Partner: Priya Vyas
TA’s: Xinyi Li and Lawrentina D’Souza
Experiment performed on September 26, 2018
BIOL 130L section 019
Wednesday 2:30PM to 5:20PM, STC 4008
Due on November 14, 2018
Enzymes are biological catalysts that moderate reactions that are needed in order for normal cell functions to occur. The function of enzymes is to speed up chemical reactions by lowering the activation energy. Lower the activation energy, higher the rate of reaction. Furthermore, enzymes perform many bodily functions including digestion of food, formation of cells and disposing waste. Without the presence of enzymes, these chemical reactions would work at a very slow speed which is not good for living species. Therefore, gaining the knowledge on enzymatic reactions and how it affects the chemical reactions that take place is crucial to deepen our understanding of how enzymes contribute to the bodily functions.
The purpose of this lab is to observe how enzyme concentration affects the reaction time of an enzymatic reaction, moreover, the effect of the concentration of reactants and products on the direction of enzymatic reactions. Since the major role of an enzyme is to speed up a chemical reaction by lowering the activation energy, a practical hypothesis is that supplying a higher concentration of an enzyme to a substrate (reactant) would increase the rate of reaction. However, the only time enzymes react is when they bind to a substrate, therefore if the enzyme concentration surpasses the amount of substrate, the remaining extra enzymes cannot behave as catalysts. Increasing the enzyme concentration cannot affect the rate of reaction, during this saturation point.
Salivary amylase reacts on starch (another reactant) as the substrate to catalyze the chemical reaction. In this reaction, alpha-1,4 linkages amongst glucose units in starch is hydrolyzed for the formation of units of maltose (a disaccharide and reducing sugar). Maltose is the energy source for our body.
Utilizing multiple concentrations of the salivary amylase solution, the effect of enzyme concentration on the time taken for the reaction to reach completion can be perceived. Inorder to know if starch was present, an iodine test is conducted. A result giving a positive iodine test means that a colour change occurred (colour changed from light yellow to a blueish black). The endpoint of a reaction can be reached if the iodine test resulted negative (colour stayed yellow, no colour change). Furthermore, Benedict’s test was conducted in order to find out whether or not maltose is present. Having a positive result means that maltose is present (colour change occurs) and there is a chemical reaction that took place. Having a negative result means that there is no maltose present (solution stays light blue) and there is no chemical reaction.
Phosphorylase enzyme was utilized in the second part of the experiment. Phosphorylase enzyme is present in plants and it breaks down starch. This enzyme is needed in order for the reaction of phosphorylase to take place. In this reaction, phosphoric acid (reactant) breaks down starch into small units. Phosphorylase enzyme acts as a catalyst between starch and phosphate.
Utilizing various starch concentrations, the enzymatic reaction can be perceived
The effect of the concentration of reactants and products on the direction of the enzymatic reaction can be observed
Iodine test was conducted in order to know whether or not the longer starch is present. A positive iodine test means that longer starch is present and the colour changes from a light yellow to a blueish- black. A negative iodine test means that short starch is present and there is no colour change.
Materials and Methods
Please refer to “Lab 7: Enzymes” on pages 54-60 in the BIOL 130L Fall lab manual for the procedure. (Department of Biology, 2018). There was one deviation in this experiment: in part 2, step 1, no cheese cloth is used to filter the homogenate.
Table 1: Iodine and Benedict’s test results for the salivary amylase solutions
Tube Test # Contents Iodine Test Benedict’s Test
1 10% amylase – –
2 5% amylase – –
3 2% amylase – –
4 1% amylase – –
5 starch + –
Table 2: Quantitative data of the reaction time when Iodine test resulted negatively towards various salivary amylase solutions in McIlvaine’s buffer
Test Tube Combination Salivary Amylase Solution Used Reaction time when
Iodine test turned
9 and 14 1% Salivary Amylase 300
8 and 13 2% Salivary Amylase 210
7 and 12 5% Salivary Amylase 105
6 and 11 10% Salivary Amylase 25
10 and 15 Water Blank N/A
Figure 1: The effect of enzyme concentration on the speed of the hydrolysis reaction
Table 3: Benedict’s test results for salivary amylase solutions in McIlvaine’s buffer (did not match expected – talk about it)
Contents Test Tube # Benedict’s Test Results
starch + 10% amylase 16 +
Starch + 5% amylase 17 +
Starch + 2% amylase 18 –
Starch + 1% amylase 19 –
Starch + water 20 –
Table 4: phosphorylase activity- before, during , and after test results
Time Test Tube #
1 2 3 4 5 6 7
0 – – – – – + +
3 – – – – – + +
6 – + – – – + +
9 – + – – – + +
12 – + – – – + +
15 – + – – – + +