Activity of Enzymes Biology Lab Report

Activity of Enzymes Lab Report

Introduction: Enzymes are catalysts that speed up chemical reactions in cells. They are important because they lower the activation energy so that certain reactions can take place faster. Enzymes are able to lower the activation energy to the extent that a small amount of heat can push reactants into a transition state. (Brooker et al. 2011) In specific, amylase is an enzyme that is found in saliva and helps digest carbohydrates. (Dugdale 2011). Amylase breaks down starch into disaccharides known as maltose. Maltose is a reducing sugar that can be found using Benedict reagent. The starches can be detected using I2KI.

The purpose of this lab was to explore the properties, indicators, activity, and control factors of enzymes; particularly amylase. Using I2KI and benedict reagent color changes are observed to indicate the presence of substrates and products. The effect of temperature, pH, and enzyme concentration are observed for variances that show the influence of certain components on enzyme activity.

Methods:

Amylase was monitored through two indicators, I2KI and Benedict Reagent. Four test tubes were obtained and marked one through four. Test tube 1 was pipetted with 1mL of the starch solution and 5 drops of I2KI; test tube 2 was pipetted with 1mL of maltose solution and 5 drops of Benedict reagent; test tube 3 was pipetted with 1mL of starch solution and 5 drops of Benedict Reagent; and test tube 4 was pipetted with 1mL of maltose solution and 5 drops of I2KI. Tubes 2 and 3 were placed into a boiling water bath for 5-10 minutes because Benedict Reagent was present. The final colors for each tube were recorded.

To test the activity of the amylase enzyme, three reaction mixtures were made. Test tube 1 contained 1mL of starch solution and 50µL of amylase solution; test tube 2 contained 1mL of starch solution and 50µL of water; and test tube 3 contained 50µL of amylase solution and 1mL of water. After the contents were combined in each tube and mixed immediately, a transfer pipet was used to place a drop of each reaction mixture on a spot plate. A drop of I2KI was added and the color was observed at one minute intervals for all three tubes. After the starch was broken down, the formation of maltose was tested by adding 5 drops of Benedict Reagent to each tube and heating them in a boiling water for 5-10 minutes. The presence (+) or absence (-) of starch and/or maltose was recorded in a table.

To observe the effect of temperature on enzyme activity, three water baths at specific temperatures were set up with a solution of pH7 starch in each to equilibrate the temperature. At the first water bath 50µL of amylase were pipetted into a test tube, mixed, and put in a rack in the water bath. After a minute, 1mL of the temperature equilibrated starch was pipetted into the tube and mixed in. After that, a transfer pipet was used to immediately take a drop of the reaction mixture to the spot plate and add 1 drop of I2KI, this step was repeated at one minute intervals eight times. The presence or absence of starch was recorded in a table. To test if maltose had formed, 5 drops of Benedict Reagent were added to each test tube and heated in the boiling water bath for 5-10 minutes, which was also recorded in the table.

To observe the effect of pH on enzyme activity, four starch solutions were buffered to specific pHs (4, 5, 6 and 7). Four test tubes with 1 mL of each starch solutions was set up and 50µL of amylase was added and mixed into each test tube. At each tube a drop was immediately taken by a transfer pipet to the spot plate, where I2KI was added. This step was repeated for one minute intervals eight times and recorded in a table. Also recorded in the table was the presence (+) or absence (-) of starch.

Finally, the enzyme concentration activity was observed by taking three test tubes and adding 1 mL of starch solution pH 7 to each tube. Added to tube 1 was 50µL of the 5% enzyme; tube 2 obtained 50µL of the 10% enzyme; and tube three obtained 50µL of the 20% enzyme. Each tube was then mixed and immediately withdrawn by a transfer pipet to the spot plate with a drop of I2KI added; this step was completed eight times at one minute intervals and recorded in a table. Two consecutive wells on the spot plate of the same color were observed indicating that the amylase reaction was completed because all the starch had been broken down. Five drops of Benedict Reagent were added to each of the tubes and heated in a water bath for 5-10 minutes to test if maltose had formed. The results were also recorded in the table.

Results:

For the indicators of amylase activity experiment, Tube 1 containing I2KI and starch turned a deep purple when mixed. Tube 2 containing maltose and Benedict's reagent turned orange when mixed. Tube 3 containing starch and Benedict's reagent turned light blue when mixed. Tube 4 containing maltose and I2KI turned pale yellow when mixed.

For the demonstration of amylase activity experiment, Tube 1 containing starch and amylase was purple at time zero, light purple at one minute, lavender at two minutes, grey at three minutes, and pale yellow from intervals four, five, six, seven, and eight. Tube 2 containing starch and water was purple at intervals zero, one, two, three, four, five, six, seven, and eight. Tube 3 containing amylase and water was light yellow at intervals zero, one, two, three, four, five, six, seven, and eight. The remaining colorless reaction mixture was then added to 5 drops of Benedict's reagent and heated. Tube 1 turned orange, while Tubes 2 and 3 turned light blue.

Figure 1. The Relationship Between Temperature and Speed of Enzymatic Activity

1. This graph shows the relationship between temperature, enzyme activity, and the time elapsed for the reaction to take place. It clearly indicates that the lower the temperature, the faster the enzymatic activity.

Figure 2. Relationship Between pH and Speed of Enzymatic Activity

2. This graph shows the relationship between pH, enzymatic activity, and time elapsed for the reaction to take place. This graph suggests that enzymatic activity performs at a faster rate with both high and low pHs.

Figure 3. Relationship Between Enzyme Concentration and Chemical Reaction Time

3. This graph shows the relationship