Enzyme and Microbial Technology 33 (2003) 537543

Research papers
Efcient production of l-phenylalanine catalyzed by a coupled
enzymatic system of transaminase and aspartase
Hong Xu, Ping Wei, Hua Zhou, Weiping Fan, Pingkai Ouyang

College of Life Science and Pharmacy, Nanjing University of Chemical Technology, No. 5, New Mode Road, Nanjing 210009, China
Received 16 September 2002; received in revised form 20 March 2003; accepted 27 March 2003
Abstract
A process for efcient production of l-phenylalanine catalyzed by a coupled enzymatic system of transaminase and aspartase with two
organisms was developed. One strain, Escherichia coli EP810 produces signicant transaminase and less aspartase under incubation in
the glucosebeef extract medium. In the presence of 50 mg/ml cells of EP810, 0.24 mol/l phenylpyruvic acid (PPA) was converted to
l-phenylalanine (l-Phe) in 8 h with l-aspartic acid as an amine donor, the conversion rate was 97%. Another strain, E. coli EA-1, a mutant
strain of ATCC11303, produces signicant aspartase and less transaminase. l-Aspartate (l-Asp), the amine donor, could be produced by
EA-1 from fumarate (Fu) and ammonia. In presence of the mixture of EP810 and EA-1 cell, l-phenylalanine was efciently produced
from PPA and ammonium fumarate. An optimum reaction condition of the coupled enzymatic system is as follows: the concentration
ratio of two cells as 0.4:1 (EA-1 to EP810), concentration ratio of two substrates (PPA to Fu) as 1:1.2 (mol). When concentration of
PPA was 0.24, 0.233 mol/l (38 g/l), l-phenylalanine acid was accumulated by the conversion rate up to 97%. l-Phenylalanine produc-
tion is more economical by the coupled enzymatic system, since one of the substrates l-aspartate was replaced by the relative cheap
fumaric acid.
2003 Elsevier Inc. All rights reserved.
Keywords: Coupled enzymatic system; l-Phenylalanine; Transaminase; Aspartase
1. Introduction
l-Phenylalanine is an important amino acid; it is widely
used in pharmaceutical and in the synthesis of sweet-
ener Aspartame. The studies on the enzymatic process
for l-phenylalanine production are quite active during
the past decades [15]. The process for the production
of l-phenylalanine via biotransformation of phenylpyru-
vic acid (PPA) and l-aspartic acid by transaminase has
demonstrated very promising (formula 1), because of high
conversion yield due to oxalacetic acid decarboxylation and
relative stable catalyst. However, this process is still ham-
pered by problems such as expensive substrates, besides
PPA, 1.2 mol l-aspartic acid is needed for high conversion
rate for 1.0 mol l-phenylalanine accumulation.
Escherichia coli EP810 was screened from soil as an
excellent producer of the transaminase in our research [6].
In the presence of EP810, l-phenylalanine was efciently
formed from PPA and l-aspartic acid. It is well known

Corresponding author. Tel.: +86-25-3587332; fax: +86-21-3325464.

E-mail address: xuh@njuct.edu.cn (P. Ouyang).
that l-aspartic acid is formed enzymatically by aspartase
from fumaric acid and ammonia (formula 2) [7,8]. An en-
zymatic process for l-phenylalanine production from PPA
and ammoniumfumarate can be established through coupled
transaminase and aspartase system (formula 3). By replac-
ing the l-aspartic acid with less expensive fumaric acid, the
cost of l-phenylalanine production would be signicantly
reduced.
The experimental results of enzymatic production of
l-phenylalanine by the coupled transaminase and aspartase
system in the presence of EP810 and E. coli EA-1 are
reported and discussed in the present paper.
(1)
Fu + NH
3
Aspartase
l-Asp (2)
PPA + Fu + NH
3
Transaminase Aspartase
l-Phe + Pyr + CO
2

(3)
0141-0229/$ see front matter 2003 Elsevier Inc. All rights reserved.
doi:10.1016/S0141-0229(03)00206-0
538 H. Xu et al. / Enzyme and Microbial Technology 33 (2003) 537543
2. Materials and methods
2.1. Chemicals
PPA, pyridoxal 5

-phosphate (5

-PLP), l-phenylalanine
(l-Phe), l-aspartic acid (l-Asp) were purchased from either
Sigma Chemical Co. or Kangda Amino Acid Co. (China).
PPA (used for substrate) was synthesized from chemical lab-
oratory of our university.
2.2. Microorganism
Escherichia coli EP810, a transaminase-producing bac-
terium, isolated from the soil samples in our lab. E. coli
EA-1, selected from the mutants of E. coli ATCC11303 with
ethyl methane sulphonate (EMS), which had high activity
of aspartase.
2.3. Cell cultures
One of looful of the EP810 cells grown overnight on an
agar slant medium were inoculated in a 500-ml Erlenmeyer
ask containing 100 ml of a sterile mediumwhich contained:
2% glucose, 1% beef extract, 2% corn steep liquor, 0.05%
MgSO
4
7H
2
O, and 0.05% K
2
HPO
4
, pH 7.0, adjusted with
NH
4
OHand aerobically cultured at 37

Cfor 24 h on a rotary
incubator with shaking at 220 rpm.
EA-1 was cultivated at the same conditions as EP810 ex-
cept medium, which contained 0.5% Na-fumarate, 1% am-
monium fumarate, 1% beef extract, 1% corn steep liquor,
0.5% MgSO
4
7H
2
O, and 0.2% K
2
HPO
4
. The pH was ad-
justed to 7.0 with NH
4
OH.
2.4. Method for production of l-phenylalanine and
l-aspartic acid
The cells were harvested by centrifugation at 8000 g
for 20 min, and then used for enzymatic reaction. The cells
were suspended to 50 ml of substrate in a 100-ml Erlenmeyer
ask to make a cell concentration of 50 mg/ml (wet weight),
and incubated for appropriate times at 37

C.
The reaction substrate for production of l-phenylalanine
with l-aspartic acid as amine donor composed of 0.10.4 mol/l
sodium phenylpyruvate, 1.2 times much l-aspartate as
sodiumphenylpyruvate, 1 mmol/l MgSO
4
7H
2
O, 0.1 mmol/l
PLP. The pH was adjusted to 8.0 with NH
4
OH; when the
production of l-phenylalanine using the coupled enzymatic
system, fumaric acid was in place of l-aspartic acid. The
reaction substrate for production of l-aspartate composed
of 0.3 or 0.7 mol/l fumaric acid, 1 mmol/l MgSO
4
7H
2
O.
The pH was adjusted to 8.0 with NH
4
OH.
2.5. Analysis
2.5.1. Transaminase and aspartase assay
The cells were cultivated and harvested by centrifuga-
tion at 8000 g for 20 min, then used for enzyme activ-
ity assay. 50 mg/ml of cells were mixed with the reaction
solution that composed of 0.2 mol/l potassium phosphate
(pH 8.5), 0.3 mol/l PPA, 0.45 mol/l l-aspartic acid, 1 mmol/l
cetylpyridinium chloride, and 0.1 mmol/l pyridoxal phos-
phate in a nal volume of 5 ml. Reactions were incubated at
37

C for 60 min with shaking at 150 rpm and the produc-

tion of l-phenylalanine was measured. Transaminase activ-
ity was expressed as units corresponding to 1 mol product
formed/min.
For measurement of aspartase activity, 1 mol/l of am-
monium fumarate solution containing 1 mmol/l Mg
2+
and
1 mmol/l cetylpyridinium chloride was used as substrate.
The pH of solution was adjusted to 8.5 with ammonium. The
reaction was initiated by adding 20 mg/ml cells to substrate
solution and incubated at 37

C for 30 min with shaking at

150 rpm. The decreasing rate of fumaric acid was measured.
Aspartase activity was expressed as units corresponding to
1 mol substrate disappeared/min.
2.5.2. Determination of substrates and products of enzyme
reaction
l-Phenylalanine and l-aspartic acid were analyzed by an
amino acid analyzer Hitachi 655 (Japan) equipped with an
integrator. PPA was estimated by Fe
3+
chromatography [9].
Fumaric acid was measured by chromatography at 240 nm
as described previously [7].
3. Results and discussion
3.1. Formation of transaminase and aspartase of strain
EP810 and EA-1
The cells of EP810 and EA-1 were cultivated and the
catalytic activities of each were determined, respectively.
The activity of transaminase in resting cells of EP810 was
26.6 U/g, which was much higher than in EA-1. On the other
hand, the activity of aspartase in resting cells of EP810
was 144.5 U/g, it was much lower than in EA-1. The activity
of aspartase in EA-1 was 850 U/g (the data was shown in
Table 1).
The cells of EP810 or EA-1 were used as raw catalysts
for the conversation of PPA and l-aspartic acid to form
l-phenylalanine, respectively. The time course of PPA to
l-phenylalanine was shown in Fig. 1. The conversion of
course of fumaric acid to l-aspartic acid was followed in a
test with 0.3 or 0.7 mol fumaric acid as reaction substrate
Table 1
Transaminase and aspartase activity of strain EP810 and EA-1
Strain Enzyme activity (U/g wet cell)
Transaminase Aspartase
EP810 26.6 144.5
EA-1 3.2 850
H. Xu et al. / Enzyme and Microbial Technology 33 (2003) 537543 539
Fig. 1. The conversion course of phenylpyruvic acid to l-phenylalanine. (A) EP810. Time proles of concentration of PPA () and l-Phe () in
0.1 mol/l PPA as substrate; PPA () and l-Phe () in 0.24 mol/l PPA as substrate; PPA () and l-Phe () in 0.4 mol/l PPA as substrate. (B) EA-1.
Time proles of concentration of PPA () and l-Phe () in 0.1 mol/l PPA as substrate; PPA () and l-Phe () in 0.24 mol/l PPA as substrate.
540 H. Xu et al. / Enzyme and Microbial Technology 33 (2003) 537543
Fig. 2. The conversion course of fumaric acid to l-aspartic acid. (A) 50 mg/ml of EP810. Time proles of concentration of Fu () and l-Asp ()
in 0.3 mol/l Fu as substrate; Fu () and l-Asp () in 0.7 mol/l Fu as substrate. (B) 20 mg/ml of EA-1. Time proles of concentration of Fu () and
l-Asp () in 0.3 mol/l Fu as substrate; Fu () and l-Asp () in 0.7 mol/l Fu as substrate.
H. Xu et al. / Enzyme and Microbial Technology 33 (2003) 537543 541
Fig. 3. Effect of temperature on l-phenylalanine production by the coupled enzymatic system.
in the presence of cells of EA-1 and EP810, respectively
(Fig. 2).
The above results showed that 0.10.4 mol/l PPA could
be transformed into l-phenylalanine in 10 h in the presence
of the cells of EP810 (Fig. 1A), because of their high cat-
alytic activity of transaminase, while 0.1 mol/l PPA could
not be transformed completely in 50 h in the presence of
EA-1 (Fig. 1B). In contrast to above, 0.7 mol/l fumaric acid
was transformed to l-aspartic in 5 h in the presence of the
cells of EA-1 (Fig. 2B), because of their high catalytic ac-
tivity of aspartase, but only about 50% fumaric acid was
transformed to l-aspartic acid in the presence of EP810
(Fig. 2A) because of their low catalytic activity of aspartase.
Based on the above results, the l-phenylalanine produc-
tion catalyzed by the coupled transaminase and aspartase
system in the presence of the cells of EP810 and EA-1
was approached, and ammonium fumarate as the substrate
in place of l-aspartic acid.
3.2. Production of l-phenylalanine by coupled
transaminase and aspartase system
3.2.1. Effect of temperature and pH
The enzymatic reaction was conducted at different tem-
perature (3050

C) and pH value (7.010.0) for 8 h with

0.24 mol/l PPA and 0.3 mol/l ammonium fumarate as the
substrate, the results were shown in Figs. 3 and 4. The opti-
mal temperature ranged from 35 to 40

C. The optimal pH
for the reaction ranged around 89.
3.2.2. Effect of concentration of fumaric acid
Although the theoretical ratio of amino donor to keto acid
precursor would be at least 1:1, an excess amount of amino
donor [10] was preferably provided in experiment. The effect
of the ratio of the concentration of fumaric acid to PPA on
the accumulation of l-phenylalanine was investigated. When
the ratio of concentration of Fu to PPA was 1.2:1 and more,
the transamination yield was highest.
3.2.3. Effect of the ratio of concentration of two cells
The effect of the ratio of concentration of two cells on
enzyme reaction was showed in Table 2. The more cells
of EA-1 were favor of the formation of l-phenylalanine
acid. The ratio of 0.4:1 (EA-1:EP810) was selected as an
economical enzyme source.
3.2.4. l-Phenylalanine production catalyzed by coupled
transaminase and aspartase system
With 0.24 mol/l PPA and 0.3 mol/l fumaric acid as the
substrate, using EP810 and EA-1 as enzyme resource, the
time course of the reaction was investigated. As Fig. 5 shows,
the formation of l-aspartic acid and the disappearance of
fumaric acid were completed in the initial 2 h of reaction. On
542 H. Xu et al. / Enzyme and Microbial Technology 33 (2003) 537543
Fig. 4. Effect of pH on l-phenylalanine production by the coupled enzymatic system.
Fig. 5. Time course of l-phenylalanine production by coupled enzymatic reaction. 50 mg/ml EP810 cells and 20 mg/ml EA-1 cells were suspended to
reaction mixture, and incubated at 37

C for 8 h.
H. Xu et al. / Enzyme and Microbial Technology 33 (2003) 537543 543
Table 2
Effect of the ratio of concentration of cells of EA-1 to EP810 on the
l-phenylalanine production in the coupled enzymatic system
Ratio of cells of
EA-1 to EP810
Reaction
time (h)
l-Phe
content (g/l)
Yield (%)
0.2:1 6 23.2 58.5
8 35.6 90
0.4:1 6 38.5 97
8 38.5 97
0.6:1 6 38.5 97
1:1 6 38.5 97
the other hand, the production of l-phenylalanine gradually
increased and concomitantly PPA disappeared in 68 h of
reaction.
4. Conclusion
Escherichia coli EP810 was as an excellent transami-
nase producer and E. coli EA-1 as a high activity aspartase
producer. With the coupled transaminase and aspartase sys-
tem, l-phenylalanine can be rapidly and efciently formed
from PPA and ammonium fumarate. The suitable reaction
temperature of transaminase and aspartase were all about
3740

C, the suitable pH value were about 8.59.0, so the

same for the coupled enzyme system, transaminase and as-
partase, were all on the favorable conditions. l-Aspartic acid
formed from fumaric acid with aspartase of EA-1 as soon
as the enzyme reaction began. The l-phenylalanine formed
from PPA and l-aspartic acid with transaminase of EP810
at almost same time. The conversion yield and reaction ve-
locity of l-phenylalanine production by coupled enzymatic
system was same as the reaction using l-aspartic acid as
amino donor. As fumaric acid was manufactured at low cost,
the l-phenylalanine production with ammonium fumarate as
the amino donor source in place of l-aspartic acid was more
protable. This process had been applied for production of
l-phenylalanine on a pilot scale of 10t a year.
Acknowledgments
This works was supported by the funds from the State
Science Commission of China (Project No. 96-A12-07-02).
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