PHR 374 BIOCHEMISTRY I

Week 12 Practical

Effect of salivary amylase on starch

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Salivary amylase is a digestive enzyme secreted by the salivary glands. It is responsible for starting the breakdown of the insoluble polysaccharide starch into soluble dextrins (oligosaccharides), maltose (disaccharide) and glucose (monosaccharide) in the mouth so that starch can be absorbed (amylon is a Greek word that means starch). Complete digestion of starch occurs in the small intestine by the action of pancreatic amylase. Salivary amylase Starch dextrin + maltose + glucose

There are three pairs of relatively large and major salivary glands: Parotid glands (located in the upper part of each cheek). Submandibular glands (located in the floor of the mouth). Sublingual glands (located beneath the tongue).

In addition to these major glands, 600 to 1,000 very tiny minor salivary glands are scattered throughout the mouth and throat. 1

Starch makes up a large proportion of our food. Chemically, starch consists of two types of molecules, amylose (20-30%) and amylopectin (7080%) which are both polymers of glucose. Amylose is a linear chain of glucose units that are linked by -1,4 bonds, whereas amylopectin is a branched molecule in which glucose units are linked by -1,4 bonds in the straight chain region in addition to -1,6 bonds at the branching points. The relative proportions of amylose to amylopectin depend on the source of the starch.

Salivary amylase hydrolyses only the -1,4 bonds that link glucose units. Therefore, starch and glycogen, which consist of -1,4 bonds, are hydrolyzed by amylase while cellulose, which consists of -1,4 bonds, is not hydrolyzed by salivary amylase.

The aim of this practical session is to: 1. Study the effect of salivary amylase on starch. 2. Study the effect of different factors (such as temperature, pH and time) on the activity of salivary amylase.

Practical
In a beaker, collect about 1 ml of your own saliva then add 9 ml of distilled water and 60 ml of 0.5% sodium chloride solution and gently mix. Use this enzyme solution to carry out the following four experiments:

I- Effect of salivary amylase on starch: Principle: If amylase is added to a solution of starch, starch will be digested. As the reaction proceeds, the progress of the reaction can be visualized by testing: 1. the disappearance of the substrate (starch). 2. the appearance of one of the products (the reducing sugar, maltose). Two simple tests, iodine test for starch detection and Benedict's test for maltose detection are used for this purpose. Procedure: 1. In a test tube, put 2 ml of 2% starch solution and carry out iodine test as follows: add 3 drops of iodine solution to the content of the test tube; a blue-black color is formed (N.B. shake starch solution well before withdrawing any volume).

(to the left: starch; to the right: starch + iodine)

2. In a test tube, put 2 ml of 2% starch solution and carry out Benedicts test as follows: add 2 ml of Benedict's reagent to the content of the test tube and put in a boiling water bath for 5 minutes. The test gives negative result where Benedict's reagent retains its clear blue color and no precipitate is formed indicating the absence of maltose (starch did not undergo any hydrolysis yet). 3

3. In a test tube, put 2 ml of the previously prepared enzyme solution and add 2 ml of 2% starch solution; mix well and leave for 10 minutes. 4. Divide the enzyme-starch mixture in 2 clean test tubes and carry out Iodine test to the first and Benedict's test to the second as follows: a. To the first test tube, add 3 drops of iodine solution; the test still gives positive result (blue-black color) indicating the presence of a remaining part of starch that did not undergo hydrolysis by salivary amylase. b. To the second test tube, add 2 ml of Benedicts reagent and put in a boiling water bath for 5 minutes. The test gives positive result (development of a precipitate with a color ranging from green, through yellow and orange to red) indicating the formation of maltose; the test depends upon the ability of the reducing sugar maltose to reduce cupric ions (blue) in Benedict's reagent to cuprous ions (green to red).

Color Blue Green Yellow Orange Red

Amount of maltose None Some More Much Most

Benedicts test

II- Effect of temperature on the activity of salivary amylase: Principle: Salivary amylase is protein in nature; the optimum temperature for its activity is 37 In extreme temperatures (cold and heat), the activity of the C. enzyme is inhibited (high temperature causes denaturation of the protein structure of the enzyme). Procedure: 1. Get 3 test tubes and number them 1, 2 & 3. 2. Put 2 ml of the previously prepared enzyme solution in each of the three test tubes. 3. To each test tube, add 2 ml of 2% starch solution and mix well. 4. Immediately put test tube 1 in an ice bath, test tube 2 in a 37 C water bath and test tube 3 in a 70 water bath, each for 20 minutes. C

5. Remove each tube to room temperature and carry out Benedicts test to check for the presence of the starch hydrolysis product maltose as follows: add 2 ml of Benedicts reagent to the enzyme-starch mixture in each tube and put in a boiling water bath for 5 minutes; Benedicts reagent will retain its clear blue color in test tubes 1 & 3 indicating the absence of maltose as a result of inhibited enzyme activity at cold and hot temperatures, while a green to red precipitate is formed in test tube 2 indicating the formation of maltose as a result of a retained activity of salivary amylase at 37 C. 5

III- Effect of pH on the activity of salivary amylase: Principle: The optimum pH for the activity of salivary amylase is the neutral pH (about 7). In the acidic pH of the stomach, the activity of salivary amylase stops. Pancreatic amylase, on the other hand, has an optimum activity in alkaline medium. Procedure: 1. Get 3 test tubes and number them 1, 2 & 3. 2. Put 2 ml of the previously prepared enzyme solution in each of the three test tubes. 3. To each test tube, add 2 ml of 2% starch solution and mix well. 4. Add 2 ml of acidic solution to test tube 1, 2 ml neutral solution to test tube 2 and 2 ml alkaline solution to test tube 3. Put the 3 test tubes in a 37 water bath for 10 minutes. C 5. Remove each tube to room temperature and carry out Benedicts test to check for the presence of the starch hydrolysis product maltose as follows: add 2 ml of Benedicts reagent to the content of each tube and put in a boiling water bath for 5 minutes; Benedicts reagent will retain its clear blue color in test tubes 1 & 3 indicating the absence of maltose as a result of inhibited enzyme activity in acidic and alkaline pH, while a green to red precipitate is formed in test tube 2 indicating the formation of maltose as a result of a retained activity of salivary amylase in neutral pH.

IV- Effect of time on the activity of salivary amylase: Principle: When a specified quantity of salivary amylase is added to a specified quantity of starch, starch is gradually digested until, by time, all starch is consumed. Procedure: 1. Put 5 ml iodine solution in a number of test tubes and number them. 2. In another test tube, put 2 ml of the previously prepared enzyme solution and add 2 ml of 2% starch solution and mix well. 3. Quickly add 5 drops of the enzyme-starch mixture to the iodine solution in the first test tube; a deep blue color is formed (zero time). 4. At one minute interval, add 5 drops of the enzyme-starch mixture to iodine solutions in successive test tubes. 5. Record the time at which iodine retains its yellow color indicating the full digestion of starch by salivary amylase; this point is known as the achromic point.

PHR 374 BIOCHEMISTRY I Week 12 Practical Effect of salivary amylase on starch

Student Name: . Student number: I. Effect of salivary amylase on starch: Test Iodine test Benedicts test Starch solution Enzyme-starch mixture

II. Effect of temperature on the activity of salivary amylase: Temperature 0 C 37 C 70 C Salivary amylase activity

III. Effect of pH on the activity of salivary amylase: pH Acidic Neutral Alkaline Salivary amylase activity

IV. Effect of time on the activity of salivary amylase: The achromic point is obtained after . minutes. 8