FSE 04 An Investigation into the Effect of Temperature on the Activity of Amylase

This experiment investigates how temperature affects the activity of amylase in breaking down starch, showing that enzyme efficiency peaks at 40℃ and decreases at lower or higher temperatures due to reduced molecular motion or denaturation.

Abstract

Enzymes are essential biological molecules that act as catalysts in living organisms. They facilitate and accelerate chemical reactions by lowering the activation energy required for the reactions to occur. Enzymes function by binding to specific molecules called substrates, which undergo a transformation to form products. The region of the enzyme where the substrate binds is known

as the active site. The active site has a unique three-dimensional structure that complements the shape and chemical properties of the substrate, allowing for precise binding and catalysis.

The process of enzyme-catalyzed reactions follows a general mechanism. Initially, the enzyme and substrate molecules collide with each other. If the substrate has a complementary shape to the active site, it can bind to the enzyme, forming an enzyme-substrate complex. This binding is highly

specific, with enzymes typically recognizing and binding only to specific substrates. The enzyme-substrate complex undergoes a series of interactions, leading to the conversion of the substrate into products. Importantly, enzymes do not undergo any permanent changes during the reaction and can be reused for subsequent reactions (Roy M. 2003).

Figure 1.1 Enzyme-Substrate Activity Diagram

One crucial factor influencing enzyme activity is temperature. Temperature affects the rate of enzyme-catalyzed reactions due to its impact on molecular motion and collision frequency. Enzymes usually have an optimal temperature range at which they exhibit maximum activity. This optimal temperature is specific to each enzyme and is influenced by its native environment. Deviating from this optimal temperature range can have adverse effects on enzyme activity. At temperatures below the optimal range, the enzyme's activity may be reduced due to insufficient molecular motion and slower collision rates. On the other hand, at temperatures above the optimal range, the enzyme may become denatured, losing its three-dimensional structure and rendering it non-functional (Roy M. 2010).

Figure 1.2 Mutated Enzyme-Substrate Activity Diagram

Mutations in enzymes—due to extreme temperatures—can disrupt their normal structure or function, leading to a range of consequences. In some cases, a mutated enzyme may lose its ability to catalyze a specific reaction, resulting in the impaired or complete loss of a particular metabolic pathway. This can lead to the accumulation of toxic substances or the deficiency of essential molecules. Alternatively, a mutated enzyme may exhibit altered activity, leading to abnormal or excessive biochemical reactions. Such dysregulation can disrupt the balance of cellular processes and may contribute to the development of diseases, including metabolic disorders, neurological conditions, or cancers (Lászlo 2004).

Research Question

How does the activity of amylase in breaking down starch change at different temperatures (20℃, 30℃, 40℃, 50℃, 60℃), and what does this indicate about the impact of temperature on enzyme activity?

Hypothesis

Since our bodies are created to be the optimal environments for enzyme activities, it is manifest that the amylase will break down starch most efficiently and effectively when the temperature is 40℃ (closest to body temperature) in this experiment.

On one hand, when the temperature is below our body temperature, the enzyme activity will be relatively slow, as the kinetic energy of the enzyme molecules decreases, causing them to move more slowly. This reduced molecular motion restricts the frequency of enzyme-substrate collisions, which are necessary for the formation of an enzyme-substrate complex.

On the other hand, in high temperatures, the rate of enzyme activities will decline and, in some cases, the enzymes may denature. As the temperature continues to rise beyond the optimal range, the enzyme’s structure and functionality is disrupted, and heat can cause the enzyme’s structure to unravel, leading to denaturation.

Variables

Table 01: Independent, Dependent, and Controlled Variables

Methodology

1. Preparation:

   1. Place spots of iodine solution into the dips on the spotting tile

   2. Using a syringe, add 5cm³ of starch suspension into one boiling tube

   3. Using a different syringe, add 5cm³ of amylase solution into another boiling tube

   4. Fill a beaker with water at 20 ℃

   
   

2. Initial Setup:

   1. Place both boiling tubes in the beaker of water

   2. Start a timer for 5 minutes

   3. Record the temperature of the water bath

   
   

3. Mixing the Solutions:

   1. After 5 minutes, pour the amylase solution into the starch suspension, leaving the tube containing the mixture in the water bath

   2. Immediately, use a pipette to take a small sample of the mixture

   3. Add the sample to the first drop of iodine solution on the spotting tile

   4. Record the color of the iodine solution

   
   

4. Testing for Starch:

1. Every 30 seconds for 10 minutes, take a sample of the mixture

2. Add the sample to the drop of iodine solution on the spotting tile

3. Record the color of the iodine solution after each addition

4. Repeat steps “a” to “c” until the iodine solution remains yellow, indicating the complete breakdown for starch

5. Repetition:

   1. Repeat the entire procedure with other temperatures: 30℃, 40℃, 50℃, 60℃

   2. Repeat the experiment at least three times to prevent any anomalies

Figure 1.3 Picture of the Experimental Setup

Processed Data

Tables:

Table 02: Color of Mixture at Different Temperatures

Figure 1.4 Effect of Temperature on the Breakdown of Starch by Amylase

Observation

Initial Color

All mixtures initially showed a blue-black color with iodine, indicating the presence of starch.

Color Change Over Time

At 40℃, the mixture quickly turned yellow, showing rapid starch breakdown.

At 20℃ and 30℃, the color changed more slowly, from blue-black to brown to yellow.

At 50℃, the color change was slower than at 40℃, progressing from blue-black to brown and then yellow.

At 60℃, the color remained blue-black the longest, indicating the least enzyme activity.

Rate Comparison

40℃ was the optimal temperature for amylase activity, showing the fastest color change.

Lower temperatures (20℃ and 30℃) and higher temperatures (50℃ and 60℃) showed slower enzymatic activity.

Consistency

Results were consistent across multiple trials, confirming the trends observed.

Conclusion

Research Question:

How does the activity of amylase in breaking down starch change at different temperatures (20℃, 30℃, 40℃, 50℃), and what does this indicate about the impact of temperature on enzyme activity?

Claim:

Amylase exhibits its highest efficiency in breaking down starch at 40℃, as shown by the quickest color change from blue-black to yellow with iodine. Enzyme activity decreases at temperatures below or above 40℃, resulting in a slower rate of starch breakdown.

Evidence:

Table 03: Color Evidence Analysis

The graph shows the quickest starch breakdown at 40℃, with much slower rates at other temperatures, particularly at the extremes of 20℃ and 60℃. The error bars in the graph do not overlap, emphasizing the significant differences in reaction rates at different temperatures and supporting the conclusion that 40℃ is the optimal temperature for amylase activity. In addition, the consistency across multiple trials further validates the data's reliability.

Reasoning:

The data demonstrates that 40℃ is the optimal temperature for amylase activity. At this temperature, the enzyme's kinetic energy is sufficient to facilitate frequent collisions with substrate molecules, leading to a faster rate of starch breakdown. This aligns with the concept that enzymes have an optimal temperature at which they function most efficiently due to the optimal balance between molecular motion and enzyme stability.

At lower temperatures (20℃ and 30℃), the enzyme's kinetic energy is reduced, resulting in fewer collisions between enzyme and substrate molecules. This slower molecular motion leads to a reduced rate of starch breakdown, as observed by the slower color change from blue-black to yellow.

Conversely, at higher temperatures (50℃ and 60℃), the enzyme begins to denature, losing its three-dimensional structure essential for substrate binding. This denaturation results in decreased enzyme activity and a slower rate of starch breakdown, as evidenced by the prolonged time taken for the mixture to turn yellow.

The lack of overlap in the error bars reinforces the conclusion that temperature significantly affects the enzyme's activity, with 40℃ being the optimal temperature for amylase. The consistency of these results across multiple trials further substantiates this finding, demonstrating the reliability and validity of the experimental data.

Evaluation

Limitations

Inconsistent Temperature Control:

It was difficult to maintain a constant temperature throughout the experiment, potentially causing slight variations in enzyme activity and affecting the results.

Subjective Data Collection:

Relying on visual observations for color changes in the iodine-starch reaction introduced subjectivity and potential inconsistencies in interpreting the data.

Narrow Temperature Range:

The experiment tested only five specific temperatures, which may not fully represent the enzyme's activity range and its exact optimal temperature.

Improvements

Better Temperature Regulation:

Use a digital water bath with precise temperature control to ensure consistent and accurate temperature maintenance during the experiment.

Objective Measurement Techniques:

Implement spectrophotometry to quantitatively measure starch concentration, providing objective and precise data on enzyme activity.

Wider Temperature Range:

Include a broader range of temperatures, such as lower (e.g., 10℃) and higher (e.g., 70℃) points, to gain a more comprehensive understanding of amylase activity and identify its exact optimal temperature.

Bibliography Citations

Daniel, Roy M., et al. "The role of dynamics in enzyme activity." Annual review of biophysics and biomolecular structure 32.1 (2003): 69-92.

Daniel, Roy M., et al. "The molecular basis of the effect of temperature on enzyme activity." Biochemical journal 425.2 (2010): 353-360.

Góth, László, Péter Rass, and Anikó Páy. "Catalase enzyme mutations and their association with diseases." Molecular Diagnosis 8 (2004): 141-149.

Fullick, Ann. Edexcel IGCSE Biology. Pearson Education, 2011.