Enzymatic synthesis of theanine from glutamic acid γ-methyl ester and ethylamine by immobilized Escherichia coli cells with γ- glutamyltranspeptidase activity

Article abstract of DOI:10.1007/s00726-010-0553-z

Theanine (γ-glutamylethylamide) is the main amino acid component in green tea. The demand for theanine in the food and pharmaceutical industries continues to increase because of its special flavour and multiple physiological effects. In this research, an improved method for enzymatic theanine synthesis is reported. An economical substrate, glutamic acid γ-methyl ester, was used in the synthesis catalyzed by immobilized Escherichia coli cells with γ-glutamyltranspeptidase (GGT) activity. The results show that GGT activity with glutamic acid γ-methyl ester as substrate was about 1.2-folds higher than that with glutamine as substrate. Reaction conditions were optimized by using 300 mmol/l glutamic acid γ-methyl ester, 3,000 mmol/l ethylamine, and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C. Under these conditions, the immobilized cells were continuously used ten times, yielding an average glutamic acid γ-methyl ester to theanine conversion rate of 69.3%. Bead activity did not change significantly the first six times they were used, and the average conversion rate during the first six instances was 87.2%. The immobilized cells exhibited favourable operational stability.

Full text of DOI:10.1007/s00726-010-0553-z

Amino Acids (2010) 39:1177–1182
DOI 10.1007/s00726-010-0553-z
ORIGINAL ARTICLE
Enzymatic synthesis of theanine from glutamic acid c-methyl
ester and ethylamine by immobilized Escherichia coli cells
with c-glutamyltranspeptidase activity
•
Fei Zhang Qing-Zhong Zheng Qing-Cai Jiao
•
•
•
Jun-Zhong Liu Gen-Hai Zhao
Received: 15 June 2009 / Accepted: 2 March 2010 / Published online: 19 March 2010
Ó Springer-Verlag 2010
Abstract Theanine (c-glutamylethylamide) is the main
amino acid component in green tea. The demand for
theanine in the food and pharmaceutical industries
continues to increase because of its special flavour and
multiple physiological effects. In this research, an
improved method for enzymatic theanine synthesis is
reported. An economical substrate, glutamic acid c-methyl
ester, was used in the synthesis catalyzed by immobilized
Escherichia coli cells with c-glutamyltranspeptidase
(GGT) activity. The results show that GGT activity with
glutamic acid c-methyl ester as substrate was about
1.2-folds higher than that with glutamine as substrate.
Reaction conditions were optimized by using 300 mmol/l
glutamic acid c-methyl ester, 3,000 mmol/l ethylamine,
and 0.1 g/ml of immobilized GGT cells at pH 10 and 50°C.
Under these conditions, the immobilized cells were con-
tinuously used ten times, yielding an average glutamic acid
c-methyl ester to theanine conversion rate of 69.3%. Bead
activity did not change significantly the first six times they
were used, and the average conversion rate during the first
six instances was 87.2%. The immobilized cells exhibited
favourable operational stability.
Keywords Theanine Á Enzymatic synthesis Á
Glutamic acid c-methyl ester Á c-Glutamyltranspeptidase
Introduction
Theanine (c-glutamylethylamide) is a characteristic, non-
proteinous amino acid in tea plant (Camellia sinensis). It is
the main amino acid component in green tea, accounting
for 50% of the plant’s total free amino acids and 1–2% of
the dry weight of tea leaves (Graham 1992). It is also the
major flavour component of green tea. Aside from the tea
plant, theanine is also found in the mushroom Xerocomus
badius and in other species belonging to the Camellia
genus (C. japonica and C. sasanqua) (Casimir et al. 1960;
Gregory and Burgunda 2006). It can cross the blood–brain
barrier and penetrate the brain, so it exhibits psychoactive
effects (Yokogoshi et al. 1998; Kimura et al. 2007). Recent
studies suggest that theanine poses multiple physiological
and pharmacological effects on relaxation (Juneja et al.
1999; Lu et al. 2004) and assists in blood pressure reduc-
tion (Yokogoshi et al. 1995; Rogers et al. 2008), anti-
obesity (Zheng et al. 2004; Maeda et al. 2005), antitumor
efficacy enhancement (Sadzuka et al. 1996, 2000), neuro-
protection (Kakuda 2002; Nagasawa et al. 2004), and
enhancement of defence against microbes (Bukowski and
Percival 2007). Due to its special flavour and versatile
physiological effects, an increasing demand for theanine is
observed in the food and pharmaceutical industries.
F. Zhang Á Q.-Z. Zheng Á Q.-C. Jiao (&) Á J.-Z. Liu Á
G.-H. Zhao
State Key Laboratory of Pharmaceutical Biotechnology,
School of Life Science, Nanjing University, Nanjing 210093,
People’s Republic of China
Since the isolation and identification of theanine in 1949
(Sakato 1949), several methods have been developed for
the production of this amino acid. One of the most effective
methods is theanine extraction from dry tea or fresh tea
plant leaves, but this procedure is laborious and time
consuming. Other methods such as tea callus cultivation
e-mail: jiaoqc@yahoo.com.cn
F. Zhang
Department of Biotechnology, Baotou Light Industry Vocational
and Technical College, Baotou 014043,
People’s Republic of China
123

1178
F. Zhang et al.
(Orihara and Furuya 1990; Matsuura et al. 1994) and
fermentation of the mushroom Xerocomus badius (Li et al.
2008) are also reported, but the production efficiency of
these procedures is low. Another effective method is
chemical synthesis (Lichtenstein 1942; Yan et al. 2003;
Gu et al. 2004). The earliest report on chemical theanine
synthesis is the preparation of c-glutamylethylamide by an
Israeli scientist in1942 in which he treated pyrrolidone-
carboxylic acid with aqueous ethylamine solutions
(Lichtenstein 1942). The chemical synthesis process is
simpler than that presented in the previously mentioned
method, and the main starting material, glutamic acid, is
inexpensive. However, synthetic methods also have limi-
tations including low yield, low purity, and a requirement
for the protection and unblocking of reactive groups.
Recently, methods for enzymatic theanine synthesis
have also been developed and intensively investigated by
several groups. In general, three enzymes, namely, gluta-
minase (EC 3.5.1.2) (Aberlian et al. 1993; Tachiki et al.
1998), glutamine synthetase (GS, EC 6.3.1.2) (Tachiki
et al. 1986; Yamamoto et al. 2005, 2008), and c-glutam-
yltranspeptidase (GGT, EC 2.3.2.2) (Suzuki et al. 1986,
2002), can generate theanine (Fig. 1a, b). The reaction
catalyzed by GS can utilize glutamic acid, a cheap chem-
ical, as a substrate, but this reaction requires a continuous
supply of ATP. In a recent study (Yamamoto et al. 2008),
110 mg/ml of theanine was produced in 48 h, and a 100%
conversion rate was achieved from coupled fermentation
with energy transfer. On the other hand, the reactions
catalyzed by glutaminase or GGT require no ATP input,
but a high concentration of ethylamine is required to drive
glutamine conversion. In a previous report (Suzuki et al.
2002), a glutamine to theanine conversion rate of 60% was
obtained using a glutamine to ethylamine mole ratio of
2:15. In addition, L-amidohydrolase (EC3.5.1.14) was also
reported to be suitable for use in DL-theanine resolution
(Li et al. 2007). At any rate, enzymatic synthesis provides
another possibility for rapidly producing theanine on an
industrial scale.
effective industrial scale processes. In addition, an immo-
bilized cell system can increase cell concentration, enhance
bead stability, and allow easier separation for reuse.
Therefore, the immobilized cell system is a desirable
technique in enzymatic theanine production. In the present
research, we optimized the conditions for theanine syn-
thesis from a novel substrate, glutamic acid c-methyl ester
(L-Glu-c-methyl ester), catalyzed by GGT (Fig. 1c). Key
factors such as substrate specificity, pH, temperature, and
substrate mole ratio were investigated.
Materials and methods
Chemicals
Theanine was purchased from Taiyo Kagaku Co., Ltd.
Glutamine, ethylamine (65%), sodium alginate, peptone,
and yeast extract were purchased from China Medicine
(Group) Shanghai Chemical Reagent Corporation. Isopro-
pyl-b-D-thiogalactopyranoside (IPTG) (purity C 98%) was
purchased from Nanjing Jitian Biotechnology Co., Ltd.,
while L-Glu-c-methyl ester and c-L-glutamylhydrazide
were prepared as described in a previous paper (Chen et al.
1994). All other chemicals were of analytical grade.
Microorganisms
The gene encoding c-glutamyltranspeptidase, ggt, was
cloned from Escherichia coli k-12 MG1655. The E. coli
strain BL21 (DE3) carrying the recombinant plasmid
pET32a-ggt was constructed in our laboratory. The strain
was cultured in LB broth containing 50 lg/ml ampicillin
and induced with 0.4 mM (final concentration) isopropyl-
b-D-thiogalactopyranoside (IPTG) at 30°C.
Immobilization of cells
A modified method from Birgisson et al. (2007) was used
for cell immobilization. To make a final concentration of
3% alginate (w/v), 5 g cells (wet wt.) were mixed with
20 ml sodium alginate water solution. After stirring the
components together, the mixture was added into 500 ml of
2% (w/v) CaCl2 solution by using a syringe to drip the
mixture from a 10 cm height. The average diameter of the
resulting beads was about 3 mm. Prior to use, the beads
were allowed to harden for 2 h at 4°C without agitation.
We find the study of Suzuki et al. (2002) relevant as it is
our view that better conversion rates at reduced substrate
costs are still possible and will be necessary for cost-
GGT activity assays
By measuring the p-nitroaniline released from c-glutamyl-
p-nitroanilide as described by Nakayama et al. (1984),
GGT activity was determined spectrophotometrically.
Fig. 1 Enzymatic theanine synthesis. a Catalyzed by glutaminase or
GGT; b catalyzed by GS; c catalyzed by GGT
123

Synthesis of theanine from glutamic acid c-methyl ester
1179
pH and temperature effect
The reaction mixture (final volume, 2 ml) containing
2.5 mM c-L-glutamyl-p-nitroanilide, 50 mM Tris–HC1
(pH 8.5), 100 mM glycylglycine, and enzymes (i.e. free or
immobilized cells) was incubated at 37°C for 5 min. The
reaction was discontinued by adding 1.5 mol/l acetic acid
(4 ml), and the amount of liberated p-nitroaniline was
determined from the optical density at 410 nm. One
enzyme unit (U) was defined as the amount of free or
immobilized cells that released 1 lmol of p-nitroaniline
per minute from c-glutamyl-p-nitroanilide at 37°C. The
specific activity is defined as units/g of free or immobilized
cells.
Enzyme activity can be affected by environmental factors,
such as pH and temperature. In this study, using L-Glu-
c-methyl ester and ethylamine as the substrates, a pH range
from 7 to 11 was used to determine the optimal initial pH.
As shown in Fig. 2, with the initial pH increasing from 7 to
10, GGT activity increased significantly and reached its
maximum at pH10. An activity decrease was observed at
pH 11. The optimal temperature for the reaction was sub-
sequently optimized. When assayed at pH 10, the optimal
temperature for the reaction was about 50°C (Fig. 3).
These results indicate that the optimal reaction conditions,
with glutamic acid c-methyl ester as c-glutamyl donor,
were similar to those with glutamine as c-glutamyl donor
(Suzuki et al. 2002).
Theanine assay
The concentrations of theanine and glutamic acid c-methyl
ester were determined by high-performance liquid chro-
matograph (HPLC) as previous described (Suzuki et al.
2002). The IR spectra of theanine in KBr was recorded on a
Perkin–Elmer 297 spectrometer calibrated with polysty-
rene. 1HNMR spectra was recorded on a Bruker 400
(400 MHz).
Effect of substrate mole ratio
In enzymatic synthesis, a frequently used means to increase
production is in excess of one substrate. In this study, the
mole ratios of L-Glu-c-methyl ester to ethylamine varied
from 1:4 to 1:12. The relationship between the conversion
rate of L-Glu-c-methyl ester and the mole ratios is shown in
Fig. 4. Ethylamine excess could significantly promote
theanine synthesis. The highest conversion rates of 96.1
and 96.3% occurred at mole ratios of 1:10 and 1:12.
Results
GGT substrate specificity
Substrate concentration effect
To investigate GGT substrate specificity, five different
c-glutamyl compounds (c-glutamyl-p-nitroanilide, L-Gln,
GSH (glutathione), L-Glu-c-methyl ester, and c-L-glutam-
ylhydrazide) were used as substrates in the enzymatic
synthesis of theanine catalyzed by GGT. As shown in
Table 1, all of these compounds could serve as c-glutamyl
donors in theanine synthesis. The GGT activity with L-Glu-
c-methyl ester as substrate was about 1.2-folds higher than
that with glutamine as substrate.
The substrate concentration effect on the conversion rate
from L-Glu-c-methyl ester to theanine was studied. The
conversion rate time course under different substrate con-
centrations is shown in Fig. 5. The results indicate that
with a substrate concentration increase, the conversion rate
120
100
80
60
40
20
0
The reaction was carried out in a 10 ml mixture con-
taining 1,000 mmol/l ethylamine and 100 mmol/l c-glut-
amyl donors, and 1 g immobilized GGT cells at pH 9 and
37°C. A gram of immobilized cells contains 550 units
of enzymes. Theanine concentrations were measured by
HPLC after 20 min.
Table 1 GGT substrate specificity
6
7
8
9
10
11
12
Substrate
Theanine (mmol/l) Relative activity (%)
pH
c-Glutamyl-p-nitroanilide 15.9
100
72.5
Fig. 2 Effect of pH on GGT activity. The reaction was carried out in
a 10 ml mixture containing 1,000 mmol/l ethylamine, 100 mmol/l
L-Glu-c-methyl ester, and 1 g immobilized GGT cells. The mixture
was kept in a plastic tube at 37°C as it was being shaken. Theanine
concentrations were measured after 20 min. The highest relative
activity of 100% denoted 15.4 mmol/l of theanine
L-Gln
11.5
14.3
13.5
7.0
GSH (glutathione)
L-Glu-c-methyl ester
c-L-Glutamylhydrazide
89.9
85.4
44.2
123

1180
F. Zhang et al.
100
80
60
40
20
100
80
60
40
20
0
0
6
12
18
24
30
36
Time (h)
30
35
40
45
50
55
60
65
Fig. 5 Conversion rate time course of L-Glu-c-methyl ester. The
reaction was carried out in a 10 ml mixture containing 1 g immo-
bilized GGT cells at pH10. The L-Glu-c-methyl ester concentrations
were 100 mmol/l (filled circle), 200 mmol/l (cross), 300 mmol/l
(empty square), and 400 mmol/l (filled triangle), respectively. The
ethylamine concentrations were tenfolds higher than those of L-Glu-c-
methyl ester, correspondingly. The mixture was kept at 50°C as it was
being shaken. The highest conversion rate of 100% denoted
100 mmol/l of theanine
t (°C)
Fig. 3 Effect of temperature on GGT activity. The mixture compo-
nents were the same as those mentioned in Fig. 2. The mixture was
kept in a plastic tube at pH 10 as it was being shaken. Theanine
concentrations were measured after 20 min. The highest relative
activity of 100% denoted 27.9 mmol/l of theanine
100
80
60
40
20
0
100
80
60
40
20
0
1:4
1:6
1:8
1:10
1:12
Mole ratio
Fig. 4 Effect of the initial mole ratios of L-Glu-c-methyl ester to
ethylamine on the conversion rate of L-Glu-c-methyl ester. The
reaction was carried out in a 10 ml mixture containing two substrates
and 1 g immobilized GGT cells at pH10. The L-Glu-c-methyl ester
concentration fixed at 100 mmol/l and ethylamine concentrations
were 400, 600, 800, 1,000, and 1,200 mmol/l, respectively. The
mixture was kept at 50°C as it was being shaken. Theanine
concentrations were measured after 8 h. The highest conversion rate
of 100% denoted 100 mmol/l of theanine
0
1
2
3
4
5
6
7
8
9
10
11
Use times
Fig. 6 Operational stability of the immobilized cells. The reaction
was carried out in a 10 ml mixture containing 300 mmol/l glutamic
acid c-methyl ester, 3,000 mmol/l ethylamine, and 1 g immobilized
cells at pH10. The mixture was kept at 50°C as it was being shaken.
Theanine concentrations were measured after 18 h
from L-Glu-c-methyl ester to theanine decreased. Reaction
inhibition was observed at a high substrate concentration.
Taking production efficiency into consideration, the opti-
mal substrate concentration would be 300 mmol/l. The
optimal reaction time was prolonged with substrate con-
centration increase. When the L-Glu-c-methyl ester con-
centration was 300 mmol/l, the optimal reaction time was
18 h.
a significant enzyme activity degradation was observed.
Upon tenth usage, the conversion rate reduced to 12.8%.
The average conversion rate during the ten-time usage was
69.3%. The immobilized cells exhibited favourable oper-
ational stability.
Isolation and identification of theanine
The reaction mixture (60 ml) containing 261.6 mmol/l
theanine was concentrated, crystallized from 80% ethanol
and dried in vacuum for 2 h to give 1.9 g (69%) of white
crystals. Above crystals were dissolved with water and
applied to a column (30 ml) of Dowex 1 9 8, which had
been prepared as the Cl- form. Theanine was eluted with
water and the fractions containing theanine were collected
and lyophilized to give 1.1 g (57%) of white crystals, IR
Operational stability of immobilized cells
In evaluating immobilized cell stability, the beads were
continuously used ten times. As shown in Fig. 6, bead
activity did not change significantly during the first six
times the beads were used, and the average conversion rate
from L-Glu-c-methyl ester to theanine during these times
was 87.2%. When the beads were used more than six times,
1
(KBr): 3,443, 3,300, 2,969, 1,647, 1,582 cm-1. HNMR
123

Synthesis of theanine from glutamic acid c-methyl ester
1181
acceptor was exceedingly more excessive over c-glutamyl
donors. Side reactions were avoided this way.
The speed of transpeptidation reaction, a nucleophilic
substitution, catalyzed by GGT is significantly affected by
pH. Generally, a high pH inhibits nucleophile ionization,
and the attack of the nucleophile, the c-glutamyl acceptor,
will be more efficient at a high pH. In our study’s result, an
acceleration of transpeptidation reaction was observed with
pH increase. The GGT activity reached its maximum at
pH10, which agreed with the results of a previous report
(Suzuki et al. 2002).
Scheme 1 GGT reaction mechanism
In previous literature, glutamine as the c-glutamyl donor
was used in the enzymatic synthesis of theanine catalyzed
by GGT, and the glutamine to theanine conversion rates
ranged from 60% (Suzuki et al. 2002) to 27% (Hung et al.
2008). The low conversion rate in previous studies could
have resulted from the low glutamine to ethylamine mole
ratio, which could be verified indirectly by our present
results. In our research, a glutamic acid c-methyl ester to
ethylamine substrate mole ratio of 1:10 enabled an
enhanced conversion of 96.1%.
(D2O): d 3.75 (t, 1H), 3.20 (q, 2H), 2.14-2.38 (m, 4H), 1.11
(t, 3H).
Discussion
Animals, plants, and microorganism have been found to
extensively contain c-glutamyltranspeptidases (GGT)
(Taniguchi and Ikeda 1998). The GGT catalyzes the
hydrolysis of c-glutamyl compounds and the transfer of
their c-glutamyl components to various primary amine
acceptors including amino acids, peptides, or other amino
compounds. As shown in Scheme 1, the catalytic mecha-
nism involves the activated oxygen atom of the side chain
of the N-terminal Thr-391 of the small sub-unit attacks the
carbonyl carbon of c-glutamyl compounds to form a
c-glutamyl-enzyme intermediate (Inoue et al. 2000; Okada
et al. 2006; Suzuki et al. 2007). When this intermediate is
exposed to a nucleophile, a c-glutamyl acceptor, a trans-
peptidation reaction takes place, and a new c-glutamyl
compound is formed. A series of c-glutamyl compounds
including c-L-glutamyl-L-DOPA, theanine, c-D-glutamyl-
L-tryptophan (SCV-07), and c-L-glutamyltaurine can be
produced in this manner (Suzuki et al. 2007).
In our study, glutamic acid c-methyl ester instead of
glutamine was used in theanine synthesis, and immobilized
Escherichia coli cells with GGT activity were used as
catalysts. The results demonstrate an improvement in
enzymatic theanine synthesis and display vast potential in
industrialization. Our results also suggest an improvement
in c-glutamyl compound preparation.
Acknowledgments We thank Ji-Bing Xiong for synthesizing L-Glu-
c-methyl ester. This work was supported by the National Technology-
Innovation Fund of P R China (02CJ-13-01-16).
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