Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration

Article abstract of DOI:10.1271/bbb.70772

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO ₃^- fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO ₃^- and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30°C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD¹.

Full text of DOI:10.1271/bbb.70772

Biosci. Biotechnol. Biochem., 72 (5), 1278–1282, 2008
ꢀ
Reverse Reaction of Malic Enzyme for HCO3 Fixation into Pyruvic
Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration
1
1
2
1;y
Yoko OHNO, Toshihiko NAKAMORI, Haitao ZHENG, and Shin-ichiro SUYE
1
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering,
University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan
2
School of Material Science and Chemical Engineering, Tianjin Polytechnic University,
Tianjin 300160, China
Received November 29, 2007; Accepted February 12, 2008; Online Publication, May 7, 2008
[doi:10.1271/bbb.70772]
þ
Malic enzyme [L-malate: NAD(P) oxidoreductase
EC 1.1.1.39)] catalyzes the oxidative decarboxylation of
L-malic acid to produce pyruvic acid using the oxidized
cially, there is currently great interest in the fixation of
CO with an enzymatic reaction system, because the
(
2
enzymatic method is an environmentally friendly proc-
1)
þ
form of NAD(P) (NAD(P) ). We used a reverse reaction
of the malic enzyme of Pseudomonas diminuta IFO
ess and that can be applied in industry.
Reverse reactions of malic enzymes have been
designed and used in CO2 fixation with coenzyme
regeneration such as photochemical and electrochemical
ꢀ
1
3182 for HCO3 fixation into pyruvic acid to produce
L-malic acid with coenzyme (NADH) generation. Glu-
cose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuco-
nostoc mesenteroides was suitable for coenzyme regen-
eration. Optimum conditions for the carboxylation of
pyruvic acid were examined, including pyruvic acid,
2
–5)
þ
reactions.
Malic enzyme (L-malate:NAD(P) oxido-
reductase (decarboxylating) EC 1.1.1.38-40) is known as
a catalyst of the decarboxylation reaction of L-malic
þ
acid, producing pyruvic acid with NAD(P) reduction.
Malic enzyme also catalyzes the fixation of carbon
þ
NAD , and both malic enzyme and glucose-6-phosphate
dehydrogenase concentrations. Under optimal condi-
6
)
dioxide, forming L-malic acid, but it has proved to be
difficult to control CO2 fixation given the unfavorable in
vitro situation. In addition, the malic enzyme reaction
consumes the coenzyme at the same rate as the substrate.
Regeneration of coenzyme is important in carrying out
the malic enzyme reverse reaction.
ꢀ
tions, the ratio of HCO3 and pyruvic acid to malic acid
ꢁ
was about 38% after 24 h of incubation at 30 C, and the
concentration of the accumulated L-malic acid in the
reaction mixture was 38 mM. The malic enzyme reverse
reaction was also carried out by the conjugated redox
enzyme reaction with water-soluble polymer-bound
We have found that Pseudomonas diminuta IFO13182
produced malic enzyme (EC 1.1.1.39), and we have
investigated its applications, focusing on the production
þ
NAD .
ꢀ
7,8)
Key words: HCO3 fixation; malic enzyme; NADH
regeneration; L-malic acid
of a reduced form coenzyme, such as NAD(P)H.
We succeeded in L-alanine production combined with
malic enzyme and alanine dehydrogenase in a NADH
9
)
Abundant atmospheric CO is one factor causing the
2
regeneration system. In this case, the malic enzyme
reaction favored a forward reaction for decarboxylation
of L-malic acid with NAD reduction. In this study, we
greenhouse effect. The concentration of CO2 is far
higher than that of other gasses, such as CH4, NOx, and
CFC (fluorocarbons). About 2,000 to 3,500 million tons
of CO2 naturally exist in the air, but 5,500 million
tons of artificial CO2 gas is annually discharged. One
important proposal to reduce atmospheric CO2 is to
develop new sources of energy, but it will take time and
money to replace the current energy system. New
technologies are quickly needed at least to reduce the
huge volume of CO2 from thermal power plants and
chemical plants. There have been various attempts to fix
CO2, including physical and chemical processes. Espe-
þ
investigated the reverse reaction of the malic enzyme
of P. diminuta IFO 131832 with enzymatic NADH
regeneration in a preliminary study of CO2 fixation, as
shown Fig. 1.
Materials and Methods
Chemicals. Glucose-6-phosphate dehydrogenase (G-
6-PDH, EC1.1.1.49, 400 unitsꢁmgꢀ ) of Leuconostoc
mesenteroides was obtained from Oriental Yeast
1
y
To whom correspondence should be addressed. Tel/Fax: +81-776-27-8914; E-mail: suye@acbio2.acbio.fukui-u.ac.jp
0
Abbreviations: G6PDH, glucose-6-phosphate dehydrogenase; HEPES, N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid; MES, 2-morpholino-
ethanesulfonic acid, monohydrate

Reverse Reaction of Malic Enzyme with Coenzyme Regeneration
1279
reverse reaction with NADH regeneration using G-6-
þ
G-6-PDH
D-Glucose-6-
phosphate
D-Glucono-
PDH contained 1.0 mmol of NAD , 100 mmol of G-6-P,
δ-lactone-
1
00 mmol of pyruvic acid, 15 mmol of MgCl , 100 mmol
2
6-phosphate
KHCO3, 0.5 unit of malic enzyme, and 0.5 unit of G-6-
PDH in 1.0 ml of 55 mM potassium phosphate buffer
NAD+
NADH
ꢂ
(
pH 7.4). The reaction mixture was incubated at 30 C.
Pyruvate
L-Malate
Preparation of water-soluble macromolecular poly-
þ
Malic enzyme
+
CO + H
2
mer immobilized NAD . Water soluble polymer (sodium
þ
þ
alginate) immobilized NAD (Alg-NAD ) was pre-
11)
Fig. 1. Design of Malic Enzyme Reverse Reaction with Coenzyme
Regeneration Conjugated with G-6-PDH.
pared as previously described. Samples of 13.8 mg of
sodium alginate and 70 mmol 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride were dis-
solved in 15 ml of water. The final pH was adjusted to
4.7, and the solution was stirred for 40 min at room
(
(
Tokyo). Pig heart lactate dehydrogenase of
ꢀ
1
EC1.1.1.27, 350 unitsꢁmg
)
was obtained from
þ
þ
Toyobo (Tokyo). NAD was from Oriental Yeast.
0
temperature. NAD was added to the solution to
N-2-Hydroxyethylpiperazine-N -2-ethanesulfonic acid
(
70 mmol. After readjustment of the pH to 4.7, the
resulting solution was stirred for 12 h at room temper-
ature. The reaction mixture was dialyzed with 10 mM
Tris–HCl buffer (pH 7.0) for 12 h, and then dialyzed
HEPES) and 2-morpholinoethanesulfonic acid, mono-
hydrate (MES) were from Nacalai Tesque (Kyoto).
The other reagents and compounds were of analytical
grade and were used without further purification.
ꢂ
þ
with water for 12 h at 5 C. Alg-NAD was recovered
by lyophilization. Lyophilized products were stored at
ꢂ
Preparation of malic enzyme. Pseudomonas diminuta
IFO 13182 was cultured in a 30-liter jar-fermenter and
prepared as a cell-free extract with partial purification,
ꢀ20 C in the dark. The coenzyme activity of Alg-
þ
NAD was determined enzymatically by the alcohol
1
2)
dehydrogenase system.
1
0)
as described previously. Malic enzyme was purified
4
4
4-fold over the cell-free extract with a final yield of
9% and a specific activity of 0.989 unitsꢁmgꢀ1 protein.
HPLC analysis. HPLC analysis of L-malic acid,
pyruvic acid, and L-lactic acid in the reaction mixture
ꢀ
was performed using a Hitachi L-7100 and Shodex
Asahipak ES-502N (100 ꢃ 7:6 mm i.d.) (Showa Denko,
Assay of malic enzyme activity for reverse and
forward reactions. The enzyme activity for the forward
reaction was measured as previously described.8) The
reverse reaction was measured in a KHCO solution as a
carbon source via the NADH-conversion of L-malic acid
from pyruvic acid. Activity was measured spectrophoto-
metrically by measuring the decrease in the absorbance
of NADH at 340 nm. The substrate mixture contained
ꢂ
Tokyo) at 50 C. The elution buffer consisted of
0.1 M potassium dihydorogenphosphate (KH2PO4)
ꢀ1
ꢂ
buffer (pH 4.2) at a flow rate of 1.0 mlꢁmin at 50 C.
The detector was a Hitachi U2010 ultraviolet/visible
spectrophotometer (Hitachi, Tokyo) at 210 nm. The
retention times of L-malic acid and pyruvic acid were
21.8 min and 11.1 min respectively.
3
6
0 mmol of pyruvic acid, 30 mmol of MgCl2, 0.6 mmol of
NADH, 60 mmol KHCO3, and 100 mmol of HEPES-
KOH buffer, pH 7.4, in a total volume of 2.0 ml. The
reaction was started by adding 1.0 ml of the enzyme
Results and Discussion
Reverse reaction activity of malic enzyme
ꢂ
solution, and the mixture was incubated at 30 C.
The reverse reaction (carboxylation) and forward
reaction (decarboxylation) activities of malic enzyme
were measured at different pH levels. As shown in
Fig. 2, the optimum pH for the reverse reaction was
at 6.0, and carboxylation was strongly dependent on
the pH value, but the reverse reaction activity was
9.7 mMꢁminꢀ , representing only 5% of the forward
reaction activity. Considering the results for the opti-
mum pH for the malic enzyme reverse reaction and the
stability of both the reduced form (NADH) and the
After exactly 2 min, 1.0 ml of 10% (w/v) sodium
dodecyl sulfate (SDS) was added to stop the reaction,
and the absorbance at 340 nm was measured. In a blank
test, 10% SDS was added before the enzyme solution.
One unit of activity was defined as the amount of the
1
enzyme that produced 1 mmol of NADH per min at
ꢂ
3
0 C. In calculating the amount of NADH produced, a
3
molar absorption coefficient for NADH of 6:22 ꢃ 10
ꢀ1
ꢀ1
M
ꢁcm was used.
þ
13,14)
oxidized form (NAD ) coenzyme,
pH 7.4 was used
Synthesis of L-malic acid by coenzyme regeneration
thereafter in malic enzyme reverse reaction mixtures.
The reverse reaction of the malic enzyme with
potassium ions was slightly better in terms of CO2
fixation (5% of the forward reaction) than with sodium
using G-6-PDH. The reverse reaction of malic enzyme
with NADH regeneration using G-6-PDH was also
carried out using glucose-6-phosphate (G-6-P) and
G-6-PDH.
ions (2% of the forward reaction). KHCO and potas-
3
The standard reaction mixture for the malic enzyme
sium pyruvate were used in further investigation.

1280
Y. OHNO et al.
1
00
7
5
2
5
0
5
0
4
5
6
7
8
9
pH
Fig. 2. Effect of pH on Reverse Reaction (carboxylation) Activity of Malic Enzyme.
Enzyme activity was measured at pH 5–8. Symbols: , MES buffer; , HEPES buffer.
50
40
30
20
10
0
50
40
30
20
10
0
0
5
10
15
20
25
Reaction time (h)
þ
Fig. 3. Effect of NAD Concentration on Malic Enzyme Reverse Reaction System.
The pyurvic acid concentration was 500 mM and the concentration of other components was the same as for the standard reaction mixture.
þ
þ
þ
Symbols: , 1.0 mM NAD ; , 0.5 mM NAD ; , 0.1 mM NAD .
Reverse reaction of malic enzyme with coenzyme
regeneration using G-6-PDH
Initially, LDH was used in the malic enzyme
combined with the reverse reaction of malic enzyme.
Two substrate systems were designed to improve
coenzyme regeneration. The G-6-PDH reaction was
used as the NADH regenerator in the system, since
G-6-PDH is useful in the regeneration of NADH from
1
5)
reaction for NADH regeneration, but L-malic acid,
ꢀ
an indicator of HCO3 fixation into pyruvic acid in the
þ
16,17)
reaction system, was not produced in the reaction
mixture (data not shown). The equilibrium of the LDH
reaction for NADH regeneration is not favorable in the
neutral pH region, and L-lactic acid causes a decrease
in the catalytic activities of the malic enzyme.3) LDH
was thus not suitable for the regeneration reaction
NAD in the neutral pH region
(Fig. 1).
The effects of several conditions on the malic enzyme
reverse reaction were investigated. The effects of the
þ
NAD concentration on the malic enzyme reverse
reaction are shown in Fig. 3. Under concentrations of
þ
0.5 and 1.0 mM NAD in the reaction mixture, 39 to

Reverse Reaction of Malic Enzyme with Coenzyme Regeneration
1281
50
40
30
20
10
0
50
40
30
20
10
0
0
5
10
15
20
25
Reaction time (h)
þ
þ
Fig. 4. Malic Enzyme Reverse Reaction System with Coenzyme Regeneration Using Alg-NAD .
The reaction was performed under standard conditions, except for the indicated Alg-NAD . The concentrations of other components were
þ
þ
þ
the same as for the standard reaction mixture. Symbols: , 1.0 mM Alg-NAD ; , 0.5 mM Alg-NAD ; , 0.1 mM NAD . The concentration
þ
þ
of Alg-NAD indicates an equimolar concentration of free NAD .
4
0 mM L-malic acid was produced.
When a reaction mixture containing 100 mM pyruvic
0.1 mM coenzyme concentration in the reaction mixture;
the NAD cycling number (moles of L-malic acid
þ
þ
acid and 0.5 mM NAD was used in the malic enzyme
reverse reaction, L-malic acid production was 38 mM
after 24 h of incubation (data not shown). The ratio of
HCO3 and pyruvic acid to L-malic acid was about
3
produce per NAD in the reaction mixture) was
þ
calculated to be 200. When 0.1 mM free NAD was
used in the reaction, the NAD cycling number was 76
(data not shown).
ꢀ
ꢀ
8%. On the other hand, the synthesis of L-malic acid
In conclusion, we carried out HCO3 fixation into
combined with malic enzyme reverse reaction and
photochemical coenzyme regeneration has been report-
pyruvic acid to produce L-malic acid in the reverse
reaction of the malic enzyme of P. diminura IFO 13182
with coenzyme regeneration. Recently, several types
of enzymatic HCO3 and CO2 fixation using the
dehydrogenase reverse reaction have been developed.
For example, several series reactions of three enzymes
5,18)
ꢀ
ed.
The ratio of HCO3 and pyruvic acid to L-malic
acid was from 1.1 to 6.6%. Using 100 mM pyruvic acid
ꢀ
þ
and 0.5 mM NAD in the reaction mixture was suitable
for the production of L-malic acid.
(formate dehydrogenase, aldehyde dehydrogenase,
and alcohol dehydrogenase) have been applied in
þ
Reverse reaction of malic enzyme with Alg-NAD as
coenzyme
1
9)
CO2 fixation into methanol. Isocitrate dehydrogen-
3)
2
0,21)
In a previous study, we successfully prepared Alg-
ase and malic enzyme can also be applied in
carboxylation. However, in these cases, CO fixed from
þ
þ
NAD coupling amino groups of NAD and the
carboxy group of alginic acid with water soluble
carbodiimide.1 Immobilization of the coenzyme as
well as the enzyme is necessary for continuous or
repeated use. We also investigated the reverse reaction
of malic enzyme with coenzyme regeneration using
2
0.007–1.1 mM of the products. In contrast, the present
study indicates 40 mM KHCO3 fixation into L-malic
acid, which is about 300 times more efficient than
in other studies. This improvement of the conjugated
coenzyme regeneration system warrants further inves-
tigation and refinement. Since G-6-P is expensive
and unstable, it is not suitable for practical use. We
also developed a modified electrode using a viologen
1)
þ
Alg-NAD as a coenzyme. As shown in Fig. 4, both
þ
cases of Alg-NAD concentration (equimolar of 0.5 and
þ
1
.0 mM free NAD ) produced L-malic acid, accumulat-
þ
þ
ing to about 33 mM. Alg-NAD is also useful in L-malic
acid production in the malic enzyme reverse reaction
derivative as mediator and Alg-NAD(P) immobilized
on the electrode surface for efficient NADH regener-
ation. Enzyme reaction conjugated with electrochemical
22)
coenzyme regeneration has been accomplished.
þ
system. We have found that the NAD moieties on
þ
Alg-NAD work as an electron transfer carrier in the
1
1)
ꢀ
conjugated two oxidoreductase reaction. In the
reverse reaction of malic enzyme using the coenzyme
HCO3 fixation into pyruvic acid combined with
electrochemical regeneration of coenzyme is now
under study.
þ
regeneration with G-6-PDH, Alg-NAD also showed
improvement in the reaction at 100 mM pyruvic acid and

1282
Y. OHNO et al.
Acknowledgments
11) Nakamura, Y., Suye, S., Tera, H., Kira, J., and Senda,
þ
M., Electron-transfer function of NAD -immobilized
alginic acid. Biochim. Biophys. Acta, 1289, 221–225
S. Suye gratefully acknowledges financial support for
this research from the Japan Securities Scholarship
Foundation (Tokyo) and The Foundation for the Earth
Environment (Tokyo).
(
2) Izumi, Y., Mishra, S. K., Ghosh, B. S., Tani, Y., and
1996).
1
þ
Yamada, H., NADH production from NAD using
formate dehydrogenase system with cells of a methanol-
utilizing bacterium. J. Ferment. Technol., 61, 135–142
(1983).
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