Bio130 Enzyme Salivary Amylase Lab Report PDF

Preview

Summary

enzyme lab report, done in first year...

Description

Introduction The purpose of this lab is to investigate how enzymes such as salivary amylase and phosphorylase interact with starch molecules by applying the Iodine and Benedict’s test on mixtures of them. The Iodine test involves a colour change in the presence of starch and glycogen, while Benedict’s test is associated with adding Benedict’s solution to mixtures, and seeing potential colour changes only after boiling. Colour change in Benedict’s test occurs by the interaction between reducing sugars, if any exist. Other objectives include learning about general enzymatic function and the conditions they require for activity. In general, enzymes are biological catalysts known to speed up biochemical reactions involving their specific complementary substrate. However, some enzymes are able to catalyze reactions linked to multiple substrates rather than a specific one. The name of the enzyme usually refers to the substrate it is associated with, such as phosphorylase which catalyzes the reaction of phosphorolysis. The way they function is by binding to substrates along their active site (Robinson, 2015). This means that substrates must have the correct shape and size in order to bind and proceed with the reaction (Robinson, 2015). In this experiment, the substrate starch, being one of the most abundant compounds found in nature, is known for its binding to salivary amylase and blue or black colour formation when tested with iodine (Marini, 2004). It also resolves in a red colour formation when its disaccharides are present during a positive Benedict's test. Salivary amylase, an enzyme produced by salivary glands in the human body, also found in pancreatic juices and breast milk, disrupts the molecular structure of starch and catalyzes the hydrolysis of starch molecules (Enemchukwu, Ubaoji, Igwilo & Udedi, 2013). The most favourable conditions for this reaction to occur are a pH values between 7.0 and 7.4 and a human

body temperature of 37 °C (Enemchukwu et. al, 2013). In these conditions, the starch substrate, becomes broken down into its disaccharides maltose and glucose due to the hydrolysis of its α (1-4) linkages (Enemchukwu et. al, 2013). Phosphorylase on the other hand, another starch digesting enzyme, converts starch and inorganic phosphate to glucose-1-phosphate by removing glucose molecules from starch (Garg & Kumar, 2008). Its optimal pH range is between 6.2 and 8.5, with a temperature range of 20 °C to 55 °C (Garg & Kumar, 2008). However, it requires phosphoric acid to participate in this phosphorolysis, hence why its enzymatic job is not called hydrolysis.

Materials and Methods Experiment was carried out as printed on pages 59 to 64 of the BIOL 130L lab manual (Department of Biology, 2019). No deviations occurred in the steps.

Results Table 1 Iodine and Benedict’s Test for Salivary Amylase and Starch Suspension Iodine Test Colour before Colour after Benedict’s Test

Test tube #1

Test tube #2

Test tube #3

Test tube #4

Test tube #5

10% salivary amylase

5% salivary amylase

2% salivary amylase

1% salivary amylase

1% starch suspension

Yellow

Yellow

Yellow

Yellow

Yellow

Very pale yellow

Pale yellow

Light yellow

Yellow

Black/Blue

Test tube #1

Test tube #2

Test tube #3

Test tube #4

Test tube #5

10% salivary amylase

5% salivary amylase

2% salivary amylase

1% salivary amylase

1% starch suspension

Colour before

Light blue

Light blue

Light blue

Light blue

Light blue

Colour after

Light blue

Light blue

Light blue

Light blue

Light blue

1

Table 1 shows the different colour changes of different solutions obtained within the Iodine and Benedict's test. In order to achieve results for the Iodine test, 1 drop of iodine was added to 1 drop of each solution, and for Benedict's test, 4mL of Benedict’s solution was added to each test tube before boiling.

Table 2 Iodine Test with Starch Suspension Mixed with McIlvaine’s Buffer

Time interval Drops until iodine test becomes negative Time elapsed

Test tube #14

Test tube #13

Test tube #12

Test tube #11

Test tube #15

1% salivary amylase

2% salivary amylase

5% salivary amylase

10% salivary amylase

water

60-second

30-second

15-seconds

5-second

30-second

6

12

4

5

-

6 minutes

6 minutes

1 minute

25 seconds

-

Table 2 represents the amount of sampling that was required at different time intervals for the iodine test to become negative (light brown). The iodine test remained positive for the water in test tube #15 even after sampling for 10 minutes.

Graph 1 Iodine Test with Starch Suspension Mixed with McIlvaine’s Buffer

2

Graph 1 represents table 2.

Table 3 Benedict’s Test with Starch Suspension mixed with McIlvaine’s Buffer Test tube #20

Test tube #19

Test tube #18

Test tube #17

Test tube #16

water

1% salivary amylase

2% salivary amylase

5% salivary amylase

10% salivary amylase

Colour before

Light blue

Light blue

Light blue

Light blue

Light blue

Colour after

Light blue

Red

Red

Red

Red

Table 3 shows the colour changes of test tubes #16-20 after adding 4 mL of Benedict’s solution to each tube and boiling for five minutes. All tubes turned red except for tube #20 which remained light blue.

Table 4 Iodine Test on Phosphorylase Associated With Different Molecules Iodine Test #1

#2

#3

#4

#5

#6

#7

Colourless

Pale yellow

Colourless

Pale yellow

Colourless

Pale violet

Dark Violet/Blue

Yellow

Pale violet

Pale violet/yellow

Yellow

Yellow

Violet

Dark violet

Colourless with bubbles

Violet with bubbles

Pale violet

Dark yellow

Pale yellow

Pale violet

Black/violet

Fresh phosphorylase + glucose-1-phosohate

Test tube #4

Pale yellow

Violet

Pale violet/yellow

Yellow

Yellow

Violet with bubbles

Violet

Pale yellow

Violet

Pale violet

Pale yellow, almost colourless

Pale yellow, almost colourless

Pale violet

Dark violet

Test tube #1 Fresh phosphorylase + glucose + starch

Test tube #2 Fresh phosphorylase + glucose-1-phosohate + starch

Test tube #3

Boiled phosphorylase + glucose-1-phosohate + starch

Test tube #5 Fresh phosphorylase + glucose-1-phosohate + potassium phosphate + starch

3

Test tube #6

Yellow

Violet

Blue/violet

Yellow with bubbles

Yellow

Violet with bubbles

Black/violet

-

-

-

-

-

-

-

Fresh phosphorylase + potassium phosphate + starch

Test tube #7 Boiled phosphorylase + potassium phosphate + starch

Table 4 shows the colour results achieved by the iodine test at 3-minute intervals on samples #1 to #7. Test tube #7 is missing results due to an error.

Discussion The obtained and expected results for the iodine test for salivary amylase and starch suspension were to observe iodine change from a yellow colour to a black or blue colour in the presence of starch as a positive test result, and remain yellow in salivary amylase as a negative test result. During Benedict’s test, however, no colour change occurred in both the salivary amylase or starch suspension as all test tubes remained blue. This was an expected negative result and is understandable as neither starch or salivary amylase contain reducing sugars for a positive test result to occur. The positive control of the iodine test was the 1% starch suspension used in tube #5, with the negative controls being the 10%, 5%, 2%, and 1% salivary amylase. Also, the reason for the differences in colour change between the different concentrations of salivary amylase, is due to that exactly. The 1% salivary amylase produces the most yellow colour change since it is the least concentrated of the four, allowing most of the colour to come from the iodine itself. This in mind, the yellow slowly becomes more pale with higher concentrations of salivary amylase. The purpose of this test was to simply see how the iodine and Benedict’s test recognizes the presence of enzymes and starch for later experiments.

4

While conducting the Iodine test for the mixtures of starch suspension and McIlvaine’s Buffer, including 2 mL of each salivary amylase solution, the expected results were achieved. The reason of this buffer is simply to maintain the optimal pH for salivary amylase. The lower the salivary amylase concentration was in the solution, the longer it took to obtain a negative (light brown) test result since the starch suspension was highly concentrated. This is especially seen in test tube #15 where each result was a positive result with a constant black colour. The negative control in this experiment was the higher salivary amylase concentration so that less starch could interact with iodine to produce produce positive results, while the positive control was the starch suspension and the low salivary amylase concentration. Test tube #11 with the highest enzyme concentration demonstrates how it interacts with its substrate by linking it via its active site and catalyzing hydrolysis of the 1– 4 glycosidic bonds present in starch, thereby obtaining the light brown colour faster (Marini, 2004). When Benedict’s test was completed with the same buffer solution, starch suspension, and salivary amylase solutions, once again the expected results were seen. Test tubes #16-19 which contained different concentrations of salivary amylase turned from a light blue colour to red. This red colour indicates that the enzyme did its job in breaking down the starch molecule to make its disaccharides present in the solution for the test to be positive. This being said, the positive control is the activated enzyme in a boiled environment. In contrast, test tube #20 does not have the enzyme to break down the starch and therefore the solution does not go through colour change, allowing a negative test result. It is clear that the negative control is the water and the absence of salivary amylase. Starch will only be degraded if its corresponding enzyme is present, otherwise it will remain as a complete molecule.

5

During the phosphorylase iodine test, a wide variety of results were obtained. The expected results were to obtain a dark blue or violet colour when phosphorolysis occured in the breakdown of starch. Test tube #1, which had a mixture of glucose and starch, resembled the expected results with a gradual change of colour from yellow to dark violet with the active enzyme catalyzing phosphorolysis. The presence of positive controls, glucose and starch, allowed phosphorylase to rupture glucose-glucose bonds and use the energy released to form glucose-1-phosphate. From there on, it attached the glucose to the primer molecule on the starch in order to increase its length for it to test positive in iodine (Kadokawa, 2018). Results for the solutions in test tube #2 to #6 were not consistent or gradual with a yellow to blue colour change. For example, in test tube #2, the second iodine test already gave a positive result, but during the fourth and fifth test the colour went back to yellow. This could be due to the presence of an already synthesised glucose-1-phosphate, allowing a faster positive test result to be obtained. The potassium phosphate in tubes #5 and #6 may have been added as an inhibitor, resulting in the on and off yellow and violet results throughout the seven tests. Its phosphate ion is known to make covalent bonds with enzymes and thus act as a mechanism of inhibition (Post, Toda & Rogers, 1974). However, at the end of all phosphorylase experiments, all tubes achieved expected results for the seventh and final iodine test meaning that they were successful in increasing the length of primer molecule on starch. Through these experiments, it is notable that with an increased enzyme concentration, enzyme reaction rate greatly increases when there is a substrate to bind to and vice versa. This can be seen in Table 2 and Graph 1 where the higher the concentration of salivary amylase contributed the fastest results, and this marks a large concept of enzymatic function. Increased

6

substrate concentration on the other hand, in this case starch, will also increases the reaction rate allowing more substrate molecules to collide with enzyme molecules. Reaction rates can be contributed in both directions of a chemical reaction, and is another important enzymatic function. Overall, these tests are a good source in seeing the relationship between enzymes and substrates and allow individuals to see their importance. Enzymes are vital for metabolic pathways in our bodies and without them, chemical reactions taking place in our bodies would be too slow and we would not be able to stay alive. A few ways to improve obtained results would have been to take more precise notes of each result and observe reactions more carefully as Table 4 is missing results for test tube #7 due to this human error. Rushing experiments in a laboratory and allowing this error to occur could be quite dangerous if exposed to harmful substances. Experiments must be done in a calm matter to prevent inaccuracy and potential injury. No other known human errors have occurred in this experiment, as most of the results achieved were as expected.

7

References  aterloo, Canada: Department of Biology. (2019). Introductory cell biology laboratory. W University of Waterloo Media.Doc

Enemchukwu, B.N., Ubaoji, K.I., Igwilo, I.O., & Udedi, S.C. (2013). Effects of Temperature, pH and Substrate Concentration on the Kinetics of Salivary Alpha- Amylase Activity among Cigarette Smokers in Awka, Anambra State, Nigeria. The Bioscientist, 1 (1), 108-113.

Garg, N., & Kumar, A. (2008). Immobilization of Starch Phosphorylase From Cabbage Leaves: Production of Glucose-1-Phosphate. Brazailian Journal of Chemical Engineering, 2 5(2), 229-235.

Kadokawa, J. (2018). α-Glucan Phosphorylase-Catalyzed Enzymatic Reactions Using Analog Substrates to Synthesize Non-Natural Oligo- and Polysaccharides. Catalysts 2018, 8 , 473.

Marini, I. (2004). Discovering an Accessible Enzyme: Salivary 𝛂-Amylase. Biochemistry And Molecular Biology Education, 3 (2), 112–116.

Post, I.R, Toda, G., & Rogers, N.F. (1974) Phosphorylation by Inorganic Phosphate of Sodium Plus Potassium Ion Transport Adenosine Triphosphatase. The Journal of Biological Chemistry, 2 50(2), 691-701

8

Robinson, K.P. (2015). Enzymes: principles and biotechnological application. Essays in Biochemistry, 59, 1-41.

9...