SALIVARY AMYLASE

Enzymes

*organic compounds

*protein in nature

*synthesized in the living cells to accelerate the rate of biological reactions.

*Factors affecting enzyme activity

1. Effect of substrate concentration

2. Effect of Enzyme Concentration

3. Effect of Co-enzyme Concentration

4. Effect of product Concentration

5. Effect of Temperature

6. Effect of pH

7. Effect of physical agents

8. Inhibitors

9. Activators

10.Oxidation, reduction

11.time

*Effect of Temperature*At 0oC: inactive (reversible)

*Increase in temperature: increase in activity (For every 10oC increase in temperature there is a double-fold increase in enzymatic activity), till the optimum temperature.*37-45oC (optimum

temperature): maximally active.*Further increase in

temperature: activity is decreased.*At 70oC: inactive (irreversible),

the enzyme is denaturated

All enzymes have the same optimum temperature All the enzymes have the same curve for temperature

*Effect of pH

*Every enzyme has an optimum pH for its maximal activity.

*Below or above this pH value, the enzyme activity is decreased.

*At the extremes of pH the enzyme is denatured.

*Salivary Amylase (Ptyalin)

Properties:*A hydrolase (splitting bonds by adding

water)

*Protein in nature as all enzymes

*Optimum temperature: 37oC

*Optimum pH: 6.8

*Activators: halogen ions especially Cl- ions.

Function:

*Present in human saliva. It begins the chemical process of digestion

*Substrate: starch

*α-1,4 glucosidase which hydrolyses the α-1,4 glucosidic bonds of starch

*Products: dextrins and maltose.

*Activity stopped by gastric acidity.

*Steps of hydrolysis of

starch by amylase amylase

*Starch amylodextrins+ maltose

amylase

*Amylodextrin erythrodrxtrins+maltose

amylase

*Erythrodextrin achrodextrins+maltose

The term, amylo-, erythro- and achro indicate the color with iodine i.e. Violet, red and yellow colors respectively

*Experiments

I. Effect of temperature on salivary amylase activity

II. Effect of pH on salivary amylase activity

first determine the achromic point of salivary amylase

the time taken by salivary amylase to hydrolyze starch into achrodextrin at optimum conditions.

*Determination of achromic point

Steps:*Prepare water bath at 37oC.

*Prepare porcelain plate and add a drop of iodine into each well.

*To 1ml of starch solution in a test tube add an equal volume of salivary amylase, mix well and place the tube immediately in the water bath.

*Record the time and take a drop of the mixture of the tube by a glass rod and mix it with the iodine drop present in the first well of the porcelain plate, the color will become blue.

*At half minute intervals, remove one drop of the starch-saline mixture and mix with iodine present in the other wells of the porcelain plate. The color, which was blue in the first well, will change to violet, then reddish and finally yellow (i.e. no changes in the color of iodine solution) in the next wells.

*Record the exact time taken for the color to be changed from blue to just yellow (which equals to the time taken by starch to be changed to achrodextrin). This time represents the achromic point.

Now, it has been identified the time taken by salivary amylase to produce achrodextrins at optimum conditions, which is 5 minutes

*Effect of Temperature on

Salivary Amylase Activity:

1. Prepare three test tubes, in each place 1ml of starch solution + 1ml of salivary amylase, mix well.

2. Put the tubes immediately in the following temperatures

Tube No.1: at 37oC water bath

Tube No.2: at room temperature (leave in the test-tube rock).

Tube No.3: at boiling water bath (boiled salivary amylase)

3. Leave the tubes for the achromic point time.

4. Cool the tubes under running tap water.

5. Add 2 drops of iodine to each of the three tubes. It will be found that iodine gives different colors in the three tubes, depending on the products of digestion of starch in each.

*The expected colors may be as follows:Tube No.1: Yellow (achrodextrin is the

product of starch digestion, salivary amylase is active, and 37oC is optimum for its maximal activity).

Tube No.2: Reddish or violet (erythrodextrin or amylodextrin may be the product of the starch digestion which is incomplete by the end of the achromic point time. Room temperature is not optimum for the enzyme activity).

Tube No.3: Blue (starch is not digested, salivary amylase is inactive and boiling causes denaturation of the enzyme).

*If Fehling test is performed on the products of starch digestion in each of the three tubes, the reduction obtained will be parallel with the coloration given by iodine,so one may expect strong reduction in the

first tube ( yellow= complete hydrolysis)

weak in the second (reddish or violet= partial hydrolysis)

no reduction in the third (blue= no hydrolysis)

*Effect of pH on Salivary Amylase

Activity:1. Prepare three test tubes, in each place 1 ml of

starch solution.

2. Add 1ml of buffer solutions of different pH as follows:

Tube No.1: 1ml of buffer solution of pH 4.5.

Tube No.2: 1ml of buffer solution of pH 6.8.

Tube No.3: 1ml of buffer solution of pH 13.

3. Add 1ml of salivary amylase into each of the three tubes, mix.

4. Put at 37oC, leave for the achromic point time.

5. Remove the tubes from the water bath, cool under running tap water and then add 2-3 drops of diluted acetic acid to tube No.3.

6. Add 2 drops of iodine to each of the three tubes and notice the color changes in each tube.

*The expected colors may be as follows:Tube No.1: Reddish or violet (erthrodextrin

or amylodextrin may be the product of starch digestion which is incomplete by the end of achromic point time, pH 4.5 is not suitable for maximal enzyme activity).

Tube No.2: Yellow (acrodextrin is the product of starch digestion, salivary amylase is active and pH 6.8 is optimum for its maximal activity).

Tube No.3: Blue (starch is not digested, salivary amylase in inactive and pH 13 is not suitable for its activity)

*Addition of acetic acid in tube No.3 will prevent the change of iodine to iodide under the effect of the alkaline pH 13. Iodide gives no color with starch while iodine give blue color with starch.

*If Fehling test is performed on the products of starch digestion in each of the three tubes, the reduction obtained will be parallel with the coloration given by iodine,so one may expect weak reduction in the first

tube ( reddish or violet= partial hydrolysis)

strong in the second (yellow= complete hydrolysis)

no reduction in the third (blue= no hydrolysis)