LAB 5: Enzymes
Pre-lab questions
Name_____________
Please read the entire lab before answering the following questions.
Fill in the blank
Enzymes are used in ___________________________________________________________________
Enzymes are __________________________________________________________________________
Two factors that can affect an enzyme’s ability to catalyze a reaction are:
1) __________________________________ 2) __________________________________
Enzymes are very specific for their substrate. The model shown below is often called a “lock and key”
model. Enzymes are very specific to the substrate that they interact with. Just like a lock, a substrate
needs the right enzyme to interact with.
Figure5. 1: The enzyme sucrase is a protein that breaks down (hydrolysis) the substrate sucrose (table
sugar) into monomers called glucose and fructose. When the enzyme and substrate are joined
together, you have momentarily formed an (ESC) Enzyme Substrate Complex. Lastly, the sucrose is
broken down into monomers (glucose and fructose). This enzyme sucrase is now able to break apart
more sucrose.
LAB 5: Enzymes
1. Use the following terms to fill in the images below: (Words may be used more than once)
Enzyme (Sucrase)
Substrate (Sucrose)
Enzyme Substrate Complex (ESC)
Products
Glucose
Fructose
Active site
Figure 2
LAB 5: Enzymes
2. Sketch a model (like in figure 5.1) that describes the equation below:
enzyme + substrate → enzyme-substrate complex → enzyme + product 1 + product 2
 Enzyme = Lactase
 Substrate = Lactose
 Products 1 & 2 = Glucose and Galactose
3. Sketch a model (like in the figure 5.1) that describes the equation below:
enzyme + substrate 1 + substrate 2 → enzyme-substrate complex → enzyme + product
 Enzyme = Maltase
 Substrates Maltose
 Product = Glucose and Glucose
LAB 5: Enzymes
Introduction
Enzymes are (proteins) large biological molecules that act as catalysts, substances that can increase the
rate of a chemical reaction. These proteins can be used over and over again without altering the shape
of these enzymes. Our bodies use enzymes for many biological processes from nerve impulses,
digestion, muscle contractions….. Without enzymes, we would not be able to survive. Each enzyme has
a specific shape and function for which substrate it catalyzes. Enzymes will best work in an optimal pH
and a specific temperature. If the environment for the enzymes is not optimal, then the protein shape
could denature and no longer function.
Vocabulary
Enzyme – large biological molecules that greatly accelerate the rate and specificity of chemical reactions
Chemical reaction – a process in which of one set of chemical substances is converted to another
Activation energy – The least amount of energy needed for a chemical reaction to take place
Substrate – A molecule upon which an enzyme acts
Active site – the part of an enzyme where the substrate binds and is converted to product
Denaturation –A structural change caused by extreme conditions. Most biological molecules lose their
ability to function when denatured.
Enzymes are large biological molecules that act as catalysts, substances that increase the rate of a
chemical reaction. These catalysts do not cause reactions to occur and they are not consumed by the
reaction, they simply speed up the rate of the reaction. In other words, they can be used over and over
again without being changed. Nearly all chemical reactions that occur in a biological systems use
enzymes, without them these reactions could not occur at rates fast enough to sustain life. Each
enzyme has a shape and activity that is specific to the particular reaction it catalyzes. In addition,
enzymes have an optimal pH and temperature specific to the environment in which they function.
Exposure to conditions outside of these optimal conditions can cause the enzyme to denature (change
shape) and no longer function.
In enzymatic reactions, the starting molecules are called substrates. These substrates are converted
into different molecules called products.
LAB 5: Enzymes
Materials for the lab group of 4 students:
13 test tubes
test tube rack
Thermometer
37 oC water bath
Wax marking pen
DI water
Chymosin solution
Amylase solution 2%Starch solution Litmus cream
Lipase powder with spatula
test tube clamp
ice bath
stop watch
500ml beaker with boiling chips
Benedict’s solution
Iodine solution
Milk
Effect of Chymosin on Milk
Overview-Chymosin is an enzyme found in a complex of enzymes produced in the stomach of many
mammals. The enzyme is produced in infants to curdle ingested milk, allowing it to remain longer in the
bowels, improving adsorption of nutrients. Purified molecules of chymosin are also utilized in the
making of cheese. The chemical reaction of chymosin with milk is as follows:
Casein
Paracasein(s)+Milk plasma
chymosin
Procedure:
1. Obtain and label two test tubes “1” and “2”
2. To tube 1, add 3 mL of milk and 3 mL of distilled water
3. To tube 2, add 3 mL of milk and 3 mL of chymosin solution (NOTE: do not use the same pipet for
the milk and chymosin solutions)
4. Mix the contents of both tubes by gently inverting each tube a few times and then place in the
37 oC water bath for 30 minutes
LAB 5: Enzymes
Effect of Temperature on Amylase Activity
Overview-Amylase is an enzyme that catalyses the breakdown of starch into sugars. In human saliva,
this enzyme begins the process of digestion. Interestingly, foods that contain high amounts of starch,
but little sugar, taste slightly sweet as they are chewed because salivary amylase turns some of the
starch into sugar in the mouth. The pancreas also makes amylase to breakdown starch into simpler
sugars to supply the body with energy.
Amylase
Starch
Maltose (disaccharide)
Procedure:
1. Obtain two tubes and add approximately 4 mL of amylase to each.
2. Label one of your amylase tubes “boiled” and place it in the boiling water bath for 20 minutes,
keep your other amylase tube on your bench at room temperature
3. Obtain six test tubes and label #1-6
4. Sugar/Benedicts test
Prepare the following tubes:
Tube
1
2
3
Contents
2 mL starch + 2 ml DI water
2 mL starch + 2 mL amylase at room temp.
2 mL starch + 2 mL boiled amylase
Allow the tubes to incubate at room temperature for 30 minutes. During the incubation, note
your hypothesis for each reaction on the post-lab report. Then, add a dropper full of Benedicts
solution to tubes 1-3 and note the color of each tube. Place the tubes in a boiling water bath
for 5 minutes. Remove tubes and allow them to cool. A change in color to orange/brown
indicates high amounts of sugar, yellow or green indicates medium amounts of sugar and no
change in color (blue) indicates no sugar.
5. Starch/Iodine test
Prepare the following tubes
Tube
4
5
6
Contents
2 mL starch +2ml DI water
2 mL starch + 2 mL amylase at room temp.
2 mL starch + 2 mL boiled amylase
LAB 5: Enzymes
Allow the tubes to incubate at room temperature for 30 minutes. During the incubation, note
your hypothesis for each reaction on the post-lab report. Add 2-3 drops of iodine solution to test
tubes 4-6. A bluish-black color indicates starch is present.
Effect of Temperature on Lipase Activity
Overview- Lipase is an enzyme that catalyzes the hydrolysis (breakdown) of lipids (fats) into fatty acids
and glycerol. Lipases perform essential roles in the digestion, transport, and processing of lipids ingested
in the diets of mammals. In this experiment we will use cream as a source of fat to observe a lipase
catalyzed reaction. The cream has a pH indicator added to it (litmus powder). When the litmus is blue,
the pH is above 7. When the litmus turns reddish pink, the pH is below 7.
Lipase
Lipids (fats)
Fatty Acids + Glycerol
Procedure:
1. Obtain three test tubes and label #1-3. Add 4 ml of litmus cream to each tube. Note the color of
the litmus cream.
2. Add a pea sized amount of lipase powder to tubes 1 & 2 and mix by gently inverting the tube
several times. Do not add any lipase powder to tube 3.
3. Put tubes 1 and 3 in the 37oC water bath (body temperature) for 30 minutes.
4. Put tube 2 on ice for 30 minutes.
5. After these 30 minute incubations compare the color of tubes 1-3.
LAB 5: Enzymes
Name:
Date:
Post lab questions
Effect of Chymosin on Milk
1. What is the substrate in the reaction?
2. What is the product in the reaction?
3. What is the enzyme in the reaction?
4. Results/Observations
Sample
Observations
Milk
Milk and
Chymosin
5. In which tube has an enzyme catalyzed reaction occurred? What results support this conclusion?
LAB 5: Enzymes
Effect of Temperature on Amylase Activity
Tube
Contents
Test
1
Starch
Sugar/
Benedicts
2
Starch +
Amylase
Sugar/
Benedicts
3
Starch +
Boiled
Amylase
Sugar/
Benedicts
4
Starch
Starch/
Iodine
5
Starch +
Amylase
Starch/
Iodine
Hypothesis
Results
Conclusions
Starch +
Starch/
Boiled
Iodine
Amylase
1. How do the results differ between the amylase experiments done with untreated enzyme and
boiled enzyme?
6
2. What can you conclude id the effect of excessive heat (boiling) on enzyme activity?
Effect of Temperature on Lipase Activity
1.
Prior to the addition of the enzyme was the litmus cream substrate acidic or basic?
2. What color is each tube after incubation?
Tube
Color after incubation
1
2
3
LAB 5: Enzymes
3. Has a reaction occurred in both the tube incubated at 37 oC and in the tube placed in the cold?
What evidence supports this conclusion?
4. a. Which tube showed the greatest amount of change?
b. Did the litmus cream indicate whether it was acidic or basic?
c.
What product caused the pH change?