BIOCHEM LAB - Enzymatic Activity of Salivary Amylase

Effect of temperature and pH on the enzymatic activity of salivary amylase Gae Khalil Rodillas, Nonia Carla Ysabel Samson, Raphael Jaime Santos* and Brylle Tabora Department of Biological Sciences, College of Science, University of Santo Tomas, Manila, Philippines Salivary amylase, found in human saliva, is an enzyme used to hydrolyze starch molecules. Its enzymatic activity is affected by several factors, such as temperature and pH. The rates of enzymatic activity of salivary amylase in different temperatures and pH were measured. Optimum temperature for the enzymatic activity of salivary amylase ranges from 32°C to 37°C and its optimum pH ranges from 6 to 7. A graph of the time reciprocal against temperature and pH both produced bell-shaped curves.

I.

Introduction An enzyme is a protein molecule that is a biological catalyst with three characteristics.

First, the basic function of an enzyme is to increase the rate of a reaction. Second, most enzymes act specifically with only one reactant, called a substrate, to produce products. The third and most remarkable characteristic is that enzymes are regulated from a state of low activity to high activity and vice versa [1]. The activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme works best at a certain pH and temperature, its activity decreasing at values above and below that point due to denaturation. For enzymes, denaturation can be defined as the loss of enough structure rendering the enzyme inactive. This is not surprising considering the importance of tertiary structure in enzyme function and noncovalent forces in determining the shape of enzymes [2]. Salivary amylase is the enzyme produced by the salivary glands. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Amylase, like other enzymes, works as a catalyst. All catalysts are enzymes, but not all enzymes are catalysts. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. Amylase digests starch by catalyzing hydrolysis, which is splitting by the addition of a water molecule. The presence and absence of starch can be confirmed by several tests such as the iodine test, Benedict’s and Fehling’s test. In general, a blue-black color indicates the presence of starch [3].

The objectives of this experiment are to examine the enzymatic activity and specificity of salivary amylase depending on changes temperature and pH. This experiment also aims to determine the narrow range of temperature and pH values at which salivary amylase exhibits its optimum activity.

II.

Materials and Methodology A. Effect of Temperature An enzyme solution was prepared by mixing one mL saliva with nine mL distilled water and

thirty mL 0.5% sodium chloride. Two mL of the enzyme solution was put in a large test tube and labeled as 4°C. Two mL of the buffered starch solution (1% stach in phosphate buffer pH 6.7) was added in a separate large test tube. Both the test tubes were incubated for ten minutes in an ice bath (4°C). The solutions were immediately mixed. Three drops of the mixture was taken quickly and two drops of the 0.001 M iodine solution was added simultaneously onto a spot plate (first well). This was the zero minute. After one minute interval (incubation continued), three drops of the mixture was taken again and two drops of the iodine solution was added simultaneously onto the second well. This was the one minute. Step 5 was repeated until a light yellow-colored solution was observed. The time (t) was noted. For the other temperatures (room temperature, 37, 50 & 70 °C), steps 1 to 6 were repeated following the desired incubation temperature. The reciprocal of time (1/time, min-1) in step 6 versus the temperature (T) was plotted. The optimum temperature of the amylase was determined. B. Effect of pH One mL of acetate buffer (pH 4) and one mL 2% unbuffered starch were mixed in a large test tube. Two mL of the enzyme solution was added in a separate large test tube. Both the test tubes were incubated for ten minutes in a 37°C water bath. The solutions were immediately mixed. Three drops of the mixture was taken quickly and two drops of the 0.001 M iodine solution was added simultaneously onto a spot plate (first well). This was the zero minute. After

one minute interval (incubation continued), three drops of the mixture was taken again and two drops of the iodine solution was added simultaneously onto the second well. This was the one minute. Step 5 was repeated until a light yellow-colored solution was observed. The time (t) was noted. For the other pH (5, 6.7, 8 & 10), steps 1 to 6 were repeated using the appropriate buffer. Acetate buffer solution for pH 5, phosphate buffer solution for pH 6.7 and 8, and bicarbonate buffer for pH 10 were used. The reciprocal of time (1/time, min-1) in step 6 versus the buffer pH was plotted. The optimum pH of the amylase was determined.

III.

Results and Discussion The effect of temperature and pH on the enzymatic activity of salivary amylase was

determined by measuring the rates of reaction in varying temperatures and pH. The 0.5% NaCl added in the enzyme solution activates the salivary amylase to perform its function – to hydrolyze starch. The hydrolysis or breakdown of starch due to the action of salivary amylase is indicated by the change in color of the starch solution from a blue-black color to a light yellowcolored solution. A. Effect of Temperature Each enzyme has an optimum temperature at which it performs best. Below or above this temperature, the enzyme loses its functionality. Table 1 shows the results obtained on how enzyme activity of salivary amylase is affected by temperature. Table 1. Results for the effect of temperature on salivary amylase activity

Temperature (T) °C 4 Room temperature, 32 37 50 70

time (t) min 1.00 11.0 6.00

1/t (min-1) 0 1.000 0.091 0.167 0

Extreme temperatures cause the native folded structure of proteins to uncoil into random configuration. As a result, the protein loses its biological enzymatic activity. This denaturization

consequently leads to loss of activity. Figure 1 shows the graph of the reciprocal of time against temperature based on the data from Table 1. 1.2

1/time (min-1)

1 0.8 0.6 0.4 0.2 0 0

10

20

30

40

50

60

70

80

temperature (°C) Figure 1. Plot of the reciprocal of time against temperature for the enzymatic activity of salivary amylase

The graph produced a bell-shaped curve with the highest peak indicating the optimum temperature for enzymatic activity. At 4°C, enzymatic reaction of salivary amylase occurs slowly or not at all due to lack of energy and heat. As the temperature increases, its enzymatic also increases up until the optimum temperature. Figure 1 shows that the optimum temperature of salivary amylase ranges from 32°C to 37°C.This applies to the human body since salivary amylase is suitable to function within these temperatures. After 37°C, the graph then steeply declines as a result of loss of activity. At 50°C and 70°C, salivary amylase is denatured. The molecular conformation of the enzyme becomes altered as the hydrogen bonds responsible for its secondary, tertiary and quaternary structures are broken [4]. B. Effect of pH Most enzymes are active only over a narrow pH range and have an optimal pH, at which reaction is the fastest. An increase or decrease in pH also causes denaturation in enzymes, thereby affecting their activity. Table 2 shows the results obtained on how enzyme activity of salivary amylase is affected by pH.

Table 2. Results for the effect of pH on salivary amylase activity

pH 4 5 6.7 8 10

time (t) min 11.0 19.0 10.0 6.00

1/t (min-1) 0 0.091 0.053 0.100 0.167

Figure 2 shows the graph of the reciprocal of time against pH based on the data from Table 2.The graph produced a bell-shaped curve and the highest peak should indicate the optimum pH for enzymatic activity.

1/time (min-1)

0.2 0.15 0.1 0.05 0 0

2

4

6

8

10

12

pH

Figure 2. Plot of the reciprocal of time against pH for the enzymatic activity of salivary amylase

At pH 4, the salivary amylase is in a too acidic environment to function. As pH decreases, certain amino acids like aspartate and glutamate are protonated, causing them to lose their net negative charge which consequently denatures the enzyme. The optimum pH for the action of salivary amylase ranges from 5.6 to 6.9 (Talwar & Srivastava, 2006). This is consistent with the peak found between pH 4 and 6 in Figure 2. However, the curved peaked highest at pH 10. Inconsistencies with the results obtained can be attributed to human error such as inaccuracies in measurement and timing during the experiment. Ideally at pH 10, salivary amylase is denatured due to high alkalinity. As pH increases, certain amino acids such as lysine and arginine are deprotonated, causing them to lose their net positive charge which also results to enzyme denaturation.

The activity of enzymes may be markedly changed by any alteration in pH, which in turn, alters electrical charges on the enzyme. Changes in charge affect the ionic bonds that contribute to the enzymes tertiary and quaternary structure, thereby changing the proteins conformation and activity. Thus, pH-activity relationship of enzymes is dependent on the amino acid side chains present in the enzyme [5].

IV.

Conclusion Several factors affect the activity of enzymes. Among these are the temperature and pH.

At optimum levels of these factors, enzymes perform their function best. Optimum temperature and pH differ from one enzyme to another. Salivary amylase is an enzyme found in human saliva which functions to break down starch to simpler compounds. Through the experiment, it was found out that the optimum temperature of salivary amylase ranges from 32°C to 37°C and its optimum pH ranges from 5.6 to 6.9. Inconsistencies of the results obtained with literature on the topic were met. These errors can be attributed to inaccuracies in measurement and timing. Due to time constraints, the experiment was not repeated to be able to compare and obtain more accurate results.

V.

References [1] Role of Enzymes in Biochemical Reactions. (2003). Retrieved on January 3, 2012 from http://www.elmhurst.edu/~chm/vchembook/570enzymes.html [2] Enzymes. (2011). Retrieved on January 3, 2012 from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html#pHandTemp [3] Sethi, R. (2009). Biology. Rachna Sagar Pvt. Ltd.: New Delhi [4] Solomon E., Berg, L. & Martin, D. (2005). Biology. Thomson Learning Inc.: USA [5] Talwar G.P. & Srivastava L.M. (2006). Textbook of biochemistry and human biology, 3rd Ed. Prentice-Hall:New Delhi