Project 2: Enzyme

OBJECTIVE

  • To investigate enzyme action on a foodstuff
  • to see the food digested before it can be absorbed in the digestive system
  • To evaluate a model for the process of digestion

INTRODUCTION

Enzymes are biological catalysts that constitute the largest and most highly specialized class of protein molecules. Enzymes act as catalysts to increase the rates of chemical reactions, but they do not cause a reaction to occur that would not proceed spontaneously without the enzyme, that is, the reaction must have ΔG < 0. The reactions of metabolism would occur at extremely slow rates at normal body temperature and pH in the absence of enzymes. An appreciation of the catalytic efficiency of enzymes can be gained by realizing that under optimal conditions, most enzymatic reactions proceed 108 to 1011 times more rapidly than the corresponding reactions without enzymes. Without the enzymes in our digestive tract for example, it would take us about 50 years to digest a single meal!

One very important property of enzymes is their specificity. Any one enzyme will only catalyze a single class of chemical reactions. Some enzymes act on one substrate only, other enzymes act on a family of related molecules. Enzymes participate in the reaction that they catalyze, but they emerge unchanged at the end of the reaction, for example they are not used up. Thus, a few enzyme molecules can go a long way.

Most enzymes are proteins with specific three dimensional shapes necessary for the proper function of the enzyme (some RNA molecules also have catalytic activity). As with any protein, the shape of an enzyme can be affected by different environmental conditions. An increase in temperature can speed up the rate of an enzymatic reaction but it can also change the shape of the enzyme causing it to become inactive because the substrate can no longer bind to it. The pH of the environment is also important to the proper functioning of an enzyme. Enzymes work best at a specific pH and a change in this value can also cause the enzyme to denature (change its shape) and become inactive.

MATERIALS

Starchy food (Milo), boiling tubes, test tubes, visking tubes, elastic bands, dimple tile, thermometer, beaker, amylase, iodine, Benedict's reagent.

METHOD

1. 3 boiling tubes was labelled 1, 2 and 3.
2. 3 model guts was set up: A wet piece of visking tubes was tied at one end.


3. The starchy foodstuff was added into the model guts using spatula until half full. 
4. The outside of the model guts was rinsed under running tap. 
5. Each model guts was placed in the boiling tubes labelled 1, 2 and 3 and secured with elastic band.
6. the boiling tube was filled with 25mL warm water.



7. One drop of warm water added was withdraw and tested with iodine in a dimple tile.
8. 5mL of water was added into model gut 1, 5mL of amylase was added into model gut 2, and 5mL of treated amylase (0ºC) was added into model gut 3. 
9. All the boiling tubes containing model guts was placed in a beaker containg water at 37ºC.
10. The incubation was set for 30 minutes. 



11. while waiting, we did the iodine and Benedict's test:

i. a grain of starchy food was place in a well of a white tile and a drop of iodine was added.

ii. some starchy food was added in a test tube. 2mL of water, and 2mL of Benedict's reagent was added. The test tube was placed in a beaker containing boiling water. The observation was recorded after 2-3 minutes. The result was recorded.

Iodine test
Benedict test



12. After 5 minutes of incubation, the iodine and Benedict test for both water in the gut model and the water in the boiling tube was repeated as follows:

i. One drop of water in model gut, and one drop of water in boiling tube was placed in a well of white tile. A drop of iodine was added. The result was recorded.

ii. 2mL of water in the model gut, and 2mL of water in the boiling tube was taken and placed in a test tube respectively. 2mL of Benedict's reagent was added. The test tubes was placed in a large beaker containg boiling water. After 2-3 minutes, the result was recorded.

13. Step 12 was repeated every 5 minutes until 30 minutes.

14. Step 12 was repeated for boiling tube 2 and 3. 

RESULT

Iodine test and Benedict test
Iodine test for each 5 minutes
Treated amylase(0ºC), water and amylase


After 5 minutes (water, amylase and treated amylase)
After 10 minutes (water, amylase and treated amylase)

After 15 minutes (water, amylase and treated amylase)
After 20 minutes (water, amylase and treated amylase)
After 25 minutes (water, amylase and treated amylase)
After 30 minutes (water, amylase and treated amylase)

 DISCUSSION
Enzyme is a specific organic molecule that speed the reaction in biological systems (acting as bio catalyst). It enables reactions to happen under relatively low temperatures, neutral pH  and atmospheric pressure, all these are conditions that common in our bodies. The vast majority of enzymes are proteins  and apart from protein component, enzymes can also contain non-protein part. They are important  for life and serve a wide range of important functions in the body, such as aiding in digestion and metabolism. Our body need enzyme to break large molecules into smaller pieces that are easily absorbed by the body. Other enzymes help to bind two molecules together to produce a new molecule. Enzymes are highly selective catalysts, meaning that each enzyme only speeds up a specific reaction.

In this experiment, we are going to determine the effect of amylase on a starchy food. We bring our own  foodstuff to perform this experiment. which is Milo. For the first 5 minutes of the experiment, we mixed the solution inside the visking tube and inside the boiling tube together, therefore the result is not accurate. For 10minutes to 15minutes, we incubate the solution for Benedict test inside the 37ºC, instead of inside boiling water, therefore the result is also not accurate. 

The Visking tubing of the model gut that we used is represents the wall of the small intestine and the water in the boiling tube outside the model gut represents blood in the blood vessels around the intestine. This model is different in real gut because in a real gut, food is moving through the tube and blood is circulating around the blood vessels to carry away the absorbed food chemicals. The gut lining is folded into villi and micro-villi, there are mechanisms that can actively absorb some food chemicals. There is also a more complicated mixture of food and enzymes in the intestine. The starch in the test tubes are still present inside the gut but also not in some cases.

In our results, the blue-black colour indicates that starch is present in the starchy food meanwhile an orange  colour indicates that reducing sugar such as glucose or a similar sugar is present. The Iodine and Benedict  test also be be repeated for both water in the gut model and water in the boiling tube. The colour of the water in boiling tube and water in gut model had changed to orange in Benedict test and turn to blue in Iodine test (shown in result part) every five minutes until 30 minutes. Beside that, the boiled amylase has had no effect on the starch in the rice because it is has been denatured by the high temperature. This means the protein molecule has lost shape and cannot longer work as a catalyst for breaking down starch to sugar.By doing this experiment, some precaution steps needed to be taken to avoid any error in the result. Visking tubing in warm water needed to be soak before hand so it is ready to use in the experiment.

 CONCLUSION
After done the experiment, we are able to know either our food sample containing starch or not. We are also learned to make a model on digestive system by using the visking tube. By doing this, we can improve our knowledge and make us understand more about the effect of amylase on the food stuff.

QUESTION

1. What does the Visking tubing of the model gut represent?
The Visking tubing of the model gut represents the wall of the small intestine.

2. What does the water in the boiling tube (outside the model gut), represent?
The water in the boiling tube outside the model gut represents blood in the blood vessels around the intestine.

3. How is this model different from the situation in a real gut?
In a real gut, food is moving through the tube, blood is circulating around the blood vessels and carrying away the absorbed food chemicals, the gut lining is folded into villi and microvilli, there are mechanisms that can actively absorb some food chemicals, there is a more complicated mixture of food and enzymes in the intestine, the pH of the gut contents is controlled by gut secretions.

4. What has happened to the starch in each of your tubes?
In each case, there is still starch inside the gut, but no starch in the water outside.

5. What has happened to sugars (detected with Benedict’s reagent) in each of your tubes?
Orange  colour indicates that reducing sugar such as glucose or a similar sugar is present.

6. Why does the body need enzymes to digest food?
The body needs enzymes to digest food because large polymer food molecules are too large to pass through the gut wall. Chemical digestion without catalysts is too slow to be useful. Specifically, we need glucose and other sugars as a ready source of energy in our bloodstream.
7. What is your result with treated amylase? Explain.
Our result with treated amylase(0ºC) is the same with amylase. This is because, the sugar is still presence in 0ºC.

REFERENCES

 1. R. K. Murry, D. K. Granner, P.A. Mayes, V. W. Rodwell, Harper’s Biochemistry, Prentice Hall International Inc., Latest Edition.
2. http://fblt.cz/en/skripta/ii-premena-latek-a-energie-v-bunce/6-enzymy/
3. http://www.nuffieldfoundation.org/standard-health-safety-guidance