Enzymatic Activity of Salivary Amylase Effects of pH

John Arcee Posadas, Bryan Rayo, *Khurt Clarence Rogando Group 6 College of Science, University of Santo Tomas, Manila Philippines

Abstract pH is one factor that affects enzymatic activity of salivary amylase. To test how pH activates enzymes, different grades of pH were tested and measured for comparison. Color change and the time it took to change is observed and recorded to show where pH works best in salivary amylase. Keywords: Enzyme - any of numerous complex proteins that are produced by living cells and catalyze specific biochemical reactions at body temperatures. Amylase - any of a group of enzymes (as amylopsin) that catalyze the hydrolysis of starch and glycogen or their intermediate hydrolysis products. Buffer - a substance capable in solution of neutralizing both acids and bases and thereby maintaining the original acidity or basicity of the solution Introduction: Starch is being catalyzed in this enzymatic reaction while salivary amylase is responsible for hydrolyzing starch into maltose. Iodine test was used to determine enzymatic activity. The rate of time for the reactions to occur was measured. Iodine is used to break down chains of polypeptides as shown in the spot plates where it turns yellowish meaning it has achieved its optimum capacity. A dark colored result however was shown in pH 4 and pH 10 meaning the enzyme remains a long chain and hasnt broken down. Methodology: A. Materials: Enzyme Solution (1mL saliva + 9mL distilled H2O + 30mL 0.5% NaCl) 2% Unbuffered Starch 0.001M Iodine Solution Spot Plates (2) Large Test Tubes

Medicine Droppers Water Bath Set at 37C Acetate Buffer Solutions (pH 4 and pH 5) Phosphate Buffer Solutions (pH 6.7 and pH 8) Bicarbonate Buffer (pH 10)

B. Method: 1mL of acetate buffer (pH4) and 1mL 2% unbuffered starch in a large test tube was mixed and in a separate large test tube, 2mL of enzyme solution was placed. Both test tubes were incubated for 10 minutes in a 37 degree Celsius water bath. Three-drops of the mixture was immediately mixed and quickly dropped and at the same time, two drops of the iodine solution was dropped onto a spot plate. This was treated as the first well and/or zero minute. After a one-minute interval while continuing incubation of the enzyme and starch mixture, three-drops of the mixture was placed in the second well and two-drops of the iodine solution simultaneously which is now your 1 minute. After each 1 min interval, when the solution turned yellow, the time it changed was noted. The whole process was repeated for other pH levels (pH 5, 6.7, 8, 10) while using the appropriate buffer. A plot was made reciprocal of time versus the buffer pH and the optimum pH of the amylase was known. Results: Five test were made to confirm the optimum pH level for promoting enzymatic activity in salivary amylase.

Group 5 6 7 8 9
Table 1: Effects of pH

pH 4 5 6.7 8 10

T (minutes) 8 6 2

Discussion: The optimum pH for enzymatic activity on salivary amylase would be 6.7. Any pH lower or higher has a slower enzymatic activity. In pH 4 and 5, they appear to be too acidic for a fast reaction to occur. At pH 8 however, it is slowly being denatured because of alkalinity level of that pH. Lastly, at pH 10, every enzyme in the salivary amylase is completely denatured. Conclusion:

In conclusion, enzymatic activity in salivary amylase has an optimum pH of 6.7 which has acidic and basophilic properties to work at its best. This experiment tells us that time is important in handling these pH levels since a mistake can change the results drastically.

Reference:

"Enzymatic Activity of Salivary Amylase." StudyMode.com. 03, 2011. Accessed 03, 2011. http://www.studymode.com/essays/Enzymatic-Activity-Of-Salivary-Amylase-

604454.html. Boyer, Rodney. Concepts in Biochemistry, Third Edition. John Wiley & Sons (Asia) Pte Ltd