N-terminal truncation of a maleate cis-trans isomerase from Rhodococcus jostii RHA1 results in a highly active enzyme for the biocatalytic production of fumaric acid

Article abstract of DOI:10.1016/j.molcatb.2013.04.002

As part of the project to develop an efficient biocatalytic process for the production of fumaric acid, a full-length putative maleate cis-trans isomerase gene from Rhodococcus jostii RHA1 was synthesized and expressed in Escherichia coli Rosetta2 (DE3) pLysS, but the protein was not soluble and showed no catalytic activity. Bioinformatics analysis of the protein sequence indicated that there were two hydrophilic and two hydrophobic amino acid clusters in an alternate arrangement at the N-terminus, and 50 extra amino acid residues at the N-terminus were not present in the known maleate cis-trans isomerases. The alternate hydrophilic and hydrophobic clusters at the N-terminus were thus truncated one by one to evaluate their effect on the gene expression and enzyme activity. Three mutants (MaiR-D41/42-304AA, MaiR-D48/49-304AA and MaiR-D52/53-304AA) without the hydrophilic and hydrophobic clusters were expressed as soluble protein with maleate cis-trans isomerase activity. Among them, MaiR-D48 was purified and its properties were studied. The purified enzyme had a temperature optimum of 40 C and a wide pH range (5.0-9.0) with the optimum pH being 8.0. The whole cells of E. coli expressing MaiR-D48 catalyzed the isomerization of maleic acid to fumaric acid at 1 M substrate concentration, showing its potential for industrial use.

Full text of DOI:10.1016/j.molcatb.2013.04.002

Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
N-terminal truncation of a maleate cis–trans isomerase from Rhodococcus jostii
RHA1 results in a highly active enzyme for the biocatalytic production of fumaric
acid
Xiangtao Liu, Qing Zhao, Jie Ren, Wenyue Dong, Qiaqing Wu∗, Dunming Zhu∗
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area,
Tianjin 300308, China
a r t i c l e i n f o
a b s t r a c t
Article history:
As part of the project to develop an efficient biocatalytic process for the production of fumaric acid, a
full-length putative maleate cis–trans isomerase gene from Rhodococcus jostii RHA1 was synthesized and
expressed in Escherichia coli Rosetta2 (DE3) pLysS, but the protein was not soluble and showed no catalytic
activity. Bioinformatics analysis of the protein sequence indicated that there were two hydrophilic and
two hydrophobic amino acid clusters in an alternate arrangement at the N-terminus, and 50 extra amino
acid residues at the N-terminus were not present in the known maleate cis–trans isomerases. The alternate
hydrophilic and hydrophobic clusters at the N-terminus were thus truncated one by one to evaluate their
effect on the gene expression and enzyme activity. Three mutants (MaiR-D41/42-304AA, MaiR-D48/49-
304AA and MaiR-D52/53-304AA) without the hydrophilic and hydrophobic clusters were expressed as
soluble protein with maleate cis–trans isomerase activity. Among them, MaiR-D48 was purified and its
properties were studied. The purified enzyme had a temperature optimum of 40 ◦C and a wide pH range
(5.0–9.0) with the optimum pH being 8.0. The whole cells of E. coli expressing MaiR-D48 catalyzed the
isomerization of maleic acid to fumaric acid at 1 M substrate concentration, showing its potential for
industrial use.
Received 11 December 2012
Received in revised form 24 March 2013
Accepted 7 April 2013
Available online 18 April 2013
Keywords:
Rhodococcus jostii
Maleate cis–trans isomerase
Fumaric acid
Biocatalytic isomerization
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
of fumarate from maleate. Despite its potential industrial applica-
tion, this enzyme has been barely studied since several reports by
The multiple functional groups of fumaric acid offer it many
potential applications in the chemical synthesis. The most impor-
tant one is its use as the starting material for the industrial
production of aspartic acid by aspartase (l-aspartate ammonia-
lyase [EC 4.3.1.1]) [1,2]. Fumaric acid can also be converted to many
other useful chemicals or materials, such as l-malic acid [2–4],
polyester resins and other biodegradable polymers [5–7]. Fumaric
acid has been widely used as a food acidulent in the bakery and
beverage industries.
Currently, industrial production of fumaric acid is based on
catalytic isomerization of maleic acid in aqueous solutions under
strong acidic conditions. Maleic acid is obtained in large volumes
by hydrolysis of maleic anhydride, which is produced via catalytic
oxidation of benzene or butane. Maleate cis–trans isomerase [EC
5.2.1.1] catalyzes the isomerization of maleate to its geometric iso-
mer fumarate with a high equilibrium constant in a broad pH range.
As such, it should serve as a promising enzyme for the production
Hatakeyama et al. around 2000 [8–10]. The growing need for replac-
ing the current production process of fumaric acid with a greener
one has stimulated our interest in the discovery of new maleate
isomerases.
In recent years, the growing microbial genomic data offers
a tremendous opportunity for discovery of new enzyme cat-
alysts with industrial application potential [11,12]. A genome
mining approach has been successfully used to identify target
enzymes [13]. As part of our project aiming to develop an effi-
cient process for the production of fumaric acid from maleic
acid catalyzed by a maleate isomerase to replace the acid-
catalyzed one, an in silico screening of DNA sequence database
(http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) for the
putative maleate cis–trans isomerase genes in microorganisms was
performed [14]. R. jostii RHA1 was isolated from lindane con-
taminated soil and utilizes a wide range of aromatic compounds,
carbohydrates, nitriles, steroids, and other compounds as sole
sources of carbon and energy [15]. It is known to catabolize a diverse
range of plant derived compounds in an O2-rich environment [16].
A putative maleate cis–trans isomerase gene (accession number
YP 700472) from R. jostii RHA1 was then selected as the target gene
∗
Corresponding authors. Tel.: +86 22 84861962; fax: +86 22 84861996.
E-mail addresses: wu qq@tib.cas.cn (Q. Wu), zhu dm@tib.cas.cn (D. Zhu).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.04.002

X. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
45
Table 1
The oligonucleotides used for PCR to obtain the truncated genes of maleate cis–trans isomerase from R. jostii.
Amino acid residue
Nde I-forward primer
BamH I-reverse primer
MaiR-D20 (21-304AA)
MaiR-D27 (28-304AA)
MaiR-D34 (35-304AA)
MaiR-D41 (42-304AA)
MaiR-D48 (49-304AA)
MaiR-D52 (53-304AA)
5ꢀ-GGAATTCCATATGCTCGCGCCACTCGGTG-3ꢀ
5ꢀ-GGAATTCCATATGACGAATCA-3ꢀ
5ꢀ-GGAATTCCATATGATACTCGTCAT-3ꢀ
5ꢀ-GGAATTCCATATGCTACGTCCG-3ꢀ
5ꢀ-GAATTCCATATGAACCGGCATGC-3ꢀ
5ꢀ-GAATTCCATATGGGCATTCATCG-3ꢀ
5ꢀ-CGGGATCCTACTGAACAGTGGTGGC-3ꢀ
The restriction sites are underlined.
[16]. However, the full-length protein was obtained in an inactive
aggregate form. Based on the bioinformatics analysis of the pro-
tein sequence, truncations of the different number of amino acid
residues at the N-terminus were performed, resulting three active
mutant maleate cis–trans isomerases. Herein we report the charac-
terization of one of these active mutant enzymes and its application
in the biocatalytic synthesis of fumaric acid.
2.4. Purification of MaiR-D48
E. coli Rosetta2 (DE3) pLysS cells carrying pET32a-MaiR-D48
were cultivated at 1 L scale and 5 g of wet cells were harvested
(9000 × g for 15 min). The cells were re-suspended in 100 mL of
Buffer A and lysed by High Pressure Homogenizer. Cell debris
was discarded after centrifugation at 30,000 g for 20 min. The
supernatant was precipitated by 45% ammonium sulfate and the
precipitate was removed by centrifugation. The resulting solution
was further precipitated by continually adding ammonium sulfate
up to 55%, and the precipitate was collected and re-suspended in
buffer A. The resulting solution was desalted and then loaded onto a
DEAE Sepharose Fast Flow column (GE Healthcare). The target pro-
tein in the column was eluted with Buffer A containing 0.25 M NaCl.
The active fractions were collected and concentrated using Amicon
Ultra-4 (10 kDa cutoff, Millipore). The concentrated sample was
then applied onto a sephacryl S-200 HR column (GE Healthcare),
which was equilibrated with Buffer A containing NaCl (0.15 M).
The entire purification procedure was followed by SDS-PAGE anal-
ysis. The concentrations of protein solution were determined by the
Bradford method using BSA as a standard, and the enzyme activ-
ity was assayed as described in the following section. The purified
enzymes were concentrated to about 1 mg/mL and stored at −80 ◦C
for use.
2. Materials and methods
2.1. Materials and strains
BactoTM Peptone, yeast extract were purchased from BD, USA.
Unless otherwise stated, all chemicals were of analytical grade
and purchased from Sigma-Aldrich or other commercial sources.
Escherichia coli Rosetta 2 (DE3) pLysS was purchased from Merck
(Bad Soden, USA).
2.2. Construction of expression vectors
The putative maleate cis–trans isomerase gene (accession
number YP 700472) from R. jostii RHA1 was synthesized by
Xuguan Biotechnology company (Shanghai, China), and cloned
into pET32a with NdeI and BamHI sites, giving plasmid pET32a-
MaiR. The plasmids of the truncated genes of MaiR were obtained
as follows: The truncated genes were amplified by PCR with
PrimerStar HS DNA polymerase (TAKARA, Japan) using primers
in Table 1 and the full-length gene as template. The PCR prod-
ucts were cloned into pET32a with NdeI and BamHI sites to
get the recombined plasmids, which were verified by restric-
tion analysis and sequencing of the plasmid DNA. The profile
computation and representation of full-length protein sequence
coded by maleate cis–trans isomerase gene was predicted by
Protein Identification and Analysis Tools on the ExPASy Server
(http://web.expasy.org/protscale/)
2.5. Activity assay
Ammonium maleate solution (1 M) was prepared by dissolv-
ing maleate in H2O and then adjusting pH with ammonia to
8.0. The reaction mixture, obtained by mixing 10 ␮L of MaiR-
D48 enzyme solution (1 mg/mL) and 20 ␮L of ammonium maleate
(1 M, pH 8.0) and then adding Buffer B (20 mM potassium phos-
phate, 5 mM of ␤-ME, pH 8.0) to 1 mL, was shaken at desired
temperature for 10 min. A sample of 500 ␮L reaction mixture was
withdrawn, and the reaction was terminated by adding 500 ␮L of
HCl solution (pH 1.37), the contents of maleic acid and fumaric
acid were measured by HPLC analysis. One unit of maleate cis–trans
isomerase activity was defined as the amount of enzyme that cat-
alyzed the formation of 1 ␮mol of fumaric acid from maleic acid in
1 min.
2.3. Expression of MaiR and the truncated MaiRs
The pET32a-MaiR or the truncated pET32a-MaiR vector (e.g.
pET32a-MaiR-D48) was transformed into E. coli strain Rosetta2
(DE3) pLysS, containing a chromosomal copy of the T7 RNA poly-
merase gene under the control of the lacUV5 promoter. E. coli
Rosetta2 (DE3) cells carrying pET32a-MaiR or truncated pET32a-
MaiR genes were grown for up to 3 h in 50 mL of LB medium
containing 100 ␮g mL−1 of ampicillin and 34 ␮g mL−1 of chloram-
phenicol at 37 ◦C and shaken at 220 rpm. When OD600 reached
approximately 0.8, cells were induced at 25 ◦C for up to 8 h by
the addition of IPTG (0.5 mM). The cells were harvested by cen-
trifugation (9000 × g for 15 min). After being re-suspended in
Buffer A (20 mM Potassium phosphate, 5 mM ␤-mercaptoethanol
[␤-ME], pH 7.2), cells were lysed by sonication on ice. Centrifuga-
tion (15,000 × g for 5 min) was performed at 4 ◦C, and SDS-PAGE
analysis was performed to monitor the soluble expression of the
genes.
2.6. Determining the temperature dependence and pH profile of
MaiR-D48
To study the effect of temperature on the activity of MaiR-D48,
the activity assays were carried out as described above at pH 8.0
and temperatures ranging from 25 to 60 ◦C. The activity assays
were also performed as described above at 40 ◦C and different pH
to determine the pH profile of MaiR-D48.
2.7. The oxidation stability of MaiR-D48
The oxidative stability of MaiR-D48 enzyme toward H2O2 was
assessed by treating a solution of the enzyme in buffer A with
0–20 mM of H2O2 (final concentration) at room temperature for

46
X. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
60 min. The activity of the H2O2-treated enzyme was assayed as
described above. To evaluate the oxidative stability of MaiR-D48
enzyme toward air, the purified protein was loaded into glass-tubes
which were 6–7 bed volume of the tested MaiR-D48. All tubes were
shaken in 40 ◦C for 0, 24, 48, 72, 96, 120, 144 h, respectively. The
remaining activity was assayed as described above.
2.8. Measurement of kinetic parameters of MaiR-D48
To determine the kinetic parameters of MaiR-D48, the activ-
ity assay was performed with different substrate concentrations
in Buffer B at 40 ◦C in the presence of certain amount of puri-
fied enzyme. The maleate ammonium solutions of different
Fig. 1. SDS-PAGE analysis for the expression of the full-length (A) and truncated MaiR genes (B and C) in E. coli. “−” and “+” mean un-induced and induced by IPTG, pellet
and supernatant mean the pellet and supernatant fractions of the lysates, respectively.

X. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
47
Fig. 2. (A) Hydrophobicity prediction of full-length MaiR with Protein Identification and Analysis Tools on the ExPASy Server (http://web.expasy.org/protscale/). (B) Schematic
domain structures of full-length MaiR and its deletion mutants.
concentrations (10, 20, 60, 100, 200, 300, 400 ␮M) were prepared
by diluting a mother solution (1 M, pH 8.0) with Buffer B. A solution
of MaiR-D48 (500 ␮L, 57.2 ng/mL) and a maleate ammonium solu-
tion (500 ␮L) were mixed and the reaction was carried out at 40 ◦C
for 10 min. The amount of product fumaric acid was determined as
described above.
3. Results and discussion
3.1. Expression of the putative maleate cis–trans isomerase gene
and its N-terminus deletion mutants
A number of attempts were made to express the full length
of putative maleate cis–trans isomerase gene ro00478 (accession
number YP 700472) from R. jostii RHA1 in Rosetta2 (DE3) pLysS,
but the encoded protein was expressed mainly as insoluble protein
(Fig. 1A) and no maleate cis–trans isomerase activity was detected.
The full-length maleate cis–trans isomerase gene ro00478 was
then analyzed by Protein Identification and Analysis Tools on the
ExPASy Server (http://web.expasy.org/protscale/) [17]. The results
indicated that there were two hydrophilic and two hydrophobic
amino acid clusters in an alternate arrangement at the N-terminus
(Fig. 2A). When the protein sequence (MaiR) was aligned with the
known maleate cis–trans isomerases from Geobacillus stearother-
mophilus (MI101) [9], Serratia marcescens (IFO3736) [8], Nocardia
farcinica (Q5YXQ1) [18] and Alcaligenes faecalis (O24766) [10], it
was found that the first 50 amino acid residues at the N-terminus
were not present in the known counterparts (Fig. 3). As such, the
hydrophilic (1-20AA)–hydrophobic (21-27AA)–hydrophilic (28-
34AA)–hydrophobic (35-41AA) alternate clusters were deleted
one by one to evaluate their effect on the gene expression and
enzyme activity (Fig. 2B). The SDS-PAGE analysis (Fig. 1B and
C) showed that the protein was in soluble form only when two
2.9. Preparative scale conversion of maleate to fumarate
catalyzed by MaiR-D48
The E. coli cells from 40 mL of fermentation broth were collected
by centrifugation (9000 × g for 15 min), and added into a maleate
ammonium solution (40 mL, 1 M, pH 8.0). The reaction mixture was
stirred at 30 or 40 ◦C, respectively. The reaction was monitored by
TLC analysis (methanol/1,2-dichloroethane = 20/80, v/v). After the
maleate disappeared, the biomass was removed by centrifugation.
The pH of the resulting solution was adjusted to 2.0 by adding HCl
solution (10 M), the product was precipitated and separated by fil-
tration. 3.83 g (83% yield) and 1.76 g (38% yield) of fumaric acid were
obtained for the reactions at 30 and 40 ◦C, respectively.
The cells (10 g wet weight) from 2 L of fermentation broth were
harvested by centrifugation (9000 × g for 15 min), and added into
a maleate ammonium solution (2 L, 1 M, pH 8.0). The reaction mix-
ture was stirred at 30 ◦C, and monitored by TLC analysis. Fumaric
acid (167 g, 72% yield) was obtained by the similar work-up proce-
dure as described above.

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X. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
Fig. 3. Alignment of amino acid sequences of MaiR with the known maleate cis–trans isomerases from G. stearothermophilus (MI101) [9], S. marcescens
(IFO3736) [8] and N. farcinica (Q5YXQ1) [18] and A. faecalis (O24766) [10]. Result from EMBL-EBI online tools, ClustalW2 of multiple sequence alignments
(http://www.ebi.ac.uk/Tools/msa/clustalw2/).
hydrophilic and two hydrophobic clusters were all removed (MaiR-
D41, MaiR-D48 and MaiR-D52), and these soluble proteins had
maleate cis–trans isomerase activity. The specific activities of both
MaiR-D41 and MaiR-D52 were about 95% of that of MaiR-D48, and
the later showed higher expression level under the same condi-
tions. As such, MaiR-D48 was chosen for the further studies. The
hydrophilic and hydrophobic clusters inducing protein aggrega-
tion is similar to that by the amphipathic peptide originating from
the Zuotin protein sequence, EAK16 (AEAEAKAKAEAEAKAK, or
(AEAEAKAK) [19], where the alternate hydrophilic and hydropho-
bic clusters were substituted with hydrophilic and hydrophobic
residues.
by the activity assay procedure described in Experimental section.
As shown in Fig. 7, the enzyme retained greater than 95% activ-
ity at 55 ◦C, while the remaining activity decreased to about 50%
at 60 ◦C. These results suggested that MaiR-D48 enzyme exhib-
ited relatively high thermo-stability, which was compatible with
that for the maleate cis–trans isomerases from S. marcescens and
B. stearothermophilus MI-102, but much higher than that for the
enzyme from A. faecalis [8–10].
When the purified MaiR-D48 enzyme was exposed to 5 mM
of H2O2 for 60 min, the remaining activity was about 60% of that
of the untreated enzyme. As the concentration of H2O2 increased
3.2. Biochemical characterization of MaiR-D48
The mutant enzyme MaiR-D48 was purified by ammonium sul-
fate precipitation, DEAE Sepharose Fast Flow column and sephacryl
S-200 HR column. The SDS-PAGE analysis for each step of the purifi-
cation procedure is shown in Fig. 4 and the yields of protein during
each step of purification are summarized in Table 2. The specific
activity of the purified MaiR-D48 reached 106.8 U/mg.
The dependence of the enzyme activity on temperature and
pH were investigated for MaiR-D48, and the results are shown
in Figs. 5 and 6, respectively. The mutant MaiR-D48 enzyme had
an optimal reaction temperature of 40 ◦C (Fig. 5) and showed high
activity in a wide range of pH from 5 to 9, with the optimum pH
being 8.0 (Fig. 6), which is a little higher than that of maleate isom-
erase from N. farcinica (pH 7.5) [18]. Maleate cis–trans isomerases
from A. faecalis and S. marcescens also had optimal reaction tem-
peratures around 40 ◦C, while the activity of the enzyme from A.
stearothermophilus MI-102 increased as the temperature increased
up to 70 ◦C [8]. The MaiR-D48 enzyme was incubated at specific
temperature for 30 min, and the residue activity was determined
Fig. 4. SDS-PAGE analysis for the purification of MaiR-D48. Lane 1, protein markers;
lane 2, crude cell-free extract; lane 3, 45–55% ammonium sulfate precipitation; lane
4, elution from DEAE column; lane 5, elution from sephacryl S-200 HR column.

X. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
49
Table 2
The purification of MaiR-D48.
Purification steps
Protein concentration (mg/mL)
Total activity (U)
Specific activity (U/mg)
Yield of activity (%)
Purification factor
Crude extract
Precipitation with (NH4)2SO4
DEAE sepharose
8.6
9.7
6.0
3.8
34,076.2
10,321.7
4912.3
39.44
35.4
68.0
100
30.5
14.5
5.8
1
0.9
1.7
2.7
Sephacryl S-200
1972.7
106.8
to 15 mM, its residue activity leveled off below 5% of the initial
activity (Fig. 8A). This is different from the observations for the
known maleate cis–trans isomerases from A. faecalis, S. marcescens
and B. stearothermophilus MI-102, which were completely inhibited
by 1 mM H2O2 [8,9]. When the purified MaiR-D48 enzyme was
exposed to air for 60 h, the remaining activity was about 50% of
the untreated enzyme (Fig. 8B).
The kinetic parameters Km and kcat of MaiR-D48 for maleate
were 149.6 9.2 ␮M and 96.7 1.3 s−1, respectively, with catalytic
efficiency kcat/Km being 6.5 × 105 M−1 s−1. The Km value was in the
range of reported data for known maleate isomerases from A. fae-
calis, N. farcinica and P. fluorescens [10,18,20]. Although the Km value
of MaiR-D48 was much higher than that of N. farcinica maleate
isomerase, they had similar catalytic efficiency kcat/Km, due to a
much higher kcat of MaiR-D48 (Table 3) [18].
100
80
60
40
20
0
20
25
30
35
40
45
50
55
60
65
Temperature/oC
Fig. 5. Effect of temperature on the activity of MaiR-D48. The experiment is repre-
sentative of 3 repeats.
A
100
100
80
60
40
20
0
80
60
40
20
0
5
6
7
8
9
0
5
10
15
20
pH
H2O2 Concentration / mM
Fig. 6. Effect of pH on the activity of MaiR-D48. The experiment is representative of
3 repeats.
B
100
80
60
40
20
0
100
80
60
40
20
0
0
20
40
60
80
100
120
140
20
25
30
35
40
45
50
55
60
65
Temperature/oC
Time / hour
Fig. 8. Effect of hydrogen peroxide (A) and air (B) on the activity of MaiR-D48. The
experiment is representative of 3 repeats.
Fig. 7. Thermal stability of MaiR-D48. The experiment is representative of 3 repeats.

50
X. Liu et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 44–50
Table 3
and a broad reaction pH range. The E. coli cells expressing MaiR-D48
catalyzed the isomerization of maleate to fumarate at the substrate
concentration of 1 M, suggesting its application potential for the
biocatalytic production of fumaric acid in the replacement of the
acid-catalyzed process.
The kinetic parameters of different maleate cis–trans isomerases.
Enzymea
kcat (s−1
)
Km (␮M)
kcat/Km (M−1 s−1
)
References
MaiR-D48
O24766
PfMI
96.7 1.3
NR
NR
149.6 9.2
40.0
300
6.5 × 105
NR
This study
[10]
[20]
NR
Q5YXQ1
2.8 0.7
4.6 0.5
6.1 × 105
[18]
Acknowledgements
NR, not reported.
a
Maleate isomerases from A. faecalis (O24766), P. fluorescens (PfMI) and N.
farcinica (Q5YXQ1), respectively.
This work was financially supported by National Basic Research
Program of China (973 Program, No. 2011CB710801), Chinese
Academy of Sciences (KGCX2-YW-203).
3.3. Biocatalytic synthesis of fumaric acid by MaiR-D48
References
The E. coli cells expressing MaiR-D48 were tested as the catalyst
for the isomerization of maleate to fumarate at 1 M substrate con-
centration. It has been found that the yield of fumaric acid was very
low when the reaction was carried out at the optimal temperature
(40 ◦C). It was due to the metabolism of fumarate by the whole cell
catalyst, as demonstrated by the observation that fumarate disap-
peared when the ammonium fumarate solution (1 M, pH 8.0) was
treated with the E. coli cells without pET-32a-MaiR-D48 plasmid
for 25 h at 40 ◦C. Under the same conditions, ammonium maleate
was not obviously metabolized. Fumaric acid was isolated in 83%
yield when the isomerization was performed at 30 ◦C, more than
two times of that at 40 ◦C, suggesting that the rate of fumarate
metabolism was reduced at lower reaction temperature.
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In order to explore the potential application of MaiR-D48 in
the biocatalytic production of fumaric acid, the isomerization was
scaled up to 2 L. The reaction was carried out at 30 ◦C with the final
concentration of ammonium maleate being 1 M. After 28 h, fumaric
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