Effects Of Ph, Temperature And Concentration On Enzyme Pepsin 1i2t6d

LAB MANUAL MP 6 – BIOPROCESS ENGINEERING 2 You are assigned to investigate about pepsin enzyme biological reaction activity. Find the effect of pepsin enzyme concentration, temperature and pH towards its activity. What is the substrate of the enzyme and are the products of the hydrolysis of the substrate.

INTRODUCTION Enzymes are biological catalysts that are made of protein. Enzymes, like all catalysts, speed up biochemical reactions without being used up, by lowering the activation energy of the reactions. Since enzymes are proteins, they are eligible to denaturation in a variety of ways, usually at extreme conditions (temperature, pH, etc). Enzymes work best at their specific optimum conditions; most enzymes have optimum activity at a neutral pH and at body temperature. Enzymes are also very specific – they only act on one substrate or one class of related substrate molecules. The reason for this is that the active site of the enzyme is complementary to the shape and polarity of the substrate. Typically, only one kind of substrate will “fit” into the active site, like a “key” fitting in a “lock”. In this experiment, we will work with enzyme pepsin. Pepsin is a protease that begins digestion of proteins, breaking them into peptides and amino acids. Pepsinogen is secreted by gastric glands of the stomach into the stomach. There, in the acid environment of the stomach, pepsinogen is converted into pepsin. Proteins are enormous molecules containing hundreds of amino acids that are linked together into a chain like structure. Complete digestion breaks the links of the protein chain freeing the individual amino acids one from another. The first stage of protein digestion begins in the stomach where the enzyme pepsin reduces the large molecular chains into many shorter chains. These smaller chains like fragments are known as polypeptides. Later the polypeptides move out of the stomach and into the small intestine where their final breakdown into amino acids takes place. In this investigation we will observe the partial digestion of a protein called albumen from an egg white. To speed the digestive process, the egg has been cooked and the egg white finely ground. Grinding decreases particle size and increases the surface area presented to the enzyme. The mixture of egg white and water has the appearance of milk but, unlike milk the particles tend to settle out, for this is not a true solution. During digestion the particles will disappear as they are broken down into soluble polypeptide fragments. In the end only a clear solution will remain and all the particles of the egg white protein will be gone. Amount of albumin remaining All Most Some None

Enzyme activity level None (0) Low (1) Moderate (2) High (3)

The following reaction is investigated for this experiment: Substrate

Enzyme Pepsin

Protein

H2O

Product

Test

Peptide Amino acid

Disappearance of egg white

Effect of Enzyme Concentration During a catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favored by a high concentration of enzyme and/or substrate. After the substrate is bound, the reaction takes place, and then the product is released. E + S <–> ES –> E + P Effect of Temperature All reactions are faster at a higher temperature. However, enzyme-catalyzed reactions become slower or stop if the temperature becomes too high, because enzymes become denatured at high temperatures. Therefore, enzymes have an optimum temperature that corresponds to maximum activity. (At higher or lower temperatures, the activity of the enzyme is lower.) The optimum temperature for pepsin is usually between 37°C (the body temperature) and 42°C. Effect of pH Each enzyme has an optimum pH. Above or below an enzyme’s optimum pH, its activity is lower. The optimum pH of a particular enzyme corresponds to the pH of its natural environment. For many enzymes, this corresponds to pH values of around 7. For pepsin, which is active in the stomach, the optimum pH is 2 (the pH of the stomach).

OBJECTIVE To study the effect of pepsin enzyme concentration, temperature, and pH on its activity.

MATERIAL AND APPARATUS Material Pepsin solution Egg white Dilute HCl Dilute NaOH

Apparatus Test tubes Dropper Water bath Blender

PROCEDURE Protein suspension preparation 1. Boil an egg to hardness, remove the shell, cut the egg in half and remove the yolk. 2. Place the hard egg white (albumin) in a blender with 100 mL of distilled water. Blend the mixture at low speed and then high speed. Add another 100 mL of distilled water and blend for several minutes. * The blending should produce a suspension of very fine egg white particles and water. 3. Filter the suspension through a strain to remove any large particles of egg white. 4. Add distilled water to the filtrate to make a volume of 500 mL of suspension.

Effect of pepsin enzyme concentration on its activity 1. Label five test tubes 1-5. Place 5 mL of white egg solution in each of the first four test tubes. Place 5 mL of pepsin solution in the fifth tube. Place all of the tubes in the 37°C water bath for 5 minutes. 2. Tube 1 will be the control and it will not contain any enzyme. Remove the tubes from the water bath momentarily, and quickly add 3 drops of the warmed pepsin solution from tube 5 to tube 2, add 6 drops of pepsin to tube 3, and add 9 drops of pepsin to tube 4. Mix the tubes quickly by shaking them gently, and immediately put them back in the 37°C water bath. Record the time at which the enzyme is added. 3. Observe and record the enzyme activity level every 5 minutes for 25 minutes.

Effect of temperature on pepsin enzyme activity 1. Place 5 mL of egg white solution in each of three clean test tubes. Place 5 mL of pepsin enzyme solution in 3 separate clean test tubes (so you will have a total of six test tubes: 3 containing albumin solution and 3 containing enzyme). 2. Place an egg white solution tube and a pepsin solution tube in the 37°C water bath. Place one tube of each in an ice-water bath, and one of each tube in a boiling water bath. Keep the tubes in their baths for 10 minutes to allow them to reach the temperature of their baths. 3. Read and record the temperature of the ice-water bath. Pour the pepsin solution into the egg white solution, mix, and put the tubes back into the bath. Record the time. 4. Repeat step 4 for the 37°C bath. 5. Repeat step 4 for the boiling water bath. 6. Observe and record the enzyme activity level every 5 minutes for 25 minutes.

Effect of pH on pepsin enzyme activity 1. Label four test tubes pH 2, 4, 7, and 10. In each tube, place 5 mL of the appropriate buffer (with the corresponding pH). Add 5 mL of pepsin solution to each of the tubes. Get 4 additional clean test tubes, and put 5 mL of egg white solution in each tube. Place all 8 of these tubes in the 37°C water bath for about 5 minutes to allow the temperature to reach equilibrium. 2. Pour the contents of each egg white test tube into a different pepsin-buffer test tube. Mix them and return the tubes to the 37°C water bath. 3. Observe and record the enzyme activity level every 5 minutes for 25 minutes.