Introduction

Enzymes are vital biological catalysts that accelerate chemical reactions within living
organisms without being consumed in the process. Among the numerous enzymes in the
human body, salivary amylase, also known as ptyalin, plays a crucial role in the initial stages
of digestion. Secreted by the salivary glands, this enzyme begins the process of breaking
down dietary starches into simpler sugars while the food is still in the mouth. This early stage
of digestion is essential for efficient carbohydrate metabolism, which provides a primary
energy source for the body.

Starch, a polysaccharide composed of glucose units, is a common component of many foods,

including grains, potatoes, and various processed foods. The digestion of starch into smaller,
absorbable molecules like maltose and dextrin is essential for the body to utilize these
carbohydrates effectively. Salivary amylase initiates this breakdown by hydrolyzing the
alpha-1,4 glycosidic bonds in starch.

The activity of enzymes is highly dependent on environmental conditions. Each enzyme

operates optimally at specific pH levels and temperatures. Understanding these optimal
conditions is important not only for basic biological research but also for various applied
fields, such as food science and medicine. In this experiment, we aim to explore how
different pH levels and temperatures affect the activity of salivary amylase. By systematically
varying these conditions and observing the enzyme’s ability to digest starch, we can gain
insights into the biochemical properties of salivary amylase and the environmental factors
that influence its activity.

Theory

Enzymes are proteins with complex three-dimensional structures that include active sites,
where substrate molecules bind and undergo chemical transformations. The specificity of
enzyme action is due to the unique shape and chemical environment of the active site.
Salivary amylase specifically catalyzes the hydrolysis of starch into maltose and dextrin.

Starch is composed of two types of molecules: amylose and amylopectin. Amylose is a linear
polymer of glucose units linked by alpha-1,4 glycosidic bonds, while amylopectin is a
branched polymer with both alpha-1,4 and alpha-1,6 glycosidic bonds. Salivary amylase
breaks the alpha-1,4 bonds, producing smaller chains of glucose molecules.

The activity of salivary amylase, like that of other enzymes, is influenced by several factors:

1. pH: Enzymes have an optimal pH at which their activity is maximized. The structure
of the enzyme and the ionization state of its active site are influenced by pH. For
salivary amylase, the optimal pH is around 7, reflecting the neutral environment of the
human mouth. Deviation from this pH can lead to changes in the enzyme’s shape or
charge properties, reducing its ability to bind to starch and catalyze its breakdown.
2. Temperature: Temperature affects the kinetic energy of molecules, which in turn
influences the rate of enzymatic reactions. As temperature increases, the kinetic
energy of the substrate and enzyme molecules increases, leading to more frequent
collisions and a higher reaction rate. However, if the temperature exceeds a certain
threshold, the enzyme may denature, losing its specific structure and, consequently,
 its activity. For salivary amylase, the optimal temperature is around 37°C, the normal
human body temperature.

In this experiment, we will monitor the breakdown of starch by salivary amylase by using
iodine, which forms a blue-black complex with starch. As the starch is broken down into
simpler sugars, the intensity of the blue-black color decreases. By observing these color
changes under different pH and temperature conditions, we can infer the activity level of
salivary amylase.

Aim

To study the digestion of starch by salivary amylase and determine the effects of different pH
levels and temperatures on the enzyme's activity.

Materials Required

 Starch solution (1% w/v)

 Salivary amylase solution (diluted human saliva, 1:10)
 Iodine solution
 pH buffers (e.g., pH 4, pH 7, pH 9)
 Water baths (set at different temperatures: 0°C, 20°C, 37°C, 60°C)
 Test tubes
 Pipettes
 Stopwatches
 Thermometers
 pH meter or pH strips
 Test tube racks

Procedure

Effect of pH on Salivary Amylase Activity

1. Label three test tubes for different pH levels (4, 7, 9).

2. Add 5 mL of starch solution to each test tube.
3. Add 1 mL of salivary amylase solution to each test tube.
4. Adjust the pH of each test tube using the respective pH buffers.
5. Incubate the test tubes at 37°C for 10 minutes.
6. After incubation, add a few drops of iodine solution to each test tube.
7. Observe and record the color change.

Effect of Temperature on Salivary Amylase Activity

1. Label four test tubes for different temperatures (0°C, 20°C, 37°C, 60°C).
2. Add 5 mL of starch solution to each test tube.
3. Add 1 mL of salivary amylase solution to each test tube.
4. Place the test tubes in water baths set at the respective temperatures for 10 minutes.
5. After incubation, add a few drops of iodine solution to each test tube.
6. Observe and record the color change.
Observation Table

Table 1: Effect of pH on Salivary Amylase Activity

Test Temperature Initial Color Final Color with Interpretation (Starch

pH
Tube (°C) with Iodine Iodine Digestion)
1 4 37 Blue-black Blue-black No digestion
2 7 37 Blue-black Yellow-brown Complete digestion
3 9 37 Blue-black Blue-black No digestion

Table 2: Effect of Temperature on Salivary Amylase Activity

Test Temperature Initial Color Final Color with Interpretation (Starch

pH
Tube (°C) with Iodine Iodine Digestion)
1 0 7 Blue-black Blue-black No digestion
2 20 7 Blue-black Light blue Partial digestion
3 37 7 Blue-black Yellow-brown Complete digestion
4 60 7 Blue-black Blue-black No digestion

Conclusion

The experiment demonstrates that salivary amylase has optimal activity at a neutral pH
(around 7) and at body temperature (37°C). Under these conditions, the enzyme efficiently
breaks down starch into simpler sugars. Deviations from these conditions, either in terms of
pH (more acidic or basic) or temperature (lower or higher), result in decreased enzyme
activity and reduced starch digestion. This highlights the importance of maintaining specific
environmental conditions for the effective functioning of salivary amylase during the initial
stages of carbohydrate digestion.

Precautions

1. Accurate Measurement: Precisely measure the volumes of starch solution, salivary

amylase, and pH buffers.
2. Fresh Enzyme Solution: Use fresh saliva or a freshly prepared salivary amylase
solution.
3. Controlled Conditions: Maintain consistent and accurate temperatures using water
baths.
4. pH Calibration: Ensure pH buffers are accurately prepared and calibrated.
5. Consistent Timing: Start timing the reaction immediately after adding the enzyme
and adhere strictly to incubation periods.
6. Avoid Contamination: Use clean and sterile equipment to avoid contamination.
7. Iodine Test: Add iodine solution in consistent amounts to each test sample.
8. Observation: Observe and record the color changes promptly after adding iodine to
minimize errors in interpretation.