.5
blue/black
8
med brown
3.5
light brown
3
med yellow
2
blue/black
10
med brown
3.5
dark yellow
2.5
med yellow
2
blue/black
Table 3: Bacterial Amylase activity
Group 1 recorded color changes for each temperature during breakdown of starch by bacterial amylase, and it was represented in numerical values by using color coding scheme presented in Figure 1
Graph 4: Fungal Amylase Activity graphical representation
Fungal amylase activity data taken from Table 4 showing that optimal temperature for this kind of amylase according to group 1 is 40 °C
Temp (°C)
0
40
60
95
Color
#
Color
#
Color
#
Color
Time (min)
0
blue/black
5
blue/black
5
blue/black
5
blue/black
2
light brown
3
dark yellow
2.5
light brown
3
blue/black
4
light brown
3
dark yellow
2.5
light brown
3
blue/black
6
light brown
3
dark yellow
2.5
light brown
3
blue/black
8
light brown
3
med yellow
2
light brown
3
blue/black
10
light brown
3
med yellow
2
light brown
3
blue/black
Table 4: Fungal Amylase Activity
Group 1 recorded color changes for each temperature during breakdown of starch by fungal amylase, and it was represented in numerical values by using color coding scheme presented in Figure 1
Temp (°C)
0
40
60
95
Time (min)
0
0
0
0
0
2
0.408248
0.258199
0
0
4
0.258199
0.258199
0.258199
0
6
0.316228
0.316228
0.418330
0
8
0.316228
0.316228
0.376386
0
10
0.516398
0.316228
0.418330
0
Table 5: Class Average Standard Deviation for Bacterial Amylase activity
From the results from Table 1, the standard deviation was taken, showing that the results collected by each group for Bacterial amylase are close to average results.
Graph 5: Class Average Standard Deviation for Bacterial Amylase activity Graphical Representation
Data from Table 5 was exposed in a graph, showing that the difference between the mean and the samples collected by each group is minimal.
Temp (°C)
0
40
60
95
Time (min)
0
0
0
0
0
2
0.408248
0.516398
0.68313
0
4
0.408248
0.408248
0.66458
0
6
0.408248
0.408248
0.68313
0
8
0.408248
0.491596
0.68313
0
10
0.408248
0.449868
0.68313
0
Table 6: Class Average Standard deviation for Fungal Amylase Activity
From the results from Table 2, the standard deviation was taken, showing that the results collected by each group for Bacterial amylase are close to average results.
Graph 6: Class Average Standard Deviation graphical Representation
Data from Table 6 was exposed in a graph, showing that the difference between the mean and the samples collected by each group is minimal
Discussion:
After evaluating the results of the experiment, present in Table 1 and 2 it can be concluded that the data provides enough evidence to support the predictions or hypothesis presented in the introduction section that when temperature is not optimal for an enzyme, it will denature or reduce its functions. The results showed that low or high temperatures have an effect in the ability of enzymes to break down starch (Graph 1 and 2). By comparing the results with color coding scheme provided (Figure 1), the optimal temperatures for both amylases were able to be determined. The optimal temperature for the enzyme had a bright yellow color, which meant that the amylase was able to breakdown the starch present in the solution; when the solution remained blue-black the enzyme is said to be denature, meaning that it was not capable of breaking down the starch( Figure 2 and 3).
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The most important parameters taken into account to get the previous results were temperature and time. Looking at the color for the reaction between starch and amylase, by using the Iodine test, it can be concluded that for bacterial amylase, the optimal temperature is 40 °C, and this occurs around the 6 minute time. Fungal amylase optimum temperature was reached at 6 minutes time and it was 60 °C. All the previous result can be observed in Figure 2 and 3, as well as in Graph 1 to 5
Table 5 and 6 show that the results of the experiment are consistent for all lab groups, because the difference between the sample data collected by each individual group and the average of that data is minimal, showing that, the results collected by each group are close very close to be accurate.
What parameters of the experimental design were important in the expected (or unexpected) results?
The expectations for the experiment concurred with the results, because a previous understanding of enzymes was given in the lab manual, however, the optimal temperatures were not exactly known because each enzyme works best depending on its environment. For future research, the range in temperature should be more variable, not only including positive values, but negative ones. Also, if enzymes sources had more variation, it will provide a better understanding of the optimal conditions and temperature of enzymes.
Literature Cited/ References:
– Alberte J., Pitzer T., Calero K. (2012).General Biology Lab Manual / Second Edition. Florida International University: The McGraw Hill Companies.
– Garcia-Viloca M., Gao J., Karplus M. Truhlar D. G.(2004). How Enzymes Work: Analysis by Modern Rate Theory and Computer stimulations. Science 303:pp. 186-195.
– Harisha S. (2006). Introduction to Practical Biotechnology. India: Laxmi Publications.
– Raven P., Johnson G. B., Mason K. A., Losos J. B., Singer S. S. (2008). Biology 8th edition. New York: The McGraw Hill Companies.
– Ringe D., Petsko G. A. (2008). How Enzymes Work. Science 320: pp. 1428.
– Whitehurst R. J., Van Oort M. (2009). Enzymes in Food Technology: Wiley-Blackwell; 2nd edition.
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