Screening of microorganisms producing cold-active oxidoreductases to be applied in enantioselective alcohol oxidation. An antarctic survey

Article abstract of DOI:10.3390/md9050889

Several microorganisms were isolated from soil/sediment samples of Antarctic Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms, 15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the (S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent enzymatic activity. These new bacteria strains were selected for optimization study, in which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction conditions. From these studies, it was observed that Flavobacterium sp. has an excellent enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C. We have also determined the growth curves of these bacteria, and both strains showed optimum growth at 25 °C, indicating that these bacteria are psychrotroph.

Full text of DOI:10.3390/md9050889

Mar. Drugs 2011, 9, 889-905; doi:10.3390/md9050889
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Screening of Microorganisms Producing Cold-Active
Oxidoreductases to Be Applied in Enantioselective Alcohol
Oxidation. An Antarctic Survey
Lidiane S. Araújo 1, Edna Kagohara 1, Thaís P. Garcia 1, Vivian H. Pellizari 2 and
Leandro H. Andrade 1,*
1
Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, SP 05508-900,
São Paulo, Brazil; E-Mails: lidianepiaui@yahoo.com.br (L.S.A); ednakb@iq.usp.br (E.K.);
thais_pelegrin@yahoo.com.br (T.P.G)
2
Instituto Oceanográfico, Universidade de São Paulo, Praça do Oceanográfico 191, SP 05508-120,
São Paulo, Brazil; E-Mail: vivianp@usp.br (V.H.P.)
* Author to whom correspondence should be addressed; E-Mail: leandroh@iq.usp.br;
Tel.: +55-11-3091-2287; Fax: +55-11-3815-5579.
Received: 20 April 2011; in revised form: 17 May 2011 / Accepted: 20 May 2011 /
Published: 24 May 2011
Abstract: Several microorganisms were isolated from soil/sediment samples of Antarctic
Peninsula. The enrichment technique using (RS)-1-(phenyl)ethanol as a carbon source
allowed us to isolate 232 psychrophile/psychrotroph microorganisms. We also evaluated
the enzyme activity (oxidoreductases) for enantioselective oxidation reactions, by using
derivatives of (RS)-1-(phenyl)ethanol as substrates. Among the studied microorganisms,
15 psychrophile/psychrotroph strains contain oxidoreductases that catalyze the
(S)-enantiomer oxidation from racemic alcohols to their corresponding ketones. Among the
identified microorganisms, Flavobacterium sp. and Arthrobacter sp. showed excellent
enzymatic activity. These new bacteria strains were selected for optimization study, in
which the (RS)-1-(4-methyl-phenyl)ethanol oxidation was evaluated in several reaction
conditions. From these studies, it was observed that Flavobacterium sp. has an excellent
enzymatic activity at 10 °C and Arthrobacter sp. at 15 and 25 °C . We have also determined
the growth curves of these bacteria, and both strains showed optimum growth at 25 °C ,
indicating that these bacteria are psychrotroph.

Mar. Drugs 2011, 9
890
Keywords: oxidoreductases; chiral alcohols; psychrophile; psychrotroph; oxidation;
deracemisation
1. Introduction
Enantiomerically pure alcohols are important precursors in the synthesis of bioactive compounds,
especially in agrochemical and pharmaceutical industries [1]. Biocatalytic transformation, an
enzyme-catalyzed reaction, is one of the techniques that have been rapidly developed in the last years
in the synthesis of chiral alcohols. The most employed enzymes for this process are alcohol
dehydrogenases, a subclass of oxidoreductases [2,3]. The biocatalytic reactions can be performed by
isolated enzymes or whole-cell system, which may be microorganisms or plant cells [4]. It is well
known that the screening of a wide variety of microorganisms living in the soil is an efficient method
to obtain the desired enzyme [5–7]. In this context, unusual sources, such as microorganisms from
extreme environments, have called the scientists attention for the possibility to find new biocatalysts or
natural products [8]. Psychrophile microorganisms contain cellular processes that are thoroughly
adapted to survival and proliferation at low temperatures and rates similar to those achieved by the
closely related species living in temperate environments. This implies that they have developed various
structural and physiological adjustments, enabling them to compensate the deleterious effects of low
temperatures. The two main physicochemical low temperatures effects are: chemical rates decrease
exponentially with decreasing temperature; temperature has also a strong effect on the viscosity of the
medium, thereby contributing to further slow down the reaction rates [9]. Several cold-adapted
enzymes from Antarctic bacterial strains have been studied [10–14]. The specific activities of these
enzymes are much higher than their mesophilic counterparts [9]. This is reflected by the significantly
lower activation energy values and the significant enzyme flexibility improvement, leading to a large
distribution of conformational isomers [15] accompanied by increased thermolability. In cold-adapted
enzymes, flexibility appears to play a crucial role in catalysis by securing the accommodation of the
substrate at low temperature, as well as by facilitating water molecules movement and products
release [9].
In this work we isolated several microorganisms from soil and sediment samples collected in the
Antarctic Continent to be applied in enantioselective oxidation of (RS)-1-phenylethanol derivatives.
The main goal of this work was to select microorganisms producing cold-active oxidoreductases to
perform alcohol oxidation in an enantioselective fashion.
2. Results and Discussion
2.1. Isolation of Microorganisms Producing Oxidoreductases
By using the enrichment technique 232 microorganisms from soils or sediments samples were
isolated (Table 1). This technique consisted of applying (RS)-1-phenylethanol (1 mM) as a carbon
source presented in the culture medium used in the isolation of microorganisms [16]. The use of this
chiral alcohol results in the isolation of microorganisms containing new oxidoreductases that can be

Mar. Drugs 2011, 9
891
applied in enantioselective alcohol oxidation. The enzymatic system able to perform the alcohol
oxidation can be consisting 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.
Table 1. Microorganisms isolated from soil and sediment samples.
Culture medium
MCBE7a
Microorganisms (soil)
Microorganisms (sediment)
103
96
17
16
MCFE7b
General conditions: 2 g soil or sediment; a pH 7, 2 days of incubation at 20 °C; b pH 5.6, 4 days of
incubation at 20 °C.
The results described in Table 1 demonstrated that the highest number of microorganisms was
obtained from soil samples and culture medium at neutral pH. The culture medium MCFE7 chemical
composition was aimed to induce the isolation of fungi. The minimal medium culture MCBE7 was
chosen to induce the isolation of yeasts and bacteria. Despite the pH difference between the culture
media, it was possible to observe bacteria and fungi growth in both media.
2.2. Evaluation of the Enzyme Activity of Microorganisms
Enantioselective oxidations of alcohols, para-substituted (RS)-1-phenylethanols, were used to
evaluate the enzyme activity (oxidoreductases, OXRED) of 232 microorganisms
psychrophile/psychrotroph previously isolated (Scheme 1).
Scheme 1. Enantioselective oxidation of (RS)-1-phenylethanol and its derivatives.
From this study, 15 microorganisms presented oxidoreductases that catalyzed the oxidation of the
(S)-enantiomer of racemic alcohols to their corresponding ketones. Therefore, the (R)-alcohols
remained intact with high enantiomeric excesses (up to >99%). It was considered as good enzyme
activity (OXRED), conversions of alcohol to ketone > 10% and enantiomeric excess >60%. Some of
the results are shown in Table 2.
As shown in Table 2, the enzyme activities of the microorganisms were very high when the R
substituent in alcohols (1–6) was a methoxy group. In these cases, considering a kinetic resolution
process, the alcohols conversions to their corresponding ketones were excellent (Entries 3, 9, 21 and
33), and in some cases the enantiomeric excesses were higher than 99% (Entries 3, 9 and 21). It is
noteworthy that, in a reaction of kinetic resolution the maximum conversion is 50%.
In reactions mediated by bacterium HK1D10 cells, in which the R substituent was halogen, low
conversions (~1%) and no selectivity were observed (Entries 34 and 35). In general, all
microorganisms produce oxidoreductases with preferential oxidation for the (S)-enantiomer from the
(RS)-alcohols (1–6)

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Table 2. Evaluation of the enzymatic activity of microorganisms isolated from
soil/sediment samples collected in the Antarctic.
Entry
Microorganism
R
Ketone/Alcohola
Alcohol e.e. (%)a
1
H
11/89
22/78
46/54
21/79
23/77
43/57
16/84
27/73
47/53
25/75
4/96
25 (R)
92 (R)
>99 (R)
89 (R)
96 (R)
57 (R)
22 (R)
97 (R)
>99 (R)
95 (R)
17 (R)
33 (R)
10 (R)
57 (R)
45 (R)
52 (R)
61 (R)
6 (R)
2
Me
MeO
Cl
3
MPS8D3
4
5
Br
6
NO2
H
7
8
Me
MeO
Cl
9
HK1D9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Br
NO2
H
34/66
6/94
Me
MeO
Cl
17/83
25/75
16/84
21/79
7/93
HK2D2
Br
NO2
H
8/92
22 (R)
93 (R)
>99 (R)
69 (R)
87 (R)
22 (R)
3
Me
MeO
Cl
17/83
44/56
15/85
19/81
21/79
2/98
MPS4D2
Br
NO2
H
Me
MeO
Cl
5/95
17 (R)
22 (R)
15 (R)
15 (R)
15 (R)
17 (R)
24 (R)
79 (R)
1
15/85
5/95
HK1D8
Br
5/95
NO2
H
5/95
11/89
9/91
Me
MeO
Cl
41/59
1/99
HK1D10
Br
1/99
2
NO2
4/96
2
Reaction conditions: (RS)-alcohols 1–6 (0.75 mmol), culture medium MCBE7 (pH 7.0, 2 mL), 20 °C,
160 rpm, 72 h. a Determined by GC using a chiral column Chiral-Dex-CB; e.e.: enantiomeric excess.

Mar. Drugs 2011, 9
893
Based on these results we decided to perform only the identification of microorganisms that showed
excellent enzyme activity. For this purpose Flavobacterium sp. (HK1D9) and Arthrobacter sp.
(MPS8D3) were selected. These bacteria were isolated from soil and sediment samples, respectively.
The chromatograms from oxidation reaction of the compound (RS)- mediated by cells of
3
Flavobacterium sp. and Arthrobacter sp. (Table 2, Entries 3 and 9) are shown in Figure 1, as an
example of the chiral analysis using GC employed for this enzyme evaluation step.
Analysis using GC.
Sample containing
a
mixture of
Figure 1.
(a)
(RS)-1-(4-methoxyphenyl)ethanol and 4-methoxyacetophenone.
Sample from oxidation
(b)
Sample from oxidation reaction of
(c)
reaction of (RS)- mediated by Arthrobacter sp.
3
(RS)- mediated by Flavobacterium sp.
3
According to the chromatogram obtained from oxidation reaction of the (RS)-3 mediated by
Arthrobacter sp. (Figure 1b) and Flavobacterium sp. (Figure 1c), it can be seen that the (S)-enantiomer
was oxidized, leaving the unreacted alcohol (R)-3 with high enantiomeric excess.

Mar. Drugs 2011, 9
894
2.3. Optimization of the Enantioselective Oxidation Reaction Using Flavobacterium sp. and
Arthrobacter sp.
Flavobacterium sp. and Arthrobacter sp. were selected to be applied in the optimization study due
to their high enantioselective performance in the oxidation reaction of the alcohols (RS)- – . Based on
1 6
the good results presented in Table 2 (Entries 2, 8, 14, 20) and by the fast GC analysis using a chiral
column, we selected (RS)-1-(4-methylphenyl)ethanol as model substrate for this optimization study.
A survey in the literature indicated that psychrophile/psychrotroph microorganisms tolerate
temperatures between 0 and 40 °C [17–22]. The temperature corresponds to one of the major
environmental factors that can influence the bacterial growth, when the temperature is increased, the
chemical and enzymatic reactions in cells tend to become faster, thus accelerating the growth rate [23].
In order to determine the ideal reaction conditions for achieving an excellent kinetic resolution
process mediated by Flavobacterium sp. (HK1D9) and Arthrobacter sp. (MPS8D3), the bacterial cells
were resuspended in phosphate buffer solution (Na2HPO4/KH2PO4, pH 7.0, 0.1 mol/L).
The selected temperatures were 5, 10, 15, 20, 25 and 30 °C (each one is tolerated by psychrotroph
microorganisms) [18]. These temperatures were used for both bacterial growth and alcohol oxidation
reaction. The results are shown in Table 3.
When the bacterial growing and reactions were performed at 15 °C and 25 °C (Flavobacterium sp.)
excellent oxidoreductase activities were observed. The best conversion of the (RS)-alcohol to ketone
2
(45%) was obtained after 24 h reaction at 25 °C (Table 3, Entry 13,). Along the reaction, there was a
few decrease in ketone concentration and an increase in (R)-1-(4-methylphenyl)ethanol concentration
and, consequently its enantiomeric excess (Table 3, Entries 13–15). In an oxidative kinetic resolution
process, the ratio of the ketone and a single alcohol enantiomer concentration should be 1:1. The above
mentioned behavior indicates the presence of ketoreductase that catalyzes enantioselectively the
reduction of the produced 4-methylacetophenone to the (R)-1-(4-methylphenyl)ethanol, resulting in a
higher (R)-alcohol concentratrion than 50%. This process can be called as deracemisation (Scheme 2).
Table 3. Oxidation of the (RS)-1-(4-methylphenyl)ethanol (2) mediated by Flavobacterium
sp. and Arthrobacter sp. at different temperatures for bacterial growth and oxidation
reaction.
Flavobacterium sp. (HK1D9)
Entry
Temp. (°C)a
Time (h)
24
Ketone/Alcoholb
Alcohol e.e. (%)b
1
2
3
4
5
6
7
8
9
nd
nd
5
48
nd
nd
72
nd
nd
24
5/95
14/86
14/86
32/68
46/54
48/52
10 (R)
21 (R)
24 (R)
42 (R)
78 (R)
91 (R)
10
15
48
72
24
48
72

Mar. Drugs 2011, 9
895
Table 3. Cont.
42/58
10
11
12
13
14
15
16
17
18
24
48
72
24
48
72
24
48
72
76 (R)
83 (R)
96 (R)
89 (R)
93 (R)
95 (R)
nd
20
25
30
41/59
49/51
45/55
40/60
33/67
nd
nd
nd
nd
nd
Arthrobacter sp. (MPS8D3)
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
24
48
72
24
48
72
24
48
72
24
48
72
24
48
72
24
48
72
-
-
5
-
-
1/99
1/99
2/98
5/95
40/60
37/63
34/66
40/60
36/64
30/70
38/62
34/66
30/70
-
-
1
10
15
20
25
30
1
11 (R)
94 (R)
96 (R)
96 (R)
95 (R)
95 (R)
96 (R)
96 (R)
95 (R)
95 (R)
-
1/99
2/98
-
4
Reaction conditions: (RS)-alcohol 2 (0.75 mmol), cells harvested from culture medium (100 mL) and
ressuspended in phosphate buffer solution (0.1 mol/L, pH 7.0, 10 mL), 5–30 °C , 160 rpm, 24–72 h;
a
b
Temperature for bacterial growth and oxidation reaction; Determined by GC using a chiral column
Chiral-Dex-CB; nd = not carried out due to very low biomass concentration.
Scheme 2. Deracemisation process mediated by Flavobacterium sp. (HK1D9).

Mar. Drugs 2011, 9
896
The Arthrobacter sp. (MPS8D3) showed good enzyme activity at 15, 20 and 25 °C (24 h), since
under these temperatures, the highest enantiomeric excess and concentration of the
(R)-1-(4-methylphenyl)ethanol were obtained (Table 3, Entries 25, 28 and 31). On the other hand, it
was observed that Flavobacterium sp. and Arthrobacter sp. exhibited very low enzyme activity at
10 °C, since the values of ketone conversions and alcohol e.e. were very low (Table 3, Entries 4–6
and 22–24).
It is noteworthy to mention that there is few deracemisation process described in the literature
performed by a single microorganism [3,24–28] or without the aid of chemical agents [29,30]. This
phenomenon is extremely interesting to be pursued in bioprospecting process.
We decided to study the oxidation of (RS)-1-(4-methylphenyl)ethanol at different temperatures
(5, 10, 15, 20, 25, 30 °C) after growthing the Flavobacterium sp. and Arthrobacter sp. at a specific
temperature, 15 or 25 °C. The bacterial growth temperatures were selected due to the good results of
conversions and enantioselectivities of the alcohol oxidation (Table 3, Entries 7–9, 13–17 and 31–33).
The main goal of this study was to evaluate the enantioselectivity of oxidoreductases produced by
bacterial growth at 15 and 25 °C in the oxidation reaction carried out at different temperatures
(Tables 4 and 5).
Table 4. Growth of Flavobacterium sp. and Arthrobacter sp. at 25 °C and oxidation of
(RS)-1-(4-methylphenyl)ethanol (2) at different reaction temperature.
Flavobacterium sp. (HK1D9)
Entry
1
Temp. (°C)a
Time (h)
24
Ketone/Alcoholb
39/61
39/61
45/55
35/65
44/56
51/49
25/75
43/57
43/57
28/72
35/65
38/62
45/55
40/60
33/67
7/93
Alcohol e.e. (%)b
39 (R)
79 (R)
95 (R)
62 (R)
95 (R)
>99 (R)
36 (R)
83 (R)
95 (R)
49 (R)
80 (R)
83 (R)
89 (R)
93 (R)
95 (R)
6 (R)
2
5
48
3
72
4
24
5
10
15
20
25
30
48
6
72
7
24
8
48
9
72
10
11
12
13
14
15
16
17
18
24
48
72
24
48
72
24
48
6/94
9 (R)
72
7/93
18 (R)
Arthrobacter sp. (MPS8D3)
19
20
21
24
48
72
40/60
44/56
43/57
88 (R)
96 (R)
96 (R)
5

Mar. Drugs 2011, 9
897
Table 4. Cont.
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
24
48
72
24
48
72
24
48
72
24
48
72
24
48
72
40/60
46/54
41/59
41/59
37/63
36/64
43/57
38/63
36/64
45/55
40/60
33/67
42/58
33/67
27/73
94 (R)
97 (R)
96 (R)
>99 (R)
>99 (R)
>99 (R)
98 (R)
98 (R)
>99 (R)
89 (R)
93 (R)
95 (R)
97 (R)
97 (R)
97 (R)
10
15
20
25
30
Reaction conditions: (RS)-alcohol 2 (0.75 mmol), cells harvested from culture medium (100 mL) and
ressuspended in phosphate buffer solution (0.1 mol/L, pH 7.0, 10 mL), 5–30 °C, 160 rpm, 24–72 h;
Temperature for bacterial growth = 25 °C; a Temperature for oxidation reaction; b Determined by GC using a
chiral column Chiral-Dex-CB.
Analyzing the data in Table 4, in which Flavobacterium sp. grew at 25 °C, we found low enzyme
activity for the oxidation of (R,S)-1-(4-methylphenyl) ethanol at 30 °C leading to low ketone
conversion and enantioselectivity (Entries 16–18). However, this bacterium showed good enzyme
activity in other temperatures (Entries 1–15). In general there was an increase in conversion and
enantioselectivity with increasing reaction time. However, the best oxidation performance was
observed at 10 °C and 72 h of reaction (Table 4, Entry 6). In this case, a perfect kinetic resolution
process was achievied, ketone conversion 50% and alcohol e.e. > 99%.
Growth of the Flavobacterium sp. and Arthrobacter sp. at 15 °C and oxidation
Table 5.
reaction of the (RS)-1-(4-methylphenyl)ethanol ( ) at different reaction temperatures.
2
Flavobacterium sp. (HK1D9)
Entry
Temp. (°C)a
Time (h)
24
Ketone/Alcoholb
Alcohol e.e. (%)b
1
2
5/95
15 (R)
5
48
16/84
30 (R)
3
72
21/79
43 (R)
4
24
19/81
21 (R)
5
10
15
20
48
34/66
65 (R)
6
72
42/58
89 (R)
7
24
25/75
36 (R)
8
48
43/57
83 (R)
9
72
43/57
95 (R)
10
11
12
24
16/84
44 (R)
48
34/66
73 (R)
72
41/59
96 (R)

Mar. Drugs 2011, 9
898
Table 5. Cont.
27/73
13
14
15
16
17
18
24
48
72
24
48
72
53 (R)
77 (R)
87 (R)
9 (R)
25
30
33/67
34/66
9/91
9/91
10 (R)
12 (R)
6/94
Arthrobacter sp. (MPS8D3)
42/58
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
24
48
72
24
48
72
24
48
72
24
48
72
24
48
72
24
48
72
90 (R)
>99 (R)
>99 (R)
95 (R)
97 (R)
97 (R)
89 (R)
97 (R)
98 (R)
98 (R)
98 (R)
98 (R)
>99 (R)
>99 (R)
>99 (R)
96 (R)
97 (R)
97 (R)
5
42/58
45/55
41/59
41/59
43/47
35/65
34/66
31/69
42/58
36/64
33/67
43/57
36/64
32/68
37/63
33/67
25/75
10
15
20
25
30
Reaction conditions: (RS)-alcohol 2 (0.75 mmol), cells harvested from culture medium (100 mL) and
ressuspended in phosphate buffer solution (0.1 mol/L, pH 7.0, 10 mL), 5–30 °C, 160 rpm, 24–72 h;
a
b
Temperature for bacterial growth = 15 °C; Temperature for oxidation reaction; Determined by GC using a
chiral column Chiral-Dex-CB.
The reactions carried out under several temperatures and mediated by cells of Arthrobacter sp.
resulted in moderate to excellent alcohol e.e. and ketone conversion (Table 4, Entries 19–36). For
example, oxidation reactions carried out at 15 to 30 °C, there was a decrease in ketone concentration
and an increase in alcohol e.e. over the reaction time (Table 4, Entries 25–36). The Flavobacterium sp.
has oxidoreductase with high activity at 10 °C and Arthrobacter sp. at 15 and 20 °C.
When the microbial growing temperature was 15 °C (Flavobacterium sp.) a low enzyme activity
was observed at 5 and 30 °C. These reactions afforded low values of ketone concentration and
enantioselectivies (Table 5, Entries 1–3 and 16–18). However, the best ketone concentration and
enantioselectivity were observed for reactions carried out at 15 and 20 °C (Table 5, Entries 9 and 12).
On the other hand, Arthrobacter sp. showed good enzyme activity at 5 and 10 °C after 48 and
72 hours, since it was obtained high enantiomeric excess for the alcohol (R)- , up to >99% (Table 5,
2
Entries 20, 21, 23 and 24). We also can see that Arthrobacter sp. showed good enzymatic activity at
reaction temperatures of 15–30 °C, in which ketone conversions and e.e. for the alcohol (R)- were
2

Mar. Drugs 2011, 9
899
moderate to excellent (Table 5, Entries 25–36). At these temperatures, the value of (R)-alcohol
concentration is higher than 50% with increasing the reaction time. As observed for Flavobacterium
sp. (Table 3 and Scheme 2), this process indicates the presence of ketoreductase that catalyzes
enantioselectively
the
reduction
of
the
produced
4-methylacetophenone
to
the
(R)-1-(4-methylphenyl)ethanol, resulting a high (R)-alcohol concentration.
In general, it was observed that Arthrobacter sp. and Flavobacterium sp. had excellent enzyme
activity with growth temperature at 25 °C.
Therefore, we decided to determine the growth curves of Flavobacterium sp. and Arthrobacter sp.
at 15, 20 and 25 °C (Figure 2). By spectrophotometric analysis of optical density (OD) of bacterial
culture it was evaluated the turbidity and estimated the relative concentration cell.
Spectrophotometric analysis of optical density (OD) of the culture of
Figure 2.
Flavobacterium sp.
and Arthrobacter sp.
. Culture medium (600 mL),
(B)
(A)
(RS)-1-(phenyl)ethanol (1 mmol/L), 160 rpm.
A
0.2
0.16
0.12
0.08
0.04
0
0
10
20
30
40
50
60
70
80
Time (hours)
20 ºC
25 ºC
B
0.160
0.120
0.080
0.040
0.000
0
10
20
30
40
50
20 ºC
60
70
80
25 ºC
Time (hours)
According to the Figure 2, it was noted that Flavobacterium sp. and Arthrobacter sp. grew well at
25 °C and exhibited excellent enzyme activity at this temperature (Table 4, Entries 13–15 and 31–33).
Then, it was concluded that these bacteria are psychrotroph since it excludes the possibility of
psychrophile, in which the maximum temperature for growth is 20 °C. This conclusion was based on
literature data about the identification of psychrotroph microorganism [17,31,32].

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3. Experimental Section
3.1. General Methods
Acetophenone, 1-(4-methylphenyl)ethanone, 1-(4-methoxyphenyl)ethanone, 1-(4-bromophenyl)
ethanone, 1-(4-chlorophenyl)ethanone and 1-(4-nitrophenyl)ethanone were purchased from Aldrich
and used without further purification. Thin-layer chromatography (TLC) was performed using
pre-coated plates (Aluminum foil, silica gel 60 F254 Merck, 0.25 mm). Silica gel (Acros Organics,
0.035–0.070 mm, 240–400 mesh) was used for flash chromatography. Low Resolution Mass Spectra
(LRMS) were recorded on a Shimadzu GCMS P5050A (70 eV) spectrometer and GC cappillary
column used was a DB-5-MS (30 m × 0.25 mm × 0.25 m, J&W Scientific), carrier gas-He, 100 kPa):
injector 270 °C ; detector 270 °C. Temperature program 100–250 °C , rate 10 °C /min. GC analyses were
performed in a Shimadzu GC-17A instrument with a FID detector using hydrogen as carrier gas
(100 kPa), autosampler AOC20i. It was used a GC chiral cappillary column, Chirasil-Dex CB
-cyclodextrin (25 m × 0.25 mm, Varian), for determination of the conversion and enantiomeric
excesses. Sterile materials were used to perform the experiments and the microorganisms were handled
in a laminar flow cabinet Fisher Hamilton Class II Biological Safety Cabinet.
3.2. Preparation of the (RS)-1-(Phenyl)ethanols (1–6) [33]
To a solution of acetophenones (7–12) (10 mmol) in methanol (20 mL) was added NaBH4
(11.1 mmol, 420 mg), and the mixture was stirred at room temperature for 4 h. The methanol was
removed by vacuum evaporation. Distilled water (20 mL) was added into the reaction mixture and the
pH adjusted to 6 with aqueous solution of HCl (1 mmol/L), then the aqueous layer was extracted with
methylene dichloride (3 × 30 mL). The organic phases were combined and dried over MgSO4. The
solvent was removed in vacuum and the residue was purified by silica gel column chromatography
eluting with a mixture of hexane and ethyl acetate (4:1) to afford compounds (RS)-1–6. The 1H NMR
and 13C NMR spectra of these compounds were in agreement with those reported in the
literature [34,35].
3.3. Procedures for Bioprospection
3.3.1. Soil and Sediment Sampling Procedure
Soil and sediment samples were collected from environmental preservation area in Antarctic
Peninsula (Admiralty Bay, King George Island, Antarctic in 2008): Peninsula Keller (21 E 427302,
UTM 3115830); (21 E 425986, UTM 3117583); (21 E 426110, UTM 3118275); Machu Picchu
(21 E 423394, UTM 3114505); Ponta Hennequin (21 E 427680, UTM 3111880); Arctowski
(21 E 423807, UTM 3106862); Ponta Demay (21 E 425211, UTM 3100473); Ponta Botany
(21 E 424432, UTM 3092107); Ponta Steinhouse (21 E 425419, UTM 3110733); The samples were
collected under clean, sterile conditions and kept frozen in polyethylene tubes at −20 °C .

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3.3.2. Culture Media
The culture media were (amount of the ingredients are indicated per liter):
-MCFE7 (pH = 5.6) = (RS)-1-(phenyl)ethanol–1 mmol, 0.122 g, 122 L), KH2PO4 (0.1%; 1.0 g),
MgSO4.7H2O (0.04%; 0.4 g), NH4Cl (0.04%; 0.4 g), Dextrose (Oxoid) (0.025%; 0.25 g), Peptone
(Oxoid) (0.025%; 0.25 g), agar (Oxoid) (2.0%; 20.0 g; when needed).
-MCBE7 (pH = 7.0)= (RS)-1-(phenyl)ethanol–1 mmol, 0.122 g, 122 L), K2HPO4 (0.2%; 2.0 g),
KH2PO4 (0.1%; 1.0 g), MgSO4.7H2O (0.04%; 0.4 g), NH4Cl (0.04%; 0.4 g), Meat Extract (Oxoid)
(0.025%; 0.25 g), Peptone (Oxoid) (0.025%; 0.25 g), agar (Oxoid) (2.0%; 20.0 g; when needed).
3.3.3. Isolation and Growth of Microorganisms
A soil suspension was prepared by adding 20 mL of sterile phosphate buffer solution (pH 7.0,
0.1 mol/L) to 2.0 g of the soil or sediments sample. It was stirred, the solution resting for about
1 minute for sedimentation and 200 µL of the supernatant were incubated (in duplicate) in a
polypropylene microtiter plate (each well consisting of a 10 mL total volume capacity; Whatman)
containing 2 mL of the appropriate culture media:
a-One microtiter plate contains MCFE7 culture medium;
b-One microtiter plate contains MCBE7 culture medium;
After 4 days of incubation for MCFE7-containing microtiter plate and 2 days of incubation for
MCBE7-containing microtiter plate at 20 °C , aliquots of 100 µL of microorganisms culture from each
well was sprayed onto the culture medium agar plates to microorganisms isolation step.
From each microorganism culture sample (100 µL) that was plated onto solid culture medium,
individual colonies appeared. Each colony was inoculated separately into 20 mL of appropriate
culture media in Erlenmeyer flasks (125 mL) and incubated in an orbital shaker during 2 or 4 days
(160 rpm, 20 °C ).
3.4. Enzyme Assays
From each pre-culture obtained previously, a 750 µL aliquot was transferred to microtubes
containing 250 µL of glycerol for cryo-preservation (−20 °C ). Another 200 µL were transferred to
inoculate in a sterile polypropylene microtiter plate containing 2 mL of culture media per well. The
plates were kept at 20 °C in an orbital shaker (160 rpm) during 2 or 4 days and then 40 µL of a
solution of each alcohol (1–6, 5 mmol/L) in dimethylformamide were added to each well of the plate.
The microtiter plates were returned to the same incubation condition as described above for 72 hour.
Each well content was extracted by stirring with tert-butyl methyl ether (2.0 mL) followed by
centrifugation (6000 rpm, 1 min.). The organic phase was analyzed by GC using a chiral capillary
column (see Section 3.5).
3.5. GC Analysis
GC conditions (carrier gas-H2, 100 kPa): injector 220 °C ; detector 220 °C . Isotherm and tR (min):
retention time of chiral compounds: (RS)-phenyl-ethanol: 107 °C, R-enantiomer 6.22 min,
S-enantiomer 6.87 min; (RS)-1-(4-methyl-phenyl)-ethanol: 113 °C , R-enantiomer 6.79 min,

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S-enantiomer 7.71 min; (RS)-1-(4-methoxy-phenyl)-ethanol: 119 °C , R-enantiomer 6.22 min,
S-enantiomer 6.87 min; (RS)-1-(4-chloro-phenyl)-ethanol: 130 °C , R-enantiomer 7.33 min,
S-enantiomer 8.19 min; (RS)-1-(4-bromo-phenyl)-ethanol: 135 °C , R-enantiomer 9.54 min,
S-enantiomer 10.52 min; (RS)-1-(4-nitro-phenyl)-ethanol: 150 °C , R-enantiomer 19.92 min,
S-enantiomer 20.30 min. The enantiomeric excess (ee) was defined as the ratio of
{([R] − [S])/([R] + [S])} × 100%, where [R] and [S] are the concentrations of (R) and (S) enantiomers,
respectively.
3.6. Microorganisms Library and Preservation
The microorganism strains with the best performance in enzyme assays were stored in a freezer at
−20 °C . They were preserved as suspensions in glycerol solution (20%) using two different culture
media described in Section 3.3.2.
3.7. Bacteria Identification
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 [36].
3.8. Effect of Temperature and Reaction Time on the Oxidation Reactions Using Flavobacterium sp.
and Arthrobacter sp.
Cultures of Flavobacterium sp. and Arthrobacter sp. were separately inoculated under sterile
conditions into 20 mL of appropriate culture media in Erlenmeyer flasks (125 mL) and incubated in an
orbital shaker during 2 days (160 rpm, 20 °C ). After that, the cultures were transferred separately to
two Erlenmeyer flasks (2000 mL) containing 600 mL of sterilized culture media and incubated again.
Then the cells were harvested by centrifugation at 20 °C on Sorvall RC-5B (Plus rotor) for 15 min at
5000 rpm. The harvested cells were resuspended in 60 mL of sterile phosphate buffer solution
(Na2HPO4/KH2PO4, pH 7.0, 0.1 mol/L), divided equally in 6 Erlenmeyer flasks (125 mL) and an
aliquot (200 µL) of substrate solution (RS)-1-(4-methylphenyl)ethanol (1 mmol/L) was added to each
flask. The mixture was kept at different temperatures (5, 10, 15, 20, 25 and 30 °C ) in an orbital shaker
(160 rpm) during 1, 2 or 3 days. The products of the total reaction volume (10 mL) were extracted by
stirring with tert-butyl methyl ether (2.0 mL) followed by centrifugation (6000 rpm, 1 min.). The
organic phase was analyzed by GC using a chiral capillary column (see Section 3.5).
3.9. Growth Measurements
Growth curve experiments were performed with Erlenmeyer flasks (2000 mL) containing 1000 mL
of sterilized culture media and 100 mL of inoculated cultures. The mixture was incubated in an orbital
shaker (160 rpm, 15, 20 and 25 °C ). Samples of culture (1 mL) were periodically removed at 120 min
intervals and the growth was estimated spectrophotometrically (and expressed as optical density
[O.D.]) at 600 nm with a Biospectro model sp-220 spectrophotometer using cells with a 1 cm
light path.
The replicate values were averaged and the O.D. was plotted against time on linear coordinates.

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3.10. Absolute Configuration
The absolute configurations of all compounds were determined by comparison of the sign of the
measured specific rotation with those in the literature [37–39].
4. Conclusions
In summary, the enrichment technique using (RS)-1-(phenyl)ethanol, as a carbon source, was a very
important tool for the selection of microorganisms producing cold-active oxidoreductases. Samples of
soil and sediment from the Antarctic provided 232 microorganisms. The enzyme activity
(oxidoreductase) of these microorganisms was evaluated in the oxidation of racemic alcohols. Fifteen
microorganisms presented oxidoreductases which catalyzed the oxidation of the (S)-enantiomer from
racemic alcohol to its corresponding ketone. Therefore, the (R)-alcohol remained intact and with high
enantiomeric excess (up to >99%). After the optimization study, Flavobacterium sp. showed excellent
enzyme activity with temperature of growth and oxidation reaction at 25 °C and 10 °C, respectively.
However, Arthrobacter sp. presented excellent enzyme activity with temperature of growth and
oxidation reaction at 25 °C. After the determination of growth curves for Flavobacterium sp. and
Arthrobacter sp., it was possible to classify them as psychrotroph, since the best temperature for
bacterial growth was 25 °C and 20 °C , respectively. Further studies regarding enzyme purification are
in progress in our laboratory.
Acknowledgments
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.
References
1. Soni, P.; Banerjee, U.C. Enantioselective reduction of acetophenone and its derivatives with a new
yeast isolate Candida tropicalis PBR-2 MTCC 5158. Biotechnol. J. 2006, 1, 80–85.
2. Matsuda, T.; Yamanaka, R.; Nakamura, K. Recent progress in biocatalysis for asymmetric
oxidation and reduction. Tetrahedron: Asymmetry 2009, 20, 513–557.
3. Voss, C.V.; Gruber, C.C.; Kroutil, W. Deracemisation of secondary alcohols via biocatalytic
stereoinversion. Synlett 2010, 7, 991–998.
4. Ishige, T.; Honda, K.; Shimizu, S. Whole organism biocatalysis. Curr. Opin. Chem. Biol. 2005, 9,
174–180.
5. Cardenas, F.; Alvarez, E.; de Castro-Alvarez, M.S.; Sanchez-Montero, J.M.; Valmaseda, M.;
Elson, S.W.; Sinisterra, J.V. Screening and catalytic activity in organic synthesis of novel fungal
and yeast lipases. J. Mol. Catal. B: Enzym. 2001, 14, 111–123.
6. Andrade, L.H.; Comasseto, J.V.; Rodrigues, D.F.; Pellizari, V.H.; Porto, A.L.M. Enantioselective
reduction of ortho-substituted acetophenones by bacterial strains isolated from medium enriched
with biphenyl or diesel fuel. J. Mol. Catal. B: Enzym. 2005, 33, 73–79.

Mar. Drugs 2011, 9
904
7. Yoon, J.; Miwa, H.; Ahmed, I.; Yokota, A.; Fujiwara, T. Rhodococcus baikonurensis BTM4c, a
boron-tolerant actinobacterial strain isolated from soil. Biosci. Biotechnol. Biochem. 2010, 74,
178–181.
8. Lo Giudice, A.; Bruni, V.; Michaud, L. Characterization of Antarctic psychrotrophic bacteria with
antibacterial activities against terrestrial microorganisms. J. Basic Microb. 2007, 47, 496–505.
9. Marx, J.C.; Collins, T.; D`Amico, S.; Feller, G.; Gerday, C. Cold-adapted enzymes from marine
antarctic microorganisms. Mar. Biotechnol. 2007, 9, 293–304.
10. Cavicchioli, R.; Siddiqui, K.S.; Andrews, D.; Sowers, K.R. Low-temperature extremophiles and
their applications. Curr. Opin. Biotech. 2002, 13, 253–261.
11. Russell, N.J. Toward a molecular understanding of cold activity of enzymes from psychrophiles.
Extremophiles 2000, 4, 83–90.
12. Kazuoka, T.; Oikawa, T.; Kuroda, I.M.S.; Soda, K. Cold-active and thermostable alcohol
dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris
KUC-1. Extremophiles 2007, 11, 257–267.
13. Feller, G.; Arpigny, J.L.; Narinx, E.; Gerday, C. Molecular adaptations of enzymes from
psychrophilic organisms. Comp. Biochem. Physiol. 1997, 118A, 495–499.
14. Velonia, K.; Tsigos, I.; Bouriotis, V.; Smonou, I. Stereospecificity of hydrogen transfer by the
NAD+-linked alcohol dehydrogenase from the antarctic psychrophile Moraxella sp. TAE123.
Bioorg. Med. Chem. Lett. 1999, 9, 65–68.
15. D`Amico, S.; Marx, J.C.; Gerday, C.; Feller, G. Activity stability relationships in extremophilic
enzymes. J. Biol. Chem. 2003, 278, 7891–7896.
16. Cripps, R.E.; Trudgill, P.W.; Whateley, J.G. The metabolism of 1-phenylethanol and
acetophenone by Nocardia T5 and an Arthrobacter species. Eur. J. Biochem. 1978, 86, 175–186.
17. Morita, R.Y. Psychrophilic bacteria. Bacteriol. Rev. 1975, 39, 144–167.
18. Feller, G.; D’Amico, D.; Gerday, C. Thermodynamic stability of a cold-active α-amylase from the
Antarctic bacterium Alteromonas haloplanktis. Biochemistry 1999, 38, 4613–4619.
19. Rashid, N.; Kikuchi, H.; Ezaki, S.; Atomi, H.; Imanaka, T. Isolation and characterization of
psychrotrophs from subterranean environments. J. Biosci. Bioeng. 1999, 87, 746–751.
20. Connaughton, S.; Collins, G.; O’Flaherty, V. Psychrophilic and mesophilic anaerobic digestion of
brewery effluent: A comparative study. Water Res. 2006, 40, 2503–2510.
21. Hoover, R.B.; Pikuta, E.V. Psychrophilic and psychrotolerant microbial extremophiles in polar
environments. Polar Microbiol. 2010, 115–156.
22. Irwin, J.A. Extremophiles and their application to veterinary medicine. Environ. Technol. 2010,
31, 857–869.
23. Cavicchioli, R.; Thomas, T.; Curmi, P.M.G. Cold stress response in Archaea. Extremophiles 2000,
4, 321–331.
24. Kroutil, W.; Mischitz, M.; Faber, K. Deracemization of (±)-2,3-disubstituted oxiranes via
biocatalytic hydrolysis using bacterial epoxide hydrolases: kinetics of an enantioconvergent
process. J. Chem. Soc. Perk. Trans. 1 1997, 24, 3629–3636.
25. Allan, G.R.; Carnell, A.J. Microbial deracemization of 1-aryl and 1-heteroaryl secondary alcohols.
J. Org. Chem. 2001, 66, 6495–6497.

Mar. Drugs 2011, 9
905
26. Gareth, C.J.; Andrew C.J.; Harald, T.; Thomas, R. Microbial deracemisation of N-(1-hydroxy-1-
phenylethyl)benzamide. Tetrahedron: Asymmetry 2004, 15, 239–243.
27. Demir, A.S.; Hamamci, H.; Sesenoglu, O.; Neslihanoglu, R.; Asikoglu, B.; Capanoglu, D. Fungal
deracemization of benzoin. Tetrahedron Lett. 2002, 43, 6447–6449.
28. Gruber, C.C.; Lavandera, I.; Faber, K.; Kroutil, W. From a racemate to a single enantiomer.
deracemization by stereoinversion. Adv. Synth. Catal. 2006, 348, 1789–1805.
29. Orru, R.V.A.; Kroutil, W.; Faber, K. Deracemization of (±)-2,2-disubstituted epoxides via
enantioconvergent chemoenzymatic hydrolysis using Nocardia EH1 epoxide hydrolase and
sulfuric acid. Tetrahedron Lett. 1997, 38, 1753–1754.
30. Kroutil, W.; Faber, K. Deracemization of compounds possessing a sec-alcohol or -amino group
through a cyclic oxidation-reduction sequence: a kinetic treatment. Tetrahedron: Asymmetry
1998, 9, 2901–2913.
31. Russell, N.J. Cold adaptation of microorganisms. Phil. Trans. R. Soc. B 1990, 326, 595–611.
32. Feller, G.; Arpigny, J.L.; Narinx, E.; Gerday, C. Molecular adaptations of enzymes from
psychrophilic organisms. Comp. Biochem. Physiol. 1997, 118, 495–499.
33. Andrade, L.H.; Polak, R.; Porto, A.L.M.; Schoenlein-Crusius, I.H; Comasseto, J.V. Application of
bioreduction by microorganisms in the enantioselective synthesis of alpha-substituted-1-
phenylethanols. Lett. Org. Chem. 2006, 3, 613–618.
34. Nakamura, K.; Matsuda, T. Asymmetric reduction of ketones by the acetone powder of
Geotrichum candidum. J. Org. Chem. 1998, 63, 8957–8964.
35. Salvi, N.A.; Chattopadhyay, S. Studies on Rhizopus arrhizus mediated enantioselective reduction
of arylalkanones. Tetrahedron 2001, 57, 2833–2839.
36. Nogales, B.; Moore, E.R.B.; Brossa, E.L.; Mora, R.R.; Amann, R.; Timmis, K.N. Combined use
of 16S ribosomal DNA and 16S rRNA to study the bacterial community of polychlorinated
biphenyl-polluted soil. Appl. Environ. Microbiol. 2001, 67, 1874–1884.
37. Naemura, K.; Murata, M.; Tanaka, R.; Yano, M.; Hirose, K.; Tobe, Y. Enantioselective acylation
of primary and secondary alcohols catalyzed by lipase QL from Alcaligenes sp.: A predictive
active site model for lipase QL to identify which enantiomer of an alcohol reacts faster in this
acylation. Tetrahedron: Asymmetry 1996, 7, 3285–3294.
38. Yadav, J.S.; Nanda, S.; Reddy, T.P.; Rao, A.B. Efficient enantioselective reduction of ketones
with Daucus carota Root. J. Org. Chem. 2002, 67, 3900–3903.
39. Kim, M.J.; Kim, H.M.; Kim, D.; Ahn, Y.; Park, J. Dynamic kinetic resolution of secondary
alcohols by enzyme-metal combinations in ionic liquid. Green Chem. 2004, 6, 471–474.
Samples Availability: Available from the authors.
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