2003 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY ANDMOLECULAR BIOLOGY EDUCATION

Printed in U.S.A. Vol. 31, No. 1, pp. 4245, 2003

Laboratory Exercises

Biopolymers, Enzyme Activity, and Biotechnology in an Introductory

Laboratory Class Experience*
Received for publication, January 17, 2002, and in revised form, September 3, 2002

Diana L. Vullo
From the Area Qumica, Instituto de Ciencias, Universidad Nacional de General Sarmiento, J.M. Gutierrez 1150,
(B1613GSX) Los Polvorines, Pdo. de Malvinas Argentinas, Provincia de Buenos Aires, Argentina

This work introduces aspects of Biological Chemistry to students of non-related careers and, in some way,
tries to develop abilities that have remained unexplored up to the moment the students face this subject.
This aim was successfully achieved as shown in the results obtained with the biological model of invertase
activity in immobilized Saccharomyces cerevisiae. By optimizing time and temperature and understanding
the hydrolysis yield, we managed to introduce sugar and biopolymer chemistry concepts.
Keywords: Cell immobilization, bakery yeast, invertase activity, calcium alginate.

The yeast-mediated sucrose inversion is a well known
biological mechanism that has been perfectly described
and studied [1, 2]. The reaction involves a hydrolytic en-
zyme activity (invertase) that cleaves the sucrose molecule
(disaccharide) into two products as follows:
Invertase
2 to industrial and home activities. The invertase activity in
SUCROSE 3 GLUCOSE FRUCTOSE
non-reducing sugar reducing sugars
The enzyme activity measurement is easy to perform. It
only consists of the quantification by analytical procedures
of the reducing sugar produced after incubating Saccha-
romyces cerevisiae cells (bakery yeast) with a sucrose
solution. The immobilization of the bakery yeast by entrap-
ment in calcium alginate improves the technique as further
product purification is not required [35].
Our University is characterized by non-conventional ca-
reers such as Urban Ecology and Urbanism as well as the
more conventional Industrial Engineering and Mathemat-
ics and Physics Professor Majors. Being non-Chemistry
majors, they learn about fundamental aspects of General,
Inorganic, Organic, and Biological Chemistry, which in-
clude topics related to environmental interactions. The aim
in our teaching is to show the direct application of every
subject in Chemistry so that the students can relate ex-
perimental events to facts that pop up daily in their work.
Our Biological Chemistry course is an introduction to the
description of natural molecules, macromolecules, and the
microbial world. The lectures are based on generalities of
Carbohydrates, Proteins and Enzyme Activities, Synthetic
* This work was developed by the financial support of the
Instituto de Ciencias, Universidad Nacional de General
Sarmiento.
To whom correspondence should be addressed. E-mail:
dvullo@ungs.edu.ar.
and Biological Polymers, Lipids, and Nucleic Acids. From
this introduction, the necessary knowledge integration is
achieved in laboratory classes to provide a better grasp of
biological processes. As a first approach toward the un-
derstanding of an experimental model and the development
of observational abilities, we have chosen a biological sys-
tem that is perfectly well described and closely relates
S. cerevisiae is the ideal model because the enzyme source
is available at any supermarket, and its measurement is
considered the best for an effective understanding.
The experimental work was planned to integrate not only
our Biological Chemistry curriculum subjects but also to
understand scientific procedures. These are as follows:
1. Recovery of the experimental data, their mathemati-
cal treatment, and construction and comprehension
of different graphics from them;
2. Biopolymer properties, such as alginic acid and ap-
plications in food industry;
3. Mechanism of S. cerevisiae entrapment in calcium
alginate, studied by visualization of the immobiliza-
tion procedure and the chelating agent effects on the
gel structure, together with a microscopic observa-
tion of both free and immobilized cell states; and
4. Introduction of hydrolysis yield, sugar reducing
power, and enzyme activity concepts by a sucrose
inversion minireactor set-up.
EXPERIMENTAL PROCEDURES
The results presented below are the experimental data from the
year 2000 course. The students worked in teams of four guided
by the author. Each team worked independently in the whole
laboratory experience.
First Laboratory Class (4 h)
Alginic Acid PropertiesAlginic acid, for which the monomer
unit is D-mannuronic acid, is a natural polymer found in marine

42 This paper is available on line at http://www.bambed.org

 43
algae. The fact that free carboxylic groups are repeated in the TABLE I
macromolecule makes them accessible to divalent cations such Calibration curve for quantitative determination of reducing
as Ca2, and the formation of coordination complexes derives in sugar by neocuproine method
the jellification process. Monovalent cation salts are soluble in
Reaction Glucose solution Destilled Solution Solution
water and commonly used to increase viscosity in the food in- tube no. (50 g/ml) water A B
dustry, so a point for the students to pay attention to is sodium
alginate solubilization and the viscosity of the solution. ml
100 ml of 0.1 M acetic acid/sodium acetate buffer pH 4.75 1 0 1 1 1
was prewarmed at 60 C, and 1.5 g of a technical purity grade 2 0.05 0.95 1 1
sodium alginate was added with continuous stirring until every 3 0.10 0.90 1 1
4 0.15 0.85 1 1
clot had disaggregated.
5 0.20 0.80 1 1
To evaluate some rheological properties, an aliquot was 6 0.25 0.75 1 1
dropped into a 0.1 M CaCl2 solution, and the gel spheres that 7 0.30 0.70 1 1
formed were picked up. The students were able observe the 8 0.35 0.35 1 1
different jellification process levels immediately and 15 min after 9 0.40 0.60 1 1
exposure to the CaCl2 solution. 10 0.45 0.55 1 1
Immobilization of Bakery Yeast by Entrapment in Calcium Algi- 11 0.50 0.50 1 1
nate Gel3.25 g of commercial bakery yeast (approximately 8
109 colony-forming units in Sabouraud agar plates/g) were sus-
pended in 100 ml of the previously prepared 1.5% sodium algi- Solution B was prepared by dissolving 0.12 g of neocuproine
nate solution. hydrochloride (Sigma) in 100 ml of distilled water and stored in the
A 20-ml glass syringe attached to a plastic tube was filled with
dark. To determine reducing sugar concentration, both solutions
the yeast suspension and then emptied, drop by drop, into the 0.1
were added to the reaction tubes (10-ml graded tubes), and after
M CaCl2 solution. This way all the suspension turned into immo-
homogeneization, they were heated for 8 12 min at 100 C (boil-
bilized yeast spheres. After 15 min, batch minireactors were
ing water bath). Then the tubes were quickly cooled under tap
prepared filling eight 5-ml glass syringes with 5 ml of sphere
water, and distilled water was added for a final volume of 10 ml in
apparent volume. Plastic tubes (3 cm long) were used as needles
each tube. Absorbance was then measured at 450 nm in a
with their corresponding clump. The volume of each drop was
Metrolab Spectrophotometer and plotted versus g of glucose to
calibrated to 0.05 ml to estimate the number of cells per sphere
get the curve slope.
and to obtain spheres large enough to fill the minireactors, avoid-
Reducing sugars in mol were plotted as a function of time as
ing the risk of losing any. The columns were washed by adding
shown in Fig. 1. Invertase activity was defined as follows: one
small volumes of 0.1 M acetic acid/sodium acetate buffer to
invertase unit (IU) is the amount of enzyme that produces 1 mol
eliminate Ca2 excess. The reactors were stored at 7 C until
of reducing sugar/min. According to the obtained results, 15 min
needed.
was the standard time determined for the enzyme reaction. Then
One volume of spheres was suspended in 2 volumes of 50 mM
the students estimated a substrate hydrolysis grade of 66.5%
EDTA (at pH 7 with NaOH 0.1 M) and stirred at 10-min intervals.
After at least 2 h, the students could disaggregate the gel and with the following expression:
release the entrapped cells. To appreciate the difference between
the immobilized and free cell states, they used a microscope % hydrolysis
(40 objective lens) to observe movement limitation of the im-
mobilized cells. mol of reducing sugar/(2 mol of total sucrose) 100
Standardization of Inversion TemperatureMeanwhile, the
Second Laboratory Class (4 h): Parameter Optimization of other four syringes were stabilized at the following temperatures:
Sucrose Inversion Process 7 C (refrigerator temperature), 18 C, 32 C, and 37 C. Then 0.1
ml of the sucrose solution was added to each column and incu-
Incubation Time for Standardization of InversionFour of the bated for 15 min. At this point, 2.5-ml fractions were recovered
eight syringes filled with the immobilized yeast beads were sta- from each syringe, and reducing sugars were determined. Results
bilized at room temperature (T 24 C), and 0.1 ml of 20% (w/w) are shown in Fig. 2. Using these data, the students determined
commercial sucrose solution was added to each of the four
optimal temperature of the process and the degree of hydrolysis
tubes. The difference between the two solution densities was
for each of the four temperatures used (Table II). After this exper-
clearly visible. Immediately after 5, 10, 15, and 20 min of incuba-
iment, the students could conclude that sucrose inversion per-
tion at 24 C, a 2.5-ml fraction was extracted from each column.
formed at 24 C gave the best yield in final products (reducing
To estimate invertase (sucrase) activity, the column eluates were
sugars in this particular case).
diluted 10-fold, and the reducing sugar measurements were
made from 20 l of this 1:10 dilution. As a control of residual
reducing sugar, the determination was also performed on 0.1 ml CONCLUSIONS
of the 20% sucrose solution. This experience introduced a number of points to the
The neocuproine method [6] was chosen to measure reducing students: enzyme activity, sugars and their reducing
sugar concentration in the eluates. This quantitative technique is
based upon the Cu(II) to Cu(I) reduction produced by the sugar-
power, biopolymer gels, cells, and biotechnology. Obvi-
reducing functional group (carbonyl) in alkaline conditions. The ously the experimental work was carried out after lectures
Cu(I) formed is rapidly and quantitatively complexed by neocu- that operated as an introduction to the subject and helped
proine (2,9-dimethyl-1,10-phenanthroline). This complex is or- them understand the different phenomena taking place in
ange and reaches absorbance peak at 450 nm. The calibration the lab. Besides elementary procedures like how to use
curve was calculated with a 50 g/ml glucose standard solution tips and micropipettes, they learned both chemical and/or
(Table I). Solution A was prepared by a simple procedure: 40 g of
biological complex mechanisms. The main point for us
Na2CO3 (anhydrous) (Merck) were dissolved in 600 ml of distilled
water, then 16 g of glycine (Sigma) were added, and once it had teachers is that they should be able to see the direct
dissolved completely, 0.45 g of CuSO45H2O (Merck) were incor- applications of theoretical knowledge in common events
porated, and distilled water is added to a total volume of 1 liter. of their daily experience.
44 BAMBED, Vol. 31, No. 1, pp. 4245, 2003
Light microscope, slides, and cover slides
General glass laboratory materials
For the second day class:
20 ml of 20% (w/w) commercial sucrose in distilled
water
50 10-ml graduated tubes
10 100-l and 100 1,000-l micropipettes with their
corresponding tips
100 ml of Solution A (neocuproine method): 40 g of
Na2CO3 (anhydrous) (Merck) are dissolved in 600 ml
of distilled water, afterward 16 g of glycine (Sigma) are
added, and when they are completely dissolved,
0.45 g of CuSO45H2O (Merck) are incorporated. Fi-
nally distilled water is added to a total volume of 1
liter.
FIG. 1. Sucrose inversion as a function of incubation time. 100 ml of Solution B (neocuproine method): 0.12 g of
neocuproine hydrochloride (Sigma) are dissolved in
100 ml of distilled water, and the solution is stored in
the dark.
10 ml of 50 g/ml glucose standard solution
Thermostatic water bath
Thermostatic incubator
Refrigerator
Spectrophotometer and cuvettes
General glass laboratory materials

REAGENT TOXICITIES AND DISCARDED MATERIAL TREATMENT

FIG. 2. Temperature optimization of sucrose inversion. IU,
invertase units.
Information on hazardous reagents can be found at
hazard.com/msds/, for example:
CuSO45H2O: hazard.com/msds/tox/f/q43/q511.html
1,10-phenanthroline: hazard.com/msds/tox/f/q88/q32.
html
or at www.jtbaker.com, for example:
CuSO45H2O: www.jtbaker.com/msds/C5918.htm
1,10-phenanthroline monohydrochloride: www.jtbaker.
com/msds/P1795.htm

TABLE II EDUCATIONAL RESOURCES ON THE WEB

Hydrolysis as a function of the process temperature Information can be found from these sites and their
Temperature Hydrolysis respective links:
C % www.molecules.org/
7 6.6 cmm.info.nih.gov/modeling/science.html
18 24
24 66.5 EVALUATING QUESTIONS
32 35.8
37 35.5 1. Why is prewarming to 60 C of the acetate buffer
solution suggested?
2. What happened to alginate and Ca2? Explain the
SUMMARY OF REAGENTS AND EQUIPMENT FOR EACH
TEAM OF WORK fact that the spheres are easily disaggregated when
just formed, while after 15 min in CaCl2 solution they
For the first day class:
are not.
150 ml of 0.1 M acetic acid/sodium acetate buffer 3. How many yeast cells can be counted per ml of the
(pH 4.75) in distilled water initial suspension?
2.25 g of sodium alginate (technical purity grade) 4. If the drop volume is 0.05 ml, calculate how many
200 ml of 0.1 M CaCl2 in distilled water cells are entrapped per sphere.
3.25 g of commercial bakery yeast (8 109 colony- 5. Why is 15 min the recommended exposure time to
forming units in Sabouraud agar plates/g) CaCl2 solution?
1 20-ml glass syringe 6. What is the EDTA effect on gel structure? Why is the
8 5-ml glass syringes pH of the EDTA solution important? What other
9 3-cm-length, 3-mm-diameter plastic tubes chemicals could be used as an EDTA substitute?
50 ml of 50 mM EDTA (pH 7) in distilled water 7. Explain the % hydrolysis mathematical expression.

8. Why is the emergence of reducing sugar the ideal
way of measuring enzyme activity?
AcknowledgmentsI especially thank Dr. Anita Zalts and
Licenciado Enrique Hughes for help in the writing of this article.
REFERENCES
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of Saccharomyces cerevisiae, Anal. Biochem. 130, 469 470.
[2] P. Babczinski (1980) Partial purification, characterization and localiza-
tion of the membrane-associated invertase of yeast, Biochim. Biophys.
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Acta 614, 121133.
[3] M. Kierstan, C. Bucke (1977) The immobilization of microbial cells,
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[5] K. Mosbach, Ed. (1976) Methods in Enzymology, Vol. 44, Academic
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[6] S. Dygert, L. H. Li, D. Florida, J. A. Thoma (1965) Determination of
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