Identification of unknown a-Amylase
through testing different temperatures and pH values to detect the absorbance of maltose.
Enzymes are biological catalysts, mainly proteins for this experiment, generated by an organism to speed up chemical reactions. They have active sites on which the substrate is attached, and then broken up or joined. For this experiment we are going to work with the enzyme a-amylase. Amylase is an enzyme that breaks starch down into sugar. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain starch like rice and potatoes. Amylase turns some of their starch into sugar in the mouth. The pancreas makes amylase (alpha amylase) to hydrolyze dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Alpha amylase in this lab is used to hydrolyze starch to break it down into maltose. Purpose of this lab is to show that both pH temperature affect enzyme actions. The optimal temperature for the enzyme from Aspagillus is 37°C, and an acidic pH of 4-6 whereas, the enzyme from the hot spring bacteria is 100°C and acidic pH of 4-6. For the enzyme from the pig pancreas, optimal temperature is 37°C and a neutral pH of 7.
Material and Methods:
Part I: Effect of Temperature on a-amylase activity
To be recorded in a notebook are which a-amylase preparation to be used (A,B,C). One test tube labeled as “blank” other five test tubes as :“4°C,”23°C, ”37°C,” “65°C,” and “100°C.” 1 ml of 1% starch and 1ml of pH 7 was added to each tube. The substrate of the reaction was starch. Of the indicated temperatures, one tube was placed in each. At room temperature the “blank” and 23°C tubes are to be left, furthermore, the 4°C tube to be placed on ice. Equilibrate the tubes at each temperature for 10 mins.100ml of the concentrated a-amylase stock solution was added to 9.9 ml of distilled water. Stocks and diluted solutions are to be kept on ice when not in use, Vortex to mix the mixture. At the 10min mark all the tubes are to be retrieved, then 1ml of distilled water is to be added to the blank tube only. For the other five tubes, timer is to be set at 12minutes as 1ml of diluted a-amylase is to be added to each of the five tubes. Vortex as to mix your mixture. Each test tube is to be returned to appropriate temperature exactly 12mins from set timer.
For the next step gloves have to be worn to handle the maltose color reagent. Proceed with the maltose detection step after the 12 minutes have run out.1 ml of maltose color reagent was added to each test tube including the “blank”. The test tubes are to be vortexed then placed at 100°C exactly 15 minutes. When the Maltose color reagent is to be added to the first test tube, timing is to be started. Enzymatic reactions are stopped when Maltose color reagent denatures all the enzymes. Tubes are to be placed on ice, cooled to room temperature after the 15minutes run out. Furthermore, 9 ml of distilled water are to be added to each test tube, then mixture is to be vortexes. The “blank” test tube is used to zero the spectrophotometer, so to obtain the measurements of all samples on the A540nm. Final results are recorded on a table.
Part II: Effect of pH on a-amylase activity
Record in your notebook which a-amylase preparation you are to use(A,B,C)one test tube labeled as “blank” other five test tubes as :“pH 5,”pH 6,”pH 7,”pH 8,” and “pH 9.” one milliliter of the appropriate pH starch is to be added to each corresponding tube. 1ml of 1% starch and 1 ml of pH 7 was added to each tube. The substrate of the reaction was starch. 100 ml of the concentrated a-amylase stock solution was added to 9.9 ml of diluted water. Stocks and diluted solution s are to be kept on ice when not in use, Vortex to mix the mixture. 1ml of distilled water is to be added to the blank tube only. For the other...