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Extended Essay Subject: Chemistry
Selective utilization of (±)-glyceraldehyde
enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Name: Mojmír Mutný Candidate number: 000771-038 Examination session: May 2012 School: Spojena skola Novohradska Word count: 3979
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
Abstract
A possible different utilization of glyceraldehyde enantiomers has been studied in two
common yeast species Saccharomyces cerevisiae and Candida utilis. This was done
by incubation of yeasts in a medium with (±)-glyceraldehyde, and subsequent
measurement of a change of optical attributes of media after 24 hours of incubation.
Yeasts were added to two solutions of (±)-glyceraldehyde and incubated for 24
hours. Two other solutions of the same content were used as a negative test, and
were incubated for less than 5 minutes, and then filtrated and cooled to stop the
utilization. The test solutions showed an optical activity with rotation of polarized light
to the right. However, in both cases of incubated solutions the optical activity
decreased. This suggests that some dextrorotatory compound was utilized or some
levorotatory created. The value of change of optical attributes was significantly higher
than expected. Therefore, other compounds such as trehalose were suggested to be
present in yeast cells which were used. Consequently, trehalose was released into
media, and influenced the optical rotation of media. The concentration of trehalose
could not be precisely determined. Thus, the extent to which trehalose influenced the
optical characteristics of the medium and how much did other compounds, namely
(+) or (-)-glyceraldehyde could not be precisely determined. Altogether, the source of
optical rotation could not be precisely identified, but it is unlikely for yeast cells to
selectively utilize (±)-glyceraldehyde based on facts from previous studies, which
were obtained from further investigation after the experiment.
Word count: 245
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
Contents
Introduction...............................................................................1
Chirality ............................................................................................ 1
Naming conventions ......................................................................... 2
Racemic solution, significance of enantiomers and asymmetric
synthesis ........................................................................................... 2
Glyceraldehyde ................................................................................. 4
Polarimetry ....................................................................................... 5
Biochemical background .........................................................5
Hypothesis ................................................................................7
Materials ....................................................................................8
Method .......................................................................................9
Results.....................................................................................10
Discussion ..............................................................................12
Data interpretation ......................................................................... 12
Limitations and Improvements ....................................................... 16
Conclusion ..............................................................................17
References ..............................................................................18
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
1
Introduction
Chirality
The era of optical isomerism started in 19th century by discovering the ability of
certain compounds to rotate the plane of linearly polarized light. It was discovered by
French physicist Jean-Baptiste Biot.(17, p. 294) This discovery was followed by the
honorable work of Louis Pasteur who discovered that one half of crystals of sodium
ammonium tartrate rotated the plane of linearly polarized light in the opposite
direction than the other half.(17, p. 296) He noticed that the two crystals were mirror
images. Such two components of a compound are now called enantiomers or optical
isomers.(17, p. 297) Pasteur also proposed the condition for a compound to have an
optical isomer. His idea was based on the inability to superimpose an image of one
enantiomer to form the other. Similarly, right hand and left hand are alike but they
cannot be superimposed. Later, his suggestions were proved, and a carbon with four
different substituents (chiral center) was identified as a source of optical isomers-
chirality (figure 1).
The term chirality is derived from the Greek word ―handed‖.(20, p. 168) Chirality is a very
interesting form of isomerism, since two isomeric forms have nearly the same
chemical properties such as boiling point, melting point, refractive index or density
except for the fact that they rotate linearly polarized light in opposite ways.(20, p. 179) In
addition, chiral substances produced by inorganic or organic reactions outside
organisms often end with a solution, which contains 50% of one isomer and 50% of
Figure 1(14)
A Picture showing two example enantiomers with the line of symmetry. Also, the fact that four substituents are a condition for the chiral center can be seen.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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the other isomer. Such solution is called racemic solution, and is optically inactive.(20,
p.185)
Naming conventions
Furthermore, to distinguish between two enantiomers naming conventions were
developed. One of the three commonly used methods is based on the manner of
rotation of polarized light, and this method is of biggest importance in this study.
When a substance rotates light to the right, it has a sign (+) before its name.
Conversely, when a substance rotates light to the left a (-) sign is before its formula.
Another system for naming the substances is according to Cahn-Ingold-Prelog
convention. This convention is based on the priorities of substituents of carbon with
four substituents. It is described in IUPAC rules in detail.(9) Last D/L method is used
mostly when naming sugars and refers to the stereochemical configuration of a
hydroxyl group on the last carbon. This convention is derived from (+)-(R)-
glyceraldehyde to which a letter D was assigned, and every sugar which is
synthesized in this manner has also the D configuration.(17, p. 980) In addition, the prefix
DL- or (±) stands for racemic solution
Racemic solution, significance of enantiomers and asymmetric synthesis
Racemic solutions occur mostly when a prochiral compound is synthesized by
inorganic means or when organic prochiral compound is synthesized outside
organisms, because both enantiomers are equally probable to form. Example of such
reaction can be seen in figure 2. In contrast, biological pathways usually process only
compounds that are enantiopure, because enzymes involved in pathways are often
specific for one particular optical isomer. Therefore, enantiomers may act differently
in an organism, and thus they may be processed in unlike metabolic pathways. Also
because of this fact, in nature we observe mostly D-sugars and L-amino acids.(17, p.
980 & 1021)
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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The significance of enantiomers can be seen in pharmaceutical industry where drugs
for humans are developed. They are mostly synthetically prepared, thus, they can
have two optical isomers where only one may be active. The other isomer can be a
source of side effects or may inhibit the active isomer. For example, Thalidomide was
a drug against morning nausea for pregnant women, which was used in 1960s. After
its introduction to the market it was discovered that it is a strong teratogen.(14)
However, further investigation showed that only one enantiomer is teratogenic, and
the other is active without any side effects. Another example of such drug can be
Ibuprofen, widely used analgesic and antipyretic, which also exists in two isomeric
forms one being active and the other being inactive and having an inhibitory effect on
the active one.(14) In contrast, most of the best selling cardiovascular drugs are sold
as enantiomers due to reduction of side effects, and are probably prepared by the
pioneering method called asymmetric synthesis where one enantiomer product is
preferred.(17, p. 734)
Asymmetric synthesis can have many approaches, but they are alike in the aspect of
creation of asymmetric environment. For example, asymmetric catalysis is approach
where chiral ligands, which hold a substance in the manner that one side is preferred
are used.(17, p. 734) For pioneering work in the area of asymmetric synthesis a Nobel
Figure 2(17, p. 312)
An example of reaction where chiral products are produced in equal proportions creating a racemic mixture. The reaction is addition of water H20 to 1-Butene.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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Prize in chemistry was awarded in 2001.(17, p. 734) On one hand this is a very good way
to produce single enantiomer, but on the other hand, chiral ligands are often
expensive and hard to develop which may sometimes discourage people from their
usage. Another way to create asymmetric conditions is by using the same
mechanism as nature does. Enzymes of a microorganism can be specific for one
optical isomer, and thus can process only one isomer. In this study the possible use
of this method was investigated using yeast cells as asymmetric environment. More
specifically, it was investigated whether the most available yeast species
Saccharomyces cerevisiae and Candida utilis can selectively utilize (±)-
glyceraldehyde in a medium.
Glyceraldehyde
Glyceraldehyde is the simplest aldose with only one chiral center. It can be seen in
figure 3.1 with its enantiomer. It is a sweat and colorless compound(1) which can be
formed by oxidation of glycerol with Fenton’s reagent(11) (figure 3.2). The reaction has
tree principal products and they are D-glyceraldehyde, L-glyceraldehyde and
dihydroxyacetone. The reaction cannot be easily stoichometrically evaluated but it
can be expected that ⅓ of the product would be dihydroxyacetone due to removal of
hydrogen from the central atom. The remaining ⅔ create DL-glyceraldehyde where
marginal hydrogen is removed. In addition, the presence of reducing sugar such as
glyceraldehyde can be confirmed via Fehling’s test. (13)
Figure 3.1.(17, p. 980 & 981) Two enantiomer forms of glycerol are
shown with all three naming conventions assigned to them.
Figure 3.2 Oxidation of glycerol with Fenton’s reagent to produce DL-(±)-glyceraldehyde and dihydroxyacetone.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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Polarimetry
Polarimetry is a method by which chiral compounds can be differentiated.
Polarimeter (figure 4.1) measures the angle of rotation of linearly polarized light after
the beam passes through the tube with measured sample. The light source is
monochromatic and usually a sodium lamp with wavelength 589.6 nm(17, p.295) is used,
because table values of specific rotation are given at this wavelength. Specific
rotation is a physical constant that tells us the angle of rotation when beam of
polarized light passes through 1 dm of a solution with unit concentration. The formula
is described in figure 4.2. For example, optical rotation of glyceraldehyde is
8.7º 𝑚𝐿
𝑑𝑚 𝑔.(17, p. 300)
Biochemical background
Asymmetric conditions can be created by a presence of yeast microorganisms that
utilize a compound by their enzymatic pathways, and only one enantiomer of a
compound is processed by enzymes due to their specificity. This method is probably
cheaper and easier than asymmetric catalysis. Therefore, a possible use of this
method was investigated in this study.
Conditions for a compound to be selectively utilized by yeasts are its ability to
transport or diffuse through yeast plasma membrane, and consequently to integrate
into yeast metabolic pathways which are specific for one enantiomer of the
Figure 4.1(20, p. 181)
The schematic overview of polarimeter components. The analyzing filter is rotated until the maximal intensity
is reached.
Figure 4.2(17, p. 295)
Mathematical formula of specific rotation constant
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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compound. Compound that was chosen to be selectively utilized by yeasts in this
study was racemic solution of glyceraldehyde. It was expected that only (+)-
glyceraldehyde would be utilized, because it satisfies both previously stated
conditions whereas (-)-glyceraldehyde would stay intact, because it cannot enter
glycolysis metabolic pathways in yeasts. In addition, (±)-glyceraldehyde utilization
does not end with chiral products either if it enters aerobic respiration pathway or
anaerobic respiration pathway.
In detail, the significant permeability of Saccharomyces cerevisiae plasma membrane
for glyceraldehyde, glycerol and other three carbon compounds was suggested in the
study by C. F. Heredia and others.(7) Further, Luyten and others(15) found that yeasts
S. cerevisiae posses a special protein channel that transports glycerol. Also,
Gancedo et. al.(6) studied glycerol uptake into Candida utilis and found that uptake
was high enough to expect that C. utilis possess glycerol transport system. Glycerol
and glyceraldehyde are different compounds but they are closely related three
carbon compounds, thus, it is possible that glycerol transport system may also
transport glyceraldehyde.
Continuing with the second condition and considering glyceraldehyde and its
utilization in C. utilis and S. cerevisiae, D-(+)-glyceraldehyde-3-phosphate is middle
product in the glycolysis metabolic pathway. Fructose-1,6-biphosprahte is divided
into D-(+)-glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. D-(+)-
Glyceraldehyde-3-phosphate is further processed by triose phosphate
dehydrogenase to create 1,3-Biphosphoglycerate.(4) In addition, D-(+)-
glyceraldehyde-3-phopshate and dihydroxyacetone phosphate are substrates for
enzyme called triosephophate isomerase which swaps between these two trioses.(4)
In order to integrate glyceraldehyde into this pathway it has to be phosphorylated by
a kinase enzyme. Molin and others(18) mention in their study that dihydroxyacetone
kinase of yeast Schizosaccharomyces pombe can phospohorylate also glycerol and
(±)-glyceraldehyde, and can be classified generally as a triose kinase. S. cerevisiae
and C. utilis may also posses this enzyme. Thus, they may be also able to integrate
glyceraldehyde into their glycolysis pathway.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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In addition, Gancedo et. al.(6) who studied C. utilis and its glycerol metabolism found
that glycerol kinase can also phosphorylate (±)-glyceraldehyde. Besides
phosphorylating (+-) glyceraldehyde, it produces also a very unstable product, which
does not seem to be further utilized, as they reported. Altogether, there are most
likely two pathways of phosphorylation of glyceraldehyde from which the glycerol
kinase pathway is rather negative and may be waste of ATP.
Hypothesis
Provided that the yeasts use primarily the dihydroxyacetone metabolic pathway, it is
expected that yeasts grown in medium with (±)-glyceraldehyde and
dihydroxyacetone will selectively utilize (+)-glyceraldehyde, and leave the (-)-
glyceraldehyde intact. The utilization of (+)-glyceraldehyde would lead to the change
of physical characteristics of the solution, and the solution may start to rotate
polarized light in left direction, because of (-)-glyceraldehyde dominancy. Expressing
in quantitative terms, provided that the oxidative reaction of glycerol proceeds as
expected and ⅓ of glycerol becomes (+)-glyceraldehyde, and all this (+)-
glyceraldehyde gets utilized the maximal possible measured change of angle would
be -0.22º (figure 5.1).
Moreover, it is also possible that L-(-)-glyceraldehyde will get phosphorylated by the
same enzymes (DHA kinase or glycerol kinase), and its specific rotation will be
different from the specific rotation of non-phosphorylated L-(-)-glyceraldehyde. If this
phosphorylated glyceraldehyde would be released by some means into cytosol or the
enzymes would be present in a solution (disrupted cells) the change may be even
greater. L-(-)-glyceraldehy-3-phosphate has the value of specific rotation -14.5º 𝑚𝐿
𝑑𝑚 𝑔
(19), whereas normal L-(-)-glyceraldehyde has only value -8.7º 𝑚𝐿
𝑑𝑚 𝑔. If dihydroxyacetone
or glycerol kinase would be present in the medium and all L-(-)-glyceraldehyde gets
phosphorylated the change in angle would be –0.15º (figure 5.2).
Hence, the expected difference in optical rotation of the medium after yeast
incubation should lie somewhere in the interval of -0.10º to -0.42º. Exact value
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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cannot be expected, since media can contain some spare chiral compounds.
Furthermore, the uptake system of glyceraldehyde is not perfectly known, the
possible phosphorylation of L(-)-glyceraldehyde in extracellular space may or may
not occur, and the oxidative reaction of glycerol is not exact, and can result in
different fraction of enantiomers than expected.
α1 =mass of a compound
volume of a water solution× α D × lenght of the cell with compound
α1 = 0.41 ∗ 44 ∗
13 g
356 ml× −8.7 × 1.5 dm = −0.22º
α2 =mass of a compound
volume of a water solution× α D × lenght of the cell with compound
α2 = 0.41 ∗ 44 ∗
13 g
356 ml× − 14.5 − 8.7 × 1.5 dm = −0.15º
Materials
85% (w/w) glycerol in water solution, 30% (w/w) hydrogen peroxide, 10 g of ferrous
(FeS04) powder, tap water, test-tubes, Buchner’s funnel, polarimeter, 90 g of
compressed Saccharomyces cerevisiae (baker’s yeast), 90 g of dry Candida utilis
(torula yeast), scales with imprecision value of 0.01g, chemical flasks with various
volumes, centrifuge and polarimeter (1,5 dm sample tube and sodium light (586nm)).
Figure 5.1 The calculation of the maximal possible angle of rotation of linearly polarized light when all (+)-glyceraldehyde (one third of the original glycerol mass) that was produced via oxidative reaction of glycerol would be utilized. It was calculated according to formula in figure 4.2, and the data were taken
from method section of this study.
Figure 5.2 The calculation of the maximal possible angle of rotation of linearly polarized light when all (-)-glyceraldehyde (one third of the original glycerol mass) would be phosphorylated by external enzymes to L-(-)-glyceraldehyde-3-phosphate (specific rotation: -14.5).
(19) It was calculated according to formula in
figure 4.2, and the data were taken from method section of this study.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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Method
a) Preparation of (±)-glyceraldehyde
1. 226.6 g of 30% hydrogen peroxide solution was added into 217.6 g of 85%
glycerol solution. Then, 10 g of ferrous salt was added into the mixture and
the solution was left to react for 3 hours (± 10 min).
2. After three hours, the ferrous powder was filtrated from the solution by
passing the solution through a filter paper.
(The final solution is expected to have 41.4% organic compounds in it,
principally, glycerol, glyceraldehydes and dihydroxyacetone.)
3. To confirm aldehyde presence, Fehling’s test was carried out.
4. The solution was left in refrigerator (6 ºC) for 5 days until next use.
b) Feeding the yeast with (±)-glyceraldehyde and dihydroxyacetone
1. Six 500 ml flasks were cleaned and prepared.
2. 44 g of the initial solution form part a) was added to every flask
3. 356 g of tap water was added to every solution.
4. Yeast cultures were added to the solutions according to Table 1. They
were cultivated at room temperature (21ºC), and were not placed in
shaker.
No. of sample
Yeast culture
Concentration of organic
molecules in medium [%]
Cultivation time
[hours] ± 10 minutes
Mass of yeast
culture [g] ±0.01g
1 Saccharomyces
cerevisiae 5 24 30
2 Candida utilis 5 24 30
3 Saccharomyces
cerevisiae (negative test)
5 0 30
4 Candida utilis (negative test)
5 0 30
Table 1 Composition of media of experimental samples, type of yeast species in medium and incubation time
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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5. After expiration of the cultivation time, the decanted yeast cells were
separated from the liquid solution, and the solution was filtrated using
Buchner’s funnel.
In case of samples 3 and 4 the decantation did not occurred, thus the
samples were only filtrated.
6. The solutions were kept in refrigerator (6 ºC) for 7 days, and then they
were centrifuged at 5000 rpm for 5 minutes.
c) Polarimetry
The polarimeter, which was used, had precision 0.005º and the tube with
tested compound had length of 15 cm.
1. The solution was poured into the cell for tested sample and sealed. The
apparatus was fixed and the analyzer filter was rotated until the maximum
intensity was reached. The observation of maximal intensity was done by
a human eye.
2. Step 1 was repeated for solutions 1 to 4.
Results
Note: The limit of reading of the polarimeter was 0.01º, but only difference of
more than 0.05º gave a clear difference in light intensity.
No. of sample
Yeast species Cultivation time [hours] ±10 min
Angle of rotation [º] ±0.005 º
1 Saccharomyces
cerevisiae 24 0.01
2 Candida utilis 24 0.07
3 Saccharomyces
cerevisiae (negative test) 0 1.00
4 Candida utilis (negative
test) 0 0.92
racemic solution
- - 0.04
Table 2.1 Angles of rotation of two yeast species with their negative tests which were incubated in the solution only for 5 minutes, and then quickly filtrated.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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0
0,2
0,4
0,6
0,8
1
1,2
1 2 3 4 racemic solution
An
gle
of
rota
tio
n [
º]
No. of sample
Angle of rotation of linearly polarized light in inspected solutions
Yeast culture Change of the angle of
rotation [º] ±0.01 º Mass of yeasts [g]
±0.01 g
Saccharomyces cerevisiae -0.99 30
Candida utilis -0.85 30
Figure 6.1 A bar chart showing angles of rotation of linearly polarized light of the
individual solutions that were measured by polarimetry. Racemic solution of the (±)-glyceraldehyde and dihydroxyacetone was also measured.
Table 2.2 The change of angle of rotation in solutions. Two different yeast species are present. The change of angle of rotation was calculated by
subtraction of the 24 hour cultivated solution from the negative control solution.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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Discussion
Data interpretation
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species
Saccharomyces cerevisiae and Candida utilis has been studied. The two yeast
species were incubated in the solution with (±)-glyceraldehyde. It was expected for
the medium to change its optical rotation characteristic, and it was expected that
negative change in angle of rotation would be observed lying somewhere in the
interval of 0.10º to 0.42º.
However, the measurements differ significantly from the expected values. To begin
with description, in figure 6.1 we can see that racemic solution has angle of rotation
0.04º. It can be argued that this value is too far from zero, and thus, it can be argued
whether the equipment had 0.005º precision or 0.025º as stated previously, or there
0,00
0,20
0,40
0,60
0,80
1,00
1,20
Saccharomyces cerevisiae Candida utilis racemic solution
An
gle
of
rota
tio
n [
º]
Name of cutlure
Angle of rotation of linearly polarized light in inspected solutions
Figure 6.2 A bar chart showing the change of angle of rotation in two different yeast cultures. The data based on which this chart was plotted were taken from the table 2.1. The first column (dark) represents the control culture without incubation time, and the
second column (light) represent angle of rotation of the yeasts which were incubated for 24 hours.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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may be a possibility that some compound contaminated the solution. Specifically,
other minor products of the oxidative reaction of glycerol may be formic acid, glyceric
acid, glycolic acid and formaldehyde.(1) All these products do not possess a carbon
atom with four different substituents, and thus, they are not chiral and they are not
possible sources of optical activity. Therefore, I suggest that this negligible deviation
is due to inaccurate measurement, or a systematic error.
Continuing in the description, media 3 and 4, which were test media with yeasts
incubated only for 5 minutes show significant optical rotation to the right (+). In case
of C. utilis the value is slightly lower compared to 1.00º in S. cerevisiae. These two
values are very similar. Therefore, it can be expected that they are of the same
origin. It can be expected that a package of baker’s yeasts and a package of torula
contains some compounds which rotate the light in positive direction. The most
obvious suggestion would be that it contains spare sugars, medium components or
conservation additives from manufacturer. Hui et. al(8) claim that baker’s yeasts are
grown in medium containing molasses, biotin, ammonium and other compounds in
smaller amounts. They also claim that additives such as sorbitans are added for
better storage.
Molasses contain mainly sucrose and glucose which may rotate the polarized light in
positive direction, and have high values of specific rotation 66.4 and 52.7(19).
However, Hui et al.(8) also claim that yeasts are grown in the medium with 0.01% of
sugar source to maintain most effective reproduction, and also Walker(21) indicates
that yeast in final stages are grown in very small concentrations of sugars to induce
storage sugar production. Thus, the angle of rotation only due to this fact can be
doubted when the value is as high as 1º. In addition, Biotin, emulsifying sorbitans and
other compounds are present in too low amounts(8) to have a significant impact on
the optical features of medium. Therefore, other factors have to be considered.
Walker(21) and Hui et al.(8) claim that to ensure protection during drying and pressing,
yeasts are incubated in the way to induce trehalose and glycogen
production/storage. These two compounds are storage sugars with very high values
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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of specific rotation. Trehalose has value of specific rotation +199(2) and glycogen has
+198.9 or +184.5.(3) This makes these two compounds, and mainly trehalose a good
candidate for explaining the change in rotation when yeast cells are damaged and
two storage compounds can enter the medium. (5) Moreover, Cerrutti and others(5) in
their study work with S. cerevisiae suggest that mass concentration of trehalose can
go up to 20% of dry yeast mass. The calculation of approximate mass of trehalose to
create the change of angle of value 1º is in figure 7.2.
𝛼 =𝑚
𝑉× 𝛼 𝐷 × 𝐿
1 =𝑚
356× 199 × 1.5
𝑚 ≈ 1.2 𝑔
If mainly only trehalose that was in a medium influenced the rotation of polarized
light, then the trehalose mass concentration in yeast cells would be 4% in dry yeasts.
In pressed yeasts, 30% of the mass of package are solid yeast cells as Hui and
others(8) suggest. Thus, mass of concentration of trehalose in pressed yeasts is
13.3%. Both these values seem to be reasonable, below maximal 20%. However, not
all cells were damaged, and not all trehalose from cells was exerted into medium
even thought there is a channel in yeast species S. cerevisiae which transports
trehalose into extracellular space.(12) Probably, most of the trehalose in the medium
was released due to damaged cell membranes which were disrupted either by
mechanical means in a factory by cutting(8) or by hydrogen peroxide. The
glyceraldehyde medium, in which yeast cells were incubated, was produced by
oxidative reaction involving hydrogen peroxide which could be still present in the
medium, and damage the yeast plasma membranes resulting into release of cytosol
into the medium.
Taking into account all previous facts and calculations, trehalose seems to be the
most likely source of rotation due to its high specific rotation and possible presence in
yeast package but on the other hand it is probable that also other substances were
involved in the change of optical characteristics. Since no information from the
Figure 7.1 Calculation of the necessary mass of trehalose in a medium to create optical rotation 1º. The same method as in figures 5.1 and 5.2 was used.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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manufacturer about trehalose concentration was found, and because I was unable to
quantify the possible disruption of yeast cells due to hydrogen peroxide, precise
concentration of trehalose in yeasts cannot be determined. Further, with trehalose
contamination/presence in solutions it is impossible to prove or disprove the
postulated hypothesis by means described in the hypothesis, because also trehalose
can be metabolized, and then one cannot decide which compound caused change in
optical rotation.
Few more words to data interpretation, yeast cultures were incubated also in media 1
and 2. They showed evident signs of respiration either aerobic or anaerobic.
Production of CO2 bubbles was observed during inspection after 12 hours of
cultivation. It can be seen also from the polarimetry results that the utilization of some
dextrorotatory substrate in the medium occurred (figure 6.2). It can be expected that
trehalose, which was probably in the medium, was utilized in both solutions, and this
resulted into lowered angle of rotation (figure 6.1). In addition, C. utilis solution shows
smaller change in angle of rotation than S. cerevisiae (figure 6.2) which can be
explained by different trehalose concentration or by different activity of dry and
pressed yeasts. Hui and others(8) propose that dry yeasts are less active then the
pressed ones even though their solid content is greater. Another fact that can explain
this phenomenon is possible utilization of (+)-glyceraldehyde by S. cerevisiae.
However, further research in biological journals resulted in finding the study of May
and others(16) which showed that yeasts S. pombe are unable to live on (±)-
glyceraldehyde as a sole medium. Further, Janson and Cleland(10) show that (+)-
glyceraldehyde blocks the glycerol utilization in yeasts, because it induces ATPase
activity of glycerol kinase. In contrast, (-)-glyceraldehyde act as a substrate. The
possible utilization of (+)-glyceraldehyde in the way presented in the hypothesis is
therefore unlikely even though the findings of May and others focus S. pombe
species only. Simply, (±)-glyceraldehyde likely cannot be separated by selective
utilization of (+)-glyceraldehyde.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
16
Alternatively, L-(-)-glyceraldehyde and L-(-)-glyceraldehyde phosphate have different
values of specific rotation -8,7 and -14,5(19), respectively. Different optical
characteristics can be induced by the release of this phosphorylated product from
cytosol. This option is unlikely, because phosphorylated products cannot leave a cell.
On the other hand, some cells were apparently lysed and their enzymatic content
was spilled in the medium. Therefore, it is possible that dihydroxyacetone kinase
(triokinase) or glycerol kinase phosphorylated L-(-)-glyceraldehyde. However, this
cannot be directly proved, if the solution contained trehalose. Therefore, the most
probable reason for the decrease is still due to trehalose utilization/storage, and no
other conclusion can be made about enantioselective utilization, mostly because of
previously unknown facts, and trehalose presence.
Limitations and Improvements
The experiment was limited by several factors, firstly it was the problem with the
hypothesis which was later suggested to be wrong by study of May and others(16), but
these information were not taken under consideration due to their absence at the
time of the hypothesis postulation. This automatically undermined the hypothesis
about selective utilization of (+)-glyceraldehyde, however the method that (-)-
glyceraldehyde can be phosphorylated by dihydroxyacetone kinase to form (-)-
glyceraldehyde-3-phosphate still remains to be a possible way to
separate/differentiate these two optical isomers.
Another problem which apparently ruined the whole experiment was trehalose
presence. Trehalose, a storage sugar, is evidently present in baker’s yeast cells and
dried torula yeasts which were sources of yeast cultures in this experiment. Its
presence in media resulted in the change in optical rotation, and I was not able to
determine the exact trehalose content in the yeasts. Therefore, the decrease of angle
of rotation of the solutions could not be precisely assigned to one inspected
compound, in our case (±)-glyceraldehyde.
In addition, (±)-glyceraldehyde was created by the oxidative reaction with Fenton’s
reagent containing H2O2 which may be dangerous for cells and may result in
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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disruption of cell membranes, mainly when present in high concentrations. This is
what apparently happened, because trehalose extracellular concentration was higher
than normal when cells are not disrupted. After addition of yeasts into media bubbles
were seen which may point to spare H2O2 presence in media. It was expected that
bubble contain released O2. A supportive argument for this may be that when the
addition was repeated to inspect this phenomenon, and a fire source was moved
near these bubbles the intensity of flame increased drastically.
To improve the experiment, yeasts with no trehalose production can be used or
simply mutant strains which have corrupted genes responsible for the trehalose
synthesis. Moreover, (±)-glyceraldehyde could be purified from a solution to ensure
that no other compounds are present in the solution. This was my intention from the
start. However, it could not have been done due to insufficient laboratory equipment.
Conclusion
Selective utilization of the (±)-glyceraldehyde enantiomers was inspected in yeast
species S. cerevisiae and C. utilis. It was found in literature after the experimentation
that yeasts probably do grow on a sole carbon source of (±)-glyceraldehyde. This
idea was not proved or disproved in this work due to trehalose presence in the yeasts
which made the experiment uncontrolled. In addition, also selective phosphorylation
of only (-)-glyceraldehyde to (-)-glyceraldehyde-3-phosphate by triose kinases which
would lead to different physical characteristics was not observed due to the same
reason.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
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References
1. American Cleaning Institute. n.d. ―Chemical Properties and derivatives of glycerine.‖
Accessed: 9th of July 2011.
<http://www.aciscience.org/docs/Chemical_Properties_and_Derivatives_of_Glycerol.pdf>
2. Bar, A. 2000. ―Trehalose produced by a novel enzymatic process.‖ Bioresco - Biorearch
Management and Consulting Ltd. Accessed: 9th of July 2011
<http://www.food.gov.uk/multimedia/pdfs/0_1.pdf>
3. Bell, J. D., Young, F. G. 1934. ―Observation on the Chemistry of Liver Glycogen.‖ Biochem J.
28(3): 882-889.
4. Campbell, N.A., Rice, J. B. et al. 2008. ―Biology.‖ 8th ed. Pages 169-170. Pearson Education
Inc.
5. Cerrutti, P. et. al. 2000. ―Commercial baker’s yeast stability affected by intracellular content of
trehalose, dehydration procedure and the physical properties of external matrices.‖ App.
Microbiol. Biotechnol. 54: 575-580.
6. Gancedo, C.. Gancedo, J. M., and Sols, A. 1968. ―Glycerol Metabolism in Yeasts.‖ European
J. Biochem. 5: 165-172.
7. Heredia, C. F., Sols, A., and DelaFuente, G. 1968. ―Specificity of the Constitutive Hexose
Transport in Yeast.‖ European J. Biochem. 5: 321-329.
8. Hui, Y.H., Corke, H. 2006. ―Bakery products: science and technology.‖ Pages 177-183. Willey-
Blackwell
9. International Union of Pure and Applied Chemistry. 1976. ―Rules for the Nomenclature of
organic chemistry: Section E Stereochemistry.‖ Pure & App. Chem. 45: 11—30. Pergamon
Press, Britain. Accessed: 10th of July 2011.
<http://www.iupac.org/publications/pac/1976/pdf/4501x0011.pdf>
10. Janson, C.A., Cleland, W.W. 1974. ―The Kinetic Mechanism of Glycerolkinase.‖ J. Bio. Chem.
249(8): 2562-2566.
11. Khanna, S.K., et. al. 2006. ―Excel With Objective Questions In Chemistry.‖ Page 247. Golden
Bells.
12. Kim, J. et al. 1996. ―Disruption of the Yeast ATH1 Gene Confers Better Survival after
Dehydration, Freezing, and Ethanol Shock: Potential Commercial Application.‖ Applied and
Environmental Microbiology. 62(5): 1563-1569.
13. Lancashire, R.J. 2006. ―Fehling’s test.‖ The Department of Chemistry, University of the West
Indie. Accessed: 9th of July 2011. <http://wwwchem.uwimona.edu.jm/courses/Fehling.html>
14. Leffingwell, J.C. 2003. ―Chirality & Bioactivity I.: Pharmacology.‖ Accessed: 9th of July 2011.
<http://www.leffingwell.com/download/chirality-phamacology.pdf>
15. Luyten K. et al. 1995. ―Fpsl, a yeast member of the MIP family of channel proteins, is a
facilitator for glycerol uptake and efflux and is inactive under osmotic stress.‖ EMBO J. 14(7):
1360-1371.
Selective utilization of (±)-glyceraldehyde enantiomers by yeast species Saccharomyces cerevisiae and Candida utilis
Mojmír Mutný Candidate number: 000771-038
19
16. May, J.W. 1982. ―Glycerol Utilization by Schizosaccharomyces pombe: Phosphorylation of
Dihydroxyacetone by a Specific Kinase as the Second Step.‖ J. General Microbiology 128:
1763-1766.
17. McMurry, J. 2008. ―Organic Chemistry.‖ 7th ed. Thomson Learning, Inc.
18. Molin, M., Norbeck J., Blomberg A., 2003. ―Dihydroxyacetone Kinases in S. cerevisae Are
Involved in Detoxification of Dihydroxyacetone.‖ J. Biol. Chem. 278(3): 1415-1423.
19. PDR-Chemical LLC. n.d. ―Advanced Laser Polarimeter (ALP).‖ Page 21. Accessed: 9th of July
2011<http://www.pdr-chemical.com/pdr_documents/alppaper2010.pdf>
20. Wade, L.G. 2006. ―Organic Chemistry.‖ 6th ed. Pearson Education, Inc.
21. Walker, G.M. 1998. ―Yeast Physiology and Biotechnology.‖ Pages 300-305. John Wiley &
Sons Ltd.