SUBMITTED BY,

MERIN MARIAM MERIN MARIAM JOSEJOSE Class:-XII A Class:-XII A Roll no: -36 Roll no: -36

It is said that a thankful heart is not only the greatest virtue but also the parent of all other virtues. My efforts bore fruit with the successful completion of this project. However there are many others who share the reward of this effort simply because it would never have been this good without this help.

First and foremost, I thank God almighty for been able to complete this project with success. I acknowledge the cooperation, encouragement, austerity of my chemistry teacher Mrs.Bindhu.P whose guidance did half the magic of keeping me thrilled throughout this project. I extend my sincere gratitude to my teammate, who worked on this project with me, friends, classmates, batch mates and also the lab assistants who have helped me whenever I got stuck and for being patient with me even at the toughest hours. Each of you played a very crucial role in the development of this project.

A special thanks to my parents and family for their support and understanding and of course for bearing with me when I was all too busy with the project.

AIM-------------------6AIM-------------------6INTRODUCTION------INTRODUCTION------77

THEORY--------------THEORY--------------8,98,9REQUIREMENTS------REQUIREMENTS------1010PROCEDURE-1--------PROCEDURE-1--------1111PROCEDURE-2--------PROCEDURE-2--------1212PROCEDURE-3--------PROCEDURE-3--------1313

OBSERVATIONS------OBSERVATIONS------14,1514,15CONCLUCONCLU SISI OO N--------N--------1616REFERENCES------17REFERENCES------17

To study the digestionTo study the digestion of starch by salivary of starch by salivary amylase and the amylase and the

effect of temperature effect of temperature and pH on it. and pH on it.

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Every Health book insists on the chewing of food. The act of chewing stimulates the excretion of saliva. Saliva mixes up with the food and helps its digestion. That is, the enzyme ptyalin or amylase present in human saliva hydrolyzes the big molecules of food into many molecules. For example, starch into mono-saccnaricies, maltose and glucose; proteins into amino acids and fats

into fatty acids and glycerol. Thus saliva not only helps in digestion of food but convert it into energy generating substances. Further, enzymes and their activity are very sensitive to temperature and pH. Even a slight variation in these two factors, can disturb the action of enzymes. In other words, digestion of food by salivary amylase is also effected by pH and temperature and can be verified experimentally.

An enzyme is a protein molecule that is a An enzyme is a protein molecule that is a biological catalyst with three characteristics. biological catalyst with three characteristics. First, the basic function of an enzyme is to First, the basic function of an enzyme is to increase the rate of a reaction. Second, most increase the rate of a reaction. Second, most enzymes act specifically with only one reactant, enzymes act specifically with only one reactant, called a substrate, to produce products. The thirdcalled a substrate, to produce products. The third and most remarkable characteristic is that and most remarkable characteristic is that enzymes are regulated from a state of low enzymes are regulated from a state of low activity to high activity and vice versa. The activity to high activity and vice versa. The activity of enzymes is strongly affected by activity of enzymes is strongly affected by changes in pH and temperature. Each enzyme changes in pH and temperature. Each enzyme works best at a certain pH and temperature, its works best at a certain pH and temperature, its activity decreasing at values above and below activity decreasing at values above and below that point due to denaturation. For enzymes, that point due to denaturation. For enzymes, denaturation can be defined as the loss of denaturation can be defined as the loss of

enough structure rendering the enzyme inactive. enough structure rendering the enzyme inactive. This is not surprising considering the importance This is not surprising considering the importance of tertiary structure in enzyme function and non-of tertiary structure in enzyme function and non-covalent forces in determining the shape of covalent forces in determining the shape of enzymes.enzymes.

(P.T.O) (P.T.O)

Salivary amylase is the enzyme produced by the Salivary amylase is the enzyme produced by the salivary glands.salivary glands. Formerly known as ptyalin, it Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. breaks down starch into maltose and isomaltose. Amylase, like other enzymes, works as a catalyst.Amylase, like other enzymes, works as a catalyst. All catalysts are enzymes, but not all enzymes All catalysts are enzymes, but not all enzymes are catalysts. A catalyst is a substance that are catalysts. A catalyst is a substance that hastens a chemical reaction but does not becomehastens a chemical reaction but does not become part of the end product. Amylase digests starch part of the end product. Amylase digests starch by catalyzing hydrolysis, which is splitting by theby catalyzing hydrolysis, which is splitting by the addition of a water molecule. The presence and addition of a water molecule. The presence and absence of starch can be confirmed by several absence of starch can be confirmed by several tests such as the iodine test. In general, a blue-tests such as the iodine test. In general, a blue-black colour indicates the presence of starchblack colour indicates the presence of starch..

1)1) Test tubesTest tubes2)2) Test tube standTest tube stand3)3) One dropperOne dropper

4)4) BeakerBeaker5)5) Starch and Iodine solutionStarch and Iodine solution6)6) Salivary amylase solutionSalivary amylase solution7)7) ThermometerThermometer8)8) Stop watchStop watch9)9) Dil. HCl and NaOH solutionDil. HCl and NaOH solution10)10) Heating arrangementHeating arrangement

1) Collection of Saliva : - Rinse mouth thoroughly with cold water and ensure that it does not contain any food particles. Now take about 20ml of lupe warm water in the mouth and gargle for about three minutes so that saliva mixes up well with it. Spit this into a beaker. Filter, if there is any suspended impurity. Clear filtrate is saliva solution and contains enzyme ptyalin.

2) Preparation of starch solution - Take about 0.5g of starch in a 100ml beaker and add enough water to make a paste. Dilute the paste by adding 50ml water and boil for about 5 min.

3) Digestion of starch a. Take 5ml of the starch solution in a test tube. Add 2 ml

of saliva solution into it. Mix the solutions well by shaking the tube carefully and start a step watch.

b. After one minute take out two drops of the mixture solution from the test tube with the help of a dropper and transfer it into another test tube containing about one ml of 1% iodine solution. Note the colour produced, if any.

c. Repeat this test after every one minute taking two drops of the mixture solution and fresh 1% iodine solution continue until the test shows no blue colour. Record the time and blue colour intensity.

Take three test tubes and label these 1, 2, and 3.Take 5ml of the starch solution, 2ml of the saliva solution and 5 ml of water in each test tube.Place test tube No. 1 in water at room temperature, test tube No.2 in a beaker containing water at 500 C and test tube No.3 in boiling water.After 5 minutes, observe the colour change on mixing two drops of the mixture of every tube with one ml of 1% iodine solution. Note the intensity of blue coloured form.

Take three test tubes and label these 1, 2, 3.Add 5ml of the starch solution, 2ml of the saliva solution in each test tube. Now add 2 ml of water in test tube No. 1, 2 ml of dil HCl in test tube No. 2 and 2ml of NaOH solution in test tube No. 3 and shake carefully. Keep the three test tubes in water at room temperature for about 10 minutes. Add two drops of the solution of each test tube with 1% iodine solution and observe the colour change.

Absence of blue colour on addition to iodine solution means absence of starch in the mixture solution. That is whole of the starch has got digested or hydrolysed.

TEMPERATURE 320C-370C(ROOM TEMPERATURE)

500C-550C 700C-850C(BOILING WATER)

COLOUR INTENSITY

DEEP BLUE LIGHT BLUE NO COLOUR

As the temperature increases, its enzymatic also increases up until the optimum temperature.From the experiment it is observed that the optimum temperature of salivary amylase ranges from 32°C to 37°C.This applies to the human body since salivary amylase is suitable to function within these temperatures. After 37°C, there is loss of activity indicated by colour change from blue-black to light yellow colour of the solution. At 50°C and 70°C, salivary amylase is denatured. The molecular conformation of the enzyme becomes altered as the hydrogen bonds responsible for its secondary, tertiary and quaternary structures are broken.

TIME PASSED AFTER MIXING

1 MINUTE 2 MINUTES 3 MINUTES 4 MINUTES

COLOUR INTENSITY

DEEP BLUE BLUE LIGHT BLUE NO COLOUR

pH Dil. HCl (5.5) Water(6.5-7) NaOH solution(9-10)

COLOUR INTENSITY

DEEP BLUE DEEP BLUE NO COLOUR

The optimum pH for the action of salivary amylase ranges from 5.6 to 6.9.This is consistent with observation between pH 4 and 6 (dil. HCl and water) in table-3. Ideally at pH 10(NaOH solution), salivary amylase is denatured due to high alkalinity. As pH increases, certain amino acids such as lysine and arginine are deprotonated, causing them to lose their net positive charge which also results to enzyme denaturation.

Several factors affect Several factors affect the activity of enzymes. the activity of enzymes. Among these are the Among these are the temperature and pH. At temperature and pH. At

optimum levels of these optimum levels of these factors, enzymes factors, enzymes perform their function perform their function best. Optimum best. Optimum temperature and pH temperature and pH differ from one enzyme differ from one enzyme to another. Salivary to another. Salivary amylase is an enzyme amylase is an enzyme found in human saliva found in human saliva which functions to breakwhich functions to break down starch to simpler down starch to simpler compounds. Through compounds. Through the experiment, it was the experiment, it was found out that the found out that the

optimum temperature ofoptimum temperature of salivary amylase ranges salivary amylase ranges from 32°C to 37°C and from 32°C to 37°C and its optimum pH ranges its optimum pH ranges from 5.6 to 6.from 5.6 to 6. 99..

Comprehensive lab manual class XIIComprehensive lab manual class XIIwww.britannica.comwww.britannica.comwww.google.comwww.google.com

Report on Study of Report on Study of effect of effect of temperature and pH on the enzymatic temperature and pH on the enzymatic activity of salivary amylase by activity of salivary amylase by Gae Gae Khalil Rodillas, Nonia Carla Ysabel Khalil Rodillas, Nonia Carla Ysabel Samson, Raphael Jaime Santos and Samson, Raphael Jaime Santos and Brylle Tabora (Department of Biological Brylle Tabora (Department of Biological

Sciences, College of Science, University Sciences, College of Science, University of Santo Tomas, Manila, Philippines).of Santo Tomas, Manila, Philippines).