Enantioselective oxidation of (±)-2-phenyl-1-propanol to (S)-2-phenyl-1-propionic acid with Acetobacter aceti: Influence of medium engineering and immobilization

Article abstract of DOI:10.1016/S0957-4166(02)00641-9

The enantioselective oxidation of (RS)-2-phenyl-1-propanol to (S)-2-phenylpropionic acid catalysed by Acetobacter aceti occurred under very mild conditions. The rate, substrate tolerance and enantioselectivity (enantiomeric ratio, E >200) can be increased

Full text of DOI:10.1016/S0957-4166(02)00641-9

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 13 (2002) 2345–2349
Enantioselective oxidation of (±)-2-phenyl-1-propanol to
S)-2-phenyl-1-propionic acid with Acetobacter aceti: influence of
medium engineering and immobilization
(
a
a
a
b
Raffaella Gandolfi, Antonella Borrometi, Andrea Romano, Jos e´ V. Sinisterra Gago and
a,
Francesco Molinari *
a
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Sezione Microbiologia Industriale,
Universit a` degli Studi di Milano, via Celoria 2, 20133 Milano, Italy
Departamento de Qu ´ı mica Org a´ nica y Farmac e´ utica, Facultad de Farmacia, Universidad Complutense de Madrid,
b
Plaza Ramon y Cajal, 28040 Madrid, Spain
Received 30 August 2002; accepted 4 October 2002
Abstract—The enantioselective oxidation of (RS)-2-phenyl-1-propanol to (S)-2-phenylpropionic acid catalysed by Acetobacter
aceti occurred under very mild conditions. The rate, substrate tolerance and enantioselectivity (enantiomeric ratio, E >200) can
be increased dramatically simply by adding cyclodextrins to the reaction medium. Cells immobilized in calcium alginate showed
good operational stability and good enantioselectivity (e.e. 94%); the catalyst can be re-used at least five times without e.e.
diminution. © 2002 Published by Elsevier Science Ltd.
1
. Introduction
deactivation of the biocatalyst is often reduced. Among
the supramolecular additives, cyclodextrins (CD) are
particularly attractive since they are commercially avail-
able and their internal hydrophobic cavities can be
suited for achieving a controlled release of organic
compounds in aqueous media. The use of cyclodextrin
to ameliorate the rate and selectivity of aqueous bio-
conversions has been successfully applied for the mod-
ification of steroids and vitamins and can be a useful
Acetic acid bacteria have been employed traditionally
for the selective oxidation of primary and secondary
alcohols. These bacteria have an incomplete oxidative
metabolism and, therefore, can efficiently perform
chemo-, regio- and stereoselective biotransformations
of primary alcohols, affording good yields of the corre-
sponding carboxylic acids, while aldehydes are not nor-
mally isolated. As a consequence, acetic acid bacteria
can also be conveniently employed for the kinetic reso-
lution of racemic alcohols affording the corresponding
1
7,8,10
tool for bioconversions in plant cell biotechnology.
We have recently reported the use of different acetic
acid bacteria for enantioselective oxidation of primary
2–5
carboxylic acid in enantiomerically pure form.
5
alcohols and diols. A strain of Acetobacter aceti 2000/
Hydrophobic substrates are often converted only slowly
in aqueous biotransformations, since their dissolution
in the aqueous medium may be the rate controlling
2
8 MIM turned out to be quite versatile and efficient as
a biocatalyst and was employed in this work for study-
ing the enantioselective oxidation of (RS)-2-phenyl-1-
propanol to (S)-2-phenylpropionic acid (Scheme 1).
6
step. Several procedures have been described to
increase the reaction rate of these substrates such as
using organic solvents or additives which help the
availability of the organic substrate in aqueous
6
–9
medium. This last solution is interesting because the
*
0
Corresponding author. Tel.: 0039-0250316695; fax: 0039-
250316694; e-mail: francesco.molinari@unimi.it
0
Scheme 1.
957-4166/02/$ - see front matter © 2002 Published by Elsevier Science Ltd.
PII: S0957-4166(02)00641-9

2
346
R. Gandolfi et al. / Tetrahedron: Asymmetry 13 (2002) 2345–2349
2
. Results and discussion
Different strategies can be used to reduce the concen-
tration of an organic substrate available for a biocata-
lyst working in an aqueous medium. These include the
addition of a second phase (for example an adsorbing
Acetobacter aceti 2000/28 MIM was selected among
recently isolated acetic acid bacteria for its oxidative
13
14
activity towards (RS)-2-phenyl-1-propanol. Fig.
1
resin or an organic solvent) to partition the sub-
strate and lower its effective concentration with respect
to the enzyme; the use of additives able to complex the
substrate reversibly (e.g. cyclodextrins); and the immo-
bilization of the whole cells on a solid support. Experi-
ments were performed in the presence of hydrophobic
organic solvents (1/1 v/v), or cyclodextrins (equimolar
ratio with respect to the substrate). Different cyclodex-
trins were employed: underivatized b-cyclodextrin (b-
CD), acetyl cyclodextrin (Ac-CD), methyl-b-cyclo-
dextrin (Me-CD), trimethylammoniumcyclodextrin
(TM-CD) and hydroxypropylcyclodextrin (HP-CD).
various amounts of cyclodextrins were tested and the
highest rates were observed with molar ratio substrate/
CD of 0.5 in all the cases. The results are reported in
Table 2.
shows the relationship between growth and molar con-
version obtained after 24 h; (S)-2-phenylpropionic acid
was always produced with enantiomeric excess of 92%.
Incomplete enantioselectivity can be due to enzymatic
racemization, which was ruled out by the observation
that enantiomerically pure (S)-2-phenylpropionic acid
added to A. aceti MIM 2000/28 was not racemized even
after 24 h.
Cells of A. aceti MIM 2000/28 showed the highest
molar conversions after 24 h of growth. Experiments
aimed at the improvement of the reaction rates and
enantioselectivity were carried out with cells grown for
2
4 h and re-suspended in different buffers and under
different reaction conditions (pH, temperature and sub-
strate concentration). While the pH and temperature
Table 1. Production of (S)-2-phenylpropionic acid by oxi-
dation of (RS)-2-phenyl-1-propanol with Acetobacter aceti
MIM 2000/28: enantioselectivity and molar conversion
after 24 h at different substrate concentrations
(
2
the best results in terms of conversion were obtained at
8°C and initial pH 7.0) did not significantly influence
the stereobias, the initial substrate concentration
strongly affected the enantioselectivity since high enan-
tiomeric excesses (>97%) could be obtained with low
substrate concentration (Table 1).
Substrate
concentration (g/L)
Molar
conversion (%)
e.e. of product
(%)
E
0
1
2
5
7
.5
.0
.5
.0
.5
45
45
41
28
12
\97
\97
92
67
46
\200
\200
46
The observation that the enantioselectivity of the reac-
tion drops with higher substrate concentrations is often
encountered when using whole microbial cells and it is
often due to the simultaneous action of different dehy-
6.5
2.9
drogenases, 1,12which
display
different
enantio-
1
0.0
\5
–
–
1
selectivities.
Figure 1. Growth of Acetobacter aceti MIM 2000/28 and molar conversion of (RS)-2-phenyl-1-propanol into phenylpropionic
acid. Molar conversion after 24 h.

R. Gandolfi et al. / Tetrahedron: Asymmetry 13 (2002) 2345–2349
2347
The addition of cyclodextrins generally reduced the
reaction times, whilst affording similar yields and an
improvement in the enantioselectivity. Modified
cyclodextrins were more efficient, which can be
attributed to the different size and hydrophobicity of
the modified cyclodextrin cavity compared to that of
unmodified cyclodextrins, thus improving the recogni-
tion of hydrophobic chiral alcohol molecules. The sub-
strate molecules are released from the cyclodextrin
cavity in a more controlled fashion, and as a result the
enzymatic machinery is not saturated, leading to similar
yields in shorter reaction times. Hydrophobic organic
solvents, working as reagent reservoirs, increased the
enantioselectivity, but low yields were achieved even
after reaction times of 24 h. This result is likely to be
due to the reduction of the activity of the cells by the
The specific activities using methyl cyclodextrin and
different substrate concentrations were compared with
those obtained without cyclodextrin (Fig. 2).
No formation of the R-enantiomer could be detected in
all the experiments performed in the presence of CD,
and the reaction occurred with good rates even at high
substrate concentrations (10 g/l) where no reaction
could be observed in experiments carried out without
additives. Experiments with cells of A. aceti immobi-
2+
2+
lized in alginate beads with Ca or Ba as counterions
were also carried out (Table 3).
Simple immobilization of the cells did not improve the
enantioselectivity or the yield compared to free whole
cells. The biotransformation was also carried out with
immobilized cells in the presence of methyl-b-cyclodex-
trin or iso-octane. The addition of iso-octane to immo-
bilized cells resulted in lowered reaction productivity
and a small improvement of the enantiomeric ratio,
while no reaction occurred in the presence of cyclodex-
trins. This finding can be explained by the observation
that alginate gels attract the cyclodextrin molecules,
which then have a reduced capacity to bind organic
9
hydrophobic organic solvent.
Table 2. Production of (S)-2-phenylpropionic acid by oxi-
dation of (RS)-2-phenyl-1-propanol with Acetobacter aceti
MIM 2000/28 in the presence of solvents or cyclodextrins.
[
substrate]=2.5 g/L
Solvent or
additive
Molar
conversion
Time (h)
e.e. (%)
E
15
molecules.
(
%)
Table 3. Production of (S)-2-phenylpropionic acid by oxi-
dation of (RS)-2-phenyl-1-propanol with immobilized Ace-
tobacter aceti MIM 2000/28
None
44
23
32
31
38
47
47
48
48
24
24
24
24
6
6
6
6
6
92
96
97
51
103
100
100
\200
\200
\200
\200
\200
Heptane
iso-Octane
Pentadecane
b-CD
97
Alginate
Solvent or Molar
Time (h) e.e. (%)
E
\97
\97
\97
\97
\97
counterion additive
conversion (%)
Ac-CD
2
+
Hp-CD
Me-CD
TM-CD
Ca
None
None
iso-Octane 25
35
32
24
24
24
94
94
97
54
50
66
2
+
Ba
Ca
2
+
Figure 2. Activity of Acetobacter aceti MIM 2000/28 towards (RS)-2-phenyl-1-propanol at different concentrations with and
without methyl cyclodextrins.

2
348
R. Gandolfi et al. / Tetrahedron: Asymmetry 13 (2002) 2345–2349
Figure 3. Re-use of free and immobilized cells for the oxidation of (RS)-2-phenyl-1-propanol.
Free and immobilized cells were re-used in successive
experiments for gaining information about the stability
of the biocatalyst (Fig. 3).
The simple methodology described in the paper can be
used in a traditional organic chemistry laboratory and
furnishes enantiomerically pure (S)-2-phenylpropionic
acid with higher yields than reported previously.
Freely suspended cells lost 50% of their activity after
two batches, while cells immobilized in calcium alginate
showed good operational stability, with high activity
after five successive batch reactions. Additionally, the
enantiomeric excess of the acid produced after repeated
batches was the same (94%) showing that immobiliza-
tion is a good tool for completion of this biotransfor-
mation under preparative conditions.
4. Experimental
4
.1. General
All the chemicals were purchased from Fluka Chemical
Co., including enantiomerically pure (R)- and (S)-2-
phenylpropionic acid. Cyclodextrins were a generous
gift from Wacker-Chemie GmbH.
3. Conclusions
4
.2. Microorganism, growth and biotransformation
The oxidation of (RS)-2-phenyl-1-propanol with A.
aceti furnished (S)-2-phenylpropionic acid under very
mild conditions. The rate, substrate tolerance and enan-
tioselectivity are dramatically increased by adding
cyclodextrins to the reaction medium. These effects
might be connected with the formation of reversible
complexes among the cyclodextrin and the substrate;
the controlled release maintains the low concentration
of the substrate in water, therefore allowing for high
enantioselectivity and lowering the tendency for sub-
strate inhibition. A four-fold increase in the reaction
rate (45% molar conversion after 6 h) was achieved by
using methyl cyclodextrin, with enantiomeric excess
always above 97%. Cells immobilized in calcium algi-
nate showed good operational stability, although the
acid was obtained with slightly lower enantioselectivity
conditions
A. aceti MIM 2000/28 from our collection (MIM:
Microbiologia Industriale Milano) was routinely main-
−
1
tained on GYC slants (glucose 50 g l , yeast extract 10
−
1
−1
−1
g l , CaCO 30 g l , agar 15 g l , pH 6.3) at 28°C.
3
The strain, grown on GYC slants for 24 h at 28°C, was
inoculated into 100 ml Erlenmeyer flasks containing 10
−
1
ml of the liquid medium GLY (glycerol 25 g l , yeast
−
1
extract 10 g l , pH 5, distilled water) and incubated on
a reciprocal shaker (100 spm); the flask liquid cultures
of A. aceti MIM 2000/28 were used for inoculating a 1
L reactor with 200 mL working volume, agitation speed
250 rpm, air flow rate 1 vvm. The dry weight was
determined after centrifugation of 100 ml of cultures,
cells were washed with distilled water and dried at
110°C for 24 h.
(
e.e. 94%).

R. Gandolfi et al. / Tetrahedron: Asymmetry 13 (2002) 2345–2349
2349
Biotransformations were completed using bacteria
grown directly inside the reaction vessel; in experiments
with two-liquid phase systems, solvents were added to
reach a 1:1 v/v ratio. Neat substrate was directly added
to suspensions. Control of the pH was performed by
continuous addition of aqueous NaOH via a multi-
channel Watson–Marlow 503 U/R peristaltic pump
connected to a pH controller (pH/ORP Controller
chloride solution. The reaction was discontinued if the
yield of the acid was less than 15% after 24 h.
Acknowledgements
This work was supported by the C.N.R. Target Project
on Biotechnology (n 97.01019. PF 115.08601). We
thank Wacker-Chemie GmbH for the generous gift of
cyclodextrins.
3
675, Jenco Electronics). The work-up of these bio-
transformation was carried according to Ref. 4.
4.3. Analytical methods
The absolute configuration of the obtained acid was
determined by comparison with the specific rotation of
authentic samples of the enantiomerically pure com-
pounds. Samples (0.5 ml) were taken at intervals
brought to pH 1 by addition of 5 M HCl and extracted
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obtained beads were rinsed with aqueous CaCl solu-
2
tion (0.1 M, 50 ml), separated by filtration and the wet
beads were stored at 4°C until use. Both free and
immobilized cells were re-used in successive cycles.
After 24 h, free cells were centrifuged and re-suspended
in fresh reaction mixture and 2-phenylethanol (2.5 g/l)
was added. Immobilized cells were filtered on paper and
re-used after washing with a 0.1 M aqueous calcium