The aim of the experiment is to monitor amylase enzymes in distinct environments and detecting each environment with the help of color changes. Enzymes are key biological agents that help in catalyzing varied chemical reactions. With a few exceptions, all the enzymes are mainly proteins and each enzyme is peculiar to a particular chemical reaction.

In order to function appropriately, enzymes must sustain a specific three-dimensional structure. In case an enzyme’s structure is impaired (by harsh chemicals or heat), it might stop to function. This denaturation (breakdown) of the enzyme’s structure can turn out to be fatal.

Amylase Enzyme

Amylase, which is normally found in saliva and germinating seeds, help to catalyze the breakdown of starch. When chemical reaction takes place between amylase and starch, the polymeric carbohydrate splits into disaccharide maltose (two connected glucose molecules). As the reaction proceeds, the amount of starch left is depleted, and maltose (sugar) is left in greater quantity.

The functions of amylase can also be observed by using iodine, as it reacts with starch to develop a purple/brown color. As said in the earlier context, amylase breaks down starch, which causes the color of the solution (if iodine is also added) to turn lighter.
Amylase reaction was observed under 4 distinct treatments:

• Effect of pH
• Effect of temperature
• Effect of enzyme concentration
• Effect of substrate concentration

The Effect of Temperature

Amylase is a vital metabolic enzyme. Its job is to help in catalyzing the hydrolysis of starch into glucose. Amylase becomes denatured at high temperatures and is no longer able to catalyze the hydrolysis process.

The Effect of pH

On the basis of these results, what is the ideal pH for amylase? Is this ideal pH considered neutral, acidic, or alkaline/basic? What causes the activity to decrease when the pH is too high or too low?

Apparatus

• Amylase Enzyme
• Starch
• Na2HP04
• KH2P04
• HCI
• Iodine
• Beaker
• Heater
• Spectrophotometer
• Falcon tube

Procedure

• Prepare a pH5 buffer solution of KH2P04 (0.27g) with 20ml water.
• Prepare a pH6 solution of KH2P04 (0.27g) with 20ml water.
• Prepare a pH7 solution of KH2P04 (0.27g) with 100ml water.
• Prepare a pH8 solution of Na2HPO4 (0.282g) with 20ml water.
• Prepare a pH9 solution of Na2HP04 (0.282g) with 20ml water.
• Prepare 20g starch with 50ml cold water.
• For testing the activity of amylase with pH difference, mix 5ml buffer solution (pH5, 6, 7, 8, 9 each is used), 5ml starch, and 1ml of amylase.
• Wait for 10 minutes and put 0.5ml of the prepared sample into 5ml of HCI.
• Measure the results in a spectrophotometer at 620nm.
• For checking the effect of temperature, mix 5ml pH7 buffer solution, 5ml starch, and 1ml amylase.
• Put the prepared sample at different temperatures of 30, 50, 70, and 90°C.
• Put 5ml HCI into 0.5ml of the prepared sample after 10 minutes.
• Add 5ml iodine into 0.5ml of the new sample after 2-3 minutes.
• Measure the absorbance of each sample with a spectrophotometer.

Observations

The purpose of this experiment was to create different environments for examining the activity of the amylase enzyme. The differences in the environment can also be created by pH variance. The experiment demonstrated the amylase activity at different pH, and amylase activity at different temperatures but constant pH.

The pH 5, 6, and 7 were prepared with K2HPO4 and pH 8 and 9 with Na2PO4. In each preparation procedure, a 5ml buffer solution, a 5ml starch, and 1ml amylase were mixed together and left for 10 minutes. Then 5ml HCI was added to 0.5ml of sample mixture after 10 minutes. Similarly, the pH7 mixture was prepared for temperature observation, and iodine was added at the end of the procedure. After that, absorbance results were taken from a spectrophotometer (at 620nm).

Buffer samples of pH with amylase:

• 0.074
• 0.027
• 0.026
• 0.043
• 0.074

Based on the results, the smallest one can be thought of as the best one. The quantity of enzyme used is a more important point. If it is the least one, it implies starch can’t be used appropriately. On the other hand, high starch quantity implies that complex amount is also high. However, the opposite one indicates the best amylase activity at the smallest concentration, and the color is more light as well (smaller absorbance could mean the best amylase activity).

Temperature samples with amylase:

• 0.064
• 0.006
• 0.192
• 0.130

The color is marginally orange at 30°C.

The color is fairly light, like iodine color at 50°C.

The color is mildly purple at 70°C.

The color is more purple at 90°C than at 30°C (where color was like orange-purple). The best activity of amylase was at 50°C at a constant pH.

Result

The purpose of the entire process centered on the activity of amylase. For detecting it, different pH from KHP04 and Na2HP04 were prepared by adding acids and bases (usage of both depends upon the interval of buffer solutions). After the preparation of buffer solutions, the absorbance level was measured using a spectrophotometer.

Absorbance gave different concentrations at different pH. If the concentration of amylase enzyme with the sample mixture is small, it implies enzymes used are more complex (and less in quantity too), and their activity is also the best among others. Comparing different pH, the smallest concentration was at pH7.

After that, the second part of the experiment was carried out by using pH7. The choice of pH7 was directly involved with the observation of the best amylase activity in the first part (which was at pH7). At the constant pH7, samples of enzyme concentration were taken at different temperatures. The smallest concentration (0.006) was recorded at 50°C, where the color was lighter like iodine color. Here starch was used with amylase, which is why the complex color was also fairly light. The best amylase activity was also recorded at 50°C, and this measurement was taken at 620nm.