Week 9 Enzymes Lab Report Final Draft (4)

Week 9: Enzymes Lab Report Jack Lacelle 21061823 TA: Marcus, Chloe, Kayla BIO130L November 6th: 2:30pm - 5:20pm STC Room 4018 Section 002 Due date: November 13, 2023

1 Introduction An enzyme is a type of protein that functions as a catalyst to lower the activation energy of a chemical reaction and speed up the reaction's rate (Newman, 2022). The purpose of this experiment is to investigate the effects of substrate concentration, reaction time, and enzyme concentration on the direction of an enzyme reaction (Department of Biology, 2023). Additionally, this lab also observes the effect of temperature on an enzyme’s activity. Enzymes facilitate reactions without affecting the equilibrium of a reaction through the following process: the substrate initially forms a complex with the enzyme, known as a substrate-enzyme complex (Cooper, 2000). Within this complex, the substrate undergoes conversion into the 'enzyme product.' Subsequently, the complex dissociates, freeing the enzyme and producing the product (Cooper, 2000). In the first part of the lab, we examined how the digestive enzyme salivary amylase affects starch by altering the enzyme concentration. Salivary amylase is crucial for breaking down starches in our food (PDBE, 2022). It accomplishes this by breaking the bonds in starch and creating maltose through a process called hydrolysis (DiMango, 2004). The second part of the lab explored how the enzyme phosphorylase behaves at various temperatures. Starch phosphorylase facilitates the transfer of glucosyl units from glucose-1-phosphate to the nonreducing end of alpha-1,4-D-glucan chains in a reversible manner, accompanied by the release of phosphate (Boehkle, 2015).

3 Table 1.2: Benedict’s Test for Salivary Amylase and Starch Suspension Test Tubes Benedict’s Test Results #1: 10% Amylase solution Negative ( - ) #2: 5% Amylase solution Negative ( - ) #3: 2% Amylase solution Negative ( - ) #4: 1% Amylase solution Negative ( - ) #5: 1% Starch suspension Negative ( - ) Table 1.2 shows the results of Benedict’s test for the solutions listed above. 4mL of Benedict’s solution was added to each test tube. Then they were mixed thoroughly, and boiled in a hot bath for 5 minutes (Department of Biology, 2023). Each solution remained bright blue. Therefore, each solution produced a negative result, therefore there is no presence of reducing sugars. Table 1.3: Disappearance of Starch in Reagent Tubes Time Key: Each interval is different for each test tube as instructed in the lab manual. Each interval for #14 is 60 seconds. #13 is 30 seconds. #12 is 15 seconds. #11 is 5 seconds. #15 is 30 seconds. Int = Time Interval. Time Test Tube# 14 Test Tube #13 Test Tube #12 Test Tube #11 Test Tube #15 Int 1 Positive + Positive + Positive + Positive + Positive +

4 Int 2 Positive + Positive + Positive + Negative - Positive + Int 3 Positive + Positive + Negative - Done Positive + Int 4 Positive + Positive + Done Positive + Int 5 Positive + Negative - Positive + Int 6 Positive + Done Positive + Int 7 Positive + Positive + Int 8 Negative - Positive + Int 9 Done Positive + Int 10 Positive + Int 11 Positive + Int 12 Positive + Int 13 Positive + Int 14 Positive + Int 15 Positive + Int 16 Positive + Int 17 Positive + Int 18 Positive + Int 19 Remained Positive Table 1.3 shows the results of iodine testing each solution at various intervals. Each test tube is placed in a water bath at 37 ॰ C to equlibrate for 5 minutes. This is the optimal temperature for salivary amylase activity (Department of Biology, 2023). Each solution was iodine tested at various intervals as stated above in the key. Test tube #11 resulted in a negative test after 10

6 Table 1.5: Benedict’s Test for Solutions #16-20 Test Tubes Benedict’s Test Results #16 Positive ( + ) #17 Positive ( + ) #18 Positive ( + ) #19 Positive ( + ) #20 Negative ( - ) Table 1.5 shows the results for Benedict’s test of solutions. Each test tube contains 4mL of Benedict’s solution. #16 consists of 2mL of 1% starch and 2mL of McIlvaine’s buffer, as well as the remaining 10% salivary amylase from Step 17 (Department of Biology, 2023). #17 consists of 2mL of 1% starch and 2mL of McIlvaine’s buffer, as well as the remaining 5% salivary amylase from Step 16 (Department of Biology, 2023). #18 consists of 2mL of 1% starch and 2mL of McIlvaine’s buffer, as well as the remaining 2% salivary from Step 15 (Department of Biology, 2023). #19 consists of 2mL of 1% starch and 2mL of McIlvaine’s buffer, as well as the remaining 1% salivary amylase (Department of Biology, 2023). Lastly, #20 consists of 2mL of 1% starch and 2mL of McIlvaine’s buffer, as well as the remaining water from Step 18 (Department of Biology, 2023).

7 PART B Table 2.1: Iodine Tests for Phosphorylase Time Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 0 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) 3 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) 6 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) 9 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) 12 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) 15 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) 18 mins ( - ) ( + ) ( + ) ( - ) ( - ) ( + ) ( + ) Table 2.1 shows the results of iodine testing for test tubes #1-7 in part B. #1 contains 1.5mL of 0.001M glucose, 1 drop of 0.2% starch, and 2mL fresh phosphorylase (Department of Biology, 2023). #2 contains 1.5mL of 0.01M glucose-1-phosphate, 1 drop of 0.2% starch, and 2mL fresh phosphorylase (Department of Biology, 2023). #3 contains 1.5mL of 0.01M glucose-1-phosphate, and 2mL of fresh phosphorylase (Department of Biology, 2023). #4 contains 1.5mL of 0.01M glucose-1-phosphate, 1 drop of 0.2% starch, and 2mL of boiled phosphorylase (Department of Biology, 2023). #5 contains 1.5mL of 0.01M glucose-1-phosphate, 0.5mL of 0.2M potassium phosphate, and 2mL fresh phosphorylase (Department of Biology, 2023). #6 contains 0.5mL of 0.2M potassium phosphate, 1.5mL of 0.2% starch, and 2mL of fresh phosphorylase (Department of Biology, 2023). Lastly, #7 contains

9 The iodine test for mixtures of 2mL 1% starch suspension and 2mL of McIlvaine’s Buffer, which also included 2 mL of each salivary amylase solution yielded the expected results. The purpose of McIlvaine’s buffer was to keep the pH optimal for salivary amylase (DBpedia, 2023). The lower the salivary amylase concentration in the solution, the longer it took to get a negative iodine test result (Breslin, 2016). This conclusion is supported by the results of this testing. It is especially noticeable in test tube #11, and test tube #15. Test tube #11 contained 10% salivary amylase– the highest concentration of salivary amylase out of all the solutions (Department of Biology, 2023). In just ten seconds, the salivary amylase broke down the starch and produced a negative test, in just the second interval of iodine testing. Test tube #15 on the other hand contained 2mL of water, rather than any salivary amylase (Department of Biology, 2023). Due to the lack of amylase the starch was unable to break down, and the tests resulted in positive iodine tests the entire way through, meaning starch remained in the solution. The results of test tubes #12-14, supported this conclusion as well as each solution gradually took longer time to produce a negative result, this is due to the lower concentrations of amylase which makes it slower for the enzyme to break down the starch (Bartleby, 2022). Test tubes #12, #13, and #14 each contained 5% salivary amylase, 2% salivary amylase, and 1% salivary amylase respectively (Department of Biology, 2023). The negative control in this experiment was the higher salivary amylase concentration to reduce starch interaction with iodine, producing positive results. The positive control was the starch suspension with low salivary amylase concentration. Our results were expected, and the test made it evident that higher concentrations of salivary amylase catalyze the hydrolysis of the 1–4 glycosidic bonds in starch. As a result, solutions with elevated enzyme concentrations will exhibit the yellowish-brown color more quickly (Agirre, 2019).

10 Benedict’s tests were completed for the same solutions found in test tubes #16-20 (Department of Biology, 2023). The results we achieved were once again expected. Test tubes #16-18 yielded positive results, changing from a bright blue color to a orangish/red solution (Aryal, 2022). Test tube #19 produced a positive result as well, however it turned a dull green, which indicates a positive Benedict’s test, however a lower concentration of reducing sugars (Aryal, 2022). The red and green color change signifies that the enzyme successfully broke down the starch molecule. The reason the Benedict’s tests were positive is due to the formation of maltose. Maltose is a sugar that is produced when the enzyme salivary amylase breaks down starch (University of Illinois, 2012). Therefore the maltose was detected by the Benedict’s test, resulting in positive results. Test tube #20 produced a negative result, this is because it has no enzyme, therefore the starch is not broken down into maltose. The positive control of this reaction is the activated enzyme in the boiled setting, while the negative is the water with the absence of salivary amylase (Hickman, 2022). In the phosphorylase iodine test, a range of results was observed. The anticipated outcome was the development of a dark blue or violet color, indicating the occurrence of phosphorolysis during the breakdown of starch (Swhimmer, 1955). A positive iodine test suggests that starch is still present in the solution. This may imply that the enzymatic action of phosphorylase has not fully broken down the starch into simpler molecules like glucose, or it could indicate that the reaction has not proceeded to completion (Brust, 2020). We achieved positive results for test tubes #2, #3, #6, and #7. I believe this is due to the presence of 1.5mL of 0.2% starch in test tubes #6 and 7. Test tubes #2 and 3 contain glucose-1-phosphate. It’s accumulation can play a role in feedback regulation, potentially slowing down further

12 phosphorylase to act upon. This can result in a slower rate of reaction and a less efficient conversion of starch to glucose (Brust, 2020). In conclusion, in these experiments, we observed that when there is a higher enzyme concentration, the reaction happens faster. This is evident in Table 1.3 and Graph 1.4, where higher concentrations of salivary amylase led to quicker results, highlighting a key aspect of how enzymes function (Breslin, 2016). Conversely, having more of the substance the enzyme acts on such as starch, also speeds up the reaction by providing more opportunities for the substances to interact (Libretexts, 2022). Reaction rates play a role in both directions of a chemical reaction, emphasizing another important function of enzymes (Keenleyside, 2019). In summary, the experiments conducted provide valuable insights into the connection between enzymes and substrates, highlighting their significance in biological processes.

13 References Agirre, J., Moroz, O. V., Meier, S., Brask, J., Munch, A., Hoff, T., Andersen, C., Wilson, K., & Davies, G. (2019). The structure of the AliC GH13 α-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the α-amylase family. Acta Crystallographica Section D: Structural Biology , 75 (1), 1–7. https://doi.org/10.1107/s2059798318014900 Aryal, S. (2022, August 10). Benedict’s Test- Principle, Composition, Preparation, Procedure and Result Interpretation . Microbiology Info.com. https://microbiologyinfo.com/benedicts-test-principle-composition-preparation-procedure -and-result-interpretation/ Boehlke, C., Zierau, O., & Hannig, C. (2015). Salivary amylase – The enzyme of unspecialized euryphagous animals. Archives of Oral Biology , 60 (8), 1162–1176. https://doi.org/10.1016/j.archoralbio.2015.05.008

15 Libretexts. (2022, July 4). Starch and iodine . Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_( Biological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine Libretexts . (2022, August 17). 18.7: Enzyme activity. Chemistry LibreTexts. https://chem.libretexts.org/Bookshelves/Introductory_Chemistry/Basics_of_General_Org anic_and_Biological_Chemistry_(Ball_et_al.)/18%3A_Amino_Acids_Proteins_and_Enz ymes/18.07%3A_Enzyme_Activity McIlvaine buffer . (2023). DBpedia. https://dbpedia.org/page/McIlvaine_buffer Newman, T. (2022, July 8). Enzymes: How they work and what they do . https://www.medicalnewstoday.com/articles/319704 PDBE. (2022). The wonders of salivary amylase | Protein Data Bank in Europe . https://www.ebi.ac.uk/pdbe/news/wonders-salivary-amylase Robinson, P. (2015). Enzymes: principles and biotechnological applications. Essays in Biochemistry , 59 , 1–41. https://doi.org/10.1042/bse0590001 Röder, P. V., Wu, B., Liu, Y., & Han, W. (2016). Pancreatic regulation of glucose homeostasis. Experimental & Molecular Medicine , 48 (3), e219. https://doi.org/10.1038/emm.2016.6

16 Shwimmer, S. (1955.) Effect of phosphate and other factors in potato extracts on amylose formation by phosphorylase. https://pdf.sciencedirectassets.com/778417/1-s2.0-S0021925818X60980/1-s2.0-S0021925 818653396/main.pdf? The effect of the concentration amylase on the rate of. . . | Bartleby . (2023). https://www.bartleby.com/essay/The-Effect-of-the-Concentration-Amylase-on-PKM8CP AQGDAX#:~:text=The%20lower%20the%20concentration%20of,twice%20as%20many %20active%20sites.&text=Enzymes%20are%20made%20of%20long,have%20an%20irr egular%20globular%20shape. University of Illionois. (2012). Disaccharides http://butane.chem.uiuc.edu/pshapley/GenChem2/B10/1.html#:~:text=Examples%20of% 20Disaccharides&text=Maltose%20is%20derived%20from%20the,disaccharide%20of% 20galactose%20and%20glucose.