Effects of Different pH Level on Amylase in Starch
Amylase, a family of proteins that differ in isoforms, is a digestive enzyme found in saliva and pancreatic fluid that helps digest starch into simple sugars. (Scannapieco et. al., 1993). Amylase is the first step in digesting starch, which is used for the intake of carbohydrates or energy in humans. (Butterworth et. al., 2011). Amylase functions in the hydrolysis of starches, which produces glucose monomers. (Karp, 2010). This is essential in glycolysis and the harvesting of ATP. Since amylase is also a protein that performs enzymatic reactions, the secondary and tertiary structures are affected by external stimuli like ...view middle of the document...
(Luesse, 2012). The amylase protein sequence was analyzed by comparing it to other eukaryotic organism’s amylase proteins and looking at their similar protein sequences.
Figure 1: Experimental Test Tubes. Test tubes 0-15. 0(blank), 1-3(pH2), 4-6(pH 6), 7-9(pH 7), 10-12(pH 8), 13-15(pH 12). Test tubes 1, 4, 7, 10, and 13 are at zero minute, 2, 5, 8, 11,and 14 are at five minutes, and 3, 6, 9, 12, and 15 are at ten minutes.
Materials and Methods
In the experiment, sixteen 10ml test tubes were used to conduct this experiment. They were labeled 0-15 with zero being the blank and the rest being different pH levels as follows: 1-3 pH two, 4-6 pH six, 7-9 pH seven, 10-12 pH eight, and 13-15 pH twelve. The sets of three are for times zero, five, and ten (Figure 1). Then 2 ml of IKI is added to all of the test tubes, 0-15. The IKI is used to stop the reaction and see how much of the starch is broken down by amylase by staining the starch. In the blank (0), 2 ml of water is added to see how much light passes through when there is no starch present in the media. (Luesse, 2012). The digest solution is made in five 30 ml tube with 5 ml of 0.1% starch and 8 ml distilled water in all of the tubes, and 2 ml of each pH buffer’s 2, 6, 7, 8, and 12 in one of the tubes. The tubes should be labeled 2, 6, 7, 8, and 12 to differentiate the different digest solution. This solution does not contain amylase so the reaction has not yet started. This digest solution is what makes the controls for each pH level. 2 ml of the digest solution for each pH level is added to 1, 4, 7, 10, and 13 respectively to create a control that is without the enzyme amylase. Have a stop watch in hand and add 5 ml of amylase solution to the each of the five digest solutions and start the stop watch. This is when the reaction started and the time should be started.
After five minutes have passed, 2 ml of the new solution should be removed from each pH level to the five minute test tubes or 2, 5, 8, 12, and 14. This represents five minutes of the reaction that has proceeded and is frozen when added to the test tubes. (Luesse, 2012). After ten minutes have passed 2 ml of the reaction solution is added again, but in the ten minute test tubes, 3, 6, 9, 12, and 15 respectively for each pH level. All the samples are now collected and 2 ml of water is added to make a total volume of 6 ml. The spectrophotometer was then used to see how much of the starch is broken down for each pH and time.
Bioinformatics was used to find amylase’s protein sequence. This was done by using the online database of the National Center for Biotechnology Information. To find the protein amylase in the system, human amylase was put into the search bar and was narrowed down to only search for proteins. When the results came up, an amylase sequence of 511 amino acids was found and saved for further used. Next, three other organism’s amylase protein sequences were found to compare to the human amylase protein...