Enantioselective reduction of 4-phenyl-2-butanone to chiral 4-phenyl-2-butanol in glycerol modified Saccharomyces cerevisiae cell culture

Article abstract of DOI:10.1016/j.molcatb.2015.11.013

A model compound 4-phenyl-2-butanone was used for studying the effect of glycerol in yeast Saccharomyces cerevisiae catalyzed reaction culture on the enantioselectivity for producing R- and S-4-phenyl-2-butanol under various reaction conditions. As the volume percentage of glycerol in the cell culture was 10%, the reaction gave a best R-enantiomeric excess (e.e.) value of 72.3% and a yield of 86.4% at the reaction conditions: a substrate concentration of 10 μg mL^-1, 25 °C, an initial medium pH 10.0 but without controlling the pH, and without aeration. As the volume percentage of glycerol in the cell culture was 30%, the reaction showed a best S-e.e. value of 96.0% and a yield of 19.0% at the reaction conditions: a substrate concentration of 100 μg mL^-1, 34 °C, pH 7.0, and without aeration. Substrate inhibition was shown with this reaction.

Full text of DOI:10.1016/j.molcatb.2015.11.013

Accepted Manuscript
Title: Enantioselective reduction of 4-phenyl-2-butanone to
chiral 4-phenyl-2-butanol in glycerol modified Saccharomyces
cerevisiae cell culture
Author: Cheanyeh Cheng Yu-Chuan Nian
PII:
S1381-1177(15)30106-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.11.013
MOLCAB 3277
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Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
10-8-2015
10-11-2015
10-11-2015
Please cite this article as: Cheanyeh Cheng, Yu-Chuan Nian, Enantioselective
reduction of 4-phenyl-2-butanone to chiral 4-phenyl-2-butanol in glycerol modified
Saccharomyces cerevisiae cell culture, Journal of Molecular Catalysis B: Enzymatic
http://dx.doi.org/10.1016/j.molcatb.2015.11.013
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Enantioselective reduction of 4-phenyl-2-butanone to chiral
4-phenyl-2-butanol in glycerol modified Saccharomyces cerevisiae cell
culture
Cheanyeh Cheng* chengce@cycu.edu.tw, Yu-Chuan Nian1 ks1528@hotmail.com
Center for Biomedical Technology, Research Center for Analysis and Identification,
Department of Chemistry, Chung Yuan Christian University, Chungli District,
Taoyuan City 32023, Taiwan, ROC
*Corresponding author at: Cheanyeh Cheng, Department of Chemistry, Chung Yuan
Christian University, 200 Chung Pei Road, Chungli District, Taoyuan City 32023,
Taiwan, ROC. Tel.: +886-3-2653322; Fax: +886-3-2653399.
1 The permanent address of second author: Yu-Chuan Nian, No. 6-1 Alley 52, Section
2, Santsung District, New Taipei City, Taiwan, ROC. Tel: +886-2-29896386.
1

Highlights
 4-Phenyl-2-butanone was used for yeast catalyzed enantioselective reduction.
 The % glycerol in yeast culture (v/v) affects the reaction enantioselectivity.
 The reaction with 10% glycerol in yeast culture was in R-4-phenyl-1-butanol excess.
 The reaction with 30% glycerol in yeast culture was in S-4-phenyl-1-butanol excess.
 The reaction shows substrate inhibition.
2

Abstract
A model compound 4-phenyl-2-butanone was used for studying the effect of glycerol
in yeast Saccharomyces cerevisiae catalyzed reaction culture on the enantioselectivity
for producing R- and S-4-phenyl-2-butanol under various reaction conditions. As the
volume percentage of glycerol in the cell culture was 10%, the reaction gave a best
R-enantiomeric excess (e.e.) value of 72.3% and a yield of 86.4% at the reaction
conditions: a substrate concentration of 10 g mL–1, 25C, an initial medium pH 10.0
but without controlling the pH, and without aeration. As the volume percentage of
glycerol in the cell culture was 30%, the reaction showed a best S-e.e. value of 96.0%
and a yield of 19.0% at the reaction conditions: a substrate concentration of 100 g
mL–1, 34C, pH 7.0, and without aeration. Substrate inhibition was shown with this
reaction.
Keyword: Enantioselective reduction; Saccharomyces cerevisiae; glycerol;
4-phenyl-2-butanone; R-4-phenyl-2-butanol; S-4-phenyl-2-butanol.
3

1. Introduction
Chiral compounds such as amino acids and carbohydrates existing in biological
organisms are in only one of their two enantiomeric forms which is physiologically
active due to the specific molecular receptor of biological organism. With the specific
designed receptor, the two enantiomers of a chiral compound show different
physiological properties thus give important applications in odorants and
pharmaceutical drugs [1-3]. In order to obtain only one of the two enantiomers in
synthetic chemistry, the asymmetric synthesis or chiral synthesis has been important
strategy in pharmaceutical industry for chiral drugs development particularly using
the technique of chiral switch [1,2].
The utilization of microbes in asymmetric synthesis is interested by chemists for
reasons: (1) the reaction is environmentally friendly; (2) the reaction conditions are
mild; (3) the reaction is substrate specificity and product selectivity; and (4) there is
no need for enzyme purification. Among various microbial biotransformation
methods, the enantioselective bioreduction of prochiral carbonyl compounds with
microbes has long been extensively used for many pharmaceutical applications which
features the advantages of in situ regeneration of cofactor, easily scaling up, and cost
effective. For examples, enantiomerically pure S-arylalkanols have been synthesized
in excellent yields through the Geotrichum candidum catalyzed reduction of
acetophenone derivatives [4]; a hydrophobic polymer (AmberliteTM XAD) was used
as controlling material for stereoselective microbial reduction of simple aliphatic and
aromatic ketones [5]. Although there are a lot of microbes can be used for the
enantioselective reduction of prochiral ketones, baker’s yeast Saccharomyces
cerevisiae has been most widely used since the beginning of 20th century [3,6]. In our
laboratory, yeast such as Candida utilis and baker’s yeast mediated enantioselective
reduction of prochiral simple aliphatic ketone or phenyl group substituted aliphatic
ketones to their corresponding chiral alcohols have also been studied for many years
[7-10]. Other ketones such as α-bromoenones have also been used for the production
of (1S,2R)-2-bromoindan-1-ol and (2S,3S)-3-bromo-4-phenylbutan-2-ol to the
implication in the synthesis of the HIV protease inhibitor indianavir [11].
One problem with the baker’s yeast mediated reduction of prochiral carbonyl
compounds is the low solubility of the substrate in aqueous reaction culture to make a
poor yield. Many endeavor have been made to solve this probelm such as the
absorbing resins in aqueous fermentation medium has been used for absorbing
substrate that changes the substrate concentration to favor the production of
S-(+)-ethyl-3-hydroxybutanoate [12] in the baker’s yeast catalyzed reduction of ethyl
acetoacetate; the hexane/water biphasic cell culture has been performed for the
4

baker’s yeast mediated bioreduction of pheny n-propyl ketone to improve the
enantioselectivity and the yield [9]; and the reduction of prochiral -keto esters and
ketones has been performed by adding yeast powder or immobilized yeast to pure
glycerol medium to give high yield and high enantiomeric excess (e.e.) value [13-15].
Recently,
different
compositions
of
deep-eutectic-solvents
(choline
chloride/glycerol)/aqueous mixtures have been used for the baker’s yeast catalyzed
enantioselective reduction of ethyl acetoacetate to manipulate the enantioselectivity of
the reaction [16].
Since glycerol is non-toxic, a solvent for poorly miscible organic compounds,
and an energy source for microorganism fermenting system, it was used for this study
as a reaction medium modifier to enhance the yeld and enantioselectivity of yeast
mediated prochiral ketone reduction. In this paper, 4-phenyl-2-butanone was used as
the model compound for the batch type baker’s yeast S. cerevisiae catalyzed
enantioselective biotransformation (Scheme 1) to study the effects of pH, temperature,
and the volume percentage of glycerol in yeast culture on the enantiomeric excess
(e.e.) value and yield of the reaction.
2. Experimental
2.1. Materials and chemicals
Freeze dried yeast Saccharomyces cerevisiae CCRC 21443 was purchased from
the Culture Collection Research Center of Food Industry Research and Development
Institute (Hsinchu, Taiwan, ROC). Reagent grade KH2PO4, K2HPO4, (NH4)2SO4,
MgSO4‧7H2O, CaCl2‧2H2O, FeSO4‧7H2O, ZnSO4‧7H2O, D-(+)-glucose, and liquid
chromatographic grade methanol (MeOH, CH3OH) were all bought from Merck
(Darmstadt, Germany). Reagent grade NaOH, H2SO4, and glycerol (C3H5(OH)3)
weresupplied by Showa (Tokyo, Japan). Liquid chromatographic grade isopropanol
(IPA, (CH3)2CHOH) was provided by Tedia (Fairfield, Ohio, USA). Liquid
chromatographic grade n-hexane (n-C6H14) was bought from Echo Chemical Co.
(Taoyuan, Taiwan, ROC). Liquid chromatographic grade anhydrous ether
(C2H5OC2H5) was got from J. T. Baker (Phillipsburg, NJ, USA). 4-Phenyl-2-butanone
(C10H12O, 98%), R-4-phenyl-2-butanol (C10H14O, >99%), S-4-phenyl-2-butanol
(C10H14O, >99%) were purchased from Alfa Aesar (Karlsruhe, Germany). Deionized
distilled water (18.2 MΩ) used for all kinds of experiments was prepared from tape
water double distilled twice and further purified with EASYpure Ⅱ RF/UV
ultrapure water system (Barnstead international, Dubuque, IA, USA).
2.2. Incubation and growth of yeast
5

Yeast S. cerevisiae grown on the agar slant were transferred to 110 mL sterilized
culture media. The culture medium was prepared by dissolving 0.3 g KH2PO4, 0.54 g
K2HPO4, 0.26 g (NH3)2SO4, 0.36 g MgSO4, 0.0175 g CaCl2, 20 μL (1.25% w/v)
FeSO4, 0.03 mg ZnSO4, and 1.2 g D-glucose in 200 mL deionized distilled water. The
inoculated culture media were incubated at 30C in a controlled temperature orbital
shaking incubator for 2 days at 150 rpm stirring speed [9].
2.3. Shaker-flask type reduction
Twelve 250 mL Erlenmyer flasks containing 0, 1.1, 2.5, 4.3, 5.0, 6.7, 10.0, 15.0,
23.3, 40.0, 90.0, and 190.0 mL glycerol, respectively, were illuminated with
ultraviolet (UV) light for 30 min. Another twelve 190 mL mixtures of water and
glycerol containing different water/glycerol volume percentages (mL/mL) of 190/0,
170.0/18.9, 150.0/37.5, 130.0/55.7, 125/61.0, 110.0/73.3, 100.0/80.0, 80/95.0,
60.0/106.7, 40.0/110.0, 20.0/80.0, and 0/0, respectively, were prepared and mixed
with previously prepared twelve glycerol solutions to make a 190 mL mixture in each
flask. Subsequently, these twelve flasks were homogenized for 10 min using the
controlled temperature orbital shaking incubator at a stirring speed of 250 rpm. Then,
they were taken out from the incubator and illuminated with UV light for 30 min.
Ten milliliters of freshly grown yeast culture described in Section 2.2 were added
to each of the twelve previously prepared 190 mL water/glycerol solutions. These
twelve 200 mL cell cultures with glycerol volume percentage of 0%, 10%, 20%, 30%,
33%, 40%, 50%, 60%, 70%, 80%, 90%, and 95%, respectively, were homogenized for
10 min using the controlled temperature orbital shaker incubator at a stirring speed of
250 rpm.
Each of these twelve 200 mL glycerol containing yeast cell cultures was evenly
divided into two 100 mL cell cultures. Then, the pH of these cell cultures were
adjusted to 10.0 using 1 M NaOH. Finally, 10 μL of 4-phenyl-2-butanone were added
individually to each of these cell cultures and they were reacted in the controlled
temperature orbital shaking incubator at 30C and a stirring speed of 150 rpm for 7
days.
2.4. Batch type reduction
According to the procedure described in Section 2.3, five 190 mL water and
glycerol mixtures with a measured volume percentage of glycerol (mL/mL) were
prepared and homogenized in the controlled temperature orbital shaking incubator for
10 min with a stirring speed of 250 rpm. After homogenization they were illuminated
with UV light for 30 min.
6

Ten milliliters of the yeast culture grown for 2 days as described in Section 2.2
were separately added to the above 5 water/glycerol solutions to make five 200 mL
glycerol modified yeast cultures at a specified glycerol volume percentage.
Subsequently, they were homogenized for 10 min in the controlled temperature orbital
shaking incubator at a stirring speed of 250 rpm. Thereafter, they were put together (a
combined total of 1 L) in the bench type bioreactor. Ten milliliters of the freshly
grown yeast cell culture were taken and filtered through a microporous membrane
filter (Nylon, 47 mm dia., 0.22 μm). The filtered membrane was dried in the oven at
30C for 24 h to measure the dry cell mass and the filtrate was analyzed by high
performance liquid chromatography (HPLC). The rest of the glycerol modified yeast
culture at a specified % glycerol (v/v) in the automatically controlled 2 L bench top
bioreactor (Eyela, Rikakial, M-100, Tokyo, Japan) was adjusted to the desired pH with 6
M H2SO4 or 6 M NaOH. Then, the reaction substrate 4-phenyl-2-butanone at a
specified concentration was added to the bioreactor for reaction. The yeast catalyzed
bioreduction was performed either at a controlled pH or uncontrolled pH condition
with a preset temperature and a stirring speed of 150 rpm.
2.5. Liquid-liquid extraction
Ten milliliters reaction cell culture was vacuum filtered through a microporous
membrane of 0.22 μm pore diameter. The filtrate was successively extracted three
times with ether and each time with 5 mL ether. The 15 mL ether extract were
vaporized to about 2 mL. Then, they were transferred to 10.0 mL volumetric cylinder
and kept evaporating until to a volume of 1.0 mL. At this moment, HPLC elution
solvent (Hexane/IPA, 93/7, v/v) was added to make a total volume of 10.0 mL which
were then transferred to a 20 mL capped sample vial for later use [9].
2.6. HPLC analysis
The instrumentation of HPLC was consisted of a dual piston solvent delivery
pump (Jasco, PU-2085, Tokyo, Japan), a dual piston solvent delivery pump (Jasco,
PU-2080, Tokyo, Japan), a sample injection valve (Rheodyne 7725i, Cotati, California,
USA) containing a 20 μL sample loading loop, an analytical column (Phenomenex lux
3u Cellulose-1 chiral OD column, cellulose tri(3,5-dimethylphenylcarbamate, 150 mm
× 4.6 mm i.d., 10 μm) coupled with a guard column (5 mm × 4.6 mm i.d.,
Phenomenex, Torrance, CA, USA), a column oven (Colbox, Kaohsiung, Taiwan,
ROC), a variable wavelength UV detector (Jasco, 2075-plus, Tokyo, Japan), and a
desktop computer equipped with chromatographic data software (SISC Corp., Taipei,
Taiwan, ROC). The analysis were performed using an isocratic mobile phase of
hexane/IPA (93/7, v/v) at an elution flow rate of 0.5 mL min-1. The column
temperature was kept at 30C and the UV detection wavelength was set at 221 nm.
7

The e.e. value and the yield were calculated by the following equations:
Smole  Rmole
(1)
e.e. 
100%
Smole  Rmole
where Smole and Rmole are the moles of produced S-4-phenyl-2-butanol and
R-4-phenyl-2-butanol, respectively.
Productmole
yield 
100%
(2)
(InitialSubstrate)mole
where Productmole is the total moles of the products S- and R-4-phenyl-2-butanol and
(Initial Substrate)mole is the initially moles of 4-phenyl-2-butanone added to the
glycerol modified yeast cell culture.
3. Results and Discussion
3.1. Shaker-flask type reaction
The results of shaker-flask type yeast catalyzed enantioselective reduction of
4-phenyl-2-butanone using cell cultures of different volume percentages of glycerol
(0% to 95% glycerol) were shown in Table 1. When the reaction cell culture was with
10% glycerol the major enantiomeric product was R-4-phenyl-2-butanol. However, as
the reaction cell culture was 0% glycerol or with glycerol volume percentage equal to
or larger than 30%, the major enantiomeric product was S-4-phenyl-2-butanol. While
for the reaction cell culture containing 20% glycerol in volume, one of the duplicate
reactions results an S-enantiomeric excess and the other reaction gives R-enantiomeric
excess which indicates a transition of enantiomeric excess for the reaction. Since there
are two types of alcohol dehydrogenase, i.e. R-alcohol dehydrogenase and S-alcohol
dehydrogenase, in S. cerevisiae [17,18], the reaction cell culture at low glycerol
content will probably assist the formation of R-enzyme thus produce an increase of
the R-alcohol enantioselectivity of the yeast cell; the reaction cell culture of high
glycerol content will probably favor the formation of S-enzyme thus help the
S-alcohol enantioselectivity of the yeast cell. However, due to an increase of the
solubility of the substrate ketone at higher glycerol content that will produce greater
substrate inhibition, the yields were thus reduced.
The pH effect of the reaction cell culture on the enantioselectivity of the yeast
catalyzed reduction was further checked for the glycerol modified yeast cell culture
with either 10% or 30% glycerol. The results shown in Table 2 indicate that a better
yield and a greater R-enantiomeric excess value were obtained for the cell culture
containing 10% glycerol volume percentage at pH 10.0 and although with a lower
yield a greater S-enantiomeric excess value was obtained for the 30% glycerol cell
8

culture at pH 7.0. Although the pH control is not ideal for the shaker-flask type
reaction, the results indicate that the pH of the cell culture indeed will influence the
reaction yield and the enantioselectivity of yeast for the two different % glycerol cell
cultures. The pH of the cell culture is able to affect both the enzyme activity and the
enantioselectivity of the corresponding enzymes producing preferentially R- and
S-enantiomers by changing their three-dimensional conformational structures. For
these shaker-flask reactions the final pH of the cell culture was all decreased to
become acid which should be due to the formation of organic acids and amino acids
via the tricarboxylic acid (TCA) cycle by the glycolysis of glucose during the growth
of yeast.
3.2. Yeast catalyzed reduction with batch type bioreactor
3.2.1. pH effect on cell culture containing 10% glycerol in volume
The results in Table 3 indicate that both the yield (75.6%) and the R-enantiomeric
excess value (43.2%) were the greatest for the reaction at room temperature and with
an initial pH 10.0 but without controlling the pH of reaction. While both the yield
(8.7%) and the S-enantiomeric excess value (13.4%) were pretty low for the reaction
with the pH controlled at 10.0 during reaction. This result demonstrates that strong
alkaline reaction cell culture (pH 10.0) is not favorable for yeast growth and inhibits
the activity of R-alcohol dehydrogenase more than S-alcohol dehydrogenase. Despite
the enzyme activity and the enantioselectivity for producing R-4-phenyl-2-butanol
was larger at pH 6.0 than those at pH 7.0, generally, the enzyme activity and the
activity of R-alcohol dehydrogenase became smaller from pH 9.0 to 7.0 and the yields
and the R-e.e. values were all smaller than those of the reaction at an initial pH 10.0
but without pH control. The results therefore show that the activity of R-alcohol
dehydrogenase will be high at high pH cell culture.
3.2.2. Temperature effect on cell culture containing 10% glycerol volume percentage
The results of the enantioselectivity of yeast catalyzed reduction of
4-phenyl-2-butanone performed at different reaction temperatures with an initial pH
10.0 but without controlling the pH during reaction were shown in Table 4. As the
temperature was controlled in the range 25-28C, the major enantiomeric product was
R-4-phenyl-2-butanol and the best R-enantiomeric excess value (52.1%) was at the
temperature of 25C but with a yield of only 18.2%; for the reaction temperature
controlled in the range 30-34C, the major enantiomeric product was
9

S-4-phenyl-2-butanol and the best S-enantiomeric excess value (55.2%) was at the
temperature of 34C with a yield of 37.6%. These results indicate that the activity of
R-alcohol dehydrogenase was high at low temperature and the activity of S-alcohol
dehydrogenase was high at high reaction temperature. The effect of reaction
temperature on the enantioselectivity of yeast catalyzed reduction should also be due
to the change of the three-dimensional conformational structure of both R- and
S-enzymes at different temperatures. Combining the results of pH effect of last section,
it seems that the effect of reaction temperature on the yeast catalyzed reduction
overrides the pH effect.
3.2.3. pH effect on cell culture with 30% glycerol in volume
The pH effect on yeast catalyzed reduction of 4-phenyl-2-butanone with yeast
culture containing 30% glycerol was performed at the temperature 34C according to
the results of previous sections. The results in Table 5 show that an S-enantiomeric
selectivity for cell culture controlled at the pH range 5.0-9.0 had the greatest
S-enantiomeric excess (96.0%) at pH 7.0 although the yield (19.0%) was not the
greatest. The results were in conformity with previous results. Except the reaction run
at pH 9.0, the yields of the yeast catalyzed reduction were in the range 19-32.4% in an
approximately descending trend from pH 5.0 to 8.0. Again, the results show that the
activity of enzymes is not favorable to the alkaline reaction cell culture for the yeast
culture with 30% glycerol.
3.3. Substrate Inhibition
The yields of in previous section with 30% glycerol cell culture under different
pH were generally not high which could be probably due to the substrate inhibition
with the high substrate concentration (100 μg mL-1) used for these reactions. The
results in Table 6 show that reactions with reduced concentrations (10, 20, 30, and 50
μg mL-1) for 10% and 30% glycerol cell culture under their optimal pH and
temperature, respectively. For the reaction with 30% glycerol cell culture, the highest
yield was 56.9% as the substrate concentration was decreased to 10 μg mL-1 and the
yields were in the range 19.0-25.7% for the substrate concentration range between 20
and 100 μg mL-1. For the reaction with 10% glycerol cell culture, the low substrate
concentration of 10 μg mL-1 also obviously reduced the substrate inhibition and
greatly increased the activity of R-alcohol dehydrogenase to give a high yield (86.4%)
and a high R-enantiomeric selectivity (e.e. = 72.3%). All of these evidences
demonstrate the existence of substrate inhibition for this yeast catalyzed reaction. Fig.
1 is the HPLC chromatograms for a typical reaction run with 30% glycerol cell
10

culture. On the other hand, an increase of the substrate concentration is advantageous
to the enantioselectivity of S-enantiomer. Therefore, the results here indicate that the
substrate inhibition effect was greater for R-alcohol dehydrogenase than for S-alcohol
dehydrogenase to produce a better S-enantiomeric excess value at high substrate
concentrations [17,18]. The HPLC chromatograms for a typical reaction run with 10%
glycerol cell culture are shown in Fig. 2.
4. Conclusion
The use of glycerol as a modifier of the aqueous cell culture has the advantage in
the enantioselectiviy for the one phase baker’s yeast mediated reduction of
hydrophobic prochiral ketone. The use of 4-phenyl-2-butanone as a model compound
for the baker’s yeast mediated enantioselective reduction shows that the
enantioselectivity of the reaction can be switched by the volume percentage of
glycerol in the cell culture. The existence of substrate inhibition for the yeast
mediated reduction still needs to be surveyed for future industrial applications.
Acknowledgments
The authors gratefully acknowledge the financial support of the work by
National Science Council under Grant Number NSC 101-2113-M-033-001.
11

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Asymmetry 17 (2006) 2043-2045.
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12

Figure Captions
Scheme
1
Yeast catalyzed reduction of 4-phenyl-2-butanone to either
R-4-phenyl-2-butanol or S-4-phenyl-2-butanol enantiomeric excess with different
volume percentage of glycerol in yeast culture.
13

Figure 1 Chromatograms of yeast mediated enantioselective reduction of 100 mg L–1
4-phenyl-2-butanone with 30% glycerol cell culture at pH 7.0, 34C, 150 rpm stirring
speed, without aeration, and a 4-day reaction period. HPLC conditions: mobile phase
= hexane/IPA (93/7, v/v), mobile phase flow rate = 0.5 mL min-1, column =
Phenomenex Lux 3u Cellulose-1 chiral OD, column temperature = 30C, injection
volume = 20 L, UV detection at 221 nm. Peaks: 1 = 4-phenyl-2-butanone, 2 =
R-4-phenyl-2-butanol, 3 = S-4-phenyl-2-butanol.
14

Figure 2 Chromatograms of yeast mediated enantioselective reduction of 10 mg L–1
4-phenyl-2-butanone with 10% glycerol cell culture at an initial pH 10.0 and without
pH controlling, 25C, 150 rpm stirring speed, without aeration, and a 4-day reaction
period. HPLC conditions: mobile phase = hexane/IPA (93/7, v/v), mobile phase flow
rate = 0.5 mL min-1, column = Phenomenex Lux 3u Cellulose-1 chiral OD, column
temperature = 30C, injection volume = 20 L, UV detection at 221 nm. Peaks: 1 =
4-phenyl-2-butanone, 2 = R-4-phenyl-2-butanol, 3 = S-4-phenyl-2-butanol.
15

Tables
Table
1
Results of baker’s yeast catalyzed shaker-flask reduction of
4-phenyl-2-butanone using reaction culture of different volume percentages of
glycerol in yeast culture.a,b
%Glycerolc
e.e.
(%)
Yield (%)
(v/v)
0
3.3
28.0
8.3
24.0 (S)
10
20
30
33
40
50
60
70
80
90
80.7 (R)
17.4 (S)d, 29.8 (R)c
89.7 (S)
21.5
11.8
9.5
77.7 (S)
68.0 (S)
6.4
56.6 (S)
6.9
59.5 (S)
6.0
61.8 (S)
4.5
49.7 (S)
3.2
35.1 (S)
a Substrate concentration 100 μg mL–1; reaction temperature = 30C; stirring speed =
150 rpm; reaction period = 7 days.
b Average result of duplicate reaction runs.
c Volume percentage of glycerol in yeast culture.
d Result for one reaction run.
16

Table 2 The pH effect on the enantioselectivity of yeast catalyzed shaker-flask
reduction of 4-phenyl-2-butanone with reaction cell culture containing either 10% or
30% volume percentage of glycerol.a,,b
%Glycerol
Initial pH
Yield
(%)
19.5
21.0
22.2
24.3
6.5
e.e.
Final pH
(v/v)
10
(%)
7.0
8.0
68.6 (R)
78.4 (R)
79.1 (R)
78.9 (R)
52.9 (S)
48.6 (S)
48.1 (S)
33.6 (S)
3.0
4.1
5.2
6.0
4.6
4.9
5.0
5.5
10
10
9.0
10
10.0
7.0
30
30
8.0
7.1
30
9.0
7.3
30
10.0
4.8
a Substrate concentration 100 μg mL–1; reaction temperature = 30C; stirring speed =
150 rpm; reaction period = 7days.
b Average value of duplicate reaction runs.
17

Table 3 Results of yeast catalyzed reduction of 4-phenyl-2-butanone with 10%
volume percentage of glycerol in the reaction cell culture of different pH.a
Initial
pH
pH
control
yes
Yield
(%)
e.e.
pH at the end of
(%)
reaction
6.0
6.0
22.4
17.4
23.2
27.0
8.7
45.9 (R)
34.7 (R)
53.8 (R)
63.5 (R)
13.4 (S)
75.6 (R)
7.0
yes
7.0
8.0
yes
8.0
9.0
yes
9.0
10.0
10.0
yes
10.0
6.8
no
43.2
a Using a 2 L bench scale batch type bioreactor containing 1 L reaction cell culture;
substrate concentration 100 μg mL–1; Reaction temperature = room temperature;
stirring speed = 150 rpm; without aeration; reaction period = 4 days.
18

Table 4 Temperature effect on yeast catalyzed reduction of 4-phenyl-2-butanone with
10% volume percentage of glycerol in yeast culture.a
Temperature
Initial
pH
pH at the end
of reaction
6.27
e.e.
Yield
(%)
(%)
(C)
25b
26c
28c
30c
32c
34c
10.01
10.04
10.03
10.03
10.03
10.03
52.1 (R)
32.8 (R)
15.8 (R)
16.2 (S)
22.4 (S)
55.2 (S)
18.2
21.4
39.0
27.4
44.8
37.6
6.23
6.41
6.60
6.64
6.43
a Reaction conditions: substrate concentration 100 μg mL–1; initial pH 10.0 with no
control of pH; stirring speed = 150 rpm; without aeration; reaction period = 4 days.
b Average value of 3 reaction runs.
c Average value of 2 reaction runs.
19

Table 5 Results of yeast catalyzed reduction of 4-phenyl-2-butanone with 30%
volume percentage of glycerol in reaction cell culture at different pH.a, b
Yield
(%)
e.e.
pH
(%)
5.0
6.0
7.0
8.0
9.0
32.4
25.7
19.0
22.2
7.4
83.2 (S)
90.0 (S)
96.0 (S)
88.0 (S)
83.8 (S)
a Reaction condition: substrate concentration 100 μg mL–1; controlled pH; reaction
temperature = 34C; stirring speed = 150 rpm; without aeration; reaction period = 4
days.
b Result of one reaction run.
20

Table 6 Results of substrate concentration effect on yeast catalyzed reduction of
4-phenyl-2-butanone with 10%a or 30%b volume percentage glycerol in yeast culture.
Substrate
concentration
(μg mL-1)
10 c
%Glycerol
Yield
(%)
e.e.
(%)
(v/v)
56.9
25.7
24.7
22.1
19.0
87.1 (S)
76.0 (S)
79.2 (S)
93.0 (S)
96.0 (S)
20 d
30 d
50 d
100 d
30 a
10b
10 c
86.4
72.3 (R)
a
Reaction condition: uncontrolled pH with an initial pH 10, temperature 25C,
stirring speed 150 rpm, without aeration, 4-day reaction period.
b Reaction condition: pH 7, 34C, stirring speed 150 rpm, without aeration, 4-day
reaction period.
c Average value of 2 reaction runs.
d Results of 1 reaction run.
21