Biocatalytic deprotection of a cetraxate ester by Microbacterium sp. strain 7-1W cells

Article abstract of DOI:10.1271/bbb.67.192

Enzymatic deprotection of the terminal ester bond of a cetraxate methyl ester was done with resting cells of Microbacterium sp. strain 7-1W, which produces an esterase catalyzing a regioselective hydrolysis reaction, as the catalyst. When 20 g of cetraxate methyl ester in 50 ml of a reaction mixture was incubated with 5 g of wet cells for 17 h, 96% of the substrate was converted to the desired product, cetraxate, quantitatively.

Full text of DOI:10.1271/bbb.67.192

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Biocatalytic Deprotection of a Cetraxate Ester by
Microbacterium sp. Strain 7-1W Cells
Kohsuke HONDAa, Keiji SAKAMOTOab, Shinji KITAab, Michihiko KATAOKAa & Sakayu
SHIMIZUa
a Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Sakyo-ku, Kyoto 606-8502, Japan
b Present address: Research Institute, Daiichi Fine Chemical Takaoka, Toyama 933-8511,
Japan
Published online: 22 May 2014.
To cite this article: Kohsuke HONDA, Keiji SAKAMOTO, Shinji KITA, Michihiko KATAOKA & Sakayu SHIMIZU (2003)
Biocatalytic Deprotection of a Cetraxate Ester by Microbacterium sp. Strain 7-1W Cells, Bioscience, Biotechnology, and
Biochemistry, 67:1, 192-194, DOI: 10.1271/bbb.67.192
To link to this article: http://dx.doi.org/10.1271/bbb.67.192
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Biosci. Biotechnol. Biochem., 67 (1), 192–194, 2003
Note
Biocatalytic Deprotection of a Cetraxate Ester
by Microbacterium sp. Strain 7-1W Cells
†
*
*
Kohsuke HONDA, Keiji SAKAMOTO, Shinji KITA, Michihiko KATAOKA
and Sakayu SHIMIZU
,
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan
Received July 2, 2002; Accepted September 9, 2002
Aspergillus niger.
3,4) The enzymes of Microbacterium
Enzymatic deprotection of the terminal ester bond of
a cetraxate methyl ester was done with resting cells of
Microbacterium sp. strain 7-1W, which produces an
esterase catalyzing a regioselective hydrolysis reaction,
as the catalyst. When 20 g of cetraxate methyl ester in
50 ml of a reaction mixture was incubated with 5 g of
sp. strain 7-1W and A. niger both act on cetraxate es-
ters, but these enzymes are quite diŠerent from each
other. The Aspergillus enzyme catalyzes the hydroly-
sis of various benzyl esters, such as cetraxate benzyl
ester and p-hydroxyphenyl propionate benzyl ester,
wet cells for 17 h, 96
% of the substrate was converted to
but not the lower alkyl esters of these compounds. In
contrast, the Microbacterium enzyme hydrolyzes the
methyl and ethyl esters of cetraxate, and the activity
toward benzyl esters relative to that of methyl esters
is low. From a practical viewpoint, the hydrolysis of
the lower alkyl esters of cetraxate, such as methyl es-
ter and ethyl ester, is more advantageous than that of
benzyl ester, because of the di‹cult synthesis of ben-
zyl ester substrates and the considerable decrease in
the molecular mass on deprotection. Here, we report
the enzymatic preparation of cetraxate through the
regioselective hydrolysis of a cetraxate methyl ester
with Microbacterium sp. strain 7-1W cells, which
were more easily prepared than the extracted en-
zymes, as the catalyst.
the desired product, cetraxate, quantitatively.
Key words: esterase; cetraxate; regioselective hydroly-
sis; Microbacterium
Cetraxate hydrochloride
(trans-4-[[4-(amino-
methyl)cyclohexyl]carbonyl]oxybenzenpropionate),
which is widely used as an antiulcer medicine, has
been industrially produced by chemical deprotection
of the terminal ester of the esteriˆed intermediate,
benzyl cetraxate1) (Scheme). But the regioselective
deblocking of the terminal ester of benzyl cetraxate is
a troublesome chemical reaction because of the
coexisting labile phenyl ester. We previously reported
the puriˆcation and characterization of a novel
esterase that catalyzes the regioselective hydrolysis of
cetraxate esters from the membrane fraction of
Microbacterium sp. strain 7-1W.2) Concerning the
enzymatic deprotection of cetraxate esters, Kuroda
et al. reported the regioselective hydrolysis of the ter-
minal ester bond of benzyl cetraxate by an esterase,
obtained from a commercial cellulase preparation of
Microbacterium sp. strain 7-1W (AKU 163,
Graduate School of Agriculture, Kyoto University)
was cultured on a medium of 2
NH4Cl, 1 yeast extract (Oriental Yeast Co., Osaka,
Japan), 0.6 K2HPO4, 0.2 NaCl, and 0.02
MgSO4 7H2O, pH 7.0, for 4 days at 28 C with shak-
z glycerol, 1z
z
z
z
z
・
9
ing. After cultivation, cells were harvested by cen-
trifugation and used in the reactions.
Scheme
†
To whom correspondence should be addressed. Tel Fax: +81-75-753-6462; E-mail: kataoka
＠
kais.kyoto-u.ac.jp
W
*
Present address: Research Institute, Daiichi Fine Chemical, Takaoka, Toyama 933-8511, Japan

Biocatalytic Deprotection of a Cetraxate Ester
193
Fig. 2. Regioselective Hydrolysis of the Cetraxate Methyl Ester
to Cetraxate with Microbacterium sp. Strain 7-1W Cells.
Fig. 1. EŠects of the Substrate Concentration on the Hydrolysis
Activity of Microbacterium sp. Strain 7-1W.
The reaction was done at 309C with stirring, with the addition
The reaction mixture contained 50 m
M
potassium phosphate
), 2.5 ),
of 5.0 g of cetraxate methyl ester at 30-min intervals four times
to the reaction mixture, 50 ml, with Microbacterium sp. strain
7-1W cells (5.0 g). The pH of the reaction mixture was con-
buŠer, pH 7.0, 80 mg ml wet cells, and 1.0
z
(

z
(

W
#
5.0
z
(

), 7.5
z
(

), or 10
z
(
) cetraxate methyl ester, in a
C with
ˆnal volume of 10 ml. The reactions were done at 30
9
trolled in the range of 6.5–7.5 with 6
methyl ester; , cetraxate.
M NaOH. , cetraxate

shaking (300 rpm), and the hydrolysis rate was measured period-
ically.

Both the cetraxate methyl ester and cetraxate were
only slightly soluble in water. So, after reactions with
high substrate concentrations, the reaction mixtures
were diluted with water, and then the hydrolysis rates
were measured by high-pressure liquid chromato-
graphy as described previously.2)
scale regioselective hydrolysis was done as follows:
5.0 g (wet weight) of Microbacterium cells was sus-
pended in 50 ml of H2O, and 5.0 g of cetraxate
methyl ester was added slowly (within 2–3 min) to the
reaction mixture. The reaction was done at 309C with
stirring. To avoid a pH drop in the reaction mixture
because of the formation of cetraxate, the pH was
The optimum temperature and pH for the
regioselective hydrolysis of the cetraxate methyl ester
controlled in the range of 6.5–7.5 with 6
M
NaOH
by Microbacterium cells were about 45
9
C and pH
at appropriate intervals. Furthermore, 5.0 g of
cetraxate methyl ester was added, at intervals of
30 min, three times to the reaction mixture. The total
8.0, respectively. However, long-term reaction at
high temperature and at pH 7.0 or above might cause
not only inactivation of the enzyme but also spon-
taneous hydrolysis of the substrate to a by-product,
3-(4-hydroxyphenyl)propionic acid. Further investi-
amount added was 20 g. After reaction for 17 h, 96
z
of the cetraxate methyl ester was converted to
cetraxate, and little of the by-product 3-(4-hydroxy-
phenyl)propionic acid was detected (Fig. 2). There-
fore, the inhibitory eŠect with the high concentration
of cetraxate methyl ester did not arise from the con-
centrations of the products, cetraxate and methanol,
but from the concentration of the substrate. The
gation was done at 30
9C and pH 7.0.
When 80 mg ml of the cells (wet weight) was incu-
W
bated with 1.0, 2.5, or 5.0
z
(w v) of the substrate at
W
30 C, pH 7.0, the hydrolysis reaction proceeded
9
e‹ciently and the substrate was almost stoichiometri-
cally converted to the desired product, cetraxate
(Fig. 1). When the substrate concentration was more
Aspergillus enzyme also is inhibited by a high (
À5z)
concentration of the substrate, the benzyl ester of
cetraxate, and the inhibition seems to be caused by
the accumulation of benzyl alcohol.4) Probably, if the
substrate was added in small portions several times,
improvement in the hydrolysis rate could not be ex-
pected.
than 5.0
z
, the enzyme activity was inhibited, and
the reaction hardly proceeded with 10
z
cetraxate
methyl ester. However, when the substrate was added
to the reaction mixture little by little every few
minutes, the hydrolysis reaction proceeded even with
10
z
cetraxate methyl ester, and after a reaction for
of the initial substrate was converted to
After the product had been collected by ˆltration
and puriˆed by recrystallization from 50z isopro-
50 min, 90
z
cetraxate. The sudden addition of large amount of
the substrate probably caused a local drop of pH in
the reaction mixture, because of the formation of
cetraxate, and this change probably inactivated the
enzyme.
panol, 17.7 g of cetraxate hydrochloride (molar
yield, 92.2 ) was obtained.
Microbacterium sp. strain 7-1W was useful as a
catalyst for the production of cetraxate, and might be
used for deprotection reactions in production proc-
esses.
z
On the basis of these observations, preparative-

194
K. HONDA et al.
Acknowledgments
References
1) Hirayama, T., Kuroda, H., and Iwase, M., Japan
Kokai Tokkyo Koho, 81167648 (Dec. 23, 1988) (Chem.
Abstr., 96, 217482a, 1982).
2) Honda, K., Kataoka, M., Ono, H., Sakamoto, K.,
Kita, S., and Shimizu, S., Puriˆcation and characteri-
zation of a novel esterase promising for the production
of useful compounds from Microbacterium sp. 7-1W.
FEMS Microbiol. Lett., 206, 221–227 (2002).
This work was supported in part by Grants-in-Aid
for Scientiˆc Research Nos. 1729 (to H.K.),
14360054 (to M.K.), and 138530009 (to S.S.), from
the Japan Society for the Promotion of Science. This
work was also done as a part of the Project for
Development of a Technological Infrastructure for
Industrial Bioprocesses on R&D of New Industrial
Science and Technology Frontiers by the Ministry of
Economy, Trade & Industry (METI), and entrusted
by the New Energy and Industrial Technology
Development Organization (NEDO).
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reactivities of cetraxate benzyl ester hydrochloride-
hydrolyzing enzyme. Chem. Pharm. Bull.
,
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2929–2932 (1989).
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