Theanine production by coupled fermentation with energy transfer using γ-glutamylmethylamide synthetase of Methylovorus mays No. 9

Article abstract of DOI:10.1271/bbb.70663

γ-Glutamylmetylamide synthetase (GMAS) of Methylovorus mays No. 9, produced by Eschericia coli AD494 (DE3) harboring pET21aGM, formed theanine from glutamic acid and ethylamine with coupling of the reaction with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn²⁺ to the reaction mixture, whereas Mg²⁺ was less effective. Increases to a certain level in the concentrations of GMAS and the substrates in the mixture were effective in increasing theanine formation, but high concentrations of ethylamine (900 mM or more) inhibited yeast sugar fermentation, and eventually decreased theanine formation. The inhibitory effect of ethylamine was restored by increasing the concentration of potassium phosphate buffer in the mixture. Approximately 600 mM (110 mg/ml) theanine was formed in 48 h in an improved reaction mixture containing 600 mM sodium glutamate, 600 mM ethylamine· HCl, 300 mM glucose, 200 mM potassium phosphate buffer (pH 7.0), 30 mM MgCl ₂, 5 mM MnCl₂, 5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of yeast cells. The yield of theanine was 100% on the substrates (glutamic acid and ethylamine) and also on the energy source (glucose consumed).

Full text of DOI:10.1271/bbb.70663

Biosci. Biotechnol. Biochem., 72 (5), 1206–1211, 2008
Theanine Production by Coupled Fermentation with Energy Transfer
Using ꢀ-Glutamylmethylamide Synthetase of Methylovorus mays No. 9
y
2;
Sachiko YAMAMOTO,1 Yosuke MORIHARA,2 Mamoru WAKAYAMA,2 and Takashi TACHIKI
1Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan
2Department of Bioscience and Biotechnology, Faculty of Science and Engineering,
Ritsumeikan University, Shiga 525-8577, Japan
Received October 16, 2007; Accepted January 31, 2008; Online Publication, May 7, 2008
[doi:10.1271/bbb.70663]
ꢀ-Glutamylmetylamide synthetase (GMAS) of Meth-
ylovorus mays No. 9, produced by Eschericia coli AD494
(DE3) harboring pET21aGM, formed theanine from
glutamic acid and ethylamine with coupling of the
reaction with sugar fermentation of baker’s yeast cells
as an ATP-regeneration system. Theanine formation
was stimulated by the addition of Mn2þ to the reaction
mixture, whereas Mg2þ was less effective. Increases to a
certain level in the concentrations of GMAS and the
substrates in the mixture were effective in increasing
theanine formation, but high concentrations of ethyl-
amine (900 mM or more) inhibited yeast sugar fermen-
tation, and eventually decreased theanine formation.
The inhibitory effect of ethylamine was restored by
increasing the concentration of potassium phosphate
buffer in the mixture. Approximately 600 mM (110 mg/
ml) theanine was formed in 48 h in an improved reaction
mixture containing 600 mM sodium glutamate, 600 mM
human beings: significant reductions in blood pressure,4)
relief from convulsions induced by caffeine,5) generation
of ꢁ-brain waves, indicating relaxation,6) and a favorable
influence on the levels of several bioactive substances
in the brain.7–9) Hence the demand for theanine has
increased as a supplement for maintaining human health.
Recently, enzymatic production of theanine with
glutamine and ethylamine as substrates was accom-
plished by ꢀ-glutamyl transfer reaction of bacterial
glutaminase10) and ꢀ-glutamyl transpeptidase,11) but this
method has certain shortcomings with respect to the
supply of glutamine, the hydrolysis of glutamine, and
the formation of other ꢀ-glutamyl derivatives.10,11)
This indicates a need for another efficient method, and
we have investigated a process of theanine production
from glutamic acid and ethylamine as substrates using a
bacterial theanine synthetase-like enzyme.12–15) In this
process, sugar fermentation of dried baker’s yeast cells
was coupled as an ATP-regenerating system (coupled
fermentation with energy transfer).
.
ethylamine HCl, 300 mM glucose, 200 mM potassium
phosphate buffer (pH 7.0), 30 mM MgCl2, 5 mM MnCl2,
5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of
yeast cells. The yield of theanine was 100% on the
substrates (glutamic acid and ethylamine) and also on
the energy source (glucose consumed).
In earlier studies,12,13) we demonstrated the principle
of theanine production by coupled fermentation with
energy transfer using glutamine synthetase (GS; L-
glutamate:ammonia ligase, ADP-forming, E.C. 6.3.1.2)
of Pseudomonas taetrolens Y-30, which has a certain
reactivity toward ethylamine. The results suggested the
possibility of theanine production in high concentrations
by the use of large amounts of GS in the reaction
mixture,13) but this possibility could not be confirmed
because of the difficulty of solving large amounts of
P. taetrolens Y-30 GS with low reactivity to ethyl-
amine.14)
Subsequently, we found a new enzyme in Methyl-
ovorus mays No. 9, that has high theanine-forming
activity.15) The isolated enzyme predominantly cata-
lyzed the ꢀ-glutamylmethylamide-forming reaction,
with glutamic acid, methylamine, and ATP as the
substrates. It also had high reactivity to ethylamine to
form theanine, but hardly catalyzed the glutamine-
Key words: theanine; ꢀ-glutamylmethylamide synthe-
tase; coupled fermentation with energy
transfer
Theanine (ꢀ-glutamylethylamide) is an unique amino
acid found in Japanese green tea leaves.1) It is
synthesized in the root of the tea plant by theanine
synthetase (L-glutamate:ethylamine ligase; ADP-form-
ing, E.C. 6.3.1.6) with glutamic acid, ethylamine, and
ATP as substrates.2) Theanine greatly contributes to the
taste of green tea,3) and the quality of the tea leaves is
dependent on its contents. In addition, theanine has
received much attention due to its many favorable
physiological functions in several animals, including
Amino acid is L-isomer unless otherwise stated.
To whom correspondence should be addressed. Tel: +81-77-566-1111 ex. 8265; Fax: +81-77-561-2659; E-mail: tachiki@se.ritsumei.ac.jp
y

Theanine Production by Coupling with Energy Transfer
1207
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Fig. 1. Theanine Formation and Effect of Divalent Cations.
A, The initial reaction mixture (see ‘‘Materials and Methods’’) containing 1 unit/ml of recombinant GMAS was incubated at 30 ꢀC for 24 h
with shaking (200 rpm). Mg2þ and Mn2þ (B), Mn2þ (C), or Mg2þ (D) was omitted from the mixture for (A). Incubation was carried out at 30 ꢀC
for 24 h with shaking (200 rpm). Symbols: , glucose; , FBP; , theanine.
forming reaction (a GS-like reaction).
contained (i) 200 mM sodium glutamate and 300 mM
.
The reactivity of the M. mays No. 9 enzyme toward
methylamine, ethylamine, and ammonia was very
similar to that of known ꢀ-glutamylmethylamide syn-
thetase16) (GMAS; L-glutamate:methylamine ligase,
ADP-forming, E.C. 6.3.4.12), indicating that the enzyme
was GMAS. In addition, its reactivity to ethylamine
was realized in a mixture of coupled fermentation with
energy transfer,16) which suggested theanine production
in high concentration using GMAS.
Since an overexpression system of the gene of
M. mays No. 9 GMAS has been constructed,17) this
paper deals with theanine production by coupled
fermentation with energy transfer using recombinant
GMAS.
ethylamine HCl (substrates for theanine synthesis by
GMAS), (ii) 200 mM glucose and 50 mM potassium
phosphate buffer, pH 7.0 (substrates for yeast sugar
fermentation), (iii) 30 mM MgCl2 and 5 mM MnCl2
(cofactors for GMAS and several enzymes in yeast sugar
fermentation), (iv) 5 mM AMP (ATP source), and (v)
enzyme preparations (recombinant GMAS and 40 mg/
ml of dried yeast cells; GMAS concentration is indicated
in the legend to each experiment). The reaction was
carried out at 30 ꢀC with shaking (200 rpm), and
terminated by immersing the reaction tube in boiling
water for 3 min. The supernatant obtained by centrifu-
gation at 1;500 ꢁ g for 10 min at room temperature was
submitted to assay.
The yield of theanine on the energy source (glucose)
in coupled fermentation with energy transfer was
calculated on the basis of the amount of ATP regen-
erated by the glucose consumed; yield (%) = {(theanine
formed, mM)/(2 ꢁ glucose consumed, mM)} ꢁ 100.13)
Materials and Methods
Enzyme preparation. Pressed baker’s yeast supplied
by Oriental Yeast (Tokyo) was dried as described
previously.18)
Recombinant GMAS produced by E. coli AD494
(DE3) harboring pET21aGM (an expression vector for
the GMAS gene of M. mays No. 9) was prepared by the
method described previously.17) One unit of GMAS was
defined as the amount of enzyme forming 1 mmol of
ꢀ-glutamylhydroxamate per min at 30 ꢀC in a reaction
mixture consisting of 50 mM sodium glutamate, 15 mM
hydroxylamine, 7.5 mM ATP, 30 mM MgCl2, 100 mM
imidazole buffer (pH 8.0), and recombinant GMAS
preparation.15,17)
Assay. Theanine, inorganic phosphate, protein,
glucose, and fructose 1,6-bisphosphate (FBP) were
determined by methods described previously.12,13)
Reagent. Glucose oxidase (glucose CII-test Wako)
was supplied by Wako Pure Chemical (Osaka), and
theanine was by Tokyo Kasei (Tokyo). The other
reagents were highest-grade commercial products.
Results and Discussion
To examine optimum pH for the theanine-forming
reaction, the reaction mixture was modified by replacing
hydroxylamine with ethylamine, and the amount of
inorganic phosphate formed was determined.
Theanine formation and effect of divalent cation
As Fig. 1A indicates, theanine was formed with a
decrease in glucose and a temporary accumulation of
FBP in the initial reaction mixture for coupled fermen-
tation with energy transfer, and that the amounts reached
to about 60 mM. When GMAS or the yeast cell was
removed from the reaction mixture, theanine formation
did not occur (data not shown), indicating that the
formation in Fig. 1A was produced by coupling of the
Reaction conditions for theanine production. A
temporary reaction mixture (dubbed the initial reaction
mixture) for coupled fermentation with energy transfer
was defined according to previous findings with P. tae-
trolens Y-30 GS.13) The mixture (1 ml in a test tube)

1208
S. YAMAMOTO et al.
theanine-forming reaction by recombinant GMAS and
sugar fermentation by the yeast cells.
yeast cells, and also to CO2 generated by yeast sugar
fermentation.19,20)
These results were nearly the same as those for
theanine formation using P. taetrolens Y-30 GS,13) but
the amount of theanine formed with GMAS (Fig. 1A,
about 60 mM) was much larger than that (8 mM) with
P. taetrolens Y-30 GS13) under the same conditions,
due to the high reactivity of GMAS to ethylamine.
The theanine formation in Fig. 1A also did not occur
when the divalent cations were removed from the
reaction mixture (Fig. 1B), whereas sugar fermentation
proceeded slowly but steadily, suggesting that divalent
cations contained in the yeast cells were effective for
sugar fermentation but not for the theanine-forming
reaction by GMAS. A similar observation was made
in the case of theanine production with P. taetrolens
Y-30 GS.13)
Several divalent cations have been reported to induce
unique structural changes in GS protein,21,22) and to vary
its enzymatic properties such as the pH-activity profile,
reactivity and affinity to substrates, etc.21,22) GMAS
has a certain homology with GS with respect to amino
acid sequence and subunit structure.17) The data in
Table 1 are probably due to cause similar to that in the
case of GS.
Effect of concentrations of GMAS and substrates on
theanine formation
Figure 2A-1, A-2, and A-3 indicate the effect of the
concentration of GMAS on theanine production in the
initial reaction mixture. The amount of theanine formed
in the mixture increased with increases in GMAS
concentrations, as observed in the reaction using
P. taetrolens Y-30 GS.13) In the mixture with 7.5
units/ml of GMAS (Fig. 2A-3), sodium glutamate
(200 mM) was converted completely to theanine (ap-
proximately 200 mM) in 24 h.
The addition of Mg2þ (Fig. 1C) or Mn2þ (Fig. 1D)
stimulated theatnine formation, and Mn2þ was more
effective than Mg2þ. Theanine formation with Mn2þ
(Fig. 1D) was stimulated to some extent by the addition
of Mg2þ to the mixture (see Fig. 1A).
The effect of Mg2þ or Mn2þ, observed in Fig. 1C and
D, appeared inconsistent with the divalent cation
dependence of GMAS with ethylamine as a substrate,
which was observed previously15) in the enzyme
reaction: the reactivity with Mg2þ was about 3 times
higher than that with Mn2þ at optimum pH (7.8) for the
Mg2þ-dependent reaction. However, detailed repeat
experiment of the enzyme reaction with Mn2þ revealed
that the Mn2þ-dependent reaction proceeded optimally
at pH 7.0 (Table 1), and the reactivity with Mn2þ at
As Fig. 2A-3 indicates, the theanine yield was 50%
based on glucose consumed, but this value is incorrect
because the concentration of sodium glutamate
(200 mM) was less than all the energy released from
200 mM glucose (corresponding to 400 mM ATP). In
order to evaluate the yield on glucose correctly, the
reaction was done again with a mixture containing
.
400 mM sodium glutamate and 600 mM ethylamine HCl.
About 300 mM theanine was formed using 7.5 units/
ml GMAS with the yield of 75% on glucose consumed
(Fig. 2B-1), and the yield reached 100% with the
formation of about 400 mM theanine (Fig. 2B-2) when
the GMAS concentration was increased to 15 units/ml.
The theanine formation, of 400 mM, was much larger
than that by P. taetrolens Y-30 GS (170 mM, 28% yield
on glucose consumed).13)
pH 7.0 was almost the same as that of the Mg2þ
dependent reaction.
-
Table 1 might explain the findings in Fig. 1C and D,
because the pH of the reaction mixture of the coupled
fermentation was maintained at neutral or slightly acidic
pH due to the buffer-like action of large amounts of
Inhibitory effect of high concentrations of substrate
on yeast sugar fermentation
Table 1. Effect of pH on Theanine-Forming Reaction with Mg2þ or
Mn2þ
To achieve theanine formation at higher concentra-
tions, the concentrations of the substrates was increased
to 300 mM (glucose), 600 mM (sodium glutamate), and
Inorganic phosphate formed (mM/min)
with Mg2þ
Buffer
pH
with Mn2þ
.
900 mM (ethylamine HCl). However, as shown in
Acetate
4.0
5.0
6.0
0.26
0.26
0.29
0.29
0.27
0.67
Fig. 3A, yeast sugar fermentation was repressed, and
eventually the final amount of theanine decreased to less
than that shown in Fig. 2B-2 (with 200 mM glucose,
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Imidazole
6.0
6.5
7.0
7.8
8.0
0.39
0.77
1.6
0.51
1.3
.
300 mM sodium glutamate, and 600 mM ethylamine
HCl).
1.5
2.5
2.3
0.77
0.71
Several experiments analyzing and coping with the
inhibition revealed that ethylamine was the main cause
of the inhibition, and that potassium phosphate buffer
restored or accelerated yeast sugar fermentation. Figure
3B-1 and 3B-2 show some of the results: yeast sugar
fermentation decreased with increases in the concen-
trations of ethylamine (Fig. 3B-1), and was restored
with increases in the concentration of potassium phos-
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Tris–HCl
8.0
8.5
9.0
1.9
1.2
0.58
0.43
0.40
0.81
The reaction mixture, consisting of 50 mM sodium glutamate, 15 mM
ethylamine, 7.5 mM ATP, 30 mM MgCl2 or 3 mM MnCl2, 100 mM each
buffer, and 0.17 units/ml of GMAS, was incubated at 30 ꢀC for 10 min. The
amount of inorganic phosphate formed was measured.

Theanine Production by Coupling with Energy Transfer
1209
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Fig. 2. Effect of Various Concentrations of GMAS and Substrates on Theanine Formation.
A, The concentration of GMAS in the initial reaction mixture with 200 mM sodium glutamate and 300 mM ethylamine HCl was 1 unit/ml
.
(A-1), 3 units/ml (A-2), or 7.5 (A-3) units/ml. B, The concentrations of sodium glutamate and ethylamine HCl in the initial reaction mixture
.
were increased to 400 mM and 600 mM respectively. The concentration of GMAS was 7.5 units/ml (B-1) or 15 (B-2) units/ml. The reaction
was carried out at 30 ꢀC for 24 h with shaking (200 rpm). Symbols: , glucose; , FBP; , theanine.
B-1
A
B-2
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Fig. 3. Effect of Concentration of Ethylamine and Potassium Phosphate Buffer on Yeast Sugar Fermentation Inhibited by High Concentrations of
Substrates.
.
A, The concentrations of sodium glutamate, ethylamine HCl, and glucose were increased to 600 mM, 900 mM ethylamine, and 300 mM
respectively in the initial reaction mixture. The concentration of GMAS was 15 units/ml. Incubation was done at 30 ꢀC with shaking (200 rpm).
.
Symbols: , glucose; , FBP; , theanine. B, GMAS was removed from the mixture for (A). The concentration of ethylamine HCl (B-1) in
the mixture was varied with a fixed concentration (50 mM) of potassium phosphate buffer. The concentration of potassium phosphate buffer
.
(B-2) was varied with the fixed concentration (900 mM) of ethylamine HCl. The glucose level in the mixture was determined. Symbols: B-1,
.
, 600 mM ethylamine HCl; , 750 mM; , 900 mM; B-2, , 50 mM potassium phosphate buffer; , 100 mM; , 200 mM.
phate buffer (Fig. 3B-2). An inhibitory effect of ethyl-
amine on sugar fermentation was also observed in
coupled fermentation with P. taetrolens Y-30 GS.13)
Increases in dried yeast cells were slightly effective in
increasing theanine formation through acceleration of
yeast sugar fermentation (data not shown). The addition
of large amounts of yeast cells does not always increase
product formation, due to unbalanced increases in
certain ATP-consuming reactions in the yeast cells.13,23)
Other components in the reaction mixture did not affect
inhibited yeast sugar fermentation (data not shown).
Theanine formation in an improved mixture
Figure 4 shows theanine formation in an improved
reaction mixture, which was designed on the basis of the
above results, and contained 300 mM glucose, 600 mM

1210
S. YAMAMOTO et al.
A
B
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40
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50
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Fig. 4. Theanine Production at High Concentrations.
.
The reaction mixture contained 600 mM sodium glutamate, 600 mM ethylamine HCl, 300 mM glucose, 200 mM potassium phosphate buffer
(pH 7.0), 30 mM MgCl2, 5 mM MnCl2, 5 mM AMP, 40 mg/ml of dried yeast cells, and 15 (A) or 30 (B) units/ml of GMAS. The reaction was
carried out at 30 ꢀC for 48 h with shaking (200 rpm). Symbols: , glucose; , FBP; , theanine.
.
(1998).
sodium glutamate, 600 mM ethylamine HCl, and
200 mM potassium phosphate buffer (pH 7.0).
7) Kimura, R., and Murata, T., Effect of theanine on
norepinephrine and serotonin levels in rat brain. Chem.
Pharm. Bull., 34, 3054–3057 (1986).
Improvement of the reaction mixture restored
yeast sugar fermentation, and also theanine formation
(Fig. 4A). Due to increasing the GMAS concentration to
30 units/ml (Fig. 4B), approximately 600 mM (110 mg/
ml) theanine was formed in 48 h with 100% conversion
from glutamic acid and ethylamine, and with 100% yield
based on the glucose consumed. The amount of theanine
formed (Fig. 4B) was about 2.5 times larger than that
formed from glutamine and ethylamine by bacterial
glutaminase (240 mM).10)
8) Yokogoshi, H., Mochizuki, M., and Saitoh, K., The-
anine-induced reduction of brain serotonin concentration
in rat. Biosci. Biotechnol. Biochem., 62, 816–817 (1998).
9) Yokogoshi, H., Kobayashi, M., Mochizuki, M., and
Terashima, T., Effect of theanine, ꢀ-glutamylethylamide,
on brain monoamines and striatal dopamine release in
conscious rats. Neurochem. Res., 23, 671–677 (1998).
10) Tachiki, T., Yamada, T., Mizuno, K., Ueda, M., Shiode,
J., and Fukami, H., ꢀ-Glutamyl transfer reactions by
glutaminase from Pseudomonas nitroreducens IFO
12694 and their application for the syntheses of theanine
and ꢀ-glutamylmethylamide. Biosci. Biotechnol. Bio-
chem., 62, 1279–1283 (1998).
11) Suzuki, H., Izuka, S., Miyakawa, N., and Kumagai, H.,
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The present paper describes the characteristics of
theanine formation by coupled fermentation with energy
transfer using recombinant GMAS from the viewpoint
of production at high concentrations.
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Theanine Production by Coupling with Energy Transfer
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