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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
-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. 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
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