Exploiting the enzymatic machinery of Arthrobacter atrocyaneus for oxidative kinetic resolution of secondary alcohols

Article abstract of DOI:10.1016/j.molcatb.2012.06.013

We evaluated Arthrobacter atrocyaneus (R1AF57) as producer of oxidoreductases for oxidative kinetic resolution of racemic secondary alcohols via oxidation reaction. This bacterium was isolated from Amazon soil samples using medium enriched with (RS)-1-(4-methylphenyl)ethanol as a carbon source. The kinetic resolution of several secondary alcohols through enantioselective oxidation mediated by resting cells and growing cells of A. atrocyaneus was efficiently achieved for the most alcohols. In general, it was possible to obtain only the (S)-enantiomer from (RS)-1-arylethanols.

Full text of DOI:10.1016/j.molcatb.2012.06.013

Journal of Molecular Catalysis B: Enzymatic 83 (2012) 23–28
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Exploiting the enzymatic machinery of Arthrobacter atrocyaneus for oxidative
kinetic resolution of secondary alcohols
a
b
a
a
a
Camila R. Silva , Juliana C. Souza , Lidiane S. Araújo , Edna Kagohara , Thaís P. Garcia ,
b
a,∗
Vivian H. Pelizzari , Leandro H. Andrade
Institute of Chemistry, University of São Paulo, Av. Prof. Lineu Prestes 748, SP 05508-900 São Paulo, Brazil
Institute of Oceanography, University of São Paulo, Pra c¸ a do Oceanográfico 191, SP 05508-120 São Paulo, Brazil
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
We evaluated Arthrobacter atrocyaneus (R1AF57) as producer of oxidoreductases for oxidative kinetic
resolution of racemic secondary alcohols via oxidation reaction. This bacterium was isolated from Amazon
soil samples using medium enriched with (RS)-1-(4-methylphenyl)ethanol as a carbon source. The kinetic
resolution of several secondary alcohols through enantioselective oxidation mediated by resting cells and
growing cells of A. atrocyaneus was efficiently achieved for the most alcohols. In general, it was possible
to obtain only the (S)-enantiomer from (RS)-1-arylethanols.
Received 6 March 2012
Received in revised form 11 June 2012
Accepted 24 June 2012
Available online 13 July 2012
Keywords:
©
2012 Elsevier B.V. All rights reserved.
Arthrobacter atrocyaneus
Oxidoreductases
Alcohol dehydrogenase
Chiral alcohols
1
. Introduction
that are able to accept non-natural substrates, and also con-
tain an appropriate cofactor for the oxidoreduction reaction [16].
Therefore, the use of an external system for cofactor regenera-
tion may be omitted because it is automatically performed by cell.
In addition, enzymes and cofactors are well protected inside cell
[17].
This work aims at the use of Arthrobacter atrocyaneus (R1AF57)
in oxidative kinetic resolution (OKR) of racemic secondary alcohols
to achieve optically pure compounds. This bacterium was isolated
by enrichment technique using soil collected from the environmen-
tal conservation area (Amazon forest, Brazil).
Biooxidations of alcohols to ketones mediated by isolated oxi-
doreductases and whole cell systems are well established [1–4].
However, a literature survey revealed that there are fewer reports
on enantioselective enzymatic oxidation of alcohols than enan-
tioselective reduction of ketones to achieve chiral alcohols. These
compounds are interesting chiral building blocks for the synthe-
sis of natural products, pharmaceutical, and agriculture chemicals
[
1,5–9].
Enzymes such as hydrolases and oxidoreductases are generally
used for kinetic resolutions, because they can transform selectively
one enantiomer from a racemic mixture into a new compound,
enabling physical separation after the reaction [10–12]. Oxidore-
ductases used in the oxidation of secondary alcohols to ketones are
classified into two categories: alcohol dehydrogenase and alcohol
oxidase [13].
2
. Experimental
2.1. General methods
Chemicals were purchased from Aldrich and used without
Alcohols dehydrogenases (ADH’s) are most commonly used for
enantioselective alcohol oxidation and ketone reduction. These
further purification. Gas chromatography (GC) analyses were per-
formed on a Shimadzu GC-17A instrument equipped with a flame
ionization detector. High performance liquid chromatography
+
+
enzymes are NAD -dependent or NADP -dependent and can cat-
alyze the oxidation of secondary alcohols to ketones, and primary
alcohols to aldehydes [14,15]. ADHs are enzymes present in all liv-
ing organisms. Microbial cells contain multiple dehydrogenases
(HPLC) analyses were performed on a Shimadzu, LC-10AD liq-
uid chromatography equipped with a variable wavelength UV–VIS
detector (SPD-10AV). HPLC grade solvents, i-PrOH and hexane (60%
n-hexane), were purchased from J.T. Baker. Sterile materials were
used to perform the experiments involving microorganisms. A lam-
inar flow cabinet Fisher Hamilton (Class II Biological Safety Cabinet)
was used to handle microorganisms.
∗ Corresponding author.
E-mail address: leandroh@iq.usp.br (L.H. Andrade).
1
381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.06.013

2
4
C.R. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 23–28
◦
2
.2. Synthesis of the racemic alcohols 1–26 [18]
(RS)-1-phenyl-ethanol (1): 107 C, tR = 6.22 min for (R) and
.87 min for (S);
6
◦
The preparation of alcohols 1–26 was according to the method-
(RS)-1-(4-methyl-phenyl)-ethanol (2): 113 C, tR = 6.79 min for (R)
ology previously established [19].
and 7.71 min for (S);
◦
(
(
(
RS)-1-(4-methoxy-phenyl)-ethanol (3): 119 C, tR = 6.22 min for
R) and 6.87 min for (S);
RS)-1-(4-chloro-phenyl)-ethanol (4): 130 C, tR = 7.73 min for (R)
2.3. General procedure for prospection of
◦
oxidoreductases-producing microorganisms
and 8.19 min for (S);
RS)-1-(4-bromo-phenyl)-ethanol (5): 135 C, tR = 9.54 min for (R)
and 10.52 min for (S);
RS)-1-(4-nitro-phenyl)-ethanol (6): 150 C, tR = 19.92 min for (R)
and 20.3 min for (S);
RS)-1-(2-methyl-phenyl)-ethanol (7): 123 C, tR = 8.00 min for (R)
and 10.0 min for (S);
◦
(
Soil samples were collected from environmental preservation
area in Monte Negro city (Amazon forest located in Rondônia State,
Brazil). The samples were collected under clean, sterile conditions
◦
(
◦
and kept frozen in polyethylene tubes at −20 C. Twenty bacterial
◦
(
strains were isolated after applying enrichment technique using
a minimal salt medium and 1-(4-methyl-phenyl)ethanol as car-
bon source (0.5 mmol/L) according to the method described in the
literature [20–26].
◦
(
(
(
(
(
RS)-1-(3-methyl-phenyl)-ethanol (8): 113 C, tR = 10.73 min for
R) and 11.77 min for (S);
RS)-1-(2-methoxy-phenyl)-ethanol (9): 129 C, tR = 8.85 min for
◦
Enzyme activity for all these bacterial strains were evaluated
according to methodology described by us [27]. The best perfo-
mance for the oxidative kinetic resolution of secondary alcohols
was observed by A. atrocyaneus (R1AF57).
R) and 9.72 min for (S);
RS)-1-(3-methoxy-phenyl)-ethanol (10): 119 C, tR = 17.93 min
◦
for (R) and 20.0 min for (S);
◦
◦
Oven temperature: 100–140 C, 3 C/min:
2.4. Culture medium
The bacteria A. atrocyaneus (R1AF57) was grown in Erlen-
(RS)-1-(2-fluor-phenyl)-ethanol (11): t = 6.39 min for (R) and
R
meyer flasks containing culture medium (nutrient broth, NB
medium = beef extract 5 g/L; peptone 3 g/L) supplemented with
6.84 min for (S);
(RS)-1-(3-fluor-phenyl)-ethanol (12): t = 5.83 min for (R) and
6.84 min for (S);
(RS)-1-(4-fluor-phenyl)-ethanol (13): t = 5.67 min for (R) and
R
(
RS)-1-(4-methyl-phenyl)-ethanol (0.5 mmol/L). The culture was
◦
incubated in an orbital shaker (160 rpm) at 30 C.
R
6
(
.13 min for (S);
RS)-1-(2-chloro-phenyl)-ethanol (14): 130 C, tR = 7.09 min for (R)
and 8.92 min for (S);
RS)-1-(3-chloro-phenyl)-ethanol (15): 130 C, tR = 7.76 min for (R)
and 8.34 min for (S);
◦
2.5. Growing cells assays
◦
(
A. atrocyaneus was inoculated in 50 mL of appropriate culture
medium in Erlenmeyer flasks (125 mL) and incubated in an orbital
◦
◦
(RS)-1-(2-bromo-phenyl)-ethanol (16): 145 C, t = 7.09 min for (R)
shaker for 2 days (160 rpm, 32 C). After that, each substrate (1–28,
0 mg diluted in 100 ␮L of dimethylformamide) was added under
R
and 9.12 min for (S);
(RS)-1-(3-bromo-phenyl)-ethanol (17): 135 C, t = 11.9 min for (R)
and 13.08 min for (S);
2
◦
◦
sterile conditions in the flasks and kept at 32 C in an orbital shaker
160 rpm) for different reaction time (Tables 2 and 5). The reaction
R
(
◦
(RS)-1-(2-nitro-phenyl)-ethanol (18): 150 C, tR = 9.61 min for (R)
content (10 mL) was treated with ethyl acetate (2 mL) followed by
centrifugation (6000 rpm, 1 min). The organic phase was analyzed
by GC or HPLC (see Section 2.7 or Section 2.8).
and 11.43 min for (S);
RS)-1-(3-nitro-phenyl)-ethanol (19): 140 C, tR = 25.28 min for (R)
and 27.25 min for (S);
RS)-1-phenylpropan-1-ol (26): 117 C, tR = 5.2 min for (R) and
.73 min for (S);
RS)-1-phenylpropan-2-ol (27): 97 C, tR = 17.34 min for (R) and
8.5 min for (S);
◦
(
◦
(
5
(
1
2.6. Resting cells assays
◦
A. atrocyaneus was inoculated in 50 mL of appropriate culture
medium in Erlenmeyer flasks (125 mL) and incubated in an orbital
◦
shaker for 2 days (160 rpm, 32 C). Bacterial cells were harvested
◦ ◦
Oven temperature: 60–85 C, 1.5 C/min, hold 3 min:
◦
by centrifugation at 4 C (5000 rpm) and resuspended in phosphate
buffer solution (50 mL, pH 7.0, 100 mmol/L). After that, each sub-
strate (1–28, 20 mg diluted in 100 ␮L of dimethylformamide) was
added under sterile conditions in the flasks and kept at 32 C in an
orbital shaker (160 rpm) for different reaction time (Tables 2 and 5).
The reaction content (10 mL) was treated with ethyl acetate (2 mL)
followed by centrifugation (6000 rpm, 1 min). The organic phase
was analyzed by GC or HPLC (see Section 2.7 or Section 2.8).
(
RS)-octan-2-ol (28): retention time of enantiomers (acety-
◦
lated) = 13.20 min and 16.94 min.
The enantiomeric excess was defined as the ratio of
{
([R] − [S])/([R] + [S])} × 100%, where [R] and [S] are the concentra-
tions of (R) and (S) enantiomers, respectively.
2.7. GC analysis
2.8. HPLC analysis
The enantiomeric excesses (ee) of the chiral alcohols 1–28 were
measured by chiral GC analysis [19,29–35]. The analysis was carried
The enantiomeric excesses of the chiral alcohols 20–25 were
out on a column ␤-ciclodextrin (25 m × 0.32 mm × 0.25 ␮m, Varian
measured by chiral HPLC analysis [36,37]. The analysis was carried
out on a column Chiralcel® OJ-H (0.46 cmϕ × 25 cm) or Chiralcel®
OD (0.46 cmϕ × 25 cm, Daicel Chemical).
CP-Chirasil-DEX CB).
◦
GCconditions(carriergas-H , 100 kPa):injector220 C; detector
2
◦
Chiralcel® OD column, n-hexane/i-PrOH (95:5), 1.0 mL min
−1
,
2
20 C.
Isotherm and retention time (tR) of the enantiomers:
254 nm UV detector; tR (min): retention time of chiral compounds:

C.R. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 23–28
25
ꢀ
RS)-1-([1,1 -biphenyl]-3-yl)ethanol (20): tR = 30.95 min for (R)
Table 1
(
Enantioselective oxidation of (RS)-1-(phenyl)ethanol (1) mediated by growing cells
of A. atrocyaneus using different amount of culture broth.
and 14.68 min for (S);
ꢀ
(
RS)-1-([1,1 -biphenyl]-4-yl)ethanol (21): tR = 13.95 min for (R)
and 12.52 min for (S);
RS)-(4-tolyl)phenylmethanol (22): tR = 12.03 min for (R) and
3.5 min for (S);
RS)-(4-isopropylphenyl)(phenyl)methanol (23): tR = 10.12 min
and 11.14 min;
RS)-(4-bromophenyl)phenyl)methanol (24): tR = 12.9 min for (R)
and 14.53 min for (S);
RS)-biphenyl-4-yl(phenyl)methanol (25): tR = 19.36 min for (R)
and 20.8 min for (S).
(
1
(
Culture broth (mL)a
Alcohol (%)b
ee (%)b
(
10
20
40
56
56
49
48
46
45
97 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
>99 (S)
(
60
80
100
2.9. Bacteria identification
◦
Reaction conditions: (RS)-alcohol 1 (20 mg/100 ␮L DMF), 32 C, 160 rpm, 72 h.
a
Culture broth containing growing cells of A. atrocyaneus.
Determined by GC analysis.
The bacteria with the best performance in enzyme assays were
identified using 16S rDNA sequencing analysis and comparison
with GenBank top hits after a BLAST search [28].
b
Table 2
Enantioselective oxidation of (RS)-1-(phenyl)ethanol 1 mediated by growing cells
of A. atrocyaneus at different reaction time.
3
. Results and discussion
Entry
Time (h)
Alcohol (%)a
ee (%)a
3
.1. Isolation of oxidoreductase-producing microorganisms
1
2
3
4
5
6
1
6
12
24
48
72
84
58
59
54
47
49
24 (S)
67 (S)
67 (S)
88 (S)
>99 (S)
>99 (S)
Twenty microorganisms from soil samples were isolated using
the enrichment technique. This technique consisted of using (RS)-
-(4-methylphenyl)ethanol (0.5 mM) as a carbon source in the
1
culture medium [26,27]. The employment of this chiral alcohol
can result in the isolation of microorganisms containing new oxi-
doreductases. The enzymatic system is able to perform the alcohol
oxidation, which can consists of a dehydrogenase or an oxidase.
However, after the enzyme purification, we will be able to elucidate
the enzyme responsible for the alcohol oxidation reaction.
Reaction conditions: (RS)-alcohol 1 (20 mg/100 ␮L DMF), growing cells of A. atro-
◦
cyaneus (20 mL), 32 C, 160 rpm, 1–72 h.
a
Determined by GC analysis.
excess >99%. Another set of reaction was performed in order to
have the appropriate time for the kinetic resolution of (RS)-1-
phenylethanol 1 (Table 2).
3.2. Evaluation of the enzyme activity of microorganisms
According to the results in Table 2, after 48 h the (R)-alcohol 1
was transformed to the corresponding ketone and leaving the (S)-
alcohol 1 unreacted with enantiomeric excess >99% (entry 5). From
these results we can assume that the (R)-enantiomer reacts faster
with an appropriate oxidoreductase from A. atrocyaneus than the
(S)-enantiomer.
Enantioselective oxidations of para-substituted (RS)-1-
phenylethanols (1–6) were used to evaluate the enzyme activity
oxidoreductase, OXRED) of the 20 microorganisms [27].
We obtained an excellent enzyme activity when we employed
R1AF57 (A. atrocyaneus) in the oxidative kinetic resolution process.
The oxidation reaction with this bacterium yielded (S)-alcohols in
up to 50% (maximum value for a kinetic resolution process) and
enantiomeric excess in up to >99%.
(
3.3. Application of the A. atrocyaneus in the oxidative kinetic
resolution of secondary alcohols (1–28)
Based on these results we decided to evaluate the enantioselec-
tive oxidation mediated by A. atrocyaneus using different reaction
conditions. Initially (RS)-1-phenylethanol was selected as a model
substrate and the ratio of bacterial cells versus substrate concen-
tration was evaluated (Table 1). This study was performed for
achieving efficient process using a minimal amount of enzymes.
The bacterial growth was initially performed in an appropriate
culture medium. Next, different volumes of medium containing the
growing cells were used in the oxidation reaction of (RS)-1.
The results presented in Table 1 show that the appropriate
ratio between culture broth and substrate is 20 mL/20 mg. In this
case, A. atrocyaneus had the ideal performance for oxidative kinetic
resolution, as the R-enantiomer was oxidized to the correspond-
ing ketone leaving the S-enantiomer unreacted with enantiomeric
Enantioselective oxidations of rac-1–6 and additional ortho-
meta- and para-substituted (RS)-1-(phenyl)ethanols (7–21,
,
Scheme 1 and Fig. 1) were used to evaluate the broad spectrum
of oxidoreductase activity from A. atrocyaneus. For this study, we
employed growing cells (Table 3) and resting cells (Table 4).
In general, the growth of A. atrocyaneus cells promoted
the oxidation of (R)-1-phenylethanol and its derivatives
to their corresponding ketones in high conversion leaving
the (S)-enantiomer intact and more efficient than reac-
tions with resting cells. As presented in Table
3 meta-
and para-substituted alcohols (1–6, 8, 10, 12, 13, 15, 17
and 19–21, entries 3, 4, 6, 7, 9, 10, 12, 13, 15, 16, 18 and
19) were transformed to corresponding ketones with 50%
Scheme 1. Enantioselective oxidation of secondary alcohols (RS)-1–28.

2
6
C.R. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 23–28
Fig. 1. Secondary alcohols 1–28 selected as substrates for oxidative kinetic resolution mediated by A. atrocyaneus.
conversion and >99% ee for the (S)-enantiomer, except for
compound 20 that showed low conversion and ee (Table 3, entry
with high concentration, which is similar to those obtained from
growing cells (Tables 3 and 4, entries 3–4, 6–7, 18–19 and 20–21).
The oxidation of alcohol (RS)-1, which has no substituent at the
aromatic ring, by growing cells showed high conversion to ketone
(51%) and excellent enantiomeric excess (>99%) for the remaining
alcohol enantiomer-(S), while resting cells gave moderate ketone
concentration (39%) and good enantiomeric excess (85%). Both
methodologies showed low product concentration (ketone) for the
ortho- substituted compounds (Tables 3 and 4, entries 2, 5, 8, 11,
14 and 17). A possible steric hindrance caused by these groups to
the hydroxyl moiety can be the reason for low or none alcohol
oxidation.
2
0).
The oxidation reactions of fluorine-, chlorine- and bromine-
substituted-1-(phenyl)ethanols mediated by resting cells exhibited
low ketone concentrations (up to 40%) and alcohol ee (up to 93%,
Table 4, entries 8–16). The additional time, 72 and 96 h, did not
improve the conversions or alcohol ee (data not demonstrated).
On the other hand, the reactions with growing cells gave better
results (up to 50% ketone yield and in up to >99% ee for the
(
S)-alcohol, Table 3, entries 8–10, 12, 13 and 15), which indicated
a good oxidative kinetic resolution process. Reactions of meta-
and para-substituted compounds, such as methyl, methoxy, nitro
and phenyl, by resting cells afforded the corresponding ketones
We also employed alcohols with two aromatic rings on car-
bon that has the hydroxyl group (compounds 22–25). However,

C.R. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 23–28
27
Table 3
Table 5
OKR of substituted (RS)-1-(phenyl)ethanols (1–21) mediated by growing cells of A.
atrocyaneus.
OKR of secondary alcohols (RS)-22–28 mediated by A. atrocyaneus.
Entry
Substrate
Time (h)
Alcohol (%)a
ee (%)a
Enzyme source
4
7
4
7
4
7
48
72
48
72
48
72
48
72
8
2
8
2
8
2
>99
>99
>99
>99
>99
>99
>99
>99
59
53
45
50
>99
>99
0
0
0
0
0
0
0
0
1
2
3
4
5
6
7
22
23
24
25
26
27
28
Entry
R
Alcohol (%)a
ee (%)a
1
2
3
4
5
6
7
8
9
H (1)
49
99
56
50
94
49
50
97
52
47
99
51
51
99
55
49
99
46
50
69
46
>99 (S)
<1
>99 (S)
>99 (S)
8
>99 (S)
>99 (S)
4
>99 (S)
>99 (S)
<1
>99 (S)
>99 (S)
<1
>99 (S)
>99 (S)
<1
>99 (S)
>99 (S)
13 (S)
>99 (S)
Growing cellsb
2-Me (7)
3-Me (8)
4-Me (2)
2-OMe (9)
3-OMe (10)
4-OMe (3)
2-F (11)
82 (S)
95 (S)
>99 (S)
>99 (S)
20 (S)
20 (S)
3-F (12)
4-F (13)
48
72
48
72
48
72
48
72
48
72
48
72
>99
>99
>99
>99
>99
>99
>99
>99
54
55
52
52
94
0
0
0
0
0
0
0
0
10
11
12
13
14
15
16
17
18
19
20
21
8
22
23
24
25
26
2-Cl (14)
3-Cl (15)
4-Cl (4)
2-Br (16)
3-Br (17)
4-Br (5)
2-NO2 (18)
3-NO2 (19)
4-NO2 (6)
3-Ph (20)
4-Ph (21)
9
10
11
12
13
Resting cellsc
81 (S)
93 (S)
92 (S)
>99 (S)
46 (S)
27
28
14
48
Reaction conditions: (RS)-alcohol 1–21 (20 mg/100 ␮L DMF), growing cells of A.
◦
Reaction conditions: (RS)-alcohols 22–28 (20 mg/100 ␮L DMF), 32 C, 160 rpm.
◦
atrocyaneus (20 mL), 32 C, 160 rpm, 48 h.
a
Determined by GC analysis.
Culture broth (20 mL).
a
Determined by GC analysis.
b
c
Cells were harvested from culture broth (20 mL) and resuspended in phosphate
buffer solution (100 mmol/L, pH 7.0, 20 mL).
Table 4
OKR of substituted (RS)-1-(phenyl)ethanols (1–21) mediated by resting cells of A.
atrocyaneus in phosphate buffer.
(1-phenyl-1-propanol) has hydroxyl and ethyl groups attached to
the benzylic carbon while the alcohol 27 (1-phenyl-2-propanol)
has one more carbon between the functional group alcohol and the
aromatic ring. These compounds enabled to evaluate the need for
the presence of the aromatic ring attached to the carbon that has
the hydroxyl group.
Entry
R
Alcohol (%)a
ee (%)a
The oxidation reaction of the compounds 26 and 27 mediated
by resting cells and growing cells afforded their corresponding
ketones, all in high conversion (∼47–50%) and high enantiomeric
excess (∼95–99%) after 72 h (Table 5). In spite of the different posi-
tion of the hydroxyl group, the conversion values and enantiomeric
excess were closer to the ideal conditions for kinetic resolution of
alcohols.
Finally, we have decided to study the oxidation reaction of an
aliphatic compound 28. In this case, the enantioselective oxida-
tion reactions mediated by resting cells were more efficient than
growing cells (Table 5, entries 8–14). It is noteworthy that in the
reaction with growing cells the 2-octanone was not detected, even
though the remaining alcohol presented 20% enantiomeric excess.
Based on this result we may have an alcohol oxidation followed
by ketone oxidation (Baeyer–Villiger reaction) or an alcohol oxida-
tion followed by ketone reduction, resulting in a deracemization
process.
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
H (1)
61
99
53
51
97
53
56
99
60
63
99
82
87
99
86
86
99
48
48
63
47
85 (S)
<1
>99 (S)
>99 (S)
4
>99 (S)
>99 (S)
<1
88 (S)
93 (S)
<1
22 (S)
24 (S)
<1
23 (S)
23 (S)
<1
>99 (S)
>99 (S)
21 (S)
>99 (S)
2-Me (7)
3-Me (8)
4-Me (2)
2-OMe (9)
3-OMe (10)
4-OMe (3)
2-F (11)
3-F (12)
4-F (13)
1
1
1
1
1
1
1
1
1
1
2
2
2-Cl (14)
3-Cl (15)
4-Cl (4)
2-Br (16)
3-Br (17)
4-Br (5)
2-NO2 (18)
3-NO2 (19)
4-NO2 (6)
3-Ph (20)
4-Ph (21)
4. Conclusion
Reaction conditions: (RS)-alcohol 1–21 (20 mg/100 ␮L DMF), cells were har-
vested from culture broth (20 mL) and ressuspended in phosphate buffer solution
◦
(
100 mmol/L, pH 7.0, 20 mL), 32 C, 160 rpm, 72 h.
Determined by GC analysis.
We have isolated the A. atrocyaneus from medium enriched with
RS)-1-(4-methylphenyl)ethanol as a carbon source. The oxidative
a
(
kinetic resolution of several secondary alcohols through enantios-
elective oxidation mediated by resting cells and growing cells of
A. atrocyaneus was efficiently achieved for most of the alcohols. In
general, we were able to obtain only the (S)-enantiomer from (RS)-
1-phenylethanols and its derivatives with high conversion values
to corresponding ketones (∼50%). We are currently studying the
we did not observe any oxidation product (Table 5). This difficulty
has already been described in the literature [38]. Unfortunately, we
have not overcome.
Then, we decided to study the influence of the hydroxyl
group position at the phenyl-propanol. The compound 26

2
8
C.R. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 83 (2012) 23–28
enzyme purification of A. atrocyaneus. These studies will indicate
the appropriate oxidorecutase for oxidative kinetic resolution.
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Acknowledgments
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18] L.H. Andrade, R. Polak, A.L.M. Porto, I.H. Schoenlein-Crusius, J.V. Comasseto,
Lett. Org. Chem. 3 (2006) 613–618.
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We thank CNPq, CAPES and FAPESP for their financial support.
The authors also would like to thank Priscila I. Ushimaru for helping
us in the bacteria identification.
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