The hydrolytic activity of salivary amylase on starch

•CLS 331

•Experiment No.1

Introduction

Amylase is a hydrolytic enzyme which breaks down many polysaccharides such as starch .

StarchIs 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

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.

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 .

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

HyperamylasemiaIncrease the secretion of amylase in blood

Some Causes of Hyperamylasemia

Acute pancreatitis, Salivary gland lesions, etc

The Assay

In any enzyme assay, the rate of the reaction can be known by measuring the amount of substrate (s) that isutilized 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.

Starch +Amylase maltose (colourless)

Starch + Amylase + Iodine blue colour (qualitative)

PrincipleAt 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

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 Achromic point ( time taken to reach the point at which the reaction mixture no longer gives a colour with iodine)

Starch + Cl+ + α amylases maltose + intermediate formation of dextrins

Iodine

Iodine

Iodine

Procedure

Part1:Stock saliva

1 ml saliva

Control 1Test1

0.5ml saliva

9.5ml H2O

5ml

Test tube1: mix then incubate at 37 CControl tube1: gently boiled for 2 min, cooled and then placed in the water bath at 37 C

Test 2Control 2

5ml starch2ml phosphate buffer1ml NaCl

Mix well, then incubate at 37⁰C X 5-10 min

Part 2

C1

T2

T1

C2

1 ml 1 ml

In incubator

After incubation,

Spotting Plate

C TAdd 1 drop of iodine solution in 2 rows

Add 3-4 drops from C2

Add 3-4 drops from C2after 1 min, mix

Add 3-4 drops from C2after 1 min or half min, mix

add 3-4 drops from C2after 1 min or half min, mix

Add 3-4 drops from C2after 1 min or half min, mix

3-4 drops from T2After 1 min or half min, mix

Add 3-4 drops from T2after 1 min min, mix

Add 3-4 drops from T2after 1 min or half min, mix

3-4 drops from T2After 1 min or half min, mix

Add 3-4 drops from T2

Result

Test tube -> blue color then reddish brown color then colorless (A chromic point 4-40 minute)?

Control tube -> blue color why?

Report

PrincipleResults; identify the time to reach the A

chromic point for each tube.Reference value.Comment on each result exercisesHazard of chemical used