206
Published on the web January 26, 2011
Production of p-Aminosalicylic Acid through Enzymatic Kolbe-Schmitt Reaction Catalyzed
by Reversible Salicylic Acid Decarboxylase
Kohtaro Kirimura,* Satomi Yanaso, Sachiyo Kosaka, Keiko Koyama, Takasumi Hattori, and Yoshitaka Ishii
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University,
3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555
(Received December 6, 2010; CL-101036; E-mail: kkohtaro@waseda.jp)
A reversible salicylic acid decarboxylase (Sdc), found in the
yeast Trichosporon moniliiforme WU-0401, is applicable for the
production of p-aminosalicylic acid (PAS) from m-aminophenol
(m-AP). For the high-yield production of PAS, used as an
antituberculous agent, we developed F195Y, a genetically
engineered Sdc mutant. We succeeded in selectively producing
PAS from m-AP through an enzymatic Kolbe-Schmitt reaction
in aqueous solution by using recombinant Escherichia coli cells
expressing the gene encoding F195Y. We found that 70 mM PAS
was produced at 30 °C in 15 h with a conversion yield of 70%
(mol/mol).
OH
OH
CO2
CO2
COOH
H N
2
H N
2
m-AP
PAS
Figure 1. Enzymatic reversible conversion of m-aminophenol
(m-AP) to p-aminosalicylic acid (PAS).
recently succeeded in generating a mutant enzyme, F195Y, more
suitable for production of PAS, by site-directed mutagenesis
toward Sdc (unpublished data).
The Kolbe-Schmitt reaction is a well-known method for
synthesizing aromatic hydroxycarboxylic acids via a carbox-
ylation reaction of phenol salts of alkali metal at high temper-
ature and pressure.1 Various salicylic acid derivatives are
industrially produced by this method for use as medicines,
herbicides, and industrial products. However, the Kolbe-Schmitt
reaction generates a large amount of by-products, which then
have to be separated; moreover, the high reaction temperature
and pressure have a negative impact on the environment.
The biological carboxylation of phenolic compounds using
enzymes is expected to be a novel ecological alternative to the
Kolbe-Schmitt reaction because regioselective and ecological
carboxylation might be possible under environmentally benign
conditions. Several decarboxylases such as 3,4-dihydroxyben-
zoate decarboxylase,2 £-resorcylic acid decarboxylase,3-5 and
pyrrole-2-carboxylate decarboxylase6 have been found to act as
non-oxidative decarboxylases, which reversibly catalyze the car-
boxylation of aromatics into aromatic carboxylic acids. Further,
we previously isolated Trichosporon moniliiforme WU-0401 as
a salicylic acid-degrading yeast, and found that both whole cells
and cell-free extract of T. moniliiforme WU-0401 could convert
salicylic acid to phenol.7 Moreover, we discovered a novel
enzyme salicylic acid decarboxylase (Sdc) in T. moniliiforme
WU-0401, which reversibly catalyzes the regioselective carbox-
ylation of phenol to salicylic acid.8 We have already reported the
purification, characterization, gene cloning, and gene expression
of Sdc in a previous paper.8 In the course of study, we confirmed
that recombinant Escherichia coli strongly expressing the gene
(sdc) encoding Sdc can be used as an efficient and convenient
biocatalyst for the selective production of salicylic acid from
phenol via a whole cell reaction.8
In this report, we describe the production of PAS through
the enzymatic Kolbe-Schmitt reaction using Sdc and F195Y
through the whole cell reaction of recombinant E. coli cells.
To the best of our knowledge, this is the first report on the
application of enzymes in the Kolbe-Schmitt reaction for
selective and high-yield production of PAS in aqueous solution
under environmentally benign conditions, i.e., at room temper-
ature and atmospheric pressure.
First, we carried out the enzymatic Kolbe-Schmitt reaction
through a whole cell reaction with recombinant E. coli cells
expressing sdc and then identified the product obtained from
the carboxylation reaction of m-AP substrate. The sdc gene was
inserted into pET-21a (Novagen, WI, USA) to generate pSdc as
described previously.8 A whole cell reaction with recombinant
E. coli BL21(DE3) harboring pSdc was then performed with
m-AP and KHCO3. High-performance liquid chromatography
(HPLC) analysis revealed a unique product in the whole cell
reaction mixture with a retention time of 10.7 min, and liquid
chromatography-mass spectrometry (LC-MS) analysis revealed
that the protonated molecule of this product was m/z 154.1
(M + H)+ (Figure 2A). Both of these findings are identical to
those of an authentic PAS sample. Based on the chemical shifts
of authentic PAS, the signals in 1H NMR and 13C NMR are
assigned as shown in Figure 2B and Figure 2C.9 Based on the
chemical shifts of authentic m-AP, the signal at ¤ 7.0 (dd) in
1H NMR was assigned to the proton at C5 of m-AP, signals in
the range of ¤ 6.25-6.31 were assigned to protons at C2 and C6.
A distinctive signal of estimated chemical shift at ¤ 7.18 (dd) in
2-amino-6-hydroxybenzoic acid, as a regioisomer of PAS, was
detected in 1H NMR, but a distinctive signal of estimated
chemical shift at ¤ 136.2 was not detected in 13C NMR. A
distinctive signal of estimated chemical shift at ¤ 164.5 of 2-
amino-4-hydroxybenzoic acid was not detected in 13C NMR.
Other signals of estimated chemical shifts of 2-amino-6-
hydroxybenzoic acid, 2-amino-4-hydroxybenzoic acid, and 3-
amino-5-hydroxybenzoic acid cannot be assigned because these
signals are similar to those of PAS and m-AP. These results
p-Aminosalicylic acid (PAS) is an important derivative of
salicylic acid and has widely been used as an antituberculous
agent. In a previous paper, we found that Sdc catalyze the
regioselective carboxylation of m-aminophenol (m-AP) to form
PAS and the decarboxylation of PAS to form m-AP, that is
reversible conversion as shown in Figure 1.8 Moreover, we have
Chem. Lett. 2011, 40, 206-208
© 2011 The Chemical Society of Japan
Kirimura, Kohtaro
