Introduction Saliva is 99.5% water and 0.5% solutes.

Forces that hold the substrate in the active site of the enzyme are noncovalent, an assortment of: hydrogen bonds ionic interactions and hydrophobic interactions

solutes are ions, including sodium, potassium, chloride, bicarbonate, and phosphate. dissolved gases organic substances, including urea and uric acid, mucus, immunoglobulin A, the bacteriolytic enzyme lysozyme, and salivary amylase

Procedure During the experiment, salivary amylase was subjected to many of the extremes that impair enzyme function, and then was placed in an environment rich in starches for the enzymes to digest. Effect of Temperature 1. 5 water baths at different temperatures 0C or cooler, 10C, 40C, 60C and boiling 2. Prepare enzyme solution 3. 5 test tube with 1 mL of enzyme solution and 5 test tube containing 1 mL starch solution 4. Pair the test tubes using rubber bands and place one pair into each water bath 5. Permit the test tube to adapt to the temperature for ~5 mins 6. Mix the enzyme and starch solutions of each pair together and place the single test tube in its respective water bath 7. After 30 secs, remove test tube from the water baths and test all five test tubes from starch on a spot plate using Lugols solution 8. Repeat every 30 seconds until the starch disappears (record the time) Effect of pH 1. Prepare three solutions as follows: Solution ApH 4.0 Solution BpH 7.0 Solution CpH 9.0 2. Prepare fresh enzyme solution 3. Mix 4 mL of a starch solution with 2 mL of buffer solution in a test tube 4. Place one drop of starch buffer-solution on a spot plate and immediately add one drop of the enzyme solution 5. Test for starch disappearance using Lugols solution and record the time when the starch disappears completely Principle Involved

Chloride ions in the saliva activate salivary amylase (AM-i-las), an enzyme that starts the breakdown of starch in the mouth into maltose, maltotriose, and -dextrin. Bicarbonate and phosphate ions buffer acidic foods that enter the mouth pH of saliva: pH 6.356.85 After mastication, Food molecules begin to dissolve in the water in saliva, an important activity because enzymes can react with food molecules in a liquid medium only. Salivary amylase, which is secreted by the salivary glands, initiates the breakdown of starch. Amylase which acts on 1-4 glycosidic bonds in starch, dextrin and glycogen. Dietary carbohydrates are either monosaccharide and disaccharide sugars or complex polysaccharides such as starches. Thus, ingested disaccharides and starches must be broken down into monosaccharides. The function of salivary amylase is to begin starch digestion by breaking down starch into smaller molecules such as the disaccharide maltose, the trisaccharide maltotriose, and short-chain glucose polymers called -dextrins. Even though food is usually swallowed too quickly for all the starches to be broken down in the mouth, salivary amylase in the swallowed food continues to act on the starches for about another hour, at which time stomach acids inactivate it. (Tortora ) Enzyme is a protein molecule that is a biological catalyst - increases rate of reaction - most enzymes act specifically with only one reactant (substrate) to produce products

The activity changes in works best temperature

of enzymes is strongly affected by pH and temperature. Each enzyme at a certain pH (left graph) and (right graph), its activity decreasing at

values above and below that point. This is not surprising considering the importance of tertiary structure (i.e. shape) in enzyme function and noncovalent forces, e.g., ionic interactions and hydrogen bonds, in determining that shape.

At higher temperature (60- 65C in humans) irreversible loss of enzyme activity occurs due to denaturation of enzymes Effect of pH Each enzyme has an optimum pH at which it attains its maximal activity Salivary amylase 6.6 to 6.9 in humans Any change of pH below or above the optimum pH decreases the rate of enzyme action due to: a) Changes in pH leads to changes in the ionization state of the substrate or the enzyme or both b) Also, the extreme changes in pH leads to denaturation of the enzyme that is protein in nature Lugols Test The -14 lingkages between carbons in the starch cause the helical structure of the polysaccharide chain. The inner diameter of the helix is big engouh for Iodine to become deposited thus forming a blue complex. The iodine gets stuck in the coils of beta amylase molecules. The starch forces the iodine atoms in a linear arrangement in the central groove of the amylase coil. There some transfer of charge between starch and iodine. That changes the way electrons are confined, and so, changes spacing of the energy levels. The iodine/starch complex has energy level spacings that are just for absorbing visible lightgiving the complex its intense blue color. An Introduction to Practical Biotechnology by S. Harisha

Changes in pH alter the state of ionization of charged amino acids (e.g., Asp, Lys) that may play a crucial role in substrate binding and/or the catalytic action itself. Hydrogen bonds are easily disrupted by increasing temperature. This, in turn, may disrupt the shape of the enzyme so that its affinity for its substrate diminishes. The ascending portion of the temperature curve reflects the general effect of increasing temperature on the rate of chemical reactions. The descending portion of the curve above (blue arrow) reflects the loss of catalytic activity as the enzyme molecules become denatured at high temperatures Kimball, J. 2011. Enzymes. [online] Available at: http://users.rcn.com/jkimball.ma.ultranet/BiologyPa ges/E/Enzymes.html#pHandTemp [Accessed: 27 Feb 2014]. Effect of Temperature At very low temperature, enzymes are inactive. Enzyme activity increases gradually with the rise in temperature until a temperature at which the enxyme attains its maximal activity (optimum temperature) In humans, 37-40 oC Optimum temperature is the temperature at which the enzyme attains its maxima activity. The rise in temperature from low temperature to optimum temperature causes an increase in the reaction rate due to: a) The rise in temperature increases the initial energy of substrate leading to a decrease in activation energy and lower the energy barrier of the reaction b) The rise in temperature increases collision of molecules (more molecules become in the bond forming or bond breaking distance) The rise in temperature above the optimum temperature leads to a decrease in the rate of enzyme activity