The Hydrolysis of Starch Using Amylase

This lab is aimed at demonstrating the hydrolysis of starch to glucose using the enzyme amylase. The enzyme is found in the saliva secreted in the pancreas. Starch is a polymer composed of numerous rings of glucose molecules linked together via a covalent bond. Starch is produce by the dehydration process and is a form of glucoses storage in many organisms. To fully make use of the energy stored in starch the large molecules have to be broken down into simple sugars. The hydrolysis process in animals is catalyzed by the enzyme amylase. The enzyme amylase convert starch into either glucose dimers or trimmers. The dimers and trimers are then converted by other enzymes to glucose that is a raw material in the cellular respiration process. This lab will evaluate the effect of enzyme concentration on the rate of conversion of starch to maltose.

Starch blockers are supplements from the common bean that inhibit the action of the enzyme amylase. The inhibitors bind to the active site of the enzyme amylase hence preventing the catalyzing of starch breakdown. This lab will evaluate the effect of the starch blockers.

The action of amylase on starch will use the IKI indicator i.e. a mixture of iodine and potassium iodide. The IKI will stain starch to a blue-black color but does not stain maltose or maltotriose. When the catalysis process has reached end-point and all the starch has been hydrolyzed then the yellow-brown color of IKI will persist. When some starch has been hydrolyzed in the intermediate stages of the reaction the color in this case will be brownish-black. Below is an expectation of the color changes as the experiment proceeds.

Altering the activity of Amylase

Enzymes are proteins in nature and their activities is dependent on their shape. Any alteration of the structure could alter the functioning of the functioning of the enzyme. In this experiment high temperature was used as inhibitor to the activity of the enzyme amylase.

Materials and Methods

Part I: Reaction Rate Vs Enzyme Concentration

One spot plate was prepared with a drop of IKI test solution in each of the two depressions. Starch was added to one of the solution and the color recorded. The other depression was left as a standard solution.

4ml of the stock starch solution were pipetted into a test tube with the initials ss and group name. The solution was swirled to obtain the desired results. The tubes were then placed in a water bath at 37 oC

A series of three different test tubes were prepared and labeled as 10%, 5% and 1% for the various amylase concentration. One ml of amylase was pipetted to the appropriate test tube. The amylase flask was swirled before pipetting. The test tubes were then placed in the 37 oC water bath and allowed to sit for five minutes so as to attain an equilibrium with the water bath temperature.

One drop of IKI solution was placed on a depression on the spot plates. The top left-hand depression was considered the first depression.

The time was recorded as the contents of the ss test tubes were poured from the water bath into the test tube labeled 10%. The solution was swirled and then tested.

A drop was obtained from the above solution and then placed in the first IKI drop spot on the plate. Drops were put in the successive depression while the time was being recorded. This was done after every 30 seconds until the color of the IKI solution was retained.

Part II: Altering the Activity of Amylase

Three tests tubes were obtained and labeled ss with the group name. 4 ml of the stock starch solution were pipetted into each test tube. One of the solution was left at 37 oC as the control.

1 ml 10% of amylase was pipetted into each test tube. The control was left at 37 oC while the others were put in the temperature being tested for. The above technique to test for the presence of starch was repeated for each test tube.

Results

Table 1: Amylase concentration

Time (min/sec)

10%

5%

1%

0.0

Y

Y

Y

0.30

Y

Y

Y

1.00

Y

Y

Y

1.30

Y

Y

Y

2.00

Y

Y

Y

2.30

Y

Y

Y

3.00

Y

Y

Y

3.30

Y

Y

Y

4.00

Y

Y

Y

4.30

Y

Y

Y

5.00

N

Y

Y

5.30

N

Y

Y

6.00

N

Y

Y

6.30

Y

Y

7.00

Y

Y

7.30

Y

Y

8.00

Y

Y

8.30

Y

Y

9.00

Y

Y

9.30

Y

Y

10.00

N

Y

10.30

N

Y

11.00

N

Y

11.30

Y

12.00

Y

12.30

Y

13.00

Y

13.30

Y

14.30

Y

15.00

Y

15.30

Y

16.00

Y

16.30

Y

17.00

Y

17.30

Y

18.00

Y

18.30

Y

19.00

N

19.30

N

20.00

N

Table 2: Data Summary

Amylase Concentration

Time to Reach Endpoint (min)

Rate of reaction (100/Time to endpoint )

10%

6

16.67

5%

11

9.09

1%

20

5

Table 3: Condition

Time (min/sec)

Control

0.0

Y

0.30

Y

1.00

Y

1.30

Y

2.00

Y

2.30

Y

3.00

Y

3.30

Y

4.00

Y

4.30

Y

5.00

N

5.30

N

6.00

N

6.30

7.00

7.30

8.00

8.30

9.00

9.30

10.00

Table 4: Data for the inhibition of amylase

Condition

Time to Reach Endpoint (min)

Rate of reaction (100/Time to endpoint )

37 oC

6

16.67

75 oC

3.30

30.30

Figure 1: Graph of Rate of reaction vs Concentration

Discussion

It evident from the graph that the test tube with the highest concentration of amylase had the highest rate of reaction. This was because there were more active sites for the attachment with the substrate and increase the catalysis rate. The test tube with the highest concentration also took the shortest time to reach endpoint.

The high temperature test was used for the inhibition of the activity of amylase. At high temperature the molecules in the solutions will be more energized and more of them will be able to react with the enzyme. Further increase in the heat will weaken the covalent bond in the enzyme causing denaturing.

Work Cited

Bernfeld, Peter. "Enzymes of starch degradation and synthesis." Advances in Enzymology and Related Areas of Molecular Biology, Volume 12 (2006): 379-428.