Activity 1

Objectives
1. Explain how enzyme activity can be assessed with enzyme assays
a. IKI assay
b. Benedict’s assay
2. Define enzyme, catalyst, hydrolase, substrate and control
3. Understand specificity of amylase action
4. Name end products of carbohydrate digestion
5. Perform chemical tests to determine whether digestion of a particular food has occurred
6. Discuss the possible effect of changes in temperature and pH on amylase activity

Introduction
 Enzymes are large protein molecules produced by body cells. They are biological catalysts that increase the rate of chemical
reaction without becoming part of the product.
o Hastens reaction by lowering activation energy needed to start reaction
 Digestive enzymes are hydrolytic enzymes, or hydrolases, which aids in the breaking down of organic food molecules
(known as substrates).
o This is done by adding water to the molecular bonds, cleaving it between subunits.
* OPTIONAL
 Amylase: an enzyme that digests starch
 Starch: a complex carbohydrate substrate/polysaccharide
 Maltose: a disaccharide substrate
 pH buffers: solutions used to adjust pH of solution
 Deionized water: used to adjust the volume so that it is the same for every reaction
 Test tubes
 Incubators: used for temperature treatments
 IKI: starch presence
 Benedict: presence of maltose and glucose (Starch digestion/reducing sugars)

Methodology
• Drag a test tube
• Add indicated substances
• Boil tube 1
• freeze tube 2
• incubate all
• Drop IKI reagent into the assay tube
– Blue/black color positive starch test
– Negative test = diluted IKI color
• Drop benedict’s reagent into test tubes in the incubation unit
• Boil
– Green to reddish-brown = present reducing sugar = positive sugar test
• Orange = more sugar than green
• Reddish brown = more sugar than orange
– No color changes/blue = negative sugar test
Test tube content
1: Amylase, starch, pH 7.0 (boiled)
2: Amylase, starch, pH 7.0 (Frozen)
3: Amylase, starch, pH 7.0
4: Amylase, Deionized water, pH 7.0
5: Deionized water, starch, pH 7.0
6: Deionized water, maltose, pH 7.0
7: Amylase, starch, pH 7.0
8: Amylase, starch, pH 9.0
Results
 IKI reagent
– Blue/black color would indicate a positive starch test
• This is shown in tubes 1, 5, 7 & 8
• Starch is present!!!
– On the other hand, a diluted IKI color indicates a negative starch test
• Starch is not present!!!
• Seen in tubes 2, 3, 4 & 6
• Benedict’s reagent
– Green to reddish-brown means that reducing sugar is present (an indication of positive sugar test)
• Reddish brown solution means more sugar present than Orange. An orange solution means more sugar
present than Green solution.
• Tubes 2, 3 & 6 are a reddish-brown solution
• While 7 & 8 are an orange solution
• This results indicate the presence of reducing sugar in the solution
– No color changes indicates a negative sugar test
• This is seen in tubes 1, 4 & 5

Conclusion
 Amylase specifically catalyzes the hydrolysis of starch converting it to maltose
 Temperature changes affect activity of amylase
o Enzymes become denatured when there is an increase in temperature
o As seen in the experiment, when test tube 1 was boiled
 pH changes affect activity of amylase
o Amylase activity is optimal at pH 7.0

Activity 2
Objectives
 Explain how enzyme activity can be assessed with enzyme assays
o IKI assay
o Benedict’s assay
 Discuss the specificity that enzymes have for their substrate
 Discuss the difference between the substrates starch and cellulose
 Explain what would be the substrate specificity of peptidases
 Explain how bacteria might aid in digestion

Introduction
 Enzymes catalyze a specific reaction only
 Act on particular substrates only
o Active site: enzyme pocket where substrate must fit into temporarily for catalysis to occur
o Non-covalent bond (ionic bonds or hydrogen bonds)

 Both starch and cellulose are polysaccharides found in plants
o Both are polymers of glucose
 But linked differently
 Starch: alpha 1-4
 Cellulose: beta 1-4
o Starch stores energy
o Cellulose provides rigidity to their cell walls
* OPTIONAL
 Amylase: an enzyme that digests starch
 Starch: a complex carbohydrate substrate/polysaccharide
 pH 7 buffer: solution used to set pH of the test tube solution
 Deionized water: used to adjust the volume so that it is the same for every reaction
 Test tubes
 Incubators: used for temperature treatments
 IKI: starch presence
 Benedict: presence of maltose and glucose (Starch digestion/reducing sugars)
  Glucose: reducing sugar that is the monosaccharide unit of starch and cellulose
 Cellulose: complex carbohydrate in the cell wall of plants
 Peptidase: pancreatic enzyme that breaks down peptides
 Bacteria

Methodology
 Drag test tube
• Add indicated substances
• Incubate
• Add soln to the assay tubes
• Add IKI reagent into the assay test tubes
• Blue/black color positive starch test
• Negative test = diluted IKI color
• Add benedict into test tube in the incubation unit
• Boil
• Green to reddish-brown = present reducing sugar = positive sugar test
• Orange = more sugar than green
• Reddish brown = more sugar than orange
• No color changes/blue = negative sugar test
Test tube content:
1: amylase, starch, pH 7.0
2: amylase, glucose, pH 7.0
3: amylase, glucose, pH 7.0
4: cellulose, pH 7.0, deionized water
5: peptidase, starch, pH 7.0
6: bacteria, cellulose, pH 7.0

Results:
 IKI reagent
– Blue/black color would indicate a positive starch test
• This is shown in tubes 3, 4 & 5
– On the other hand, a diluted IKI color indicates a negative starch test
• Seen in tubes 1, 2 & 6
• Benedict’s reagent
– Green to reddish-brown means that reducing sugar (like glucose) is present (an indication of positive sugar test)
• Reddish brown solution means more sugar present than Orange. An orange solution means more sugar
present than Green solution.
• Tubes 1, 2 & 6 are a reddish-brown solution
• This results indicate the presence of reducing sugar (like glucose) ain the solution
– No color changes indicates a negative sugar test
• This is seen in tubes 3, 4 & 5

Conclusion:
 Amylase specifically catalyzes the hydrolysis of starch
o Starch to maltose
o Amylase cannot hydrolyze cellulose, as seen in test tube 3
 The substrate for peptidase is protein
o It cannot hydrolyze starch
o As seen in test tube 5
 Bacteria aided in the digestion of cellulose
o Through the enzyme cellulase
o An enzyme that converts cellulose to glucose