Experiment

Enzymes
Margaret Vorndam, M.S.
Version 42-0053-00-01

Lab Report Assistant

This document is not meant to be a substitute for a formal laboratory report. The Lab Report
Assistant is simply a summary of the experiments questions, diagrams if needed, and data tables
that should be addressed in a formal lab report. The intent is to facilitate students writing of lab
reports by providing this information in an editable file which can be sent to an instructor.

Observations
Note: The observed well colors should be recorded according to the following symbols. Collect
data until the well plate yields an amber color. Then, discontinue testing that test tubes contents.
++ = Black or blue-black, Starch present
+ = Light Blue or Light blue-black, Some starch present
- = Amber, Orange, or Red, No starch present

Data Table 1: Effect of Temperature on Amylase Enzyme Conversion of Starch to Sugar

Time,
Well/Test Tube 1
Well/Test Tube 2
0C
37C
minutes
++
++
0
++
++
5
+
+
10
+
15
+
20
25
30
Place test tubes that still contain starch in 37C water bath.
Continue timing for 30 minutes.
-

Well/Test Tube 3
Boiling (100C at STP)

++
++
++
++
++
++
++

60
Add 1 cm of alpha-amylase solution to test tubes that still contain starch.
Place in 37C water bath. Continue timing for 30 minutes.
-

90

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Experiment
Data Table 2: The Effect of Concentration on Amylase Enzyme Conversion of Starch to
Sugar
Time in minutes

Test Tube #1

Concentration of
Amylase:
Fill in % per
Test tube
#1 example

0.5 cm 2 cm,
or 1:4
= 25%

0
5

Test Tube
#2

Test Tube
#3

16% 12.5%

++

10 %

Test Tube
#5

8.3%

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

++

+
++
++
10
+
+
15
+
+
20
25
30
If test tube(s) still contains starch, let sit for 30 minutes and retest.
60

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#4

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Experiment
Data Table 3: The Effect of pH on Amylase Enzyme Conversion of Starch to Sugar
Time, in minutes
pH ( #1 example )
Student to fill in
0
5
10
15
20
25

Test Tube #1

Test Tube #2

Test Tube #3

Test Tube #4

pH 3.5

pH 5.0

pH 6.8

pH 11.5

++

++

+/-

++

++

++

++

+/-

++

++

++

++

++

++

++
++
30
If test tube(s) still contains starch, let sit for 30 minutes and retest.
No change
No change
60

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Experiment
Exercise 1: Enzymes and Temperature
Questions
A. Which color indicated that starch was still present in the test tube? Which color indicated that
the starch was no longer present?
Any color of blue represents starch is present. It ranges from light blue to blue/black. When
starch is absent, the color is amber orange, or red.
B. When the starch disappeared as indicated by the color change, what happened to it?
The color changed from blue to amber, red or orange.

C. What is the variable in this exercise? The variables are the temperature of the water

What could be considered the control treatment? The control is the amylase and starch solutions. They are
the same in every test tube.

D. Based on the results recorded in Data Table 1, which temperature(s) is/are optimal for enzyme
conversion activity? The temp of 37C appears to be the optimal temp since it had more of an
amber color than the other temps.

Which temperature facilitated the conversion first? Second?

The first would be a temp of 37C, and the second would be a temp of 0C.

E. Did other temperatures facilitate enzyme activity at all? No the temp of 100C still did not after 60 minutes.
It had to be transferred to the 37C bath.

Why or why not? The molecular activity was out of range for the enzymes to react with the solutions.

F. Why was the water bath temperature of 37C chosen as the mid-range temperature? Hint:
What is the oF equivalent of this oC temperature?
It was chosen because it is the same temp as a normal body temp. 37C is equal to 98.6F. This allows us to see what would
happen in our own bodies.

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Experiment
G. Why was it important to immediately observe the color of the test tube contents/iodine in
the microplate well? The color will change as more time is allowed. This will give us accurate
results if the color is read immediately. This allows for the experiment to be the same each
time it is conducted due to the time intervals.

H. Why didnt you add the iodine indicator directly to the test tubes?
In order to not skew the results. It had to be added at a specific time
to keep the tubes free of the solution. If it had been added at the
beginning it would have made the results different.

I. What happened when you incubated the test tube(s) that still contained amylase/starch at
37C?
It changed as the solution cooled off. It actually did not take the entire time for it to change to a
negative state.

Did both test tubes indicate the disappearance of starch after the incubation? If not, why? Only one
had to be transferred, because the other was negative already. They all ended up being negative at
the end
J. What happened after you added 1 cm of alpha-amylase solution to the test tube(s) that still
contained starch, and incubated them at 37C? The starch was diluted and was easier changed
to sugar.

Why did this occur after the second addition of amylase, but not before?
The amylase concentration was higher and allowed for the starch to be
broken down quicker .

K. Construct a graph that presents your findings from Table 1 with time on the x-axis, and relative
concentration of starch on the y-axis (++ is highest, +, or is lowest). Include the graph in the
report.

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Discussion
A. Was the initial hypothesis supported or refuted? Explain. It was supported. Based on my studies of
enzymes, I knew that the amylase would break down the starch. I knew that the temp of zero degrees
would still allow for the conversion to take place
B. What have you learned from this experiment? I have learned that temp and concentration have a
direct effect of the enzyme actions that will take place. Temp can affect the reaction regardless of any
other stimuli present. The enzyme will just speed up the process.
C. What practical applications could this knowledge have?
They can be applied when working in a lab setting where you will be testing blood or products for enzymes or the reactions that they will
have on a specific substance. Whether you are testing for the actual enzyme or for the reaction that the enzyme will cause.

Exercise 2: Enzymes and Substrate Concentration

Questions
A. What is the variable in this exercise? The concentration of amylase.

B. Which test tube has the highest concentration of alpha-amylase in the starch? Test Tube one with
25%.
C. How could this experiment be improved? There was no change between the 30 and 60 minute
mark. The experiment could be stopped at 30 minutes.

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Experiment
D. Construct a graph that presents your findings in Table 2 with time on the x-axis, and relative
concentration of starch on the y-axis (++ is highest, +, or is lowest). Use five different colors to
represent the concentration of amylase. Include it in the report.

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Experiment
Discussion
A. Was your initial hypothesis supported or refuted as to which test tube starch contents would
convert to sugar first? Yes, the initial hypothesis was supported due to the fact that the highest
concentration changed first. The changes noted supported the hypothesis made.

B. What have you learned from this experiment? I learned that the concentration in the beginning does not
really make a difference. Timing was not the same in most situations. The higher the concentration the faster
the change, but with the different concentrations the time was different. Concentration and time are not
equal with each.

C. What practical applications could this knowledge have? In order to have a high concentration, this will
reduce the amount of time needed to make the reaction happen.

D. Describe another way to do this experiment that would test the same concept. If you had the same amount
of amylase and different amounts of starch. The reaction would be the same, but the time would be different,
due to the higher amounts of starch. The highest amylase/less starch would still change first.

Exercise 3: Enzymes and pH

Questions
A. What is the variable in this experiment? The different levels of pH of the buffers.
B. In which test tube(s) did the amylase convert starch to sugar? The tubes containing 11.5 pH and 6.8
pH.
Why might you expect this outcome? The higher the buffer level the enzyme was allowed to
convert the starch into sugar.

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Experiment
C. Did any test tubes NOT exhibit a color change? Tube 1 and 2 did not show any change.

Why?

They did not show change due to the high concentrations of starch and the low pH level. This caused the enzymes to not be able to
work as they should. No matter the situation if the pH is incorrect, than the enzyme cannot do its job like it should.

Discussion
A. Did the results support or refute your hypothesis as to which test tube starch contents would
|+}convert to sugar first?
It was refuted, I hypothesized that all the tubes would change.

Laboratory Summary
A. What other conditions that may affect the action of enzymes? The substrates being present to act on the
enzymes. The more of an enzyme the faster the reaction will take place.

B. How might you propose to test this? Have lower levels of amylase in 2 different tubes and test the higher
pH of 6.8 and 11.5.

C. What have you learned from this laboratory? The higher alkali solution the faster the reaction will take
place.

D. What practical applications might this knowledge have? In order to clean up acidic chemical or an orgainic
spill such as blood or vomit. You would need to have an alkaline cleaner to neutralize the solutions.

Copyright 2008 by Margaret E. Vorndam, 1413 County Road 671, Rye, CO 81069
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