Lab 7

Lab 7: Enzymatic Processes
Prapti Patel
20723924
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

Introduction
Enzymes are biological catalysts that moderate reactions that are needed in order for normal cell functions to occur (Alberts et al., 2014). The function of enzymes is to speed up chemical reactions by lowering the activation energy (Alberts et al., 2014). 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 (Alberts et al., 2014). Without the presence of enzymes, these chemical reactions would work at a very slow speed which is not good for living species (Alberts et al., 2014). 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 (Artioli, 2008). 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 (Artioli, 2008). 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 (Artioli, 2008). Increasing the enzyme concentration cannot affect the rate of reaction, during this saturation point (Artioli, 2008).
Salivary amylase reacts on starch (another reactant) as the substrate to catalyze the chemical reaction (Alberts et al., 2014). 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) (Alberts et al., 2014). Maltose is the energy source for our body (Edwards et al., 2011).
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 (Edwards et al., 2011). Inorder to know if starch was present, an iodine test is conducted (Edwards et al., 2011). A result giving a positive iodine test means that a colour change occurred (colour changed from light yellow to a blueish black) (Petrucci et al., 2017). The endpoint of a reaction can be reached if the iodine test resulted negative (colour stayed yellow, no colour change) (Petrucci et al., 2017). Furthermore, Benedict’s test was conducted in order to find out whether or not maltose is present (Petrucci et al., 2017). Having a positive result means that maltose is present (colour change occurs) and there is a chemical reaction that took place (Petrucci et al., 2017). Having a negative result means that there is no maltose present (solution stays light blue) and there is no chemical reaction (Petrucci et al., 2017).
Phosphorylase enzyme was utilized in the second part of the experiment. Phosphorylase enzyme is present in plants and it breaks down starch (Sanader ; DiGiuseppe, 2012). This enzyme is needed in order for the reaction of phosphorylase to take place (Sanader ; DiGiuseppe, 2012). In this reaction, phosphoric acid (reactant) breaks down starch into small units (Sanader ; DiGiuseppe, 2012). Phosphorylase enzyme acts as a catalyst between starch and phosphate. By utilizing various concentrations of starch, the effect of the concentration of reactants and products on the direction of the enzymatic reaction can be observed (Sanader ; DiGiuseppe, 2012).
Iodine test was conducted in order to know whether or not the longer starch is present (Sanader ; DiGiuseppe, 2012). A positive iodine test means that longer starch is present and the colour changes from a light yellow to a blueish- black (Sanader ; DiGiuseppe, 2012). A negative iodine test means that short starch is present and there is no colour change (remains yellow) (Sanader ; DiGiuseppe, 2012).

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.

Results
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 1 shows the results of the Iodine and Benedict’s tests conducted on different percentages of salivary amylase concentrations. A positive result for the Iodine test (has presence of starch) has a colour change from navy blue/violet to black. Test tube with the starch (test tube 5) has a positive result meaning starch is present A negative result for the Iodine test (no starch present) has no colour change and thus the colour remains yellow. Test tubes 1 to 4 has negative results meaning no starch is present. A positive result for the Benedict’s test means that maltose is present and there should be a colour change from transparent blue to a dark yellow, brown or red. None of the test tubes has maltose in it and all of the test tubes remained transparent blue in colour thus, resulting negative for all of the test tubes. A negative result for the Benedict’s test means that no maltose is present.

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
negative (seconds)
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
Table 2 shows the time (in seconds) that the reactions in the test tubes took in order to become negative (light brown) during the Iodine test for different salivary amylase solutions used. Furthermore, it shows the results of the Iodine test performed at various time interval after mixing the amylase solution of different concentrations including 1% starch suspension. Test tube 14 took 300 seconds to turn into a yellowish- brown colour, test tube 13 took 210 seconds to turn into a dark brown colour, test tube 12 took 105 seconds to turn into a brown colour, and test tube 11 took 25 seconds to turn into a yellowish- brown colour.

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
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 3 shows the Benedict’s test results (of test tubes 16 to 20) for the different amylase concentrations, after boiling the test tubes in boiling water bath for approximately 5 minutes. Test tube with starch and 10% amylase tested positive for the Benedict’s test meaning a colour change took place. The colour changed from a yellow brown to a dark blue. Test tube with starch and 5% amylase also tested positive for the Benedict’s test and a colour change appeared from brownish- red to a dark blue. Rest of the test tubes (18 to 20) remained blue black therefore, they tested negative for the Benedict’s test.

Table 4: Iodine Test Results on Phosphorylase- before, during , and after test results
Time Iodine Test Results for Test Tube #
1 2 3 4 5 6 7
0 – – – – – + +
3 – – – – – + +
6 – + – – – + +
9 – + – – – + +
12 – + – – – + +
15 – + – – – + +

Table 4 shows the Iodine test results on the phosphorylase activity at 3 minute time intervals upto 15 minutes. A positive Iodine test should have a colour change to a dark blue/ black. Test tubes 2, 6, and 7 all result in a colour change to a dark blue/ black thus, giving a positive result for the Iodine test. Test tubes 1,3,4, and 5 all result in no colour change (stayed yellow) thus, giving a negative result for the Iodine test.

Discussion
Reaction occurring inside the cells are assisted by enzymes that increases the rate of reaction (Alberts et al., 2014). Biological catalysts that function by binding themselves to substrate molecules and lowering the Ea of a reaction are known as enzymes (Alberts et al., 2014). Enzymes are highly specific and highly efficient, making them a crucial part of life. The purpose of this lab is to analyze how the different concentrations of enzymes affect the rate of reaction. Moreover, to examine how the substrate concentration, product, and an enzymes can affect direction of an enzymatic reaction (Alberts et al., 2014).

The first part of the experiment conducted is by using salivary amylase to analyze how having various concentrations of this enzyme can impact on the rate of reaction with starch. Salivary amylase is also known as a digestive enzyme which can be found in saliva. It deteriorates starch by breaking maltose. Since this reaction needs water, it is called a hydrolytic reaction or hydrolysis.

To find out if starch is present or not in a substance we conduct the Iodine test. 1% starch solution, 1% salivary amylase, 2% salivary amylase, 5% salivary amylase, and 10% salivary amylase solutions are used to conduct the Iodine test for the presence of starch. The solutions that had different concentrations of salivary amylase, all tested negative for the Iodine test, thus indicating there is no starch present in them. Furthermore, salivary enzymes do not contain any starch component so the results are expected to be negative for the different concentrations of the salivary amylase solutions.

1% starch solution tested positive for the Iodine test because it contains starch. The colour change gave a dark blue- black colour and it is produced when there is a reaction between amylose, starch, and iodine. Starch consists of 20% amylose and 80% amylopectin.
Benedict’s test was conducted to find out whether or not reducing sugars is present in salivary amylase solutions. If sugar is present, it shows that the enzymatic reaction is to reach completion along with starch. We are to add 4 mL of Benedict’s solution in each of 4 test tubes that have different concentrations of salivary amylase in water. All test tubes with different concentrations of salivary amylase and the test tube with starch gives a negative results, meaning that no reducing sugar is present. Benedict’s solution has cupric sulphate, sodium citrate, and sodium carbonate. Since it has no reducing sugars in it, and there is no reducing sugar present in the different concentrations of salivary amylase, the Benedict’s test gives a negative result.

Benedict’s test results for salivary amylase solutions in McIlvaine’s buffer gave a positive results for the starch + 10% salivary amylase and starch + 5% salivary amylase (Petrucci et al., 2017). This did not match the expected results, as it should be negative.

To find out how the different concentrations of salivary amylase affects rate of reaction, at which starch breaks down into maltose, the Iodine test is used (Ritter et al., 2002). Different concentrations of salivary amylase in water are to be mixed with starch and McIlvaine’s buffer (Ritter et al., 2002). Buffer is necessary in order to maintain pH of the enzymatic reaction taking place. It is expected that salivary amylase acts on starch (substrate) to catalyze the reaction in order to produce maltose (Ritter et al., 2002). So then when the reaction reaches completion, no starch is present, at different time (in seconds) the test tubes with starch and different concentrations of salivary amylase began to change colour from dark blue- black to a orangish yellow (Alberts et al., 2014). This colour change means that starch is broken into maltose. The time was different for solution because they have different concentration of salivary amylase present within them. The test tube with water remains the same after performing the Iodine test on it thus it is not incorporated in the graph.

Phosphorylase is the enzyme that acts on starch by breaking glucose molecules. Additionally, this is done by disrupting the glucose – glucose bonds (Alberts et al., 2014). When the glucose- glucose bonds are disrupted, energy is released, and this gets trapped within the system (Alberts et al., 2014).. This energy is used to form glucose- phosphate bond in G-1-P (energy is not released from the system) (Alberts et al., 2014). Instead of consuming water, phosphorylase consumes phosphoric acid this process is known as phosphorolysis (Alberts et al., 2014). Water does not play a major role in phosphorolysis because it is neither a product nor a reactant, furthermore, it does not affect the reaction like it did in salivary amylase (Alberts et al., 2014). The concentrations of products and reactants diagnoses the direction of reactions moderated by phosphorylase (Alberts et al., 2014). According to the results established in the experiment, test tube with glucose 1 phosphate, fresh phosphorylase, and primer starch only (test tube 2), test tube with potassium phosphate, fresh phosphorylase and starch (test tube 6), and test tube with potassium phosphate, starch and boiled phosphorylase (test tube 7) gave a positive result after conducting the Iodine test. This means that molecules of starch are present and they are long which forms the dark blue/ black colour. Test tube with glucose, fresh phosphorylase, and primer starch (test tube 1), test tube with glucose 1 phosphate and fresh phosphorylase (test tube 3), test tube with glucose 1 phosphate, primer starch, and boiled phosphorylase (test tube 4), and test tube with potassium phosphate, primer starch, and fresh phosphorylase (test tube 5), all gave a negative result to the Iodine test performed. No colour change was seen and the colour stayed yellow, this means no starch is present (Alberts et al., 2014).

Overall, the purpose of this lab is to examine 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 (Artioli, 2008). This was achieved by performing the salivary amylase experiment and the phosphorylase experiment. We got to know that if the concentration is increased, then the rate of reaction is decreased, meaning that more time is required to reach completion for the reactions (Alberts et al., 2014). Moreover, factors like temperature, pH, enzyme concentration, substrate concentration, product concentration, and energy of activation, all affect the rate of reaction (Ritter et al., 2002).

References
Alberts, B. (2014). Essential cell biology (4th edition). New York: Garland Science.

Artioli, Y. (2008). Enzymatic processes. In Encyclopedia of Ecology, (1), p. 1377. Oxford, UK: Newnes.

Carter-Edwards, T., Abdulnour, S., Arora, A., ; Fan, C. (2011). McGraw-Hill Ryerson biology 12. Toronto, ON: McGraw-Hill Ryerson.

Department of Biology. (2018). Introductory cell biology laboratory. Waterloo, Canada: University of Waterloo Media.doc

Petrucci, R. H., Herring, F. G., Madura, J. D., ; Bissonnette, C. (2017). General chemistry principles and modern applications. Toronto, Ont.: Pearson.

Ritter, B., Adam-Carr, C., ; Fraser, D. (2002). Nelson biology 11. Toronto: Nelson Thomson Learning
Sanader, M., ; DiGiuseppe, M. (2012). Nelson chemistry 12. university preparation. Toronto: Nelson Education.