Salivary Amylase

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

*Effect of

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

Temperature

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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.

II.

Effect of temperature on salivary amylase activity 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

* 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

1.

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

2.

Put the tubes immediately in the following temperatures Ø

Ø

Ø

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)

5.

Leave the tubes for the achromic point time.

6.

Cool the tubes under running tap water.

7.

Add 2 drops of iodine to each of the three tubes. It will be found that iodine gives different colors

* The expected colors may be as follows:

ØTube No.1: Yellow (achrodextrin is the product

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

Øso one may expect strong reduction in the first tube ( yellow= complete hydrolysis) Øweak in the second (reddish or violet= partial hydrolysis) Øno reduction in the third (blue= no hydrolysis)

*Effect of pH on Salivary

Amylase Activity:

1.

2.

5.

6.

7.

Prepare three test tubes, in each place 1 ml of starch solution. Add 1ml of buffer solutions of different pH as follows: Ø

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

Ø

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

Ø

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

Add 1ml of salivary amylase into each of the three tubes, mix. Put at 37oC, leave for the achromic point time. Remove the tubes from the water bath, cool

* The expected colors may be as follows:

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

Øso one may expect weak reduction in the first

tube ( reddish or violet= partial hydrolysis) Østrong in the second (yellow= complete hydrolysis) Øno reduction in the third (blue= no hydrolysis)