Highly enantioselective reduction of acetophenone by locally isolated Alternaria alternata using ram horn peptone

Article abstract of DOI:10.1016/j.tetasy.2007.06.013

Enantiomerically pure compounds are important building blocks in the synthesis of natural products. In this study, the reduction of acetophenone to the (S)-isomer of 1-phenylalcohol with a high enantiomeric excess (ee) by locally isolated Alternaria alter

Full text of DOI:10.1016/j.tetasy.2007.06.013

Tetrahedron: Asymmetry 18 (2007) 1529–1532
Highly enantioselective reduction of acetophenone by locally isolated
Alternaria alternata using ram horn peptone
a,
b
c
a
*
Esabi B. Kurbanoglu, Kani Zilbeyaz, Namudar I. Kurbanoglu and Mesut Taskin
a
Department of Biology, Faculty of Arts and Sciences, Ataturk University, 25240 Erzurum, Turkey
Department of Chemistry, Faculty of Arts and Sciences, Ataturk University, 25240 Erzurum, Turkey
Department of Primary Science Education, Hendek Faculty of Education, Sakarya University, 54300 Sakarya, Turkey
b
c
Received 30 March 2007; accepted 14 June 2007
Abstract—Enantiomerically pure compounds are important building blocks in the synthesis of natural products. In this study, the reduc-
tion of acetophenone to the (S)-isomer of 1-phenylalcohol with a high enantiomeric excess (ee) by locally isolated Alternaria alternata
using ram horn peptone (RHP) was investigated. Ten strains of A. alternata were isolated from different plant samples. These isolates
were evaluated for the reduction of acetophenone (ACP) to 1-phenylethanol (PEA). Glucose, yeast extract and RHP in a shake flask and
fermenter for growth of A. alternata cultures were used. A. alternata EBK-4 isolate was found to be an effective biocatalyst for the enan-
tiomeric bioreduction of acetophenone. Conversions of up to 100% with excellent enantiomeric excesses (>99%) were obtained. Produc-
tion of PEA was achieved via a fermenter. The yield was calculated as 86%. This is the first report on the enantioselective reduction of
ACP by A. alternata using ram horn peptone from waste material.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
future. Synthetic chiral catalysts are being developed for
this purpose, as well as new product separation techniques.
Another possible option is to use biocatalysts, such as puri-
fied enzymes or microbial cells, since these can result in the
production of mostly a single enantiomer.
Chiral alcohols are useful intermediates for many pharma-
ceutical and chemical compounds. The need for optically
active drugs has increased in pharmaceutical and agro-
chemical fields in the recent years and, therefore, chiral
alcohols are under increasing demand. There have been
many attempts to construct bioreduction systems for the
industrial production of chiral alcohols. There has also
been much interest in the use of fungus or yeast for enan-
tioselective reductions of aromatic ketones. There are many
advantages to using microorganisms as biocatalyst instead
of purified enzymes. Microorganisms are generally much
less expensive, and in some cases, enzymes are more stable
within the cell, thus extending the life of the biocatalyst.
The use of microbial cells is particularly advantageous
for carrying out the desired reduction, since they do not re-
On the other hand, biotechnology opens future prospects
in the chemical field for the synthesis of complex com-
pounds and combines inexpensive raw materials with envi-
1
,2
2
,5,6
ronmentally friendly processes.
Recently, ram horn peptone (RHP) has been utilized as a
source of peptone for microbial growth media. One impor-
tant option is the production of ram horn protein hydroly-
sate, which can be prepared by the hydrolysis of ram horns
using proteolytic chemicals. The RHP also provides a rich
source of nutrients for microbiological growth, and is
1
–4
7
quire the addition of cofactors for their regeneration. In
addition, there is a need for alternative microorganisms for
producing enantiomerically pure pharmaceuticals, because
the racemic mixtures made today may not be allowed in the
excellent for microbial growth.
Herein, we produced the highly enantiomerically enriched
(S)-1-phenylethanol from acetophenone by locally isolated
Alternaria alternata with a process and clarified the use of
RHP from waste material for microbial reduction. We also
optimized the reaction conditions for high enantiomeric
excess (ee).
*
Corresponding author. Tel.: +90 442 231 4342; fax: +90 442 236 0948;
e-mail: ekurbanoglu@yahoo.com
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.06.013

1530
E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 1529–1532
O
OH
Table 2. Effect of incubation period on the reduction of acetophenone by
A. alternata EBK-4
Alternaria alternata EBK-4
CH
3
CH
3
pH 6.5, 28 oC, 26 h,
Time (h)
Conversion
ee (%)—Config.
1
50 rpm, 0.4 vvm,
2
48
7
96
4
44
62
70
90
87 (S)
78 (S)
56 (S)
30 (S)
Acetophenone
10 mmol) scale: 1L
(S)-1-Phenylethanol
2
(
ee = > 99%
Yield = 86%
Reaction conditions: substrate 1 mmol, temperature 25 ꢁC, pH 7, agitation
50.
1
2
. Results and discussion
The ACP reduction was carried out in two stages. In the
first stage, the reduction was studied in the flask culture
in order to find the optimum reaction conditions; in the
second stage, the total production of PEA under optimum
conditions were performed in the fermenter. Ten different
strains of A. alternata were isolated from plant samples
and evaluated for the reduction of ACP to PEA, using glu-
cose, yeast extract and RHP nutrients as substrate in shake
flask and fermenter. These cultures were found to produce
results, shown in Table 3, indicate that the fermentation
pH clearly has a significant effect on conversion and ee.
The conversion and ee increased when the pH of the
growth medium was increased from pH 4.5 to pH 6.5
and then decreased. The optimum pH for the reduction
activity was found to be 6.5. When pH 6.5 was used, the
conversion and ee were 90% and 93%, respectively. The
conversion and ee were lower when the pH was over 7.0.
The A. alternata was not able to grow at the initial pH of
8.5. Under the optimum conditions (pH 6.5, 24 h), the ef-
fect of different incubation temperatures was investigated
in order to determine whether it could increase the conver-
sion and ee of acetophenone reduction. The results are
shown in Table 4.
(
S)-1-phenylethanol, ranging from 60% to 78% ee. The best
results for the bioreduction were obtained when A. alter-
nata EBK-4 was used. The ee and conversion were 78%
and 62%, respectively (Table 1). The enantioselectivity ob-
served for this isolate is in accordance with Prelog’s rule.
Acetophenone was also reduced with the other isolates in
moderate conversion and enantioselectivity. Since the high-
est enantioselectivity was observed by using A. alternata
EBK-4 as biocatalyst, it was selected for further studies.
Table 3. Effects of different pHs on the reduction of acetophenone by
A. alternata EBK-4
pH
Conversion (%)
ee (%)—Config.
Table 1. Screening of locally isolated A. alternata strains for the
bioreduction of acetophenone
4
5
6
7
8
8
.5
.5
.5
.5
.0
.5
20
80
90
20 (S)
84 (S)
93 (S)
30 (S)
—
Isolates
Conversion
ee (%)—Config.
57
EBK-1
EBK-2
EBK-3
EBK-4
EBK-5
EBK-6
EBK-7
EBK-8
EBK-9
EBK-10
33
25
37
62
38
40
52
44
48
54
64 (S)
71 (S)
62 (S)
78 (S)
67 (S)
74 (S)
74 (S)
76 (S)
75 (S)
60 (S)
Weak growth
No growth
—
Reaction conditions: substrate 1 mmol, temperature 25 ꢁC; time 24 h,
agitation 150 rpm.
As can be seen from Table 4, a variation in temperature re-
sulted in different conversion and ee levels on the reduction
of ACP. Notably, the temperature has a significant effect
on the ee and conversion. The highest ee (99%) was ob-
tained from 28 ꢁC with 100% conversion. Temperatures
over 30 ꢁC had an inhibitory effect on ee. For example,
the lowest ee (60) was obtained at 36 ꢁC. These results sug-
gest that an increase in temperature had a negative effect on
the ee. Conversely, the high temperature did not have a
Reaction conditions: substrate 1 mmol, temperature 25 ꢁC, time 48 h, pH
, agitation 150 rpm.
7
Different incubation times were chosen to monitor the pro-
gress of the bioreduction (Table 2). The best ee (87%) was
observed after 24 h. When the ee of the PEA was observed
after 72 and 96 h, the ee was lower. Contrary to these
results, the conversion increased up to 90% for 96 h. On
the basis of these observations, we suggested that A. alter-
nata EBK-4 may express different ees and conversions
depending on the growth conditions. The purpose of
increasing the enantioselectivity of the PEA was studied
with different pH. The best conditions found were used
for further optimization of conversion and ee.
Table 4. Effect of temperature on the reduction of acetophenone by
A. alternata
Temperature (ꢁC)
Conversion (%)
ee (%)—Config.
2
28
30
3
3
3
6
96
96 (S)
99 (S)
93 (S)
86 (S)
70 (S)
60 (S)
100
100
100
100
100
2
4
6
Effect of the initial pH of the used growth medium on the
reduction of ACP is shown in Table 3. A significant
amount of work has been carried out concerning the effect
Reaction conditions: substrate 1 mmol, pH 6.5, time 24 h, agitation
150 rpm.
8
of pH on enzymatic reactions in an aqueous phase. The

E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 1529–1532
1531
negative effect on the conversion of ACP. The conversion
of ACP for 36 ꢁC was 100%, although the ee was rather
low. The optimum conditions for the high enantiomeric
excess of PEA with temperature studies were found. As a
result, other fermentation parameters such as agitation
and initial substrate concentration were not studied. These
fermentation conditions are time 24 h, pH 6.5, temperature
and its derivatives.2,4,9–11 They are used as a reducing agent
as they are easily available and inexpensive. The fermenta-
tion medium can represent almost 30% of the cost for a
microbial fermentation, with micronutrients representing
the most significant cost of production. By-products can
supply unique micronutrients to replace expensive peptone
and yeast extract. The consistency of the ingredients used
in the commercial medium formulations and significant
increase in product yield or cost reduction are critical for
2
8 ꢁC and agitation 150 rpm. The ee and conversion under
these conditions were at their maximum. Therefore, we
continued the research with optimum fermentation condi-
tions for further studies.
1
2
the industrial fermentation utilization of any byproduct.
Therefore, an inexpensive substrate, such as ram horn
waste for microbial growth in this study was successfully
used. The RHP for microbial growth in the previous study
was compared with some standard peptones. It was found
that the RHP could be utilized instead of some standard
The bioreduction of ACP for the total production of PEA
by A. alternata EBK-4 was performed in a fermenter. These
results are summarized in Figure 1. The ee for all incuba-
tion times was >99%. The best results for the bioreduction
were obtained after 26 h incubation. The reaction occurred
with good conversion (100%) and excellent ee (>99%). In
addition, the amount of PEA produced with the observed
enantioselectivity was 8.6 mmol/L. (S)-1-Phenylethanol
was produced by A. alternate via a fermenter under opti-
mum growth conditions. This fungus was firstly used for
the production of a chiral alcohol. Therefore, this microor-
ganism must be useful for biotechnological purposes such
as microbial reductions. Many papers report with different
microorganisms the microbial reduction of acetophenone
7
peptones.
3. Conclusion
This report has dealt with the first microbial reduction of
acetophenone by A. alternata on a preparative scale. It
seems more adequate to use raw materials such as waste
material from the food industry as the basis of the micro-
bial culture media. We believe that selectivity is valuable
in organic synthesis. Therefore, a detailed mechanism for
the high selectivity of the reduction of acetophenone deriv-
atives by altering the reaction conditions and microorgan-
isms is under investigation in our laboratory.
a
(+)-(R)
(-)-(S)
4. Experimental
(-)-(S)
4.1. Materials
1
2
14
12
14
Ram horns were obtained from the slaughterhouse of
Erzurum, Turkey. The other components of the culture
media and the chemical reagents were obtained from
Merck and Sigma in the highest purity available. Produc-
tion of ram horn peptone was carried out with the method
Ee = >99%
b
[
D 2 2
α] = -52 (c 0.184, CH Cl )
Conversion = 100%
Yield = 86%
9
7
5
3
1
7
of Kurbanoglu and Kurbanoglu.
4.2. Isolation of microorganisms, identification and
inoculation
The microorganisms used in this study were isolated from
plant samples, such as sunflower, grapes and apple
collected from the region around Erzurum, Turkey. The
isolation process was performed by serial dilution of the
13
samples according to standard techniques. Taxonomic
identification of filamentous fungi was identified in-house
by using mature cultures on a standard potato dextrose
agar (PDA) in order to ensure a good development of taxo-
nomically relevant features, and following the identifica-
4
8
12 16 18 22 26 30 34
Incubation time (h)
1
4
15
tion keys provided by Von Arx and Domsch et al.
These cultures were maintained on PDA slants, incubated
at 25 ꢁC and stored at 4 ꢁC. The conidia from 10 days
old cultures were used for inoculation. The conidial suspen-
sion was prepared in sterilized 10 mL distilled water by
gently scratching conidia with a sterile wire loop and then
it was shaken vigorously for breaking the clumps of
conidia.
Figure 1. Chiral analysis of the product: (a) Production on a preparative
scale of (S)-1-phenylethanol via fermenter by A. alternata EBK-4. (b)
Conversion, yield and polarimetric value after 26 h incubation. Reaction
conditions: pH 6.5, temperature 28 ꢁC, agitation 150 rpm, substrate
10 mmol, aeration 1.0 v/v/m. The yield was calculated based on the
following: Yield (%) = 100 · PC/ISC, PC and ISC are the concentrations
of product and initial substrate, respectively.

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E. B. Kurbanoglu et al. / Tetrahedron: Asymmetry 18 (2007) 1529–1532
4
.3. Culture conditions and reduction of acetophenone
Acknowledgements
The fermentation medium per liter contained (g/L): glucose
0, yeast extract 3 and RHP 4. The initial pH of the culture
medium was adjusted to 7.0 with 1 M HCl and 1 M NaOH
and sterilized at 121 ꢁC for 15 min. All the cultures were
grown in 250 mL flasks containing 100 mL of medium.
This study was supported by a grant from the Research
Funds Appropriated to Ataturk University, Erzurum, Tur-
key. The authors thank Professor Ismet Hasenekoglu for
his help in identification of fungus.
2
1
mL of conidial suspension was added to each flask.
Flasks were incubated on a reciprocal shaker at 150 rpm,
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to dryness. For analysis, a small fraction of the product
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1
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1
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