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

Introduction

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

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

Hyperamylasemia Increase 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 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.

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

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

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

Procedure

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

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

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

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