Bacterial Amylase Lab Report

Abstract
Enzymes are proteins that act as a catalyst to bring about a specific biochemical reaction. Enzymes contain an active site that allows the substrate to bind to the enzyme and form the enzyme substrate complex and then release the products. An enzymes main function is to increase the rate of chemical reactions. There are many different types of enzymes, each containing a specific set of optimal temperatures. These temperatures represent the set of conditions at which the enzyme works best in. Enzymes are used in our everyday lives in order to break things down, for example starch. Fungal and bacterial amylases are the two enzymes tested in this experiment to see their ability to break down starch. To determine how temperature affects

the activity of amylase enzyme and the optimal temperature for fungal and bacterial amylase, the activity of these enzymes were tested at different temperature and times. Iodine was used to show whether the starch was still present. In the experiment, bacterial amylase achieved hydrolysis at 25°c and at 55°c after 2 minutes. Fungal amylase achieved hydrolysis at 55°c after 4 minutes. The experiment conducted indeed determined that temperature does affect the speed of enzymatic activity in both bacterial and fungal amylase.
Introduction
Enzymes act as biological catalysis to speed up chemical reactions. Enzymes are three-dimensional proteins that fit perfectly molecules they act on. This allows them to bring two substrates together in order for the enzyme to lower the activation energy required for new bonds to form. Only a small part of the enzyme is used during the reaction, it is not either neither changed around the nor consumed. An enzyme could be used over and over again. Different types of cells that contain different sets of enzymes cause structural and functional variations among cells.
Active sites are pockets on the surface of enzymes, where substrates bind to the enzymes forming a substrate complex. A substrate complex modifies the substrate’s chemical bond and initiates a series of chemical reactions resulting in the formation of a product. The active site is reshaped by the interaction of he amino acid side chains with the substrate molecule, when a substrate binds to an enzyme. The active site determines the specificity of the enzyme. There are two different model used to show this the lock and key model and the induced fit model. Lock and key model where the substrate, the key, fit perfectly into the lock, the enzyme. The induced fit model represents the fit of substrate molecules by the enzymes molding their shape.
There are many factors that influence enzyme activity such at pH, substrate concentration, salt concentration, and the presences of inhibitors, activators, and cofactors. Enzymes tend to work better in specific conditions and temperatures, which is known as their optimal temperature. Temperature affects the rate at which the substrate and enzyme molecules collide. Increasing the temperature increases the rate of the reaction because additional heat increases molecular movement. The rate of enzymatic activity can only reach optimum temperature. If it reaches above optimum temperature, it becomes too weak to maintain the enzyme’s shape causing the enzyme to denature. Denaturation is the process in which a protein unfolds and loses its shape causing it to be biologically inactive.
The objective of this experiment was to different temperature affect enzymatic activity and to determine the optimal temperature for amylase. Higher and lower temperatures are predicted to cause the enzymes to slow or denature when put in starch for both bacterial and fungal amylase.
Methods
To set up the experiment, place a napkin under two spot plates and across the top write Temperature (0°, 25°, 55°, 85° Celsius) and on the side write the Time (0,2,4,6,8,10 minutes). Remember to have two set ups of the spot plates for each amylase, one for bacterial and one for fungal. Obtain four test tubes and label each with a different temperature and enzyme source, bacterial or fungal. Collect another four test tubes and label with different temperatures, enzyme source, and a letter S for starch solution. For the test tubes labeled with S, add 5 mL of 1.5%starch solution.
To begin the experiment, add 1 mL of amylase to the test tubes that do not contain starch. Place bacterial amylase in the test tubes labeled bacterial and place the fungal amylase to the test tubes labeled fungal. Place one test tube containing starch, one test tube containing placed each bacterial amylase, and one test tube containing fungal amylase into the respected temperatures. One test tube of are each are in 0° C into the ice bath, 25°C water bath, 55°C water bath, and 85°C water bath. Let the test tubes reach the temperatures they are placed in for five minutes.
While you are waiting, place 2-3 drops of iodine to each well in the spot plate at the 0 minutes row.

At the end of the equilibrium process, without removing the test tubes from their water baths, using temperature labeled pipettes transfer 4 drops of the starch solution from each temperature to the first row of the spot plate corresponding to the time 0 minutes. Within each temperature treatment, pour the starch into the tube containing amylase. Set the timer for 2 minutes at the moment of amylase addition. Add 2-3 of iodine to each well at the 2 minutes row. This step will be repeated before the transfer of each starch-amylase mixture to the spot

plates.
After 2 minutes, using the correct transfer pipette for each temperature remove some drops for the starch-amylase mixture from each test tube into the second row on the spot plate under the corresponding temperature. Write down the color change that occurred in a data chart for each amylase according to time and temperature. Repeat these steps after each additional 2 minutes until you reach 10 minutes. When you reach 10 minutes, write down at what temperature did 100% hydrolysis occur.
Results
Bacterial Amylase
Table 1
Temp (°C) 0 25 55 85
Time (min)
0 5 5 5 5
2 5 3 2 5
4 4 3 2 5
6 4 3 2 5
8 4 3 2 5
10 4 3 2 5
OPTIMAL TEMP. 25°C-55°C

Fungal Amylase
Table 2
Temp (°C) 0 25 55 85
Time (min)
0 5 5 5 5
2 4 4 3 5
4 4 4 3 5
6 4 4 3 5
8 4 4 3 5
10 4 3 2 5
OPTIMAL TEMP. 55°C

Bacterial Amylase

Table 3
Temp (°C) 0 25 55 85
Time (min)
Group 1 5 5 5 5
Group 2 5 5 5 5
Group 3 5 5 5 5
Group 4 5 5 5 5
Group 5 5 5 5 5
Group 6 1 2 2 5
0 minutes
Group 1 4 2 1 5
Group 2 5 4 3 5
Group 3 5 3 2 5
Group 4 4 2 2 5
Group 5 3 1 3 5
Group 6 1 2 2 5
2 minutes
Group 1 3 2 1 5
Group 2 5 3 3 5
Group 3 4 3 2 5
Group 4 4 2 2 5
Group 5 2 1 1 5
Group 6 1 2 2 5
4 minutes
Group 1 2 2 1 5
Group 2 4.5 3 3 5
Group 3 3 2 2 5
Group 4 4 2 2 5
Group 5 2 1 1 5
Group 6 1 2 2 5
6 minutes
Group 1 2 2 1 5
Group 2 4.5 3 3 5
Group 3 3 2 2 5
Group 4 3 2 2 5
Group 5 2 1 1 5
Group 6 1 2 2 5
8 minutes
Group 1 2 1 1 5
Group 2 4 3 3 5
Group 3 3 2 2 5
Group 4 3 2 2 5
Group 5 1 1 1 5
Group 6 1 2 2 5
10 minutes
OPTIMAL TEMP. 25°C-55°C

Fungal Amylase
Table 4
Temp (°C) 0 25 55 85
Time (min)
Group 1 5 5 5 5
Group 2 5 5 5 5
Group 3 5 5 5 5
Group 4 5 5 5 5
Group 5 1 3 1 4
Group 6 2 2 2 3
0 minutes
Group 1 3 4 2 5
Group 2 3.5 2.5 2.5 5
Group 3 3 3 3 5
Group 4 4 4 3 5
Group 5 1 3 1 4
Group 6 2 2 2 3
2 minutes
Group 1 3 4 2 5
Group 2 3.5 2 2 5
Group 3 3 3 3 3
Group 4 4 4 2 5
Group 5 1 3 1 4
Group 6 2 2 2 3
4 minutes
Group 1 3 4 2 5
Group 2 3.5 2 2 5
Group 3 2 2 2 5
Group 4 4 3 2 5
Group 5 1 3 1 4
Group 6 2 2 2 3
6 minutes
Group 1 3 4 2 5
Group 2 3.5 2 2 5
Group 3 2 2 2 5
Group 4 4 2 2 5
Group 5 1 3 1 4
Group 6 2 2 2 3
8 minutes
Group 1 3 4 2 5
Group 2 3.5 2.5 2.5 5
Group 3 2 2 2 5
Group 4 3 2 1 5
Group 5 2 3 1 4
Group 6 2 2 2 3
10 minutes
OPTIMAL TEMP. 55°C

In table 1, the results of bacterial amylase enzyme activity are shown. The enzyme worked best in temperatures 25°-55° Celsius making this its optimal temperature. In table 2, the results of fungal amylase enzyme activity are shown. The enzyme worked best in 55° Celsius making this its optimal temperature. In table 3, the data of the class for bacterial amylase is shown. Through experimentation the class data determines the optimal temperature to be from 25°C-55°C. In table 4, the data of the class for fungal amylase is shown. Through this table you can determine that the optimal temperature is 55°. In each table the numbers one through five represent the color the spot plate reflected. One being yellow, two being a dark yellow, three being a yellowish brown, four being a brown color and five being black, these colors indicated the presence of start and the determination of whether the enzyme works or not. As seen in the tables above, the results are similar in terms of optimal temperatures, in what temperature the enzyme broke down the starch, and how long it took the enzyme to work in each temperature.
Discussion
Based on the procedures done, the outcome of the experiment supported the original hypothesis which stated that, higher and lower temperatures are predicted to cause the enzymes to slow down or denature when put in starch for both bacterial and fungal amylase.

According to the data in the tables, when the enzyme was placed in cold temperatures nothing happened the starch was still present in both bacterial and fungal amylase. The results stayed the same throughout the whole 10 minutes for the temperature of 0° Celsius. In bacterial amylase you began to see the enzyme break down the starch but not completely in the temperature of 25° Celsius. In fungal amylase you began to see the enzyme break down the starch but not as much as bacterial amylase in the temperature of 25° Celsius. In 55° Celsius, both bacterial and fungal amylase had accomplished 100% hydrolysis. The difference is that starch broken down more meaning the enzymatic rate was faster in bacterial amylase. For the temperature of 85° Celsius, the enzymes denatured in both bacterial and fungal amylase. The denaturation in both the bacterial and fungal amylase caused the color on the spot plates to turn a dark blue-black. This occurred because the temperature was too hot and higher temperatures tend to slow down the enzymatic reactions and eventually denature the enzyme.
The effect of temperature on the enzymatic activity of bacterial and fungal amylase was determined by measuring the rates of reaction in varying

temperatures. The hydrolysis or the breakdown of starch due to the action of bacterial and fungal amylase is indicated by the change in color of the starch solution.
The results that were collected after the experiment was conducted were expected. From prior knowledge, we know that enzymes do not work well in temperatures that are above or below their optimal temperature. The reason in conducting is experiment was exactly that, to determine each amylases optimal temperature.
There are a couple factors that could have gone wrong during this experiment that could have altered the results. To begin, the fungal and bacterial amylase test tubes could have gotten mixed up due to labeling after the amylase was placed into the test tubes. Another issue could be not letting the test tubes reach the temperatures they are placed into in the beginning of the lab. Lastly, the two-minute cycle could have been changed. Meaning the amylase was added to the spot plate at different times and not on a steady routine. This could affect the results by not being able to see a change occur over a consistent period of time. Also not letting the amylase go through the process of reaching their corresponding temperatures could ruin the rate of enzymatic activity.