What Influences Enzyme

Activity?
Presented by
Deb Semmler
St. Josephs High School
dsemmmler@saintjoehigh.com

Objective of Lab
Students will investigate the activity of the
enzyme diastase, and examine the effects of
enzyme concentration, temperature, and pH
on the ability of diastase to digest starch.

Background
Catalysts help to control the rate of chemical
reactions by either speeding them up or
slowing them down.
Although they are an important factor in the
ability of a chemical reaction to take place,
they are unchanged and therefore a small
amount of catalyst can be used and reused in
a reaction.

Enzymes
Enzymes are protein molecules that act as
catalysts of biological systems.
They are, however, extremely specific and
tend to act on only one substrate (the
substance altered by an enzyme) changing it
to only one product.
This is called the lock and key model

Examples of digestive enzymes

lactase breaks down lactose (milk sugars)

diastase digests vegetable starch
sucrase digests complex sugars and starches
maltase digests disaccharides to monosaccharides (malt
sugars)
pepsin breaks down proteins into peptides
peptidase breaks down small peptide proteins to amino
acids
trypsin derived from animal pancreas, breaks down
proteins
lipase breaks down fats found in most dairy products,
nuts, oils, and meat
cellulase breaks down cellulose, plant fiber; not found in
humans

How enzymes work

Affecting enzyme performance

Concentration of enzyme how much enzyme is
available
Temperature and pH -Since the lock and key
principle is dependant on the shape of the
enzyme molecule and their ability to fit together
with the molecule of the substrate, anything that
affects that shape affects the rate of reaction.
Since enzymes are proteins they are highly
dependant on temperature and pH.

Concentration of enzyme how

much enzyme is available
- at a constant concentration of enzyme, the
reaction rate increases with increasing substrate
concentration until all of the available enzyme
is joined with substrate.
- the reaction rate is maxed out and adding
more enzyme or substrate will have no further
effect on the rate

Temperature
As the temperature is raised or lowered , the shape
of an enzyme is altered slightly. As a result of this
change in shape, it can become either more or less
effective as it may fit together better or worse with
the substrate.
This gives the enzyme a range of temperature
where it can effectively facilitate the reaction.
There is also a maximum temperature, above
which the enzyme is denatured, or deactiviated.

pH
Like temperature, enzymes are very
sensitive to changes in pH. Acidic and basic
ions, which occurs in varying
concentrations as the pH of a solution
changes, react with enzyme molecules.
These reactions change the shape of the
enzyme much like a change in temperature
does, with similar effects on enzyme
activity.

Background for the lab we are doing

Starch is a common nutrient used by many organisms, yet
it is insoluble, and cannot be effectively absorbed.
It must therefore be broken down to sugars, which are used
to produce energy in living systems.
Although this reaction would occur on its own, the time it
would take to occur spontaneously is so prohibitive that it
is not a possibility for organisms.
Diastase is an enzyme that facilitates this conversion of
starch to sugar and will be used in this lab to demonstrate
the principles of enzyme activity.

Activity 1 effect of enzyme

concentration on reaction rate
Materials: per group
Spot plate
Small cups
Pipettes
Clock w. second hand

Shared materials: class

Starch indicator solution
Starch solution
Diastase solution
Distilled water

Procedure: Activity 1
1. Obtain 10 mL of starch solution, 10 mL of
diastase solution and 10 mL distilled water
and place them into their own small cup.
Keep track of which is which and
designate a pipette to use for each.
2. Place one drop of diastase into each well
on your spot plate followed by four drops
of water into each well.

3. Now the wells are set. Before you go on, be ready

with some starch indicator solution in another cup
with a pipette and have a timing device handy.
4. The starch indicator solution will show you if any
starch is in the well or if the enzyme has broken it
down. If starch is still there you will see a blueblack color. Once the starch is broken down, you
will see an orange or yellow color.
5. You are going to run this whole experiment 5
times with a different amount of enzyme in each
well. This first time only has 1 drop of enzyme in
the well. You will all wells again with 2,3,4 and 5
drops of enzyme.
6. So lets get this one running.

Run the experiment

7. Very quickly put one drop starch into all 12 wells. Just as
quickly, add one drop of starch indicator to the first well
and note the color. This is your time zero. Start watching
the clock.
8. Every 30 seconds, add starch indictor to the next well, note
the color and go on, adding starch indicator to each
successive well every 30 seconds until you go from a blueblack color to an orange/yellow color. Once youre
achieved the color change you want, stop and count up the
number of wells youve used to accomplish this.
9. Since each well worked for 30 seconds, you can figure
out how long it took the one drop of enzyme to change the
starch into sugar. Record that time in table #1 for 1 drop of
enzyme. Rinse out your spot plate and shake out the water
and we will set for the others.

Repeating for 2,3,4,& 5 drops of

enzyme
10. Reset you spot plate but this time add 2 drops of
enzyme and 3 drops of distilled water. Proceed the
way you did in the first experiment.
11. Reset with 3 drops enzyme/2 drops water and run
again.
12. Reset and run with 4 drops enzyme/1 drop water.
13. Reset and run with 5 drops enzyme/ no water.
Record all results and graph.

Activity 2 Effect of pH on Diastase

Activity
Objective : to learn the effect of varying pH
on enzyme and particularly diastase activity
Materials: use the same materials as activity 1
but share a dilute hydrochloric acid
solution, and dilute sodium hydroxide
solution

Procedure
1. Place drops of enzyme and 3 drops of water
into each of three wells.
2. Add one drop of hydrochloric acid (acidlow pH) into the first well, one drop of
water (neutral middle pH) into the second
and one drop of sodium hydroxide (basehigh pH) into the third. Now the tray is set
to run.

To run experiment:
3. Quickly drop 2 drops of starch solution into each
well.
4. After two minutes has passed, add one drop of
starch indicator to each of the wells.
5. Observe the color change in each well and record
your observation in the table below.
Note: the sodium hydroxide has a tendency to bind
with the indicator solution and decolorize it.
Therefore, a lighter blue color will indicate a
positive test for starch in the presence of a base.

Activity 3- effects of Temperature on

Enzyme Activity
Objective: to learn the effects of varying
temperature on enzyme and particularly
diastase activity.
Materials: same as activity 1 but the enzyme
solutions will now be at different
temperatures

Procedure
1.This time add two drops of starch solution to
each of the four corner wells.
2.Take the spot plate over to the bench and
quickly add five drops of enzyme solution to
each well from four different sources: ice
bath (0-10 degrees C); room temp. (20-25
degree C); body temp. (35-45 degree C);
near boiling (90-100 C). Start timing

3. Allow reaction to continue for one minute

and add a drop of starch indicator solution
to each well.
4. Observe the wells and record you
observations in the table below.

Activity 4- Starch digestion

Objective: to observe and understand the
production of glucose from the digestion of
starch
Materials: same
Glucose test strip

 Bayer diastix for diabetics (find at any

local pharmacy- call ahead)

Procedure
1.
2.
3.
4.

5.

Place five drops of distilled water in two wells of your spot

plate.
Add two drops of starch solution in each of the two wells.
Add a drop of enzyme solution to only one of the wells. The
other well without enzyme added to it will be your control.
After 3 minutes, test for the presence of sugar by dipping the
test strips into each well. Wait 10 seconds, then observe and
record any color change in Table 4. The indicator changes color
in varying degrees, from the initial pink to dark purple in the
presence of increasing concentrations of glucose. A color
change after ten seconds is indicative of glucose production
from the digestion of starch.
After testing for glucose, add one drop of starch indicator to
both wells to test for the presence of starch. Record your
observations in Table 5.