Deracemization of aryl secondary alcohols via enantioselective oxidation and stereoselective reduction with tandem whole-cell biocatalysts

Article abstract of DOI:10.1016/j.molcatb.2010.01.031

Deracemization of racemic 1-phenylethanol, i.e., stereoinversion of (R)-1-phenylethanol to (S)-1-phenylethanol, has been successfully realized via concurrent enantioselective oxidation and stereoselective reduction employing whole-cell biocatalysts of an alcohol dehydrogenase and a ketone reductase with opposite stereoselectivity in one-pot. One biocatalyst is Microbacterium oxydans ECU2010 which catalyzes stereoselective oxidation of (R)-secondary alcohols to corresponding ketones and another is Rhodotorula sp. AS2.2241 which reduces the ketones to (S)-secondary alcohols. Each of the whole-cell biocatalysts has its own in vivo cofactor regeneration system so that there is no need to add the expensive cofactor and/or the oxidoreductase for the cofactor regeneration. To explore the generality of this method, a broad range of racemic aryl secondary alcohols were efficiently deracemized to their (S)-enantiomers by combination of the two microorganisms, affording optically pure secondary alcohols in high yields (86.5-99%) and excellent optical purity (>99% ee). Our method represents an easy going, cheap and environmentally benign way for the biocatalytic synthesis of chiral aryl secondary alcohols from their racemates.

Full text of DOI:10.1016/j.molcatb.2010.01.031

Journal of Molecular Catalysis B: Enzymatic 64 (2010) 48–52
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Deracemization of aryl secondary alcohols via enantioselective oxidation and
stereoselective reduction with tandem whole-cell biocatalysts
Yun-Long Li, Jian-He Xu∗, Yi Xu
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road,
Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Deracemization of racemic 1-phenylethanol, i.e., stereoinversion of (R)-1-phenylethanol to (S)-
1-phenylethanol, has been successfully realized via concurrent enantioselective oxidation and
stereoselective reduction employing whole-cell biocatalysts of an alcohol dehydrogenase and a ketone
reductase with opposite stereoselectivity in one-pot. One biocatalyst is Microbacterium oxydans ECU2010
which catalyzes stereoselective oxidation of (R)-secondary alcohols to corresponding ketones and
another is Rhodotorula sp. AS2.2241 which reduces the ketones to (S)-secondary alcohols. Each of the
whole-cell biocatalysts has its own in vivo cofactor regeneration system so that there is no need to add
the expensive cofactor and/or the oxidoreductase for the cofactor regeneration. To explore the generality
of this method, a broad range of racemic aryl secondary alcohols were efficiently deracemized to their
(S)-enantiomers by combination of the two microorganisms, affording optically pure secondary alcohols
in high yields (86.5–99%) and excellent optical purity (>99% ee). Our method represents an easy going,
cheap and environmentally benign way for the biocatalytic synthesis of chiral aryl secondary alcohols
from their racemates.
Received 10 December 2009
Received in revised form 26 January 2010
Accepted 26 January 2010
Available online 4 February 2010
Keywords:
Deracemization
Stereoinversion
Aryl secondary alcohols
Microbacterium oxydans
Rhodotorula sp.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
tion of intermediates in multiple-step synthesis [16]. In nature,
multiple biocatalysts are common within a microorganism for
Optically pure secondary alcohols are widely used in pharma-
ceuticals, flavors, agricultural chemicals and specialty materials.
Nevertheless, only one enantiomer (A) is usually needed for the
synthesis of a final product, and there is little (or no) use for the
opposite enantiomer (B), it has to be regarded as “waste” and
thus represents a considerable economic burden [1]. Therefore,
stereoinversion of undesired enantiomer (B) to the other (A) or der-
acemization of racemates (A/B) are both two ideal ways to produce
the desired optically pure enantiomer (A). Since the first compre-
hensive review by Carnell [2] on deracemization of chiral secondary
alcohols by stereoinversion, an impressive number of reports [3–9]
appeared recently and several recent reviews have touched this
topic in part [1,10–12].
The aim to obtain a highly valuable enantiopure product in
100% yield and with 100% ee (enantiomeric excess) from a cheap
racemic substrate or the “waste” enantiomer in a one-pot pro-
cess with multiple catalysts is currently a hot topic [3,13–15].
Catalytic (asymmetric) tandem reactions are processes in which
multiple catalysts operate concurrently in one pot, circumventing
the often time-consuming, yield-reducing isolation and purifica-
metabolizing natural compounds such as glucose. Nevertheless,
a microorganism often does not have the appropriate active and
selective enzymes for desired multiple-step chemical synthesis
when non-natural compounds are used as substrates [17]. Alterna-
tively, the best enzyme for each reaction step may be selected from
different microorganisms, mixed in different forms, and adjusted
to the desired ratio for efficient tandem transformation. It is per-
haps most important that the different catalytic reactions should
not interfere with one other. Hummel and Riebel [18] had reported
a stepwise route to enantiomerically pure alcohols from the cor-
responding racemates by employing two stereocomplementary
alcohol dehydrogenases. The stereoinversion and deracemization
of sec-alcohols by free oxidoreductases were also reported by
Kroutil and his coworkers in their recent publication [16]. However,
external addition of both the cofactor and the isolated or commer-
cially purified enzymes were necessary in these reports. Comparing
with isolated enzymes, the application of the whole-cell biocata-
lysts for biotransformation brought about more advantages, during
which the enzymes are often more stable due to the presence of
their natural environment inside the cell and the cost is much lower
because of no requirements for tedious separation of enzymes
and the regeneration of expensive cofactors (NADH/NADPH). There
were only limited reports on the deracemization of secondary
alcohol or hydroxyl acid by using growing or resting cells as biocata-
∗
Corresponding author. Tel.: +86 21 6425 2498; fax: +86 21 6425 2250.
E-mail address: jianhexu@ecust.edu.cn (J.-H. Xu).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.01.031

Y.-L. Li et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 48–52
49
lysts, such as sec-alcohol [19], mandelic acid [20] and 1,2-octanediol
[21]. In the former case, the result was not satisfactory for the der-
acemization of 1a and the product (R)-1a was obtained in only 80%
yield and 85% ee [19].
In this paper, a tandem biocatalysts system was successfully
developed for the stereoinversion of (R)-1-phenylethanol and der-
acemization of various aryl secondary alcohols by using highly
selective biocatalyst for each step and mixed them in suitable forms
for efficient tandem transformation. A variety of enantiopure (S)-
alcohols were successfully prepared in a one-pot procedure in good
yield and excellent ee.
Tris–HCl buffer (10 mL, 50 mM, pH 8.5). The reaction mixtures
were incubated in a screw-capped 100-mL flask, shaken at 30 ◦C
and 180 rpm for time necessary to obtain an appropriate conver-
sion. The biotransformation was stopped by addition of the same
volume of ethyl acetate and extracted for two times. After cen-
trifugation at 12,000 rpm for 10 min, the organic phase was dried
over anhydrous Na2SO4. Chemical yield and ee of the products were
determined by GC or HPLC analysis. The product was identified
by 1H NMR analysis. The absolute configuration was determined
by the specific rotation and comparison with the literature or
by chiral GC or HPLC analysis and comparison with known race-
mates.
2. Experimental
2.4. Assignment of the absolute configuration of the alcohols
1a–1g
Gas chromatographic (GC) analyses were performed using a chi-
ral GC-column (Betadex-120, 30 m × 0.25 mm × 0.25 ␮m, Supelco,
USA). HPLC analyses were performed using a chiral column (Chi-
ralcel OD, Ø0.46 cm × 25 cm, Daicel Chemical Industries, Japan).
Compound (S)-1a: [˛]D25 = −58.2 (c 1.03, CHCl3) {lit. [22] [˛]25
=
=
=
=
=
=
=
D
−55.1 (c 1.63, CHCl3), S}.
Compound (S)-1b: [˛]D25 = −31.9 (c 1.2, EtOH) {lit. [23] [˛]2D5
−29.7 (c 2.59, EtOH), S}.
2.1. Cultivation of microbes
Compound (S)-1c: [˛]D25 = −29.5 (c 1.11, CHCl3) {lit. [24] [˛]25
D
−39.5 (c 1.21, CHCl3), S}.
Microbacterium oxydans ECU2010, which was deposited in China
General Microbiological Culture Collection Center with an acces-
sion number of CGMCC No. 1875 was grown in a medium consisting
of glucose (15 g/L), beef extract (30 g/L), peptone (20 g/L), KH2PO4
(0.5 g/L), K2HPO4 (0.5 g/L), NaCl (1 g/L) and MgSO4 (0.5 g/L) while
Rhodotorula sp. AS2.2241, which was also deposited in China Gen-
eral Microbiological Culture Collection Center with an accession
number of CGMCC No. 1735 was grown in another medium consist-
ing of glucose (15 g/L), yeast extract (5 g/L), peptone (5 g/L), KH2PO4
(0.5 g/L), K2HPO4 (0.5 g/L), NaCl (1 g/L) and MgSO4 (0.5 g/L). After
adjusting to pH 7.0 using 2 M NaOH solution, 100 mL of the medium
was placed in a 500-mL Erlenmeyer flask, sterilized (121 ◦C, 20 min)
and inoculated with the preincubated culture, shaken at 30 ◦C and
180 rpm for 24 h.
Compound (S)-1d: [˛]D25 = −43.1 (c 1.15, CHCl3) {lit. [22] [˛]25
D
−37.9 (c 1.13, CHCl3), S}.
Compound (S)-1e: [˛]D25 = −30.6 (c 0.98, CHCl3) {lit. [25] [˛]25
D
−27.3 (c 0.53, CHCl3), S}.
Compound (S)-1f: [˛]D25 = −25.1 (c 0.41, CHCl3) {lit. [26] [˛]25
D
−30.3 (c 0.996, CHCl3), S}.
Compound (S)-1g: [˛]D25 = −39.9 (c 1.07, MeOH) {lit. [27] [˛]2D5
−37.2 (c 1.01, MeOH), S}.
The 1H NMR spectra of these compounds were in agreement
with those reported in the literatures [28–30].
3. Results and discussion
2.2. Selective oxidation of aryl secondary alcohols with M.
oxydans ECU2010
In our previous work, a novel microbial strain was isolated
from soil and identified as M. oxydans ECU2010 [31], which
could enantioselectively catalyze the dehydrogenation of (R)-
enantiomer of rac-1-phenylethanol rac-1a to acetophenone 2a
in an anti-Prelog mode via a NAD+-dependent (R)-alcohol dehy-
drogenase (ADH) initiated pathway, retaining (S)-1-phenylethanol
(S)-1a in high enantioselectivity. A red yeast strain, Rhodotorula sp.
AS2.2241, was also successfully isolated from soil samples, which
could stereoselectively reduce 2a to yield (S)-1a in excellent ee
via a NADPH-dependent (S)-stereoselective ketoreductase (KER)
[30,32,33].
Fresh cells (0.02 g) of M. oxydans ECU2010 and substrate
(0.02 mM) were suspended in a screw-capped 10-mL test tube
containing Tris–HCl buffer (1 mL, 50 mM, pH 8.5) and shaken at
180 rpm for 24 h at 30 ◦C. Then the biotransformation was stopped
by addition of the same volume of ethyl acetate and extracted
for two times. After centrifugation at 12,000 rpm for 10 min, the
organic phase was dried over anhydrous Na2SO4. Conversion and
ee of products were determined by GC or HPLC analysis.
2.3. Deracemization of various aryl secondary alcohols by
combination of resting cells of M. oxydans ECU2010 and
Rhodotorula sp. AS2.2241
3.1. Deracemization of rac-1-phenylethanol by whole-cell
biotransformation
We attempted to put two kinds of biocatalysts together to real-
ize the stereoinversion of (R)-1a to (S)-1a via a cascade one-pot
process involving the enantioselective oxidation of (R)-1a to 2a
catalyzed by M. oxydans ECU2010 followed with the asymmetric
To obtain enantiopure aryl secondary alcohols (S)-1, rac-1
(20–40 mg) and resting cells of M. oxydans ECU2010 (wcw: 0.2 g)
and Rhodotorula sp. AS2.2241 (wcw: 1.8 g) were suspended in
Table 1
Biocatalytic stereoinversion or deracemization of (R)-1-phenylethanol through a tandem stereoselective oxidation–reduction sequence with fresh cells of Microbacterium
oxydans ECU2010 and Rhodotorula sp. AS2.2241.
Entry
Substrate
Concentration (mM)
M. oxydans ECU2010 (g L−1
)
Rhodotorula sp. AS2.2241 (g L−1
)
Time (h)
(S)-1aa (%)
eeb (%)
1
2
3
4
(R)-1a
(R)-1a
rac-1a
rac-1a
30
50
30
70
20
20
20
30
180
180
180
270
32
48
24
32
97.2
85.1
98.2
96.7
>99
92.9
>99
>99
a
The relative amount was determined by GC analysis.
The enantioselectivity was determined by GC analysis, ee = ([S] − [R]/[S] + [R]) × 100%, where [S] and [R] denote the concentrations of (S)-1a and (R)-1a.
b

50
Y.-L. Li et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 48–52
reduction of 2a to (S)-1a catalyzed by Rhodotorula sp. AS2.2241 in
tandem.
In initial explorative experiments, we attempted to combine
the lyophilized cells of M. oxydans ECU2010 and Rhodotorula sp.
AS2.2241 for stereoinversion of (R)-1a to (S)-1a. But the result was
not satisfactory: only moderate yield and ee value were obtained.
Similar results were given using lyophilized cell free extract (data
not shown). As the lyophilization process will partially permeabi-
lize the cell membrane, the in vivo cofactor regeneration system
inside the cells may be affected. Further study revealed that the
cell free extract (CFE) of M. oxydans ECU2010 can catalyze not only
the oxidation of (R)-1a to 2a using NAD+ as cofactor, but also the
reduction of 2a to (R)-1a with NADPH as cofactor. The CFE (cell
free extract) of Rhodotorula sp. AS2.2241 can catalyze the reduc-
tion of 2a to (S)-1a and also the oxidation of (S)-1a to 2a at the same
time. The reaction rate of reduction was faster than that of oxida-
tion. When using the freshly harvested cells as biocatalyst, only one
direction of reaction happened, that is, M. oxydans ECU2010 only
catalyze the selective oxidation of (R)-1a to 2a and Rhodotorula sp.
AS2.2241 only catalyze the asymmetric reduction of 2a to (S)-1a.
Indeed, the combination of freshly harvested cells of M. oxydans
ECU2010 and Rhodotorula sp. AS2.2241 finally led to a successful
system for concurrent stereoinversion.
Due to different catalytic activities, the resting cells of M. oxy-
dans ECU2010 (8.9 U g−1 wet cell weight (wcw)) and Rhodotorula
sp. AS2.2241 (0.15 U g−1 wcw) were combined at different ratios of
wcw for stereoinversion of (R)-1a (Table 1). (R)-1a (30 mM) could
be transformed into (S)-1a (>99% ee) in 97.2% yield with only a very
little amount of 2a (Table 1, entry 1) within 32 h. A typical time
course of the stereoinversion process is shown in Fig. 1A, indicat-
ing that the oxidation step is the faster step of the overall reaction.
When increasing the substrate concentration to 50 mM, (S)-1a was
obtained in 85.1% yield with 92.9% ee (Table 1, entry 2).
Fig. 1. Relative amounts of enantiomers of 1a in (A) stereoinversion of (R)-1a
(30 mM) and (B) deracemization of rac-1a (30 mM) with tandem whole-cell bio-
catalysts of ECU2010 and AS2.2241.
As the stereoinversion process was quite efficient, we applied
this method to the deracemization of rac-1a. It can be seen from
Fig. 1B that the amount of (R)-1a decreased rapidly to zero at 8 h
due to the oxidation; 2a was formed and further transformed to
Table 2
Selective oxidation of various aryl secondary alcohols catalyzed by Microbacterium oxydans ECU2010.
.
Entry
Substrate
Time (h)
Conversiona (%)
eea (%)
Configurationc
1
2
3
4
5
6
7
rac-1a
rac-1b
rac-1c
rac-1d
rac-1e
rac-1f
rac-1g
24
24
24
24
24
24
40
>49.9
>49.9
>49.9
>49.9
>49.9
>49.9b
>49.9
>99
>99
>99
>99
>99
>99b
>99
S
S
S
S
S
S
S
a
Determined by GC analysis.
Determined by HPLC using Chiralcel OD-H column.
Absolute configurations were assigned by comparison of the specific rotation with the literature values.
b
c

Y.-L. Li et al. / Journal of Molecular Catalysis B: Enzymatic 64 (2010) 48–52
51
Table 3
Deracemization of various aryl secondary alcohols through a tandem stereoselective oxidation–reduction sequence with fresh cells of Microbacterium oxydans ECU2010 and
Rhodotorula sp. AS2.2241.
.
Entry
Substrate
S-1a,b (%)
eea (%)
Configurationd
1
2
3
4
5
6
7
rac-1a
rac-1b
rac-1c
rac-1d
rac-1e
rac-1f
rac-1g
95.1 (56.3)
98.2 (73.6)
>99 (68.1)
98.1 (84.3)
98.3 (42.6)
86.5c (53.6)
>99 (77.2)
>99
>99
>99
>99
>99
>99c
>99
S
S
S
S
S
S
S
a
Determined by GC analysis.
Isolated yield in parentheses.
Determined by HPLC using Chiralcel OD-H column.
b
c
d
Absolute configurations were assigned by comparison of the specific rotation with the literature values.
(S)-1a; the amount of (S)-1a increased with time until 16 h; and
nearly nothing happened between 16 h and 32 h. Finally, (S)-1a was
obtained in 98.2% yield with >99% ee (Table 1, entry 3).
acemization of various aryl secondary alcohols with resting cells
of M. oxydans ECU2010 and Rhodotorula sp. AS2.2241. A variety
of enantiopure (S)-aryl alcohols were successfully prepared in a
one-pot procedure with good yield and excellent ee. Our method
represents an easy going, cheap and environmentally benign way
for the biocatalytic synthesis of chiral aryl secondary alcohols from
their racemates.
To achieve higher product concentration, deracemization of
70 mM rac-1a with higher concentrations of M. oxydans ECU2010
cell (30 g L−1) and Rhodotorula sp. AS2.2241 cell (270 g L−1) was per-
formed, giving (S)-1a in 96.7% yield and >99% ee (Table 1, entry
4). The specific activity for the reduction with Rhodotorula sp.
AS.2.4421 cells was rather low (0.15 U g−1 wcw), as compared to the
oxidation with M. oxydans ECU2010 cells (8.9 U g−1 wcw). There-
fore, engineering and use of a recombinant strain expressing highly
active KER (ketoreductase) may be considered in the future to fur-
ther improve the productivity of such a tandem biocatalysts system.
Acknowledgements
The financial supports from National Natural Science Foun-
dation of China (Grant Nos. 20402005 and 20773038), Ministry
of Science and Technology (Grant Nos. 2006AA02Z205 and
2007AA02Z225) and China National Special Fund for State Key
Laboratory of Bioreactor Engineering (Grant No. 2060204) are
gratefully acknowledged.
3.2. Selective oxidation of aryl secondary alcohols by M. oxydans
ECU2010
To examine the generality of this method, several aryl secondary
alcohols 1a–1g were subjected to the enantioselective oxidation
reactions catalyzed by M. oxydans ECU2010 in an anti-Prelog mode.
The results are summarized in Table 2. M. oxydans ECU2010 dis-
played high yield (>49.9%) and excellent enantioselectivity (>99%
ee) for all the substrates tested (1a–1g).
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In summary, a tandem biocatalysts system was successfully
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