Enzyme Lab

Digestion is a vital process that breaks down complex macromolecules into smaller components, allowing the body to absorb and utilize nutrients effectively. Enzymes play a crucial role in the digestive system by catalyzing chemical reactions that break down various types of molecules. We will explore the digestion of carbohydrates, proteins, and fats using specific digestive enzymes: salivary amylase for carbohydrate digestion, gastric protease (pepsin) for protein digestion, and pancreatic lipase (pancreatin) for fat digestion.

Experiment I, focuses on the digestion of carbohydrates. We will investigate the effects of enzymes, pH variations, and high temperatures on the breakdown of starch, a polysaccharide, into maltose, a disaccharide. By observing the enzymatic activity of salivary amylase under different conditions, we can gain insights into how pH and temperature affect carbohydrate digestion.

Experiment II, delves into the digestion of proteins. Here, we will examine the influence of enzymes, pH variations, and low temperatures on the breakdown of albumin, a protein found in egg white. By utilizing gastric protease (pepsin), we can observe the effects of pH and temperature on protein digestion and identify the optimal conditions for efficient enzymatic activity.

Experiment III, investigates the digestion of fats. We will examine the impact of pancreatic lipase (pancreatin), an enzyme responsible for fat breakdown, and the presence of emulsifiers on the digestion of triglycerides. By observing the effects of lipase and bile salts on the breakdown of butter, we can gain insights into the importance of emulsification in fat digestion.

Throughout the experiments, we will employ various tests to assess the progress of digestion, including Lugol's test and Benedict's test for carbohydrate digestion, as well as Sudan III staining for fat digestion. These tests allow us to identify the presence or absence of specific molecules and monitor the effectiveness of enzyme activity under different experimental conditions.

By conducting these experiments, we aim to deepen our understanding of the digestive process and the role of enzymes in breaking down macromolecules. This knowledge can provide insights into the optimal conditions required for efficient digestion, which is crucial for proper nutrient absorption and overall health.

Objective

The objective of this lab is to investigate the effects of enzymes, pH variations, and temperature changes on the digestion of different macromolecules using specific digestive enzymes. The three experiments will focus on the digestion of carbohydrates (starch), proteins (albumin), and fats (triglyceride).

Materials

·         16 Test tubes

·         Water bath set to 37 °C

·         1% Amylase solution

·         HCl solution

·         1% Starch solution

·         Lugol's iodine solution

·         Benedict's reagent

·         1% Pepsin solution

·         Albumin

·         NaOH solution

·         Ice

·         1% Lipase (pancreatin) solution

·         Heavy Cream

·         Bile salts

·         Sudan III

Experiment I: Chemical Digestion of Carbohydrate

1.       Obtain four test tubes and label them 1-4.

2.       Add the following solutions to the four tubes:

a.       Tube 1: 3.0 ml water

b.       Tube 2: 3.0 ml amylase solution

c.        Tube 3: 3.0 ml amylase solution and 10 drops of HCl solution

d.       Tube 4: 3.0 ml amylase solution, then place the tube in boiling water for 5 minutes.

3.       Add 5.0 ml of starch solution to each tube.

4.       Place all four tubes in a 37 °C water bath for 1 hour to incubate.

5.       After incubation, obtain another set of four test tubes labeled 1-4. Split the four incubated solutions evenly among the two sets of tubes. One set of solutions will be tested for the presence of undigested starch (Lugol's test), whereas the other will be tested for the presence of maltose (Benedict's test).

6.       Lugol's Test:

a.       Add a few drops of Lugol's iodine solution to each of the four tubes in one set of solutions.

b.       If the solution turns amber (like the Lugol's reagent), there is no starch present (-).

c.        If the solution turns blue or black, starch is present in the solution (+).

7.       Benedict's Test:

a.       Add 10 drops of Benedict's reagent to each of the four solutions.

b.       Place the four tubes in boiling water for 3 minutes.

c.        If the solution remains blue (like the Benedict's reagent), there is no maltose in the solution (-).

Experiment II: Chemical Digestion of Protein

1.       Obtain a set of five labeled test tubes.

2.       Add 0.1g of albumin (found in egg white) to each of the five tubes.

3.       Add the following solutions to the tubes:

a.       Tube 1: 5 ml pepsin solution and 10 drops of water

b.       Tube 2: 5 ml pepsin solution and 10 drops of HCl

c.        Tube 3: 5 ml pepsin solution and 10 drops of HCl, then place on ice

d.       Tube 4: 5 ml water and 10 drops of HCl

e.       Tube 5: 5 ml pepsin solution and 10 drops of NaOH

4.       Place Tubes 1, 2, 4, and 5 in a 37 °C water bath to incubate for 90 minutes.

5.       Observe any digestion that occurs and note any color changes in the solution.

Experiment III: Chemical Digestion of Lipid

1.       Obtain a set of three test tubes.

2.       Add 2ml of heavy cream to each tube.

3.       Add the following to each tube:

a.       Tube 1: 5 ml water and a few grains of bile salts

b.       Tube 2: 5 ml lipase solution

c.        Tube 3: 5 ml lipase solution and a few grains of bile salt

4.       Shake the tubes rigorously to mix the contents.

5.       Place the tubes into the 37 °C water bath for incubation for 1 hour.

6.       After incubation, add 10 drops of Sudan III to each test tube.

Data Analysis:

Record and analyze the results of each experiment, noting the presence or absence of digestion and any color changes observed. Explain the results below.

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