EXPERIMENT 5-HYDROLYSIS OF STARCH BY α-AMYLASE

Starch is mainly composed of two parts:

- Amylose: a linear polymer made up of α-1-4 bound D-glucose units. This

polysaccharide is making up approximately 20-30% of the structure.

- Amylopectin: a polysaccharide with highly branced polymer of α-1-6 bound

glucose units.

Amylase is an enzyme that hydrolysis alpha-bonds of large alpha-linked polysaccharides

such as starch and glycogen, yielding maltose and dextrin. It is the major form of

amylase found in humans and other mammals.

Amylase is found in saliva. This form of amylase is also called “ptyalin”. The enzyme

randomly affects α-1-4 bonds, belonging to the amylose structure of starch, and maltose

units form. Amylase does not affect the α-1-6 bonds that belong to the amylopectin

structure of starch. As a result of hydrolysis carried out with α- amylase, besides the

maltose and glucose units, a large branched dextrin structure is formed in the media.

The optimum conditions for α- amylase:

- Optimum pH: 5.6-6.9

- Human body temperature: 37 0C

- Presence of certain anions and activatore:

o chloride and bromide- most effective

o iodide- less effective

o sulphate and phosphate- least effective

Procedure for Part A:

- Add 1 mL of 1% starch solution and 1 mL phosphate buffer solution (pH=6.8)

into 4 test tubes.

- Place each test tube into water baths at 00C, 250C, 370C and 950C, respectively.

- Wait for a few minutes and add 1 mL diluted (1/10) saliva into each test tube.

- Place 1 drop of iodine solution on 4 watch glasses and take 1 drop from each test

tube to observe if there is any starch left (Starch gives blue color with iodine

solution).

- Record the time for the hydrolysis of 1 mL starch in each test tube.

Questions for Part A:

What is the aim of Part A?

What is the aim of using phosphate buffer in each starch solution?

Explain the significance of each temperature.

Calculate the rate of hydrolysis reaction in each test tube.

Compare your reaction rate results at each temperature. Explain briefly.

Procedure for Part B:

- Take 7 test tubes and prepare the following solutions.

Material 1 2 3 4 5 6 7 1% Starch Solution

0.2 0.5 1 2 3 4 5

Distilled Water

4.8 4.5 4 3 2 1 -

Phosphate Buffer

1 1 1 1 1 1 1

Saliva (1/20

diluted)

1 1 1 1 1 1 1

- Shake each test tube thoroughly. Check each mixture for color with iodine per

minute.

- Record color changes.

Questions:

What is the aim of Part B?

What are the difference between the solutions 1 to 7?

Explain your color observations briefly by giving reasons.

The iodine test is used in both parts. Explain the reason of using this test by giving the

structures if necessary.