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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



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



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.



2

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)



3

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.



4

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.



5

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



6

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.



7

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



8

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.



9

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



10

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.



11

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.



12

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.



13

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



14

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.



15

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.



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



17

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.



18

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