The Effect of Temperature on the Digestion of Starch by Activity of Enzyme α-Amylase: Observation of Rate of Starch Disappearance through Iodine Test Introduction
An enzyme is a type of protein that, through its own structure including hydrogen bonds, acts like a biological catalyst and is able to accelerate the biochemical reaction rate by lowering the activation energy of the whole process, without which cells could hardly practice any physiological functions within human bodies (Sizer, 1943). Found in the saliva and pancreatic secretions of animals including human beings as well as the plant seeds, bacteria and fungi (Siddiqui et al., 2010), the enzyme α-amylase that was studied during the experiment has significant impact on the hydrolysis of starch. By breaking the alpha, 1-4 glycosidic linkages in the carbohydrates, amylase hydrolyzes the starch, a polysaccharides that is stored in plants and cannot be directly digested by animal cells, into maltose, a disaccharide that later generate two units of glucose to undergo metabolisms and provides necessary energy (Slaughter et al., 2001). The enzymatic activity of α-amylase is facilitated by calcium and chloride ions during the hydrolysis (Marini, 2006 and Siddiqui et al., 2010). The complete digestion of starch and formation of maltose and glucose can be examined through the iodine test when I2KI reagent is added into the solution and remains brown instead of turning into dark blue, marking that all the molecules of starch have been fully hydrolyzed (Hanes, 1932).
While amylase effectively activates the hydrolysis of starch, the efficiency of the catalytic process is influenced by several factors including temperature, pH level and the concentration of the substrates etc. In this experiment, as the α-amylase is a type of protein, the efficiency of enzyme is highly related to its hydrogen bonds which are affected by the temperature. Though the enzyme is collected from the porcine pancreas, due to its structural similarities to amylase in human bodies, the behaviors of two amylases should resemble each other. Given that under extreme temperature enzymes will be denatured and unable to function and the constant temperature of pigs is around 39°C, the hypothesis of this experiment is that at 37°C amylase will catalyze the hydrolysis with the highest speed, followed by amylase at 22°C. Amylase at 0°C will react extremely slowly due to the crystallization of hydrogen bonds and at100°C, amylase will lose its function since it will be denatured.
Materials and Methods
Four test tubes were marked from A1 to A4. Then, 2mL of 1% starch solution from Carolina Biological Supply Company, 4mL of deionized water and 1mL of 6.8 hydrion buffer from VWR International/ Micro Essential Laboratories were added into each tube. Another four test tubes were also labeled from B1 to B4 and added 1mL of 1% α-amylase from porcine pancreas from Sigma Aldrich. Eight tubes were paired according to the same number (A1and B1 etc.) and assigned to environments at different temperature: Tube A1 and B1 were placed into a water bath at 100°C; Tube A2 and B2 were placed into a water bath at 37°C; Tube A3 and B3were placed on the tube rack (at about 22°C); Tube A4 and B4 were placed into an ice bath at 0°C. All test tubes were kept at different temperatures for 10 minutes. Meanwhile, a control group of starch solution was prepared without amylase. (Bio Lab Manual, 2013)
At the same time, a test plate was added 2 drops of I2KI reagent (1% Iodine and 2% KI) from Carolina Biological Supply Company per well. After 10 minutes, when test tubes were still in the original environments, solutions in Tube A1 with B1 were mixed and a timer was started. At each 30-second-inteval, a drop of the mixture was released into the well on the test plate until the solution in the plate did not change into dark blue and remained brown, indicating the end of the reaction by showing no presence of starch...
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