Digestion breaks down food molecules to smaller subunits that could be used by the body to getting the necessary nutrients and energy. Many fluids and enzymes are secreted by the digestive system to aid in the digestion of different foods within the gastrointestinal tract (Tomasik & Horton, 2012). They help ingest, breakdown and extracts essential nutrients from food while removing waste. These enzymes may be substrates or hydrolases through the method they use to break down the food molecules either by breaking down the bond or adding fluid to weaken the bond (Tomasik & Horton, 2012). As such, they are essential in speeding up the reaction on food within the digestive tract to help an individual extract the essential nutrient.
Enzyme amylase is a hydrolytic enzyme that breaks down starch to glucose mostly in the mouth. Different foods substances are digested within a specific part of the gastrointestinal tract either the mouth, stomach or small intestines through the action of a particular enzyme (Pandey & Singh, 2012). The effect of the catalyst depends on the prevailing condition of the part within which they act defined by the temperature and the Ph levels. However, even with the action of the enzyme amylase in the digestion of starch, there are different monosaccharides, and its work on them differs (Tomasik & Horton, 2012). Hydrolysis of starch to glucose, maltose or galactose depends on specific factors exhibited by the enzyme. Conducting an enzyme assay helps identify the action of the enzyme on starch through the use of IKI and Benedict’s assay test.
The objective of the experiment was to investigate the hydrolysis of starch to maltose by salivary amylase. To help understand the specificity of the action of amylase appropriate chemical tests were used. Additionally, the experiment investigated the possible effects of pH and temperature on the activity of enzyme amylase on starch and maltose.
Materials and methods
- The assays used in the experiment were IKI that turns blue-black in starch and Benedict’s solution that changes color from bright blue to green, orange and reddish brown in the presence of increasing amounts of maltose.
- pH buffers
- Deionized water
- 8 Test tubes
The eight test tubes were placed in the assay incubator and filled with different reagents. Drops of enzyme amylase were placed in the first three test tubes with starch and pH 7.0 buffers. In the fourth test tube were added enzyme amylase, deionized water, and a buffer at pH 7.0. In the fifth tube, deionized water, starch and pH 7.0 buffer solutions were added. The sixth test tube included deionized water, maltose, and a buffer solution. The seventh test tube included amylase, starch and buffer solution at pH 2.0 while in the last container, amylase, starch and a buffer solution at pH 9.0 were used. The temperature of the incubator was set at 37oc for 60 mins to help mix the content in the test tubes and allow for the necessary reactions to occur. The test tubes were then removed from the incubator after the time had elapsed and observation made by the changes that had happened.
Additionally, different tubes were taken through various heat treatments to observe the change that would occur over some time through the color change. They were either placed under a room temperature of 37oc, frozen or boiled to enhance the reaction that took place in the test tubes. The procedure was repeated twice, first using IKI reagent and second using benedicts solution and also at different temperatures to test the concentration of the enzyme in the various test tubes.
Table 1: Results of the Reactions that Occurred in the Experiement
|Tube No||reagent 1||reagent 2||reagent 3||treatment||time||temp||IKI||Benedict’s|
|1||Amylase||starch||pH 7.0 Buffer||boiled||60 mins||37oc||+||–|
|2||Amylase||starch||pH 9.0 Buffer||frozen||60 mins||37oc||+||+|
|3||Amylase||maltose||pH 7.0 Buffer||none||60 mins||37oc||–||++|
|4||Amylase||deionized water||pH 7.0 Buffer||none||60 mins||37oc||–||–|
|5||deionized water||starch||pH 7.0 Buffer||none||60 mins||37oc||–||–|
|6||deionized water||maltose||pH 7.0 Buffer||none||60 mins||37oc||–||–|
|7||amylase||starch||pH 7.0 Buffer||none||60 mins||37oc||–||–|
|8||amylase||starch||pH 2.0 Buffer||none||60 mins||37oc||–||–|
The results of the experiment indicated a change in color for IKI assay in the reaction between amylase and starch at pH 7.0 boiled, pH 2.0 and pH 9.0 frozen. Also, there were definite results indicated when the contents of the reaction between amylase and starch were observed at pH 9.0. More so, there were positive results when Benedict’s solution was added to a mixture of amylase and maltose found through a red-brown color at pH 7.0 at a room temperature. However, no positive result was seen in all the other test tubes after adding drops of the IKI and Benedict’s reagent. The IKI reagent and Benedict’s solution did not show any color change after being placed in the test tubes that did not contain starch or maltose.
The indicator experiment used two reagents, IKI that indicated the presence of starch in the solution and Benedict’s solution used to identify the presence of maltose (Pandey & Singh, 2012). In the test tubes that the mixture of the substrates was boiled no reaction occurred and therefore the positive results observed through IKI assay. In this way, the action of enzyme amylase is affected as it does not occur at high temperatures. On the other hand, the frozen mixture of amylase and starch indicated a positive reaction for both IKI and Benedict’s reagent suggesting a slow down of the reaction at lower temperatures. Where no temperature or treatment of heat was used, the response of starch and amylase indicated strong results through the change in color of Benedict’s reagent indicating the presence of a pleasant heat in the action of the enzyme (Pandey & Singh, 2012).
The other test tubes that did not show positive results with the reagents noted that it was essential for starch and amylase to be mixed for reaction to occur. They were therefore used as control experiments. The results with Benedict’s reagent were only positive in the test tube that contained maltose that reacted with enzyme amylase. No reaction occurred at pH 2.0 since the action of amylase does not occur at low pH. More so, the result increased as the temperature of the temperature of the reagents was raised to 37oc which is the standard body temperature (Pandey and Singh, 47). However, there was no difference in the reaction of the enzyme amylase at different pH, and it was not evident at the best temperature for the reaction to occur. As such, it is indicated that the activity of the enzyme amylase is affected by both temperature and pH.
The action of amylase enzyme in the digestion of starch is affected by both pH and temperature. There is evidence from the present experiment that when the substrates are boiled amylase does digest the starch to produce maltose and therefore the negative result with Benedict’s reagent. Consequently, the enzyme is seen to function better at a higher pH (pH 9.0) even in lower temperatures in their action on starch. Enzyme amylase is also observed to digest maltose to smaller molecules through the results identified in the color change of Benedict’s reagent.
Pandey, Sandeep, and S. P. Singh. “Organic solvent tolerance of an α-amylase from haloalkaliphilic bacteria as a function of pH, temperature, and salt concentrations.” Applied biochemistry and biotechnology 166.7 (2012): 1747-1757.
Tomasik, Piotr, and Derek Horton. “Enzymatic conversions of starch.” Advances in carbohydrate chemistry and biochemistry 68 (2012): 59-436.