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58
Chemistry Letters 2002
A One-Pot, Two-Step Enzymatic Synthesis of L-Lactic Acid from Acetaldehyde
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Masaya Miyazaki, Kazuya Ogino, Mitsukuni Shibue, Hiroyuki Nakamura, and Hideaki Maeda
Micro-space Chemistry Laboratory, AIST Kyushu, National Institute of Advanced Industrial Science and Technology (AIST),
07-1 Shuku, Tosu, Saga 841-0052
8
(Received April 22, 2002; CL-020352)
A one-pot, two-step enzymatic synthesis of L-lactic acid from
acetaldehyde and carbon dioxide has developed. The reaction was
performed using a combination of reverse reaction of pyruvate
decarboxylase and hydrogenation of pyruvate by lactic dehy-
drogenase. The maximum yield was obtained at pH 9.5.
Recent interest of the problems of environmental pollution
forces to develop a green chemistry process for the chemical
1
industry. Several environmental friendly processes have been
developed. The biocatalytic processes such as fermentation and
enzyme reactions have attracted attentions as environmentally
2;3
safe chemical process.
Scheme 1. Synthetic process of L-lactic acid.
The biodegradable polymers have also been interested for
environmental safety.4 Among the biodegradable plastics, the
poly(L-lactic acid) has been widely studied. Several methods have
;5
and optical rotation value were identical to the commercially
13
6
{9
been developed to produce the lactic acid from many sources.
available L-lactic acid.
However, most of them produce racemic form and require
relatively longer time and multiple steps to obtain pure L-lactic
acid. These disadvantages are problematic not only for large-
scale production but also for the environmental safety. Therefore,
a simple method, which can produce optically pure L-lactic acid,
is desired.
We are interested in the development of novel enzymatic
reactions and reactor systems having environmentally safe
chemical processes. Pyruvate decarboxylase (EC 4.1.1.1) is
known as a catalyst of the decarboxylation reaction of pyruvic
acid to produce acetaldehyde, and has been utilized as a catalyst
for C–C bond formation such as carboligase for synthesis of chiral
Because the reverse reaction of pyruvate decarboxylase
proceeds at higher pH, the yield should become better than that of
neutral pH. Therefore, the effect of pH (7 to 10.5) on the reaction
was examined, first. The results were summarized on Table 1
(entries 1 to 8). In the present study, the yield of lactic acid was
maximized at pH 9.5. Much higher pH gave lower yield of L-lactic
acid and gave relatively better recovery of pyruvic acid. This
result can be explained as follows. The first step proceeds at
higher pH, whereas the second step, hydrogenation by L-lactic
dehydrogenase, gave lower yield at much higher pH, yielded
decrease of production of L-lactic acid and therefore the recovery
of pyruvic acid was increased (Table 1).
ꢀ
organic and pharmaceutical chemistry.
-hydroxy ketones, which are versatile building blocks for
We investigated the
Because the reverse reaction of pyruvate decarboxylase
prefers higher concentration of bicarbonate buffer, we also
examined the effect of concentration of bicarbonate buffer at pH
9.5. As shown on Table 1 (entries 6, 9–13), higher concentration
of bicarbonate gave better yield, as expected. The maximum yield
was obtained at 500 mM bicarbonate buffer (51%). Our previous
study of the reverse reaction of pyruvate decarboxylase yielded
about 43% of pyruvic acid production in 500 mM bicarbonate
buffer at pH 9.5. In the present study, the combined yield of lactic
acid and pyruvic acid exceeds 65%, meaning the yield of the
reverse reaction of pyruvate decarboxylase was improved than
that by alone. This might result from change of equilibrium
condition of the reaction, namely pyruvic acid consumption by
the second step might promote better yield of the first step. Further
studies are required to solve the detail of this mechanism.
Recent interest of poly(L-lactic acid) demands the effective
method to produce the material, L-lactic acid. The fermentation
method has been focused as effective solution, because it can
10;11
reverse reaction of pyruvate decarboxylase and found that the
reverse reaction can be achieved in the basic condition.12 Because
pyruvic acid can be easily hydrogenated asymmetrically by L-
lactic dehydrogenase, it is possible to produce L-lactic acid from
acetaldehyde and carbon dioxide by combining these enzyme
reactions (Scheme 1). Here we report a one-pot, two-step
enzymatic synthetic procedure for L-lactic acid from acetalde-
hyde.
The experimental was performed as follows. To a solution of
acetaldehyde (0.1 ꢁM), thiamine pyrophosphate (0.1 ꢁM),
NADH (0.2 ꢁM) in various concentration and pH of NaHCO3-
Na2CO3 buffer. The reaction was initiated by adding pyruvate
decarboxylase (1 unit) and L-lactic dehydrogenase (1 unit) at
room temperature. The reaction was performed 1 h, then the
mixture was analyzed by RP-HPLC. The amount of each
compound was estimated by peak area calibrated by commer-
cially available standards. The absolute configuration of lactic
acid was confirmed by the optical rotation, and the yield was
calculated based on acetaldehyde. For confirmation, the yielded
6
{9
produce relatively pure L-isomer. Our method utilizes enzy-
matic reaction, which proceeds in shorter times and gives the
product in higher purity than that by fermentation. Although
further studies are required to establish efficient pilot-scale
1
lactic acid was subjected to the analysis. The H-NMR spectra
Copyright Ó 2002 The Chemical Society of Japan
Miyazaki, Masaya
