Biological Chemistry
Enzymatic Activity of Salivary Amylase Ellicia Vern Mendoza, John Michael Joseph Napa, Nicanor Olanka, *Maria Christina Paine Group 6, 3Bio-3 Department of Biological Sciences, College of Science, University of Santo Tomas, España, Manila 1055 Date Submitted: December 11, 2010
Abstract
The enzymatic activity of the salivary amylase is being affected by several factors. These factors include the temperature and pH. This experiment discusses how temperature and pH take role in activating activating the enzymes. To explain explain how this happens, the rates of enzymatic enzymatic activity of salivary salivary amylase in varying temperatures and pHs were measured and compared. A plot in the end shows the results and comparison.
I. Introduction
Enzymes are natural biological catalysts that speed up the reactions occurring in living organisms, both animals and plants. The use of a specific enzyme makes a typical metabolic reaction reaction a million million times faster. faster. Enzymes are being designed specifically specifically for a certain certain chemical chemical reaction in the system. This is through the DNA, in the cell’s nucleus, which encodes instructions for the cells to synthesized specific enzymes. An enzymatic activity consists of substrate, active site & enzymes. Enzymes act on substrates. The specific site where enzymatic activity takes place is the active active site. site. The substance formed formed by the reaction reaction is called called product. product. The salivary salivary amylase, amylase, produced by salivary salivary glands, is a type of a hydrolase hydrolase enzyme (amylase). (amylase). Hydrolase Hydrolasess catalyze hydrolysis reactions where there is an addition of a water molecule to a bond resulting in bond breakage. Amylase breaks down starch and turns it into maltose, a disaccharide. These reactions have an important role in digestive process. The experiment has the main objective of
examining the enzymatic activity and specificity of salivary amylase depending on changes in pH and temperature.
II. Materials
Enzyme solution (1 mL saliva + 9 mL distilled H2O + 30 mL 0.5% NaCl) Buffered starch (1% starch in phosphate buffer pH 6.7) 0.001 M Iodine Solution Spot plates Test tubes Medicine droppers Constant temperature bath (4oC, 37 oC, 40 oC, 50 oC, 70 oC) 2% Unbuffered starch Water bath set at 37 oC Acetate buffer solutions (pH 4&5) Phosphate buffer solutions (pH 6.7 & 8) Bicarbonate buffer (pH 10)
III. Methodologies
In this this experim experiment ent,, two effect effectss on the enzymat enzymatic ic activi activity ty of saliva salivary ry amylas amylasee were were observed (effect of temperature & effect of pH). The first activity was the observation of the effect of temperature. First, 2 mL of the enzyme solution was put in a large test tube and labeled as 4 oC. Next, 2 mL of the buffered starch solution was put in a separate large test tube. Both test tubes were incubated incubated for 10 minutes minutes in an ice bath at 4 oC. The two solutions were immediately mixed. Three drops of the mixture were quickly taken and two drops of the iodine solution were
simultaneou simultaneously sly added onto the first first well of a spot plate. That has been the zero minute. After a one-minute interval with continuous incubation, three drops again of the mixture were taken and two drops of the iodine solution were added simultaneously onto the second well. That has been the one minute. The step 5 was repeated until a light yellow-colored solution was observed. The final time was noted. For the other temperatures (37 oC, 40 oC, 50 oC & 70 oC ), the steps 1 to 6 were repeated following the desired incubation temperature. Finally, the reciprocal of time (1/t, min-1) in step 6 versus the temperature (T) was plotted and the optimum temperature of the amylase was determined. The last activity was the observation of the effect of pH. First, 1 mL of acetate buffer (pH 4) and 1 mL 2% unbuffered starch were mixed in a large test tube. 2 mL of the enzyme solution was added in a separate large test tube. Both test tubes were incubated for 10 minutes in a 37 oC water bath. The two solutions were immediately mixed. Three drops of the mixture were quickly taken and two drops of the iodine iodine solution solution were added simultaneou simultaneously sly onto the first first well of the spot spot plat plate. e. That That has has been been the the zero zero minu minute te.. Afte Afterr a one-m one-min inut utee inte interv rval al with with cont contin inuou uouss incubation, three drops of the mixture were taken again and two drops of the iodine solution were simultaneously added onto the second well. That has been the one minute. The step 5 was repeated until a light-yellow colored solution was observed. The final time (t) was noted. The steps 1 to 6 was repeated for the other pH (4, 5, 6.7, 8, 10) using the appropriate buffer. Finally, the reciprocal of time (1/t, min-1) in step 6 versus the buffer pH was plotted and the optimum pH of the amylase was determined.
IV. Data & Results
A. Effect Effect of Temper Temperatu ature re
Temperature (T)
Time (min)
1/t (min-1)
4oC
∞ mins
1/∞ / 0 mins-1
37 oC
5 mins
1/5 / 0.2 mins-1
40 oC
11 mins
1/11 / 0.09 mins-1
50 oC
∞ mins
1/∞ / 0 mins-1
70 oC
∞ mins
1/∞ / 0 mins-1
B. Effe Effect ct of pH pH
V. Discussion
Time (min)
1/t (min
-1
)
4
∞ mins
1/∞ / 0 mins-1
5
19 mins
1/19 / 0.05 mins-1
6. 7
14 mins
1/14 / 0.07 mins-1
8
29 mins
1/29 / 0.03 mins-1
10
∞ mins
1/∞ / 0 mins-1
Optimum pH (6.7, 0.07
The graph shows the reaction rate of the salivary amylase (reciprocal of time versus temperature/pH). The rate of an enzyme catalyzed reaction as the temperature increases but this is only up to a point. Enzymes work best at a certain temperature wherein the reaction rate will be at a maximum. This is proved by the bell-shaped curve in the graph. This certain temperature is called the optimum temperature. The results show that the peak of temperature-related reaction rate is at 37oC. This is because the optimum temperature of this enzyme is at 37oC where the enzymes and substrates are working at its best. This applies in the human body. At 4 oC, there is little energy which makes the reaction slower. At 40oC, the enzymes and substrate are working a
lot quicker but due to the high temperature, some enzymes are denatured. At temperatures 50 oC and above, all the enzymes are denatured due to destruction of secondary and tertiary structures. At this temperature, the enzyme molecule vibrates too much to the extent that the enzymes lose their three-dimensional structure. This destroys, as well, the active sites. Thus, the activity of the enzyme is reduced to zero. If there is an optimum temperature, there is also an optimum pH. Similar to temperature, pH also has a peak where the enzymatic reactions are at its greatest. An enzyme activity requires a certain level of acidity acidity and alkalinity alkalinity to work at its best. The pHs higher or lower than the optimum optimum pH makes an enzyme activity slower slower and reduced. reduced. The optimum optimum pH of the enzyme is pH 6.7 which agrees to the rule of pH 6.7-7 as the optimum optimum pH of salivary salivary amylase. The pHs 4 and 5 are too acidic for a fast enzymatic reaction. At pH 8, the enzymes work fast but are slowly being denatured denatured due to much alkalinity alkalinity.. Enzyme Enzyme activity activity at pH 10 will have all the enzymes enzymes denatured. In this activity, activity, starch starch is the one being catalyzed catalyzed by the enzyme solution. solution. As mentioned mentioned earlier, salivary amylase is a type of enzyme that breaks down starch and turns it into maltose, a disaccharide. To test the presence of an enzyme activity between the two, iodine test was performed. In this test, the rate of loss of reactant or increase of product is being observed. Activators and inhibitors also affect the enzyme activity. Sodium chloride was added to the enzyme solution to hasten the reaction and make it more possible. Chloride ions are important in activating the salivary amylase. How does boiling affect the enzyme activity? Boiling increases the the temp temper erat atur uree whic which h dest destro roys ys the the prope properr shape shape of the the enzy enzyme mes. s. The The enzym enzymes es becom becomee denatured hindering the activity to be performed.
VI. Conclusion & Recommendation
In conclusion, the experiment highlights the importance of knowing the factors that affect the enzyme activity. Inability to maintain the optimum temperature and pH of a certain enzyme can deter the assurance of accurate and reliable results. Enzymes are very important in the systems of the living and a simple mistake can alter the process. During the experiment, the required time involved in the experiment should be carefully followed. followed. The laboratory laboratory glasswares glasswares should be maintained maintained clean to avoid mixing mixing of unwanted chemical that may affect the results.
VII. References
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