Investigation of Enzyme Concentration Effects on Amylase Activity

Testing the effects of enzyme concentration, substrate concentration and reaction time on Salivary Amylase and Phosphorylase Sukhman Sodhi 20847011 Lab Partner: Michaela Zanette TA’s: Dan Basilla, Sarah Kowalczyk & Savera Lodhy BIOL 130L Section 036 STC 4008 Conducted on November 17, 2023, from 2:30 pm - 5:20 pm Due date: November 24, 2023

Introduction The human body is composed of trillions of cells and continuously undergoes chemical reactions to survive. Each enzyme is specific to its action, meaning there are thousands of different enzymes needed to assist the reactions that are necessary for cell function. The primary reactions in the body are endothermic. The role of an enzyme is to ensure an energetically unfavourable reaction occurs, by reducing the activation energy ( Alberts et al., 2014). Enzymes bind to substrates (a reactant in the chemical reaction) and form a substrate-enzyme complex, which further reduces into an unaltered enzyme and a product. In addition, all reactions can be reversible however it heavily depends on the surrounding conditions. A few well-known enzymes include salivary amylase and phosphorylase. Salivary amylase is a glucose-polymer cleavage enzyme that is created in the salivary glands. The enzyme’s role is to break the maltose molecules off starch molecules (Peyrot des Gachons & Breslin, 2016). Phosphorylase catalyzes a reaction that breaks the glucose-glucose bonds in starch which further phosphorylates glycogen to glucose-1-phosphate – necessary for the generation of metabolic energy (Johnson, 1989). In this experiment, the effects of substrate concentration, reaction time and enzyme concentration will be investigated on salivary amylase and phosphorylase. The experiment uses the iodine test and the benedict test. The iodine test will test for the presence of starch, and a positive result will lead to a black-and-blue colour. If disaccharides are present during Benedict’s test, a positive result would lead to a red pigment. Materials and Methods The experiment was carried out following pages 54 – 61 of the BIOL 130L lab manual (Department of Biology, 2023). No deviations were made to the protocols.

Results Table 1 : Iodine and Benedict’s test for Salivary Amylase Test Tube # Sample Iodine Test Benedict’s Test 1 10% salivary amylase - - 2 5% salivary amylase - - 3 2% salivary amylase - - 4 1% salivary amylase - - 5 1% starch suspension + - (-) represents negative control, (+) represents positive control Table 1 highlights the results of each sample during the iodine test and Benedict’s test. The Iodine solution has a pale yellow colour, therefore the samples that are a negative control indicate there was no colour. If starch was present in the solution, the solution would have a black-blue pigment, indicating a positive control. Benedict’s solution originally has a blue pigment, if there are any deviations from this colour, it indicates there’s the presence of a reducing sugar (positive control).

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Table 2 : Iodine test for Salivary amylase mixed with starch suspension and McIlvaine’s buffer Test Tube # Sample Interval (s) Time elapsed (s) 11 10% salivary amylase 5 15 12 5% salivary amylase 15 90 13 2% salivary amylase 30 360 14 1% salivary amylase 60 1,380 15 Water 30 - (-) represents a negative control did not form Table 2 highlights the reaction rate of salivary amylase while it was incubating at 37 o C. All the samples underwent the test but with varied intervals until the sample became a negative control, meaning there was a colour change from dark blue to light brown.

Figure 1 : Graphs depicting the time it takes to reach the Iodine test endpoint in contrast to the concentration of Salivary amylase Figure 1 illustrates the correlation between the concentration of salivary amylase and the time it took for it to reach the first negative control. The time is on the Y-axis and is labelled in seconds, and the concentration is on the x-axis labelled in percentages.

Table 3 : Observations from Benedict’s tests on Salivary Amylase Test Tube # Sample Observations Control Type 16 10% salivary amylase Dark black-blue colour, precipitate formed + 17 5% salivary amylase Dark black-blue, precipitate formed + 18 2% salivary amylase Dark black-blue, precipitate formed + 19 1% salivary amylase Dark black-blue, precipitate formed + 20 water Blue - (-) represents negative control, (+) represents positive control Table 3 illustrates the colour changes in various concentrations of salivary amylase and water. The colour of the benedict’s solution is bright blue, meaning if there were any deviations from this result, the sample was a positive control, proving the presence of a reducing sugar. If the sample remained bright blue, the sample was a negative control. The samples also contain 1% starch suspension and 2mL of McIlvaine’s buffer.

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Table 4 : Observations from Iodine test using Phosphorylase samples Test Tube # Sample Result at t 0 (0 min) Result at t 5 (15 mins) 1 0.01M glucose, 0.2% starch suspension (primer), fresh phosphorylase - - 2 0.01M glucose-1-phosphate, 0.2% starch suspension (primer), fresh phosphorylase + + 3 0.01M glucose-1-phosphate, fresh phosphorylase - - 4 0.01M glucose-1-phosphate, 0.2% starch suspension (primer), boiled phosphorylase - - 5 0.01M glucose-1-phosphate ,0.2M potassium phosphate, 0.2% starch suspension (primer), fresh phosphorylase - +/- 6 0.2M potassium phosphate, 0.2% starch suspension (excess), fresh phosphorylase + +/- 7 0.2M potassium phosphate, 0.2% starch suspension (excess), boiled phosphorylase + +/- (-) represents negative control, (+) represents positive control Table 4 illustrates the results of phosphorylase samples at t 0 (0 minutes) and t 5 (15 minutes). The test was conducted for a total of 15 minutes. The actual colour of the iodine solution is a pale yellow colour, therefore if the solution changes its colour it would indicate the presence of state (thus being a positive control).

Discussion Enzymes are proteins that act as biological catalysts that increase the rate of chemical reactions within cells by reducing their activation energy ( The cell: A molecular approach. 2nd edition, 2000) . This lab was conducted to analyze the impact of enzyme concentration, enzyme reaction rate and substrate concentration on salivary amylase and phosphorylase, as well as how it impacts the direction of the enzymatic reaction. The two tests used in this experiment include the iodine test and the Benedict’s test. Due to the nature of what these tests are identifying, it was easy to hypothesize the types of results that would be observed. Table 1 highlights the results of the iodine and Benedict’s tests for several salivary amylase samples (test tubes 1-4), and the fifth test tube being starch. It could be deduced that the positive control in this test would be sample five, 1% starch suspension, therefore being the positive control in this test. Test tubes 1 -4 were salivary amylase that ranged in concentration from 10%, 5%, 2% and 1%. All four test tubes were negative control because there was no observable colour change. Therefore, this means there is no amylose and amylopectin in the sample to bind with iodine (Brust, Orzechowski, & Fettke, 2020). The five samples with differing concentrations of salivary amylase and 1% starch suspension would also be tested using Benedict’s test. All five of these samples were negative controls, meaning, no metal ions (such as Ca + ) were reduced, and there was no presence of reducing sugars (Hernández-López et al., 2020). Enzymes are proteins; therefore they should be negative controls under the iodine test and Benedict’s test. Therefore, this confirms that this test is accurate. Maltose or malt sugar is characterized as a disaccharide which is bound by an α-(1,4’) glycosidic bond. Maltose forms from an enzymatic hydrolysis of amylose, by salivary amylase. Maltose further changes to two molecules of glycose my maltase which hydrolyzes the

glycosidic bond (Ouellette & Rawn, 2014) . As characterized in Table 2, all the test tubes contained 1% starch suspension and 2mL of McIlvaine’s buffer. These were added to the substrate starch and water could bind to an active site and form maltose molecules. As starch was added to the complex, this indicates why all samples – whether they had a higher concentration or lower concentration of salivary amylase – were positive controls during the iodine test. As illustrated in Table 2, the sample with 10% salivary amylose only took 15 seconds to lead to negative control, the sample with 5% salivary amylose took 90 seconds to reach the endpoint, the sample with 2% salivary amylose took 390 seconds (6.5 mins) to produce a negative control, and lastly, 1% salivary amylase took 1,380 seconds (23 minutes) to reach its endpoint. The water sample continually remained to be a positive control as there was no salivary enzyme available to conduct the reaction. The water produced a dark blue-black colour, this is because of the 1% starch suspension added to the water. All tests were incubated at 37 o C, which allowed the reaction to occur quickly, however, the temperature wasn’t high enough for protein denaturation to occur (Peterson, Daniel, Danson, & Eisenthal, 2007). Overall, this illustrates that the higher the enzyme concentration, the faster the reaction can occur, and the lower the concentration, the slower this process is. This relationship can also be viewed in Figure 1, where the highest concentration of salivary amylase resulted in the fastest reaction time. Once this reaction was complete, Benedict’s test was conducted on the same salivary amylase samples and water. The purpose of this test was the identify the presence of maltose molecules after the reaction of starch, salivary amylase, and water. Due to the nature of this test, and prior understanding of how maltose molecules are formed, it was hypothesized that all samples with salivary amylase – regardless of concentration – would be labelled as a positive control. This hypothesis was indeed validated when Benedict’s test occurred. Therefore ions

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such as Cu + were reduced, producing a dark blue-black pigment and a red precipitate. On the other hand, Water continually remained a negative control since there was no enzyme in the sample to produce maltose with the starch suspension. The results from the final experiment are outlined in Table 4. The experiment tests the effects of active and inactive phosphorylase. Active phosphorylase kinase has the ability to catalyze the reaction of breaking off a phosphate from ATP and transferring it to glycogen phosphorylase b, further changing it to phosphorylase b. This begins the process of degrading glycogen to glucose-I-phosphate (Blanco & Blanco, 2017) . The reverse reaction of phosphorylase leads to the production of starch. This occurs when glucose-I-phosphate is added to a solution with an already existing starch molecule, further resulting in the loss of an inorganic phosphate. In order for starch to be synthesized, it’s vital for primer to be added. Test tube 1 contains 0.01M glucose, starch, primer and fresh phosphorylase. This mixture paves the way to having an eventual colour change to dark violet. Phosphorylase is able to break glucose bonds, and further uses the energy released from ATP to form glucose-1-phosphate, to attach the glucose molecule to the primer on starch to become a positive control. As seen in Table 4, test tubes 1, 2, 4 and 5 contain starch primer, and test tubes 6 and 7 have starch excess. Test tubes 2,3,4 and 5 contain glucose-I-phosphate. Initially, Test tubes 1,3,4,5 are displayed as a negative controls. Test tubes 2,6 and 7 are displayed as a positive control. The initial positive controls only make sense for test tubes 6 and 7 because they consist of potassium phosphate and excess starch, whereas test tube 2 contains starch primer. After 15 minutes of the reactions taking place, test tube 2 tested positive and test tubes 5,6, and 7 were labelled as “+/-, “ these samples provided a lighter blue colour, rather than a dark blue colour. The test tubes without glucose-1- phosphate were not able to undergo the reverse reaction, however, because they contained starch

excess, the forward reaction occurred. Overall, after 15 minutes, the reaction displayed the expected results. These experiments have shown how enzyme concentration and enzyme reaction rate can vary when it’s bound to a substrate or not. As viewed in Figure 1 and Table 2, the higher the enzyme concentration in a sample is, the faster a reverse reaction can occur. In addition, it’s also important to note the impact of pH on enzyme behaviour. Every enzyme has its optimal pH range, and if it changes, it will slow the enzyme activity (Frankenberger & Johanson, 1982). For example, Salivary amylase’s optimum pH for enzyme activity is from 6 – 7 (Rosenblum, Irwin, & Alpers, 1988). Phosphorylase’s optimum pH activity is 6.7 – 6.9 (Chen & Segel, 1968). Temperature also plays a vital role in enzyme activity, increasing temperature speeds up a reaction, whereas lowering the temperature slows down the reaction. However, it’s important to be wary about not increasing the temperature too much, otherwise, the enzyme may become denatured ( Alberts et al., 2014). Relating this back to the experiment, in Table 2, the samples were incubated at 37 degrees, which is higher than normal temperature, and this in fact did speed up the rate of reaction. Overall, this experiment tested the effects of enzyme concentrations, substrate concentration and reaction rate on salivary amylase and phosphorylase. Several factors were also taken into account such as temperature and pH. A few improvements could have been made, for example, conducting the experiment in a more relaxed manner would’ve reduced the opportunity for error. For example, in Table 2, the 1% salivary solution reaching its endpoint should have occurred earlier than 23 minutes. This may have been due to human error, for example, the time was not measured accurately, and the samples were not sitting in the incubator for the entire

interval (60s). In addition, test tube 2 in Table 4 should not have displayed a positive result at t 0 . This should have been a negative control, again, this may have been due to human error, and extra starch may have been added. In conclusion, the majority of the results accurately communicated the expected results from this experiment and showed the relationship between enzyme concentrations, substrate concentration and reaction rate on salivary amylase and phosphorylase.

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Reference List Blanco, A., & Blanco, G. (2017). Medical biochemistry . London, United Kingdom: Academic Press, an imprint of Elsevier. Brust, H., Orzechowski, S., & Fettke, J. (2020). Retrieved from https://www.mdpi.com/2218- 273X/10/7/1020/htm Chen, G. S., & Segel, I. H. (1968). Purification and properties of glycogen phosphorylase from escherichia coli. Archives of Biochemistry and Biophysics , 127 , 175–186. doi:10.1016/0003-9861(68)90214-2 Department of Biology. (2023). Introductory cell biology laboratory. Waterloo, Canada: University of Waterloo Print + Retail Solutions Frankenberger, W. T., & Johanson, J. B. (1982). Effect of ph on enzyme stability in soils. Soil Biology and Biochemistry , 14 (5), 433–437. doi:10.1016/0038-0717(82)90101-8 Hernández-López, A., Sánchez Félix, D. A., Zuñiga Sierra, Z., García Bravo, I., Dinkova, T. D., & Avila-Alejandre, A. X. (2020). Quantification of reducing sugars based on the qualitative technique of Benedict. ACS Omega , 5 (50), 32403–32410. doi:10.1021/acsomega.0c04467 Johnson, L. N. (1989). Glycogen phosphorylase: A multifaceted enzyme. Carlsberg Research Communications , 54 (6), 203–229. doi:10.1007/bf02910457

Ouellette, R. J., & Rawn, J. D. (2014). Retrieved from https://www.sciencedirect.com/book/9780128007808/organic-chemistry Peterson, M. E., Daniel, R. M., Danson, M. J., & Eisenthal, R. (2007). The dependence of enzyme activity on temperature: Determination and validation of parameters. Biochemical Journal , 402 (2), 331–337. doi:10.1042/bj20061143 Peyrot des Gachons, C., & Breslin, P. A. (2016). Salivary amylase: Digestion and metabolic syndrome. Current Diabetes Reports , 16 (10). doi:10.1007/s11892-016-0794-7 Rosenblum, J. L., Irwin, C. L., & Alpers, D. H. (1988). Starch and glucose oligosaccharides protect salivary-type amylase activity at acid ph. American Journal of Physiology- Gastrointestinal and Liver Physiology , 254 (5). doi:10.1152/ajpgi.1988.254.5.g775 The cell: A molecular approach. 2nd edition . (2000). Sinauer Associates, Inc.

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