Qualitative determination of Amylase activity

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• CLS 331
• Experiment No.1

The hydrolytic activity of salivary amylase on
starch
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Introduction
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Amylase is a hydrolytic enzyme which breaks down
many polysaccharides such as starch. Starch Is
a polymer of D- glucose units linked by a-1, 4
glycosidic bonds. The starch is made up of two
polysaccharides Amylopectin (branched-chain
polysaccharide).. Amylose (unbranched-chain
polysaccharide
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• 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 a and ß-
  amylases.
• The ß- amylases rapidly hydrolyse the amylose
  portion of starch to maltose by acting on
  residues at the non reducing terminals.

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They hydrolyse a-1, 4 glycosidic links in
polysaccharides so as to remove successive
maltose units from the non-reducing ends of the
chains. The a amylases, in contrast to the ß-
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 a amylases catalysed reaction than in the
ß- amylases catalysed one.
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Types of amylase amylases human amylase -a ß-
amylases bacteria and plant amylase Source of
amylase 1- Pancreas 2- Salivary
gland Secretion Serum or urine
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Hyperamylasemia Increase the secretion of amylase
in blood Some Causes of Hyperamylasemia Acute
pancreatitis, Salivary gland lesions, etc
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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.
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Starch Amylase maltose
(colourless)
Starch Amylase Iodine blue
colour
(qualitative)
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Principle
At a pH of about 6-7 and in the presence of
chloride ions, a amylases catalyses the
hydrolysis of starch to maltose with the
intermediate formation of various dextrins.
Dextrins are polysaccharide produced by the
hydrolysis of starch
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Starch Cl a amylases
maltose intermediate

formation of dextrins
Steps of the reaction 1. Starch a 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
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Procedure
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Part1
Control 1
Test1
Stock saliva
1 ml saliva
9.5ml H2O
5ml
0.5ml saliva
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
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Part 2
Control 2
Test 2
5ml starch 2ml phosphate buffer 1ml NaCl
Mix well, then incubate at 37C X 5-10 min
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After incubation,
C1
T1
1 ml
1 ml
T2
C2
In incubator
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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
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Result

• Test tube -gt blue color then reddish brown
  color then colorless (A chromic point 4-40
  minute)?
• Control tube -gt blue color why?

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Report

• Principle
• Results identify the time to reach the A
  chromic point for each tube.
• Reference value.
• Comment on each result
• exercises
• Hazard of chemical used