Two-step enzymatic synthesis of tyramine from raw pyruvate fermentation broth

Article abstract of DOI:10.1016/j.molcatb.2015.11.024

Tyramine, as a metabolite of tyrosine, is an important intermediate in synthesizing some drugs and medicinal materials. In this study, an efficient method for producing tyramine was developed by a two-step biocatalytic reaction with recombinant tyrosine phenol-lyase whole cells and tyrosine decarboxylase immobilized cells. Raw pyruvate fermentation broth was used as substrate of tyrosine phenol-lyase to economically produce l-tyrosine. l-tyrosine was catalyzed by immobilized tyrosine decarboxylase cells to effectively synthesize tyramine. The conditions of two-step enzymatic catalysis reactions were optimized separately, and the influence of immobilization on tyrosine decarboxylase activity was investigated. In a scale up study, 94.3% l-tyrosine was obtained from raw pyruvate fermentation broth under the optimal conditions. l-Tyrosine was decarboxylated to tyramine with a high yield 91.2%. The total yield of tyramine could reach approximately 86% by this two-step biocatalytic reaction. This study provides us with a green strategy for efficient preparation of tyramine.

Full text of DOI:10.1016/j.molcatb.2015.11.024

Journal of Molecular Catalysis B: Enzymatic 124 (2016) 38–44
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Two-step enzymatic synthesis of tyramine from raw pyruvate
fermentation broth
Hongjuan Zhanga,b, Yang Lub, Siping Wub, Yu Weib, Qian Liub, Junzhong Liub,∗
,
Qingcai Jiaob,∗
a School of Pharmacy, Nanjing Medical University, Nanjing 211166, China
b State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing 210093, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Tyramine, as a metabolite of tyrosine, is an important intermediate in synthesizing some drugs and
medicinal materials. In this study, an efficient method for producing tyramine was developed by a two-
step biocatalytic reaction with recombinant tyrosine phenol-lyase whole cells and tyrosine decarboxylase
immobilized cells. Raw pyruvate fermentation broth was used as substrate of tyrosine phenol-lyase to
economically produce l-tyrosine. l-tyrosine was catalyzed by immobilized tyrosine decarboxylase cells to
effectively synthesize tyramine. The conditions of two-step enzymatic catalysis reactions were optimized
separately, and the influence of immobilization on tyrosine decarboxylase activity was investigated. In a
scale up study, 94.3% l-tyrosine was obtained from raw pyruvate fermentation broth under the optimal
conditions. l-Tyrosine was decarboxylated to tyramine with a high yield 91.2%. The total yield of tyramine
could reach approximately 86% by this two-step biocatalytic reaction. This study provides us with a green
strategy for efficient preparation of tyramine.
Received 23 July 2015
Received in revised form
19 November 2015
Accepted 29 November 2015
Available online 2 December 2015
Keywords:
Tyramine
Pyruvate fermentation broth
Immobilization
Tyrosine phenol-lyase
Tyrosine decarboxylase
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
cost reduction of l-tyrosine and enzyme should be made in order
to utilize this green method, because l-tyrosine is expensive and
Tyramine [2-(p-hydroxyphenyl) ethylamine],
a
significant
purified enzyme as catalyst is another cost-added factor [20]. Con-
sequently, pyruvate fermentation broth and recombinant whole
cells were employed to prepare tyramine by two-step biocatalytic
reaction in the present work.
metabolite of tyrosine, has gained tremendous interest in recent
years as a catecholamine drug and an intermediate of medici-
nal materials and some drugs [1–6]. Tyramine is mainly prepared
through chemical synthesis [6–11]. Compared with chemical syn-
thesis, enzymatic synthesis has its advantages for its mild reaction
conditions, eco-friendly and high enantio-, regio- and chemoselec-
tivity. Tyramine could be produced by enzymatic decarboxylation
of l-tyrosine, a reaction catalyzed by tyrosine decarboxylase (TDC,
EC 4.1.1.25) [12–19]. Nevertheless, based on tyramine as the
metabolite of l-tyrosine, this enzymatically synthetic route was
more utilized to study the metabolic diversion of tyramine in plant
[12], and the effect of tyramine on food safety and spoilage due to
the health problem caused by high quantities tyramine [13–17].
By far, there is no detailed reference to investigate the enzymatic
synthesis of tyramine by TDC immobilized cells, neither for the
industrial production with the method. Furthermore, the efforts in
Tyrosine can be produced from phenol, pyruvic acid and ammo-
nia by tyrosine phenol-lyase (TPL, EC 4.1.99.2) [21–24]. The cost of
pyruvic acid accounts for a considerable proportion in enzymatic
synthesis of l-tyrosine. Fermentative production of pyruvic acid as
one of the most competitive method has many advantages such
as cheap source of glucose, mild condition, environmental and low
cost [25,26]. However, it is still difficult to purify pyruvic acid from
pyruvate fermentation broth. Pyruvic acid is generally separated
with filtration, decolorization, concentration and distillation, and
ion exchange resin is always used for further purification [25]. If the
fermentation broth could be directly used to manufacture down-
stream products to avoid the tedious operations, it will greatly
decrease the cost and stimulate the development of pyruvate fer-
mentation production.
On the other hand, immobilization of whole cells could further
improve the potential of biocatalyst for application. Since immo-
bilized cells can easily be retrieved from the reaction mixture
and reused, biotransformation with immobilized cells superiorly
∗
Corresponding authors at: State Key Laboratory of Pharmaceutical Biotechnol-
ogy, School of Life Science, Nanjing University, Nanjing 210093, China.
E-mail addresses: junzhongliu2414@163.com (J. Liu), jiaoqc@163.com (Q. Jiao).
http://dx.doi.org/10.1016/j.molcatb.2015.11.024
1381-1177/© 2015 Elsevier B.V. All rights reserved.

H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 38–44
39
performed in cell recovery and greatly declined the cost [27,28].
In l-tyrosine decarboxylation reaction, immobilized recombinant
cells were consequently used as biocatalysts because they are very
competitive and operationally stable compared with free cells.
In the present study, enzymatic synthesis of tyramine was
established by two-step biocatalytic reaction (Scheme 1). Firstly,
l-tyrosine was prepared from raw pyruvate fermentation broth
under the catalysis of TPL. Pyruvate fermentation broth was sim-
ply centrifuged, and then the supernatant was diluted and used
as substrate to produce l-tyrosine. Secondly, l-tyrosine was col-
lected as starting material to synthesize tyramine by immobilized
TDC cells from Lactobacillus brevis. Pyridoxal phosphate was the
common coenzyme for these two enzymes.
In order to explore the feasibility of bioconversion, the optimiza-
tion of enzymatic synthesis conditions of l-tyrosine such as pH,
temperature, substrate concentration and surfactant were consid-
ered. During the production of tyramine, the reaction conditions
were optimized with immobilized cells and the advantages of
immobilized cells were investigated in comparison with free cells.
Bioconversion efficiency and tyramine yield were simultaneously
determined under the optimal conditions.
ammonium chloride concentrations (0–1500 mM) were optimized
respectively. The effects of four kinds of surfactants on enzymatic
activity, Triton X-100, Tween-80, cetyltrimethyl ammonium bro-
mide (CTAB) and Emulsifier OP-10, were examined at pH 7 and
37 ◦C. Concentrations of Triton X-100, Tween-80, CTAB and OP
ranged from 0 to 0.05% (v/v) respectively. All assays were per-
formed in triplicates. TPL activity was determined by measuring
l-tyrosine generated from the reaction by HPLC.
2.4. Optimization of l-tyrosine decarboxylation
2.4.1. Preparation of immobilized TDC cells
Based on the protocol of our lab [30], immobilized cells were
prepared as follows: 2 g lyophilized cells were mixed with 100 mL
sodium alginate water solution (3%, m/v), the mixture were stirred
thoroughly and then extruded into 500 mL 2% (m/v) stirring CaCl2
solution via a syringe to form calcium alginate beads from a 10 cm
height. The beads were immersed in the CaCl2 solution to harden
for 5 h at 4 ◦C. The beads were washed with sodium acetate buffer
(0.2 M, pH 5.5) three times and stored in buffer at 4 ◦C. The bead
size varied with flow rates and always kept at 2.6 0.2 mm. When
the immobilized cells were used, they were taken out and blotted
with tissue to remove the excess buffer on the surface of beads. The
weight of immobilized beads for 2 g lyophilized cells was generally
maintained at 38.6 1.5 g.
2. Materials and methods
2.1. Chemicals
Pyruvate fermentation broth (pH 6) as raw material was pro-
vided by Shandong Yangcheng Biotech Co. Ltd., (Shangdong, China).
Pyruvate fermentation broth was only disposed by centrifugation
and pyruvate content in the light yellow supernatant is 6.1% (m/v)
by HPLC. Pyridoxal-5ꢀ-phosphate (PLP) was purchased from Sigma
(St. Louis, MO, USA). All other chemicals and reagents used in
this work were of analytical grade and purchased from Sinopharm
Chemical Reagent Co. Ltd., (Shanghai, China).
2.4.2. Effect of pH, temperature and l-tyrosine load
TDC activity was detected by measuring tyramine via HPLC.
When one influential factor was inspected, the other conditions
were fixed.
TDC immobilized cells (2 g) were added to the reaction mixture
(final volume, 10 mL) containing l-tyrosine, 20 ␮L PLP (1%, m/v),
20 ␮L TritonX-100 (10%, v/v). l-Tyrosine was added by five batches
at intervals of 0.5 h. The reactions proceeded for 4 h after l-tyrosine
was added completely. When l-tyrosine concentration was inves-
tigated, it was varied as follows: 0.3 g, 0.5 g, 0.7 g, 0.9 g, 1.1 g. The
mixture was shaken at 50 rpm and 40 ◦C for determination of the
optimal pH at pH 3, 4, 5, 5.5, 6, 7, 8 respectively. And then, the
mixture was shaken at pH 5.5 for determination of the optimal tem-
perature at 30 ◦C, 37 ◦C, 40 ◦C, 45 ◦C, 50 ◦C, 60 ◦C respectively. All of
the reaction solutions were collected and determined by HPLC.
2.2. Microorganisms and shake flask fermentation
According to the protocols established by Zhang et al. [29], all
the enzyme genes were amplified by PCR and inserted into cor-
responding plasmids, and the recombinant cells were constructed
by cloning plasmids into Escherichia coli BL21 (DE3). The tyrosine
phenol-lyase gene from Citrobacter koseri was cloned using plasmid
pETDuet as vector. Plasmid pET28a was utilized as vector to carry
the tyrosine decarboxylase gene from L. brevis.
Based on the protocols developed by Zhang et al. [29], the recom-
binant cells were inoculated in LB medium and fermented in the
sterilized fermentation media. Fermented broth was collected at
the end of fermentation process and centrifuged at 2800× g, 4 ◦C for
10 min to obtain wet cells in the pellet, which were then lyophilized
for a minimum of two days (Martin Christ GmbH, Germany) and
stored at −20 ◦C.
2.4.3. Comparison of enzymatic activity before and after
immobilization
Enzymatic activities were determined to assess the difference
between immobilized cells and free cells. l-Tyrosine (0.7 g), 20 ␮L
PLP (1%, m/v) and 20 ␮L TritonX-100 (10%, v/v) were added into
sodium acetate buffer (10 mL, pH 5.5) at 40 ◦C under the catalysis of
100 mg lyophilized free cells and 2 g immobilized cells respectively.
l-Tyrosine was added by five batches at intervals of 0.5 h.
2.4.4. Repeated batch bioconversion for immobilized cells and
free cells
2.3. Optimization for synthesis of l-tyrosine
The experiments were operated as follows: after each cycle of
6 h conversion, the beads and free cells were collected and washed
with sodium acetate buffer (0.2 M, pH 5.5) three times, the catalysts
were reused in a subsequent cycle and reacted with the substrates
at the same conditions. Enzymatic activities of each cycle were
detected by HPLC.
The general procedure of synthesizing l-tyrosine was shown
as follows: TPL cells (50 mg) were added to capped falcon tube
and mixed with reaction mixture containing pyruvate fermenta-
tion broth (3%, m/v, 10 mL), 350 mM ammonium chloride, phenol
(71 mM), 20 ␮L PLP (1%, m/v) and 20 ␮L Triton X-100 (10%, v/v).
The remnant phenol was added at intervals of 1 h and total phenol
reached 284 mM (4 × 71 mM). The reactions were carried out at pH
7, 37 ◦C and 200 rpm for 4 h after all of phenol was added. When
the influential factors were investigated, the enzymatic activities
were tested with varying each bioconversion condition one at
a time. The influential factors including pH (6–11), temperature
(25–60 ◦C), pyruvate substrate concentration (1.0–6.0%, m/v) and
2.4.5. Immobilized biocatalyst stability testing
The stability of the immobilized biocatalyst for long-term stor-
age at 4 ◦C in sodium acetate buffer (0.2 M, pH 5.5) was measured.
The stability testing was performed in standard biotransforma-
tion at 2, 4, 6, 8, 12 and 14 weeks respectively. The immobilized

40
H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 38–44
Scheme 1. Two-step enzymatic synthesis of tyramine. l-Tyrosine was prepared from raw pyruvate fermentation broth by TPL, and decarboxylated by immobilized TDC cells
to obtain tyramine. TPL = tyrosine phenol-lyase from Citrobacter koseri, TDC = tyrosine decarboxylase from Lactobacillus brevis, PLP = pyridoxal phosphate.
biocatalyst was washed with sodium acetate buffer before starting
the biotransformation.
2.5. Scale up fermentation, bioconversion and isolation of
l-tyrosine and tyramine
Based on the protocols [31,32], the fermentation of recombi-
nant whole cells was carried out in a stirred tank 10 L fermentor
(East biotech., Zhengjiang, China) which was charged with media
7 L containing 1.5% (m/v) peptone, 5% (m/v) corn plasm, 1.5% (m/v)
lactose and (0.1% v/v) polypropylene glycol (PPG, Sigma) solution
as an anti-foam agent. At the end of fermentation process, fermen-
tation broth was centrifuged to obtain wet cells. The wet cells were
lyophilized and employed to catalyze the corresponding substrates
under the optimum reaction conditions for bioconversion in 20 L
reactor (ZF-20, Jiangsu, China).
The bioconversion of tyrosine from pyruvate fermentation broth
was shown as below: TPL (75 g) were mixed with 15 L pyruvate fer-
mentation broth (3%, m/v), 350 mM ammonium chloride, phenol
(71 mM), 30 mL PLP (1%, m/v) and 30 mL Triton X-100 (10%, v/v)
under the optimum reaction conditions of pH 7, 37 ◦C. The remain-
ing phenol (3 × 71 mM) was added separately at intervals of 1 h.
The reaction was stirred at 220 rpm for 6 h after all of phenol was
added. With the reaction time prolonging, more and more tyro-
sine precipitated. The reaction mixture was centrifuged, and then
the collected precipitate was adjusted pH to 11 using NaOH (10%,
m/v) to dissolve it, the solution was heated to 80 ◦C and 300 g acti-
vated charcoal was added for decoloration. After stirring at 80 ◦C
for 30 min, the charcoal was removed by filtration. The filtrate was
cooled and pH was adjusted to 6 with 6 M HCl. The precipitated
white crystals was collected and dried in vacuum.
In order to produce tyramine, l-tyrosine and immobilized TDC
cells (3 kg) were mixed with 30 mL PLP (1%, m/v) and 30 mL Triton
X-100 (10%, v/v) at sodium acetate buffer (0.2 M, pH 5.5) and 40 ◦C
for 15 L gross volume. A total of 1050 g l-tyrosine was divided to
5 parts and added sequentially after every half hour. The reaction
proceeded for 8 h after l-tyrosine was completely added. The con-
version fluid was cooled and filtrated. The activated carbon (250 g)
was added into the filtrate for decoloration at 70 ◦C for 30 min.
And then the solution was filtrated again, the pH of filtrate was
adjusted to 1 with 6 M HCl. The reaction solution was concentrated
to 5 L, and then crystal was deposited and filtrated. While filtering,
the crystal was washed with the cold water. The washing liquor
and filtrate were merged with the next centrifuged supernatant for
further collection. The collected white crystal was dried in vacuum.
Fig. 1. Effect of surfactants on TPL activity. TPL cells were mixed with the reaction
mixture containing the pyruvate fermentation broth, ammonium chloride, phenol,
PLP at pH 7 and 37 ◦C, and activities were monitored while varying the concentra-
tions of Triton X-100, Tween-80, CTAB and OP from 0 to 0.05% (v/v) respectively.
The highest relative activity of 100% denoted 49.5 g/l of l-tyrosine in the presence
of 0.02% Triton X-100.
Fig. 1 shows the highest conversion rate (96.2%) was observed in
the presence of 0.02% Triton X-100. For high concentration of CTAB
and Tween-80 (e.g., 0.04% and 0.05%), TPL activity diminished in
comparison to the lack of surfactants. Lauchnor and Semprini have
recently shown that phenol as substrate will result in the inhibition
to enzyme to some extent [33]. For TPL, the problem of inhibition
has been successfully solved through adding phenol by fed batch
in our lab [34]. In this study, the inhibition was not also observed
and the conversion rate could reach 96% through adding phenol
four times. With pyruvate being consumed during the reaction, the
excessive phenol could not be used up, and could even bring some
trouble of post-treatment. Therefore, the molar ratio of pyruvate
and phenol substrate (1:1) was fixed.
As described in Fig. 2A, the optimal ammonium chloride concen-
tration was 350 mM, and TPL activity was greatly inhibited when
ammonium chloride was more than 750 mM (Fig. 2A). In fact, there
was a large amount of ammonium in pyruvate fermentation broth
because pH decreased and consequently adjusted with aqueous
ammonia to maintain the optimal condition while pyruvic acid was
continuously produced during fermentation. That is, ammonium
pyruvate was the main product in the fermentation broth. Thereby,
the conversion ratio (78%) was still high even though ammonium
chloride was not added (Fig. 2A).
3. Results and discussion
3.1. The optimization of TPL activity
Fig. 2B indicates the conversion rate of pyruvate decreased along
with an increase of substrate concentration. Since the pyruvate con-
tent in the raw fermentation broth was 6.1% (m/v), the influence of
different concentration of pyruvate was investigated. At high con-
centration of pyruvate (5% and 6%, m/v), tyrosine phenol-lyase was
totally inhibited. Taking production efficiency into consideration,
3% pyruvate was the optimal choice for application of pyruvate
fermentation broth (Fig. 2B).
Enzymatic activity was influenced by different bio-
conversion conditions, so the reactions were optimized
and the factors including pH, temperature, substrate concen-
tration and surfactants were investigated respectively. Pyruvic
acid, tyrosine and tyramine were all analyzed by HPLC [29], and
the enzymatic activities were obtained according to the HPLC
results.

H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 38–44
41
Fig. 2. Effect of the concentrations of ammonium chloride (A) and pyruvate fermentation broth (B) on TPL activity. TPL cells were added to reaction mixture containing
pyruvate fermentation broth, ammonium chloride, phenol, PLP and Triton X-100 at pH 7 and 37 ◦C, and activities were tested with changing pyruvate substrate concentration
(1.0–6.0%, m/v) and ammonium chloride concentrations (0–1500 mM). The optimal concentrations were 350 mM ammonium chloride and 3% pyruvate fermentation broth
respectively.
Fig. 3. Effect of pH (A) and temperature (B) on TPL activity. TPL cells were added to the reaction mixture containing the pyruvate fermentation broth, ammonium chloride,
phenol, PLP and Triton X-100, and activities were tested while varying pH from 6 to 11, temperature from 25 to 60 ◦C with pH 7 and 37 ◦C gave the highest conversion rate
(96.2%).
As described in Fig. 3A and B, TPL activity for the production of
l-tyrosine was maximal at pH 7 and 37 ◦C. When pyruvate fermen-
tation broth was used as substrate, TPL was sensitive to pH change
and the conversion rate diminished greatly, especially at pH 5, 6
and 10, 11. TPL activity changed little between 30 ◦C and 50 ◦C, but
the conversion rate of pyruvate was equal to zero at 60 ◦C.
amount of l-tyrosine substrate was 0.7 g for 2 g TDC immobilized
cells in 10 mL reaction system with 97.6% conversion rate (Fig. 4C).
3.2.2. The influence of immobilization on TDC activity
Fig. 5A indicates the change of TDC activities over the long-
term storage in buffer condition. Immobilized TDC cells remained
high conversion (97.6%) even after eight weeks storage. It has
been demonstrated that alginate was a good immobilized mate-
rial for enzymatic activity recovery, mechanical strength and
operability [27,30]. It is advantageous to produce the TDC immo-
bilized cells in bulk batches and then store the immobilized
enzyme for use when needed, but this requires that the bio-
catalyst to maintain sufficient activity over time. Immobilized
TDC cells were stored in sodium acetate buffer (0.2 M, pH 5.5)
with ampicillin (50 mg/L). Irrespective of TDC activity declined
after the longer-term storage (e.g., fourteen weeks), the approx-
imate 5% decrease was still low. Nevertheless, immobilized
cells do not need be laid aside for so long time in order to
3.2. Optimization of TDC catalyzed reaction
3.2.1. The effect of bioconversion conditions on TDC activity
The optimal pH and temperature for tyrosine decarboxylation
reaction were set at 5.5, 40 ◦C respectively (Fig. 4A and B). Though
l-tyrosine has a very poor solubility at pH 5.5, the decarboxylation
reaction could be promoted because tyramine can dissolve in pH 5.5
aqueous system. l-Tyrosine was added by fed batch to avoid exces-
sive tyrosine adhering to the surface of immobilized beads. Even
though the optimal pH was kept, more tyramine will cause inhi-
bition to TDC to cut down enzyme activity. Therefore, the optimal

42
H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 38–44
Fig. 4. Effect of pH (A), temperature (B) and l-tyrosine concentrations (C) on immobilized TDC cells activity. The activities of immobilized cells were monitored while varying
the pH from 3 to 8, temperature from 30 to 60 ◦C, or l-tyrosine concentrations from 0.3 g to 1.1 g. 0.7 g l-tyrosine, pH 5.5 and 40 ◦C gave the highest conversion rate of 97.6%.
Fig. 5. Investigation of storage stability for immobilized TDC cells (A) and TDC activities difference between immobilized cells and free cells (B and C). TDC immobilized cells
(2 g) were added to 10 mL reaction mixture containing 0.7 g l-tyrosine, 20 ␮L PLP, 20 ␮L TritonX-100 at pH 5.5 and 40 ◦C, and activities were monitored through varying
TDC immobilized cells stored for different time. While investigating TDC activities difference between immobilized cells (2 g) and free cells (100 mg), the reactions were
performed at the same conditions except catalyst. Immobilized TDC cells remained high conversion (97.6%) even after eight weeks storage. To gain the highest enzymatic
activity, immobilized cells took 6 h and could be reused six times under the optimal conditions, compared with 4 h and only one batch for free cells.
accelerate the production. Therefore, immobilization of whole cells
with alginate offers an effective method due to its improvement in
the stability and reusability of TDC.
From Fig. 5B, free cells just took 4 h to reach the high conversion
rate (97.6%) compared with 6 h for immobilized cells. This figure
illustrates that the exchange of l-tyrosine and tyramine became
more difficult in immobilized cells than in free cells [35]. Another
influential factor may be the worse solubility of l-tyrosine which
further increased the difficulty of l-tyrosine permeating the cell
wall and membrane in immobilized cells.
Repeated batch mode of l-tyrosine transformation was per-
formed to examine and compare the operation stability of
immobilized and free cells (Fig. 5C). Continuous batch conversion
was operated under the standard conditions for 2 g immobilized
cells and 100 mg free cells respectively. As described in Fig. 5C, the
activity of TDC free cells quickly decreased. The conversion rate
already fell to about 78% even in the second batch, which could be
resulted from the leakage of soluble enzyme due to the damage of
cell wall and membrane. Therefore, the free cells biocatalyst cannot
be reused for many times at all.
Though immobilization of whole cells has many advantages, it
was not optimal for producing l-tyrosine by immobilized TPL cell
catalysis. l-Tyrosine quickly precipitated from the reaction system
due to its poor solubility and always adhered to the immobilized
beads which hindered the progress of reaction. At the same time,
the reaction system containing l-tyrosine and immobilized cells
needed to be adjusted to pH 11 to dissolve l-tyrosine for its sep-
aration. Additionally, a long stir was done to completely separate
l-tyrosine from the beads. These operations disrupted the stability
of TPL immobilized cells, and their repeated batch number was less
than two times to guarantee the high conversion rate (data was not
shown). Therefore, TPL cells instead of TPL immobilized cells were
utilized to synthesize l-tyrosine.
3.3. Scale up fermentation and bioconversion, identification of
tyramine
The lyophilized whole cells of TPL and TDC for 7 L working vol-
ume were collected after fermentation with the weight 46.6 1.2 g,
43.7 1.4 g respectively.
Under the catalysis of TPL, 727.9 g l-tyrosine was obtained from
a 15 L reaction volume. The calculation formula of the yield is Y =
[m/(n × Mw)] × 100% (Y: yield (%); Mw: molecular weight of the
In case of immobilized cells, the reaction system could be run six
consecutive batches simultaneously to guarantee the high enzyme
activity. The experiments had demonstrated that the immobilized
cells possessed much better operation stability than free cells.

H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 124 (2016) 38–44
43
efits of immobilization including increased productivity, enhanced
enzyme stability, and reduced adverse effects, the most significant
advantage of TDC immobilized cells would be the overall cost-
effectiveness, since it is probably the most important concern in
industrial production [35]. No matter the synthesis or the decar-
boxylation of l-tyrosine, the post-treatment of the product could be
conveniently performed. Furthermore, the purity of l-tyrosine and
tyramine were both approximately 99%. The total yield (86%) and
purity (99.9%) of tyramine would then satisfy the demand of pro-
duction. The scale-up study demonstrated that this synthetic route
was very economical when the raw pyruvate fermentation broth
was used as the source to prepare l-tyrosine and immobilized TDC
cells were used to catalyze l-tyrosine.
4. Conclusions
In the present study, the two-step biocatalytic reaction for
efficient synthesis of tyramine was investigated. In order that
enzymatic synthesis for tyramine is competitive, we combine the
advantages of asymmetric catalysis using immobilized recom-
binant whole cell, operation in aqueous media to circumvent
hazardous organic solvent and especially raw pyruvate fermenta-
tion broth as source. Low cost pyruvate fermentation broth and
immobilized TDC cells made enzymatic bioconversion become an
efficient mean to economically produce tyramine. Through scal-
ing up bioconversion, pyruvate fermentation broth was efficiently
converted to l-tyrosine with a high yield 94.3%. Additionally, l-
tyrosine was availably decarboxylated to tyramine by immobilized
TDC cells. The total yield of tyramine could reach approximately
86% by the two-step biocatalytic reaction. Conclusively, a green and
cost-efficient two-step reaction for production of tyramine at a high
yield from pyruvate fermentation broth was established.
Fig. 6. Change of the concentrations of l-tyrosine and tyramine in scale up study.
After reacted for 10 h, l-tyrosine was converted to tyramine with a conversion rate
of 97.6% under the optimal conditions.
product; n: the theoretical molar quantity of the product (mol); m:
the actual weight of the product).
According to the formula, the yield of tyrosine was 94.3%. Due to
the poor solubility of l-tyrosine, the precipitated l-tyrosine could
be purified further by adjusting pH to 6 after decolorization at pH
11, and its specific rotation was
˛
[ ]D
20 = −11.8◦ (c = 5, 1 M HCl), as
detected by the spectropolarimeter (WZZ-2B, Shanghai, China).
Tyramine hydrochloride crystal (917.6 g) was produced from
1050 g l-tyrosine in scale-up reaction. The yield of tyramine
was 91.2%. Since l-tyrosine used as the original material was of
99.5% purity, and no other side-products were produced in this
enzyme catalyzed system, the further purification could be sim-
ply performed as shown in Section 2.5. The purity of tyramine
hydrochloride was detected by HPLC which was approximately
99.9%. The identification of tyramine hydrochloride was as follows:
MS (EI) (Micromass, Britain) m/z : 137.1 (M+-HCl). IR (KBr pellet)
(Nexus 870, US): 3092, 1614, 1518, 1494, 1255, 1220, 1141 and
Acknowledgements
We thank Dongyang Wang and Yongfu Pan (Shandong
Yangcheng Biotech Co. Ltd., Shangdong, China) for their good coop-
eration. This work was supported by National Natural Science
Foundation of China (21302100) and the Open Fund of State Key
Laboratory of Pharmaceutical Biotechnology of Nanjing University,
P.R. China.
836 cm−1 1HNMR (D2O) (Bruker DRX400, Germany) ı: 7.12(d, 2H),
.
6.81(d, 2H), 3.15(t, 2H), 2.83(t, 2H).
It is important to maintain pH at 5.5 during the produc-
tion of tyramine. Though l-tyrosine has a very poor solubility
at pH 5.5, the decarboxylation reaction could be promoted
because tyramine can dissolve in pH 5.5 aqueous system. How-
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