Enzymatic Activity of Salivary Amylase

Enzymatic Activity of Salivary Amylase Ong, Janela Rose I. ; Paguia, Maria Tricia C. ; Placente, Dax Daven A. ; Posadas, Grace Catherine A. 3Bio3-Group 8 Department of Biological Sciences, College of Science University of Santo Tomas, Espana, Manila 1008 Abstract This experiment aims to examine the enzymatic activity and specificity of salivary amylase depending on the changes in pH and temperature; and determine the optimum temperature and pH of the amylase. EXPERIMENTAL In the enzymatic activity of salivary amylase, 1ml saliva, 9ml distilled water and 30ml of 0. % NaCl made up the enzyme solution. One percent starch in phosphate buffer pH 6. 7 was the buffered starch. The experiment was comprised of two parts. For the first part (effect of temperature), 2 ml of the enzyme solution was placed in a large test tube and labelled as 4?. In a separate large test tube, 2 ml of the buffered starch solution was added. Both test tubes were incubated for 10 minutes in an ice bath with a tempe rature of 4?. The two test tubes were IMMEDIATELY mixed after 10 minutes, and three-drops were QUICKLY taken from the mixture. The three-drops of the mixture simultaneously added with two drops of iodine solution were dropped onto the first well of a spot plate and was labelled as the zero minute. Incubation should be continued and after a one-minute interval, three-drops of the mixture simultaneously added with two drops of iodine solution were dropped onto the second well of a spot plate and was labelled as one minute. The test tube of the iodine solution should not be exposed. It should be covered with a cork and wrapped around with aluminium foil. The same step of taking three-drops of the mixture simultaneously added ith two drops of iodine solution was repeated until a light yellow-colored solution was observed. Time should be noted. The first part of the procedure was not only focused on 4? but also other temperatures such as room temperature, 37? , 50? , 60? and 70?. All temperatures were incubated at the desired incubation temperature. The reciprocal of time (1/t, min-1) was plotted versus the tempe rature (T) and the optimum temperature of the amylase was determined. For the second part (effect of pH), 1 ml of acetate buffer with ph 4 and 1 ml of 2% unbuffered starch were mixed in a large test tube. In a separate large test tube, 2ml of the enzyme solution was added. Both test tubes were incubated for 10 minutes in a 37? water bath. The two test tubes were IMMEDIATELY mixed after 10 minutes and three-drops of the mixture were QUICKLY and simultaneously added with two drops of the iodine solution onto the first well of a spot plate. This was labelled as the zero minute. Incubation should be continued and after a one-minute interval, three-drops of the mixture simultaneously added with two drops of iodine solution were dropped onto the second well of a spot plate and was labelled as one minute. The test tube of the iodine solution should not be exposed also and it should be covered with a cork and wrapped around with aluminium foil. The same step of taking three-drops of the mixture simultaneously added with two drops of iodine solution was repeated until a light yellow-colored solution was observed. Time should be noted. The same steps for the second part of the experiment should be followed for other pH such as 5, 6. 7, 8 and 10 — using the appropriate buffer. The reciprocal of time (1/t, min-1) was plotted versus the buffer pH and the optimum pH of the amylase was determined.