Escherichia coli BioH: A highly enantioselective and organic solvent tolerant esterase for kinetic resolution of sec-alcohols

Article abstract of DOI:10.1016/j.tetlet.2010.09.135

Escherichia coli BioH, which is obligatory for biotin synthesis, was found to be an organic solvent tolerant esterase with high enantioselectivity for the kinetic resolution of sec-alcohols using free enzyme powder. With this esterase, a variety of racemic sec-alcohols were efficiently resolved with ee values of up to 99%.

Full text of DOI:10.1016/j.tetlet.2010.09.135

Tetrahedron Letters 51 (2010) 6360–6364
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Escherichia coli BioH: a highly enantioselective and organic solvent tolerant
esterase for kinetic resolution of sec-alcohols
⇑
Bo Wang, Xiaoling Tang, Ji Liu, Hongwei Yu
Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Escherichia coli BioH, which is obligatory for biotin synthesis, was found to be an organic solvent tolerant
esterase with high enantioselectivity for the kinetic resolution of sec-alcohols using free enzyme powder.
With this esterase, a variety of racemic sec-alcohols were efficiently resolved with ee values of up to 99%.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 August 2010
Revised 25 September 2010
Accepted 29 September 2010
Available online 7 October 2010
Keywords:
Escherichia coli BioH
Esterase
Kinetic resolution
Enantioselectivity
Organic solvent tolerance
Esterases are able to be produced from prokaryote sources,
which is much easier and economic in cloning and expression com-
pared with lipases which are mainly from eukaryotic microorgan-
isms. In addition, the source of esterases is broader than lipases.
Despite the progress made in the genetic area, the supply of syn-
thetically useful esterases remains inadequate. Therefore, the
search for new esterases exhibiting high activity and enantioselec-
tivity, especially stability in organic solvents, and meanwhile those
capable of accepting a wide variety of non-natural substrates is al-
ways highly desirable.
secondary alcohols with vinyl acetate in organic solvents using free
enzyme powder. Applying this esterase, a variety of sec-alcohols
were well resolved and the selectively acylated alcohols were
afforded with ee values of up to 99% under mild conditions.
5
In our previous work, we have demonstrated E. coli BioH as a
hydrolase with high enantioselectivity and activity in the hydroly-
sis of 1-phenylethyl acetate. Coincidently, we found this esterase
exhibiting good performance in the kinetic resolution of racemic
1-phenylethanol in organic solvents and (R)-1-phenylethyl acetate
was obtained in 47% yield with ee values of 93%. The result
Enantiomerically pure secondary alcohols are very important as
they are pivotal compounds in organic synthesis.1 Of the conven-
tional methods, enzyme-catalyzed kinetic resolution (KR) of sec-
ondary alcohols is a standard procedure and is still the most
Table 1
Evaluation of reaction conditions for the KR of racemic 1-phenylethanol applying
E. coli BioH as the biocatalyst
a
2
Ratiob
Conversionc
(%)
eed (%)
practical. To date, a large variety of enzymes have been reported.
Entry
Solvent
Temp
°C)
Time
(h)
(
Nevertheless, the availability of true esterase for kinetic resolution
in organic solvents is limited only as they get inactivated or give a
very low reaction rate in non-aqueous media. For example, Pig Li-
ver Esterase (PLE) showed no activity in organic solvents with vinyl
acetate, in order to confer activity to PLE, the addition of an organic
1
2
3
4
5
6
7
8
9
—
—
30
30
30
35
35
35
35
35
35
24
24
24
24
32
28
28
28
28
46
39
47
48
51
>49
44
42
92
93
92
93
91
93
94
90
89
Toluene
1:3
1:1
1:3
1:3
1:3
1:5
1:3
1:1
n-Hexane
n-Hexane
n-Hexane
n-Hexane
iso-Octane
Ethyl acetate
Acetone
3
polymer is required. Therefore, an organic solvent tolerant biocat-
alyst is being applied in wide applications for the synthesis of
4
important products.
33
Herein, we wish to report a new organic solvent tolerant ester-
ase-Escherichia coli BioH (lotus tag b3412 from gene bank), which
showed high activity and enantioselectivity in the acylation of
a
Reactions were performed on a 6 mL scale: 1-phenylethanol, 0.4 mmol, organic
solvent, and vinyl acetate 6 mL, crude enzyme powder of E. coli BioH, 12 mg (the
powder contents 12% of E. coli BioH esterase).
VA/Sol (v/v), ‘VA’ represents vinyl acetate, and ‘Sol’, organic solvent.
Conversions and ee values of (R)-1-phenylethyl acetate were determined by GC
analysis.
b
c, d
⇑
Corresponding author. Tel.: +86 571 8759 1873.
E-mail address: yuhongwei@zju.edu.cn (H.W. Yu).
0
040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.09.135

B. Wang et al. / Tetrahedron Letters 51 (2010) 6360–6364
6361
OH
OAc
CH3
OH
be catalyzed to form the corresponding (R)-esters. All of these four
substrates bore an -hydroxyethyl group (Fig. 1, the group was in
blue dashed frame) at the end of the chain. For substrates with
other hydroxyalkyl groups like 1q, 1r, 1s, and 1t no acylation
was observed.
E. coli BioH/Vinyl acetate
n-Hexane
+
a
R
CH3
R
R
CH3
OH
(
1a, 1e, 1o-t)
2
3
OH
CH
Based on the above results, it was concluded that E. coli BioH
C2H
5
(NR)
1
1
a =
3 (conversion: >49%; ee of 2: 89%) 1q =
could only accept alcohols bearing an
a-hydroxyethyl group. Sub-
OH
CH3 (conversion: 49%; ee of 2: 96%) 1r =
OH
C3H7 (NR)
strates bearing -hydroxyalkyl groups of more than two carbon
a
e =
atoms could not be accepted by this esterase.
H3C
As was indicated in Table 1, 1-phenylethanol was well resolved
in several organic solvents with E. coli BioH, thus we could draw a
conclusion that E. coli BioH was an organic solvent tolerant ester-
ase. We assumed that it is the property of organic solvent tolerance
mainly due to the scattered hydrophobic areas on the molecular
surface and around the entrance to an active pocket (Fig. 2). Mean-
while, the presence of disulfide bonds close to the surface also con-
ferred its organic solvent tolerance (Supplementary data, Fig. S1).
OH
CH3
OH
CH3
OH
C2H5 (NR)
OH
C2H5 (NR)
1
1
o =
p =
(conversion: 25%; ee of 2: 80%) 1s =
C3H7
C3H7
1t =
(conversion: 25%; ee of 2: 83%)
C7H15
C5H11
Fig. 1. The type screening of sec-alcohols catalyzed by E. coli BioH (here, yield and
ee values were of esters, ‘NR’ in brackets represents no reaction)
7
E. coli BioH was reported as a carboxylesterase. Hitherto, its
application in biocatalysts has been very limited. The function of
the enzyme is derived from its molecular structure. E. coli BioH
8
2
235
intrigued us to make a further exploration of substrate scope of
E. coli BioH. Different organic solvents, reaction times, ratios (vinyl
acetate: organic solvent), and temperatures were investigated to
evaluate the effect of different reaction conditions on yields and
ee values catalyzed by E. coli BioH, and the results were summa-
rized in Table 1. It was indicated that under the conditions: n-hex-
ane/vinyl acetate, 1:3, temperature 35 °C, and reaction time, 28 h.
esterase has a catalytic triad composed of Ser , His , and
2
07
Asp . The active site contains a small pocket and a large pocket
(Fig. 3), which is buried between two domains and is sufficiently
large to accommodate big groups like long linear aliphatic moie-
ties, aromatic rings, or biphenyl rings within the large pocket. Nev-
ertheless, only small groups whose sizes are not bigger than a
methyl group could be accommodated in the small pocket, for
groups whose sizes are bigger than a methyl group could not or
partly be fitted in the small pocket, leading to its hydroxyl group
not being accessible to the catalytic site and hence could not be
acylated. With its three hydrogen atoms in the hydroxymethyl
group being fully replaced by fluorine atoms, no acylation was
(R)-1-Phenylethyl acetate was afforded in the yield of >49% with
ee values of 89% (Table 1, entry 6).
Various types of sec-alcohols were then further investigated un-
der the optimized conditions. The results indicated that among
these eight substrates, the acylation of only four of them could
Fig. 2. Hydrophobic areas distributed on the molecular surface of BioH in the interval. (a) The selected side contains the active pocket (marked by oval in yellow). (b–e) Side
views from different directions, respectively (the hydrophobic areas were in red).

6
362
B. Wang et al. / Tetrahedron Letters 51 (2010) 6360–6364
Fig. 3. Microview of the active pocket of E. coli BioH binding with a molecular of (R)-1-phenylethyl acetate (1a, 1b), and (S)-1-phenylethyl acetate (2a, 2b). (a) Seen from the
right side of the entire active binding pocket. (b) Local view from the right side of the active pocket.
observed. It was assumed that the formed F–H bonds by fluorine
atoms from substrate 1b and hydrogen atoms from the amino
acids in the wall of active pocket were more stable, which resulted
in the occupation of the active pocket or the entrance to the active
pocket, leading to no other molecules of substrate 1b going in and
out of the active pocket. This might be the reason why substrates
like 1b, 1q, 1r, 1s, and 1t could not be accepted by E. coli BioH.
Kinetic resolution of a series of racemic 1-arylethanols and two
were summarized in Table 2. When group R were bulky aromatic
rings or long linear aliphatic chains, (R)-esters were obtained in
low yields (Table 2, entries 5–8, 11, and 12), to obtain the desired
products in high yields, longer reaction times, or higher tempera-
tures were required.
Enantiomeric excess of (R)-esters from the corresponding alco-
hols with aliphatic chains especially short aliphatic chains were
lower than those with aryl rings and long chains, respectively. This
was probably due to the size difference between R and methyl
2
-alkylalcohols were conducted by the enzyme and the results
Table 2
Results of kinetic resolution of sec-alcoholsa
OH
OAc
CH
OH
E. coli BioH/Vinyl acetate
n-Hexane
+
R
CH3
R
3
R
CH3
1
2
3
c
Ed
Entry
1
Substrate
1
Ester
2
Yield b (%, 2/3)
ee
p
(%)
s
ee (%)
Abs. config. of 2c
OH
OAc
CH3
CH3
1a
2a
2b
45/47
89
—
97
0
R
72
OH
CF3
OAc
CF3
2
3
1b
1c
0/93
—
—
OH
CH
OAc
CH
3
3
2c
45/48
96
96
93
94
R
R
>150
Cl
Cl
OH
CH
OAc
CH
4
3
1d
3
2d
45/49
>180
H
3
C
3
H C

B. Wang et al. / Tetrahedron Letters 51 (2010) 6360–6364
6363
Table 2 (continued)
c
Ed
Entry
Substrate
OH
CH
CH
1
Ester
2
Yield b (%, 2/3)
ee
p
(%)
ee
s
(%)
Abs. config. of 2c
OAc
CH
CH
5
3
1e
1f
3
2e
37/55
99
96
96
66
82
63
R
R
R
>700
>120
93
3
3
OH
OAc
H
3
C
C
3
H C
6
7
CH
3
CH
3
2f
39/47
37/52
H
3
H C
3
OH
OAc
CH3
CH3
1g
2g
H
3
C
CH
OH
CH
3
H
3
C
CH
3
OAc
8
9
3
1h
1i
CH
3
2h
2i
43/55
44/48
43/47
95
99
95
75
99
92
R
R
R
80
O
2
N
O
2
N
N
OH
CH3
OAc
CH3
O N
O
2
2
>1000
>100
OH
CH
OAc
CH
10
11
12
3
1j
3
2j
Br
Br
OH
CH
OAc
CH
3
1k
1l
3
2k
44/48
92
96
R
>100
H CO
H
3
CO
3
OH
CH
OAc
CH
3
3
2l
39/47
37/55
93
99
65
66
R
R
54
C H O
C H O
2
5
2
5
OH
CH3
OAc
CH3
1
3e
1me
2m
>400
OH
OAc
CH
OAc
CH3
1
4
1n
1o
2n
2o
21/69
23/68
80
83
28
22
R
R
11
13
C3H7 CH3
OH
C
3
H
7
3
1
5
C
7
H
15
CH
3
C H
7 15
a
Reactions were performed on a 6 mL scale (sec-alcohols, 0.4 mmol; crude BioH esterase powder, 12 mg; vinyl acetate, 1.5 mL; n-hexane, 4.5 mL; reaction time, 28 h at
5 °C). Unless otherwise stated, ee values were determined by GC-analysis with a chiral column (Cyclodex-B, 30 m  0.32 mm  0.25 m, Agilent Technologies).
3
l
b
c
Isolated yields
Absolute configuration of esters were determined by comparison of the sign of their optical rotations with the data published in the literature.6
d
e
E values were calculated from ee
ee values were determined by HPLC analysis with a chiral AD-H column.
s
and ee
p
. E = ln [1 À c (1 + ee
p
)]/ln [1 À c (1 À ee
p s s p
)], c = ee /ee + ee .
group which was not so bigger according to Kazlauskas’ rule.8
Small size R group enabled (S)-alcohols to be fitted into the active
pocket leading to its hydroxyl group close to the catalytic site
forming (S)-esters. Figure 2 (2a, 2b) showed the active site binding
with a (S)-1-phenylethyl acetate molecular by molecular docking
method, part of the active pocket entrance was utilized as the large
pocket. Due to small size of the entrance aperture, only short linear
aliphatic alcohols or no substituted aryl rings could be accommo-
dated within it. For those with groups in big size, the chance of
forming (S)-configuration esters was less. For example, when sub-
strate 1n was applied in this reaction, the acylated 2n was ob-
tained with ee value of 93%, however, E value decreased to 53.
Nevertheless, when substrate 1m was employed, ee value of 2m
was 99% and E value was >300 (Table 2, entries 13 and 14). With
the sizes of R groups becoming bigger, ee values of the correspond-
ing esters became higher (Table 2).
tial applications to take the place of lipases for producing enantio-
merically pure chemicals.
Acknowledgments
This work was financially supported by the National High-tech
R&D Program (863 Program, Grant No. 2010AA101502) and Qianji-
ang Ren Cai Project (Grant No. 2009R10039). We thank all the
members of Professor Yu’s group.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.09.135.
References and notes
In conclusion, we have found a new organic solvent tolerant
esterase with high enantioselectivity for the kinetic resolution of
sec-alcohols simply by free enzyme powder, no immobilization,
or addition of organic polymers (like PLE) were required to exhibit
its activity. And the corresponding esters were efficiently resolved
with ee values of up to 99%. We believe that E. coli BioH has poten-
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