Amylase acts on starch by breaking off maltose (CLC 2H22011) molecules from the end of starch chains (Did Giuseppe, 2002). Every time a bond is broken in a starch chain, a water molecule is consumed. This process is referred to as a hydrolysis reaction which requires water to digest a substrate, in this case starch (Walsh, 2002). Phosphoresce is also an enzyme that acts on starch by removing glucose molecules (Stick and Williams, 2009). Phosphoresce breaks down the bonds between adjacent molecules in starch by consuming phosphoric acid instead of water like amylase (Stick and Williams, 2010).
The energy released in this coercing reaction is used to create a glucose phosphate bond in glucose-I-phosphate. Water is a medium n this reaction, not a product or reaction. The concentration of water will not affect the direction of the reaction because it is not directly in use. A starch primer molecule is necessary for a positive iodine test to be achieved because the enzyme can only act by making the primer starch molecule longer (Stick and Williams, 2009). The iodine test is used to test for the presence of starch molecules.
This reaction is the result of the formation of polyploidy chains from the reaction of starch and iodine (Hirsch, 2002). The straight chains of starch for helices where iodine molecules assemble, resulting in a positive test with the colors purple/black (Hirsch, 2002). A negative test would occur is a transparent liquid is produced or there is not a change to the solution when the iodine test is added (Hirsch, 2002). The second test performed is the Benedicts test, which allows for the detection of sugars with a free alluded or ketene group, known as reducing sugars (Freeman and Harrington and Sharp, 2011).
All incarcerations are reducing sugars and have a free reactive carbonyl group. Starches are non-reducing sugars and will provide a negative test; the solution will stay the blue color of the Benedicts solution. A positive test occurs when the copper sulfate present in the Benedicts solution reacts with the electrons from the alluded or ketene group of the reducing sugars to form a red-brown precipitate (Hirsch, 2002). Materials and Methods: For the following experiment, refer to the Department of Biology BILL LOLL Lab Manual, Fall 2014. Experiment 5, Enzymes. Performed October 31, 2014.
Pages 39-45. Deviations include: 1. A ml pipette was used instead off Mil pipette. (Department of Biology, 2014) Results: Table 1. Iodine and Benedicts Tests for Test Tubes 1-5 Test Tube # Contents of Test Tube Iodine Test Benedicts Test 10% Salivary Enzyme Negative, Non-viscous, yellow, transparent liquid. Positive, Orange-green precipitate, opaque, 2 5% Salivary Enzyme Positive, faint orange precipitate, blue-green solution color, opaque. 3 2% Salivary Enzyme Negative, no change to solution after boiling 4 1% Salivary Enzyme 5 1% Starch Solution Positive, Purple-black opaque liquid is formed.
The above table represents the results of test tubes 1-5 when using the iodine test for starch and Benedicts solution to check for reducing sugars. Graph 1 . Concentration vs.. Time of Amylase Enzyme The above graph shows the correlation between the concentration of salivary enzyme and the time it takes for the enzyme to break down the substrate. Table 2. Using an iodine test to test disappearance of starch for test tubes 11-15 Test Tube # Contents First Positive Iodine Test First Negative Iodine Test Total Time Elapsed 15 ml of water from test tube #10, ml of 1% starch suspension and ml Mescaline’s buffer.
Interval : Positive, purple-black opaque liquid formed No change ever because there is no active enzyme 14 ml of 1% salivary enzyme from test tube #9, ml of 1% starch suspension and ml of Mescaline’s buffer. Interval : Positive, purple-black opaque liquid formed Interval #10. Negative, non-viscous, yellow, transparent liquid 600 seconds (10 intervals 60 seconds apart) 13 ml of 2% salivary enzyme from test tube #8, 2 ml of 1% starch suspension and 2 ml of Mescaline’s buffer. Interval #1 : Positive, purple-black opaque liquid formed Interval #9.
Negative, non-viscous, yellow, transparent liquid 270 seconds (9 intervals 30 seconds apart) 12 ml of 5% salivary enzyme from test tube #7, ml of 1% starch suspension and ml Interval #8. Negative, non-viscous, yellow, transparent liquid 120 seconds (8 intervals 5 seconds apart) 11 ml of 10% salivary enzyme from test tube #6, ml of 1% starch suspension and ml Interval #11. Negative, non-viscous, yellow, transparent liquid 55 seconds (1 1 intervals 5 seconds apart) The above table shows how many intervals at a given concentration for the test to go from a positive result to a negative result keeping all other variables constant.
Table 3. Benedicts test for test tubes 16-20 16 Remainder of ml of water from test tube #10, ml of 1% starch suspension and Mescaline’s buffer that was no used in previous steps. Negative. No change to elution after boiling with Benedicts solution. 17 Remainder of ml of 1% salivary enzyme from test tube #9, ml of 1% starch suspension and ml of Mescaline’s buffer that was not used in previous steps. Negative. No change to solution after boiling with Benedicts solution. 18 Remainder of ml of 2% salivary enzyme from test tube #8, ml of 1% starch Positive.
Red precipitate formed, changed from dark blue to a turquoise color over time. 19 Remainder of ml of 5% salivary enzyme from test tube #7, ml of 1% starch Positive. A more dark orange precipitate formed, the color changed from dark blue o turquoise over time. 20 Remainder of ml of 10% salivary enzyme from test tube #6, ml of 1% starch Positive. Dark orange precipitate formed, changed from dark-blue to a turquoise color over time. The above table shows the results from a Benedicts test after the contents of each test tube were incubated at 37 degrees Celsius.
Table 4. Iodine test using phosphoresce as an Enzyme. Final Result of Iodine Test ml of phosphoresce, 1. Ml of . 01 M glucose, 1 drop of . 2% starch suspension. Negative. Non-viscous, yellow, transparent liquid. ml of phosphoresce, 1. Ml of . 1 M glucose-I-phosphate, 1 drop of. 2% starch suspension. Positive. Purple-black opaque liquid formed. 3 ml of phosphoresce, 1. Ml of . 01 M glucose-I-phosphate. ml of boiled phosphoresce, 1. Ml of . 01 M glucose-I-phosphate, 1 drop of . 2% starch suspension. Negative. Non-viscous, yellow, transparent liquid. ml of phosphoresce, 1. Ml of . 01 M glucose-I-phosphate, . Ml of. MM potassium phosphate, 1 drop of . 2% starch suspension. Positive. Purple-black opaque liquid formed. 6 ml of phosphoresce, . Ml of . MM potassium phosphate, 1. Ml of . 2% starch 7 ml of boiled phosphoresce, . ml of . MM potassium phosphate, 1. Ml of . 2% starch suspension. Negative. Non-viscous, yellow, transparent liquid. The above table represents the results of an iodine test preformed when phosphoresce is used as the enzyme in different types of solutions.
Discussion: The original samples of test tubes 1-5 were tested using iodine and Benedicts tests, and the results obtained were expected based on the contents of the test tubes. Tubes 1-4 produced a negative iodine test because there were no traces of starch molecules in them, while 5 produced a positive iodine test because it was imposed of starch and water. For the Benedicts test it was the opposite, as 1-4 produced a positive test because the amylase enzyme contains reducing sugars that are readily available. Test tube 5 produced a negative Benedicts test result because starch is made up of reducing sugars that are linked together.
As a whole, starch will not yield a positive result because there is no detection of reducing sugars. The iodine test was used to detect the presence of starch at different intervals to test how the concentration of different enzymes affects the rate at which it is acted n a substrate. The first solution tested, 1% salivary amylase, provided a positive result until a negative result was obtained at interval number 10, which was 600 seconds after the commencement of the iodine test. The concentration of the enzyme in this test tube was not very high, which is why it took a long time for a negative result to occur.
A negative result occurred because amylase eventually broke down the starch, leaving nothing for the iodine to bind to (Stick and Williams, 2009). The second solution consisted of 2% salivary amylase, with the same amount of 1% starch suspension and Mescaline’s buffer as all other test tubes, making these the controlled variables. The solution was tested at 30 second intervals, providing a negative test on interval number 9, which was 270 seconds into the iodine test. The time taken was less than the previous step, as expected. This is because the concentration of the enzyme is greater, increasing the rate at which the starch is digested.
The third solution was 5% amylase, which was tested at 1 5 second intervals because the concentration is substantially larger than the preceding tests. The test became active on the 8th interval, 120 seconds into the test. Since the concentration is much greater, the time for a negative test result to occur is significantly reduced. The final test tube contained a 10% salivary amylase solution, and it reached end point the quickest because the concentration was the highest. The solution was tested in 5 second intervals, as it demonstrated the greatest rate of breaking down the starch.
It reached the end point on the 1 lath interval, which was 55 seconds into the test. The relationship of time and concentration is shown by graph 1, as concentration is increased, the time decreases. By increasing the amount of concentration there are more active sites available for the substrate to bind to, allowing the reaction to be catcalled in a quicker fashion. The last iodine test conducted was a control; water was added to the starch solution, which will always produce a positive test because there are no enzymes actively working on the starch molecule.
The Benedicts test was used to examine the amount of reducing sugars in each of the test tubes. Test tubes 16-18 produced positive results, the contents of the, were 10%,5% and 2% salivary amylase. The color produced was an orange-red precipitate with an orange tint throughout the solution. The most positive test tube was the one that contained the largest amount of salivary amylase because it broke the starch down the quickest, therefore more individual monomers were in solution.
The other test tubes, 19 and 20, produced negative results because there are very low amounts of reducing sugars. In test tube 19, there is only 1% amylase, which means not much starch has been broken down into individual monomers. While in test tube 20, there was no change because there are no enzymes present. By boiling the enzyme phosphoresce, it deactivates the enzyme, making the Benedicts test negative while the iodine test is made positive. The phosphoresce that was not boiled produced a positive Benedicts test and iodine test because the enzyme is active.
Test tubes 1 and 3 produced a negative iodine test because there was no detectable starch. This is true because the contents of the test tubes did not include a starch primer. Test tubes 2,5,6 all provided positive iodine tests because they had starch, phosphoresce and either glucose-I-phosphate or potassium phosphate. This allowed for the synthesis of starch molecules that were long enough for the iodine test to detect them and create a positive test. The final test tubes, 4 and 7 had no change to them because the enzyme was not active after being boiled.
This produced a negative test result with iodine solution. Enzymes have the ability to work in both directions of a reaction, usually with a favorable and unfavorable reaction (Walsh, 2002). By increasing the amount of the enzyme and keeping all other variables constant, the time is directly related to the concentration of the enzyme. Based on the results of the graph, as concentration increases the time decreases. This means more products are being produced per unit time but will not shift the chemical equilibrium of the reaction (Freeman et al. 2011). There were a few sources of error such as; the substances may be contaminated because the spot plate wells were very close together for the iodine testing and a lot of tests were taking place. Also, the lid was not always shut on the water bath, allowing the temperature to equilibrate to the temperature of the room. Even with these sources of error, the results obtained from the lab make sense, as he concentration of an enzyme increase, time decreases and by modifying an enzyme, it can become active or inactive.
