JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION, December 2016, p. 490-491
DOI: http://dx.doi.org/10.1128/jmbe.v17i3.1180
Supplemental Materials
for
Exploring Catalase and Invertase Activity Using Sodium
Alginate-Encapsulated Yeast (Yeast Spheres)
Pamela J. Bryer
Biology Department, Bowdoin College, Brunswick, ME 04011
Table of Contents
(Total pages 13)
Appendix 1: Sample data
Appendix 2: Preparation background and supplies
Appendix 3. Sample instructions for testing catalase activity
Corresponding author. Mailing address: Biology Department, Bowdoin
College, 6500 College Station, Brunswick, ME 04011. Phone:
207-725-3072. Fax: 207-725-3405. E-mail: pbryer@bowdoin.edu.
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Appendix 1: Sample data.
rate (cm/sec)
3
2
1
65
43
37
25
2
0
temperature oC
a)
rate (kPa/min)
6
4
2
0
0
20
40
temperature
60
oC
b)
Figure 2. Effect of temperature on the rate of yeast (Saccharomyces cervevisiae) catalase activity. Catalase
activity was measured by either a) the time it took yeast spheres to rise to the top of cylinders containing different
temperatures of 0.3% H2O2 or b) using gas pressure sensors with yeast spheres in different temperatures of 1.5%
H2O2. Results in both methods showed that as temperature increased the enzyme activity significantly increased
until 43OC where the rate then slowed (ANOVA, p<0.0001). Error bars represent SEM.
1
rate (cm/sec)
6
4
2
0.
9
0.
6
0.
3
0.
12
0
H2O2 concentration (%)
a)
rate (kPa/min)
10
8
6
4
2
0
0
1
2
3
H 2O2 conc (%)
b)
Figure 3. Effect of substrate (H2O2) concentration on the rate of yeast (Saccharomyces cervevisiae) catalase
activity at room temperature. Catalase activity was measured by a) the time it took yeast spheres to rise to the
top of cylinders containing different concentrations of H2O2 or b) or using gas pressure sensors with yeast spheres
in different concentrations of H2O2. Results in both methods showed as concentration of H2O2 increased the
enzyme activity significantly increased (ANOVA, p<0.0001). Error bars represent SEM.
2
a)
rate (Δabs/sec)
0.15
0.10
0.05
0.00
0
20
40
temperature
60
80
oC
b)
Figure 4. The effect of temperature on yeast (Saccharomyces cervevisiae) invertase activity as measured by
glucose production. Yeast spheres were immersed in 2.5% sucrose solutions at temperatures ranging from 270oC. The amount of glucose present in solution over time was measured using either a) urine analysis test sticks
for glucose at 15 minutes or b) glucose oxidase at minute intervals. Results in both methods showed an increase
in activity as the temperature increased.
3
a)
rate (Δabs/sec)
0.15
0.10
0.05
0.00
0
5
10
sucrose conc (%)
b)
Figure 5. The effect of sucrose concentration on yeast (Saccharomyces cervevisiae) invertase activity as
measured by glucose production. Yeast spheres were immersed in sucrose solutions at room temperature. The
amount of glucose present in solution over time was measured using either a) urine analysis test sticks for
glucose at 15 minutes or b) glucose oxidase at minute intervals. Results in both methods showed increase in
concentration of glucose as sucrose concentration increased.
4
Appendix 2: Preparation background and supplies.
Preparation background and supplies
1.
Yeast spheres
a.
Preparation of yeast spheres
i. Sodium alginate is a non-toxic hydrophilic polysaccharide that is used as a
thickening agent in foods such as ice cream, yogurt, and cake mixes because it
helps to emulsify oil and water and give a smooth texture to foods. It may be
purchased from Flinn (S0445, 25g/$13), online from Amazon (400g/$25), or it
may be found in gourmet supply stores because some chefs use it to make flavor
“pearls” or “caviar” and it is also used to encapsulate yeast in the production of
wine. I have found no difference in either longevity of spheres or activity
between spheres made with the different sodium alginate powders.
ii. I make a 2% sodium alginate solution (for 20 ml, use 0.4g/20mldH2O) a day or
two day before it is to be used because it takes a long time to get into solution. I
just weigh out the amount I need into a beaker or plastic cup, add dH2O
(preferable to tap), stir, and leave out at room temperature. I used to put it in the
refrigerator but forgot once and it was fine for days at room temperature.
Because the solution is so viscous I would recommend making a separate
beaker/cup for each group instead of making a big batch and trying to aliquot it
out the next day.
iii. I use Fleischman’s RapidRise bread yeast (Saccharomyces cerevisiae) to make a
10% yeast solution in water (10g yeast/100 ml warm water, tap is fine). Make
this up about 5-10 minutes before it will be used.
iv. Hydrogen peroxide may be found in the health and beauty section at most
grocery or department stores, or any drugstore. It comes as a 3% solution. Be
sure to use fresh as containers opened for long periods of time can lose their
effectiveness.
5
v. CaCl2 is available from any science supply company. Make up enough 0.15M
CaCl2 (11g/500 ml dH2O) so each group will have 50 ml.
vi. Syringes (without needles) may be purchased from any Biological supply
company (Carolina Biological Item #697780, Flinn #AP173). I use 30 ml
syringes, but any size 5 ml or above will work.
b.
Yeast sphere trouble shooting
i. When sodium alginate, a hydrophilic polymer, comes in contact with CaCl2,
sodium ions are replaced with calcium. This leads to cross-linkages between the
polymer chains and an insoluble gel is formed. If spheres with “tails” form
when the yeast-sodium alginate solution comes in contact with the CaCl2 the
yeast-sodium alginate solution may be too thick. If this is the case just thin it
out with a bit of H2O.
ii. Yeast spheres of different sizes are usually due to an uneven pressure on the
syringe plunger when dropping the solution into CaCl2 so it is important to use
even pressure when dropping into CaCl2. It is important for students to use
spheres that are close in size for their trials.
iii. Discard any floating spheres. These are usually due to an uneven suspension of
the yeast-sodium alginate solution.
iv. Once the yeast spheres are made they can be stored either in a plastic baggie
without water, or in a beaker or plastic bag with tap water or dH2O in the
refrigerator. I’ve stored them all three ways to see if activity diminishes and it
doesn’t for several days no matter what way they were stored. I even left some
out overnight in all three conditions and they were fine. Just be sure they don’t
dry out.
2.
Experiments
a.
Graduated cylinder method for catalase activity
6
i. Substrate concentration: I used 0.6% H2O2 (10 ml H2O2 + 40 ml dH2O), and
0.3% H2O2 (5 ml H2O2 + 45 ml dH2O) and 0.12% (2 ml H2O2 + 48 ml dH2O).
Students can start with the 0.3% they used for their initial trial and decide what
concentrations to use based on the timing of that trial.
ii. Temperature: make sure the H2O2 and the yeast spheres are to the temperature to
be tested before the experiment proceeds. Make sure students work quickly
when looking at different temperatures so the temperature of the spheres or the
H2O2 doesn’t get back to room temperature before they are done. The spheres
may be put in water at the desired temperature to equilibrate before the
experiment is conducted.
iii. pH: I soaked the spheres for 5-10 minutes in the pH buffer to be tested and made
the H2O2 dilutions in the pH buffer. At high and low pH the spheres may start to
disintegrate if left in the buffer to equilibrate for too long.
iv. Data analysis: Students may graph data either by the time it takes the yeast
spheres to rise to surface or they may convert to rate of reaction by dividing the
distance the spheres rose by the time. Statistical analysis (ANOVA or t-Tests)
showed there was a significant difference in the reaction rate for temperature
changes (rate increased as temperature increased to a point), substrate
concentration (rate increased as the substrate concentration increased), and pH
(rate increased as pH increased to a point)
b.
Gas pressure sensor method for catalase activity – Vernier gas pressure sensor and
LoggerPro software was used
i. Substrate concentration: I used 0.3% H2O2 (5 ml H2O2 + 45 ml dH2O), 0.6%
H2O2 (10 ml H2O2 + 40 ml dH2O), 0.9% (15 ml H2O2 + 35 ml dH2O), 1.5% (25
ml H2O2 + 25 ml dH2O), 2% (33 ml H2O2 + 17 ml dH2O), and 3% (50 ml H2O2).
Five grams of yeast spheres were added to an Erlenmeyer flask containing 50 ml
7
of the H2O2 concentration to be tested, the gas pressure sensor put in and data
collection started.
ii. Temperature: Five grams of yeast spheres were added to an Erlenmeyer flask
containing 25 ml of water, pre-equilibrated to the temperature to be tested, for 5
minutes (the Erlenmeyer was in a container containing water at the temperature
to be tested). 25 ml of 3% H2O2, also pre-equilibrated to the temperature to be
tested was then added to the flask (final concentration H2O2 = 1.5%). The gas
pressure sensor put in and data collection started.
c.
Urine analysis test stick method for invertase activity – Glucose test strips were
purchased from Flinn Scientific (T0004, 100 strips for $19.90). Alternatively Diastix
Reagent Strips for Urine Analysis may be purchased online or in a drugstore (50 strips
for approximately $11)
i. Sucrose concentration: five different sucrose concentrations were used (0, 1, 2.5,
5, and 10%). One gram of yeast spheres was added to 5 tubes, each containing
10ml of a different sucrose concentration. At 15 minutes after the addition of the
yeast spheres a test strip was dipped into the solution surrounding the spheres,
withdrawn and after 2 minutes the color was compared to the color chart
included with the test strips. If cost is not an issue, the amount of glucose
present at various time intervals may also be done (I did 1,3,5,10, and 15
minutes).
ii. Temperature: one gram of yeast spheres was put into a tube containing 5 ml of
water, pre-equilibrated to the temperature to be tested, for 5 minutes before
adding to 5 ml of 5% sucrose, also pre-equilibrated to the temperature to be
tested (final concentration sucrose = 2.5%). The tubes were in a Styrofoam cup
containing water at the temperature being tested. At 15 minutes after the
addition of the yeast spheres a test strip was dipped into the solution surrounding
the spheres, withdrawn and after 2 minutes the color was compared to the color
8
chart included with the test strips. If cost is not an issue, the amount of glucose
present at various time intervals may also be done (I did 1,3,5,10, and 15
minutes).
d.
Glucose oxidase method for invertase activity – Glucose oxidase may be purchased from
VWR (#10022-658, 100 ml for $21.64).
i. Sucrose concentration: five different sucrose concentrations were used (0, 1, 2.5,
5, and 10%). One gram of yeast spheres was added to 5 tubes, each containing
10ml of a different sucrose concentration. At 1, 3, 5, 10, and 15 minutes after the
addition of the yeast spheres 0.2 ml of the solution surrounding the spheres was
withdrawn and added to a cuvette containing 1.8ml dH2O and 1.0ml glucose
oxidase. Five minutes after the addition of the sample an absorbance reading
was taken with a spectrophotometer set @500nm.
ii. Temperature: one gram of yeast spheres was put into a tube containing 5 ml of
water, pre-equilibrated to the temperature to be tested, for 5 minutes before
adding to 5 ml of 5% sucrose, also pre-equilibrated to the temperature to be
tested (final concentration sucrose = 2.5%). The tubes were in a Styrofoam cup
containing water at the temperature being tested. At 1, 3, 5, 10, and 15 minutes
after the addition of the yeast spheres 0.2 ml of the solution surrounding the
spheres was withdrawn and added to a cuvette containing 1.8ml dH2O and 1.0ml
glucose oxidase. Five minutes after the addition of the sample an absorbance
reading was taken with a spectrophotometer set @500nm.
iii. Data analysis – graphs may be made of activity over time (abs@500nm vs time)
for each condition and/or the rate (Δabs/sec) vs concentration or temperature.
3.
Safety
a.
Both yeast and sodium alginate are considered to be non-hazardous so the yeast spheres
can safely be disposed of in the trash. BSL-1 recommendations should be followed.
9
b.
Calcium chloride is considered to be a mild body tissue irritant and slightly toxic by
ingestion. Excess calcium chloride solution may be safely rinsed down the drain with
plenty of water.
10
Appendix 3: Sample instructions for testing catalase activity.
Sample Instructions for testing for catalase activity
Preparing the yeast-sodium alginate solution
1.
Add 20 ml of a 10% yeast (Saccharomyces cerevisiae) solution to 20 ml of a 2% sodium alginate
solution (this solution is very viscous). Mix well with a glass rod or the end of an inoculating
loop.
2.
Draw up the yeast -sodium alginate solution into a 30 ml syringe. Carefully wipe off all excess
liquid from the syringe tip.
Making the yeast spheres
1.
Hold the syringe or plastic pipet containing the yeast-sodium alginate solution over a beaker or
cup filled 1/3-1/2 full (a minimum depth of 3 cm) with 0.15M CaCl2. Very slowly depress the
plunger so that a drop of the yeast-sodium alginate solution falls into the beaker. A sphere should
form as the drop comes in contact with the CaCl2 solution and fall to the bottom of the beaker.
2.
Continue releasing yeast-sodium alginate drops in a steady stream into the 0.15M CaCl2 solution.
The actual size of the spheres is not as important as having all spheres of uniform size. The size of
the spheres may vary due to the pressure exerted on the plunger. A little practice and most spheres
will be the same size.
3.
The spheres should remain in this solution for about 5 minutes to harden.
4.
Dispose of any floating spheres.
5.
Obtain a small strainer and hold it over a clean beaker or cup. Very carefully pour the contents of
the beaker containing the yeast-sodium alginate spheres into the strainer.
6.
Once drained, carefully rinse the spheres under a slow running faucet or with tap water from squirt
bottle.
7.
Pour the spheres into a cup or beaker. If not using immediately add water so the spheres don’t dry
out.
11
Testing the catalase reaction
1.
Pour 50 ml of a 0.3% hydrogen peroxide (H2O2) solution into a 50 ml graduated cylinder. If this
solution is not pre-made for you add 5 ml of 3% H2O2 (the concentration of purchased H2O2) to 45
ml dH2O.
2.
Using forceps, or the loop end of the inoculating loop, gently remove one yeast sphere from the
cup or beaker.
3.
Drop the sphere into the graduated cylinder. Decide when to start timing: as soon as the sphere
touches the surface of the hydrogen peroxide or as soon as it touches the bottom of the cylinder.
Keep timing until the sphere reaches the surface again.
4.
Dispose of the yeast sphere.
5.
Do this with a few more spheres to get the timing down. Be sure to use spheres that are all the
same approximate size.
Designing an experiment
1.
Decide what variable to test. Possibilities include substrate (H2O2) concentration, temperature,
and pH.
2.
Write out your experimental design. Things to think about: What is the control? What
concentration(s) of substrate will be used? What temperature(s)? How many trials? How will
data be displayed?
3.
If using statistics to analyze your data what type(s) of statistical test should you use?
12