1. The hydrolytic activity of salivary amylase on starch
CLS 331
Experiment No.1
The hydrolytic activity of salivary amylase on starch

2. Introduction

3. Amylase is a hydrolytic enzyme which breaks down many polysaccharides such as starch.
Is a polymer of D- glucose units linked by α-1, 4 glycosidic bonds.
The starch is made up of two polysaccharides:
Amylopectin (branched-chain polysaccharide)..
Amylose (unbranched-chain polysaccharide

4. The hydrolytic effect of amylase on starch results in yielding maltose (composed of two D-glucose molecules ) as the end product.
There are two broad groups of amylases: α and β- amylases.
The β- amylases rapidly hydrolyse the amylose portion of starch to maltose by acting on residues at the non reducing terminals.

5. They hydrolyse α-1, 4 glycosidic links in polysaccharides so as to remove successive maltose units from the non-reducing ends of the chains.
The α amylases, in contrast to the β- amylases, cause a rapid loss of the capacity of amylase to give a blue colour with iodine, also the rate of appearance of maltose is much slower in the α amylases catalysed reaction than in the β- amylases catalysed one.

6. Types of amylase:
amylases – human amylase -α
β- amylases – bacteria and plant amylase
Source of amylase:
1- Pancreas
2- Salivary gland
Secretion:
Serum or urine

7. Hyperamylasemia
Increase the secretion of amylase in blood
Some Causes of Hyperamylasemia
Acute pancreatitis, Salivary gland lesions, etc

8. The Assay
In any enzyme assay, the rate of the reaction can be
known by measuring the amount of substrate (s) that is
utilized or the amount of product (s) that is formed in unit
time, it also called enzyme unit.
In the case of amylase,
The substrate is starch (colourless)
The product is maltose (colourless)
They can be converted to coloured products by specific
chemical reactions.

9. Starch +Amylase maltose (colourless)
Starch + Amylase + Iodine blue colour (qualitative)

10. Principle
At a pH of about 6-7 and in the presence of chloride ions, α amylases catalyses the hydrolysis of starch to maltose with the intermediate formation of various dextrins.
Dextrins are polysaccharide produced by the hydrolysis of starch

11. Starch + Cl+ + α amylases maltose + intermediate
formation of dextrins
Steps of the reaction:
1. Starch + α amylase + Higher dextrins blue colour
2. Intermediated dextrins reddish brown colour
3. Lower dextrins + maltose colourless
This point known as Achromic point ( time taken to reach the point at which the reaction mixture no longer gives a colour with iodine)
Iodine
Iodine
Iodine

12. Procedure

13. Part1: Test tube1: mix then incubate at 37 C
Stock saliva
Test1
Control 1
1 ml saliva
9.5ml H2O
0.5ml saliva
5ml
Test tube1: mix then incubate at 37 C
Control tube1: gently boiled for 2 min, cooled and then placed in the water bath at 37 C

14. Part 2 Control 2 Test 2 Mix well, then incubate at 37⁰C X 5-10 min
5ml starch
2ml phosphate buffer
1ml NaCl
Mix well, then incubate at 37⁰C X 5-10 min

15. After incubation, C1 T1
1 ml
1 ml C2 T2
In incubator

16. Add 1 drop of iodine solution in 2 rows
Spotting Plate
Add 1 drop of iodine solution in 2 rows C T
Add 3-4 drops from C2
Add 3-4 drops from T2
Add 3-4 drops from T2
after 1 min min, mix
Add 3-4 drops from C2
after 1 min, mix
Add 3-4 drops from C2
after 1 min or half min, mix
Add 3-4 drops from T2
after 1 min or half min, mix
Add 3-4 drops from C2
after 1 min or half min, mix
3-4 drops from T2
After 1 min or half min, mix
add 3-4 drops from C2
after 1 min or half min, mix
3-4 drops from T2
After 1 min or half min, mix