Characterization of theanine-forming enzyme from Methylovorus mays No. 9 in respect to utilization of theanine production

Article abstract of DOI:10.1271/bbb.60590

For development of theanine production from glutamic acid and ethylamine by coupling yeast sugar fermentation as an ATP-regenerating system, several strains were selected from among about 200 methylamine- and/or methanol-assimilating bacteria depending on the theanine-forming activity of their permeated cells. The amount of theanine formed by the cells of the selected strains was much larger than that by the cells of Escherichia coli AD494 (DE3) expressing Pseudomonas taetrolens Y-30 glutamine synthetase (GS), which has been found to be a usable enzyme for theanine production. A GS-like enzyme responsible for the theanine-forming reaction was obtained from an obligate methylotroph isolate, Methylovorus mays No. 9. The enzyme was induced by methylamine in the culture medium. A molecular mass of 410-470 kDa was obtained by gel filtration of the enzyme, and 51 kDa by SDS-PAGE analysis. The enzyme showed high activity toward methylamine rather than ammonia, which indicates that it is similar to known γ-glutamylmethylamide synthetase. The isolated enzyme also had high reactivity to ethylamine in a neutral pH range, and formed theanine from glutamic acid and ethylamine in a reaction mixture containing a yeast sugar fermentation system for ATP-regeneration.

Full text of DOI:10.1271/bbb.60590

Biosci. Biotechnol. Biochem., 71 (2), 545–552, 2007
Characterization of Theanine-Forming Enzyme from Methylovorus mays
No. 9 in Respect to Utilization of Theanine Production
y
Sachiko YAMAMOTO, Mamoru WAKAYAMA, and Takashi TACHIKI
Department of Bioscience and Biotechnology, Faculty of Science and Engineering,
Ritsumeikan University, Shiga 525-8577, Japan
Received October 23, 2006; Accepted November 27, 2006; Online Publication, February 7, 2007
[doi:10.1271/bbb.60590]
For development of theanine production from gluta-
mic acid and ethylamine by coupling yeast sugar
fermentation as an ATP-regenerating system, several
strains were selected from among about 200 methyl-
amine- and/or methanol-assimilating bacteria depend-
ing on the theanine-forming activity of their permeated
cells. The amount of theanine formed by the cells of the
selected strains was much larger than that by the cells of
Escherichia coli AD494 (DE3) expressing Pseudomonas
taetrolens Y-30 glutamine synthetase (GS), which has
been found to be a usable enzyme for theanine pro-
duction. A GS-like enzyme responsible for the theanine-
forming reaction was obtained from an obligate meth-
ylotroph isolate, Methylovorus mays No. 9. The enzyme
was induced by methylamine in the culture medium. A
molecular mass of 410–470 kDa was obtained by gel
filtration of the enzyme, and 51 kDa by SDS–PAGE
analysis. The enzyme showed high activity toward
methylamine rather than ammonia, which indicates
that it is similar to known ꢀ-glutamylmethylamide
synthetase. The isolated enzyme also had high reactivity
to ethylamine in a neutral pH range, and formed
theanine from glutamic acid and ethylamine in a
reaction mixture containing a yeast sugar fermentation
system for ATP-regeneration.
ity with low yield and/or the low purity of the pro-
duct.7–9) Enzymatic production of theanine with gluta-
mine and ethylamine as substrates has been done suc-
cessfully to some extent using the ꢀ-glutamyl transfer
reaction of glutaminase10) or ꢀ-glutamyl transpepti-
dase11) despite certain shortcomings with respect to
supply of glutamine and to formation of byproducts.
In order to develop more efficient method, we
investigated theanine production from glutamic acid
and ethylamine as substrates by coupled fermentation
with energy transfer using bacterial glutamine synthe-
tase (GS, L-glutamate:ammonia ligase, ADP-forming,
E.C.6.3.1.2; glutamic acid + ethylamine + ATP !
theanine + ADP + inorganic phosphate).12–14) In this
method, a catalytic amount of another substrate, ATP,
is regenerated by sugar fermentation of baker’s yeast.
Previous papers12,13) explain that GS of Pseudomonas
taetrolens Y-30, a methylamine-assimilating isolate, is
usable for theanine synthesis in coupled fermentation
with energy transfer. The results also imply that pro-
duction of theanine in high concentrations should be
realized by increasing the amounts of GS in the reaction
mixture,13) but production in high concentrations was
not achieved due to the low reactivity of P. taetrolens
Y-30 GS to ethylamine, and due to the difficulty of
solving large amounts of the enzyme.14) This indicates
the necessity of searching for a new enzyme with high
reactivity to ethylamine.
It is known that some bacteria, which grow on
methylamine as a sole carbon and nitrogen source,
generate ꢀ-glutamylmethylamide (GMA) as an inter-
mediate in their methylamine metabolism.15,16) Syn-
thesis of GMA is performed with glutamic acid, meth-
ylamine, and ATP as substrates by GMA synthetase
(GMAS; L-glutamate: methylamine ligase, ADP-form-
ing, EC 6.3.4.12). GMAS was isolated from Methyl-
ophaga sp. AA-30. The enzyme has been described
as active to methylamine rather than to ammonia, and
also as showing high reactivity to ethylamine,17) sug-
gesting that it might be usable for theanine production
Key words: theanine-forming enzyme; Methylovorus
mays No. 9; coupled fermentation with
energy transfer
Theanine (ꢀ-glutamylethylamide) is an unique amino
acid that renders the gracefully sweet taste of Japanese
tea,1) and the quality of tealeaves is influenced by its
contents. Recently, the demand for theanine has become
great not only as a food-additive to enhance flavor, but
also as a supplement to improve and/or maintain human
health because of its favorable physiological functions
toward several animals, including human beings.2–6)
Many researchers have tried to prepare theanine, but
their methods are probably impractical due to complex-
y
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

546
S. YAMAMOTO et al.
by coupled fermentation with energy transfer. But the
detailed information on it is insufficient in respect to the
theanine-forming reaction.
method using permeated cells was established prelimi-
narily with E. coli AD494 (DE3) harboring pET21aGS
(overexpression system of P. taetrolens Y-30 GS).14)
The reaction was terminated by immersing the
reaction tube in boiling water for 3 min. The super-
natant, obtained by centrifugation at 1;500 ꢀ g for 10
min at room temperature, was submitted to the assay of
theanine concentration.
(ii) ꢀ-Glutamylhydroxamate-forming reaction (GS
reaction): The activity of the enzyme responsible for
theanine-forming reaction was also measured by the GS
reaction using a previous mixture,12) consisting of 50
mM sodium glutamate, 15 mM hydoroxylamine, 7.5 mM
ATP, 100 mM imidazole buffer (pH 8.0), 30 mM MgCl2,
and a suitable amount of enzyme preparation. The GS
reaction was used as an expedient assay method for
purification and evaluation of theanine-forming enzyme.
One unit of the enzyme was defined as the amount
forming 1 mmol of ꢀ-glutamylhydroxamate per min.
(iii) ꢀ-Glutamylmethylamide-forming reaction: This
reaction was used to characterize the isolated enzyme. A
reaction mixture consisting of 50 mM sodium glutamate,
This paper first describes the isolation of a methyl-
otroph that produces an enzyme catalyzing the theanine-
forming reaction. By the use of a new screening method,
an obligate methylotroph, Methylovorus mays No. 9,
was selected as a test organism. Secondly, an enzyme
responsible for theanine formation was isolated from
M. mays No. 9, and the theanine-forming enzyme is
characterized from the viewpoint of its use in theanine
production.
Materials and Methods
Isolation of methylotrophic bacteria (first step in
screening). Soil samples were suspended in sterilized
water, and 0.1 ml of the upper phase was inoculated into
.
HCl and 0.25% methanol as sole carbon and/or nitrogen
5 ml of medium A, consisting of 0.25% methylamine
.
source, 0.2% NaCl, 0.1% KCl, 0.03% MgSO4 7H2O,
0.005% KH2PO4, 0.005% K2HPO4, and 1 ꢀ 10ꢁ7
%
.
cyanocobalamine, pH 7, in a test tube. Medium A was
defined by modifying a previous medium12) to increase
the probability of isolating a methylotroph: omission of
yeast extract and addition of methanol.
15 mM methylamine HCl, 7.5 mM ATP, 30 mM MgCl2,
100 mM imidazole buffer (pH 7.75), and a suitable
amount of the enzyme was incubated at 30 ꢂC, and the
amount of inorganic phosphate formed was determined.
This condition or the mixture is occasionally referred
to as ‘‘the standard reaction condition’’ or ‘‘the standard
reaction mixture’’ in the text.
After 5–14 d of cultivation at 30 ꢂC on a shaker (200
rpm), part of the turbid culture was transferred into
fresh medium A and cultivated for a further 5–14 d. This
procedure was repeated 3–5 times, and then the culture
was streaked on a medium A agar plate. Bacterial
colonies that appeared after incubation at 30 ꢂC for 3–7 d
were purified, and microorganisms were isolated. The
isolates were cultivated for 3–7 d in medium A supple-
mented with 0.03% yeast extract. The microorganisms,
which gave turbidity at 610 nm of 0.1 and more, were
submitted for a second screening.
16S ribosomal DNA analysis. 16S ribosomal DNA of
the isolated bacterium was amplified by polymerase
chain reaction (PCR) using the eubacterial primers 25F
(50-AGTTTGATCCTGGCTC-30) and 1510R (50-GGC-
TACCTTGTTACGA-30) according to the description of
Lane.18) The sequence of the amplified PCR product was
analyzed with an ABI-Prism Big Dye determinator cycle
sequencing ready reaction kit and an ABI-prism 377
sequencer (Applied Biosystems Japan Ltd., Tokyo,
Japan). A homology search of the DNA sequence was
done with the BLAST program of the NCBI homepage
(http://www.ncbi.nlm.nih.gov/BLAST/).
Theanine formation by permeated cells (second step
in screening). The cells cultivated as above were
collected, and washed with 10 mM potassium phosphate
buffer (pH 6.0) containing 10% glycerol, 1 mM MgCl2,
and 1 mM 2-mercaptoethanol. The washed cells were
suspended in the same buffer, and the suspension was
used as an enzyme preparation to estimate theanine-
forming activity according to the method described
below. Incubation was carried out at 30 ꢂC for 5 h.
Purification of theanine-forming enzyme. All oper-
ations were carried out at 0–10 ꢂC, and 10 mM potassium
phosphate buffer (pH 6.0) containing 10% glycerol,
0.1% Triton X-100, 0.1 mM EDTA–Na, 1 mM MgCl2,
and 1 mM 2-mercaptoethanol (buffer A) was used unless
otherwise specified.
(i) Cell-free extract: M. mays No. 9 was grown on
5 ml of an optimum medium (medium B, see Fig. 1
under ‘‘Results and Discussion’’) in a test tube at 30 ꢂC
with shaking for 1 d. The culture was inoculated to 1 l of
the same medium in a 2-l Sakaguchi flask, and cultivated
for 1 d.
Enzyme assay. The activities of three reactions were
measured.
(i) Theanine-forming reaction: A reaction mixture
containing 50 mM sodium glutamate, 150 mM ethyl-
.
amine HCl, 7.5 mM ATP, 30 mM MgCl2, 100 mM imid-
azole buffer (pH 7.75), and a suitable amount of enzyme
preparation was incubated at 30 ꢂC.
In the case of the reaction with the cell-suspension as
an enzyme preparation, 0.1 mg/ml cetyltrimethylammo-
nium bromide (CTAB) was added to the mixture. The
The washed cells from 5 l of culture medium were
suspended in buffer A without Triton X-100, and stored
at ꢁ20 ꢂC until use. The frozen cells were thawed, and

Characterization of Theanine-Forming Enzyme of M. mays No. 9
547
Table 1. Theanine-Forming Activity of Permeated Cells of Isolated Bacteria
Activity (mM) dependent on
Identity of 16S rDNA
Strain
Mg2þꢃ1
Mn2þꢃ2
Bacterial name
(%)
100
34b
24c
24a
34a
9
0.676
2.40
2.47
1.84
3.82
4.04
0.20
0.48
0.51
0.66
1.97
2.75
Agrobacterium tumefaciens
Paracoccus aminovorans
Paracoccus aminovorans
Paracoccus yeeii
98.0
98.0
98.5
99.2
98.3
Methylovorus mays
Paracoccus aminovorans
6
E. coli AD494 (DE3)
/pET21aGS
0.085
0.45
ꢃ1The reaction mixture described in ‘‘Materials and Methods’’ was used with cell-suspension of O.D. 1.0 at 610 nm. The reaction was carried out at 30 ꢂC for 5 h, and
theanine formed was determined.
ꢃ2Mg2þ in the reaction mixture was replaced with 3 mM Mn2þ
.
disrupted by sonication (20 khz for 20 min). The cell
debris was centrifuged off (12;000 ꢀ g for 30 min), and
the supernatant was used as the starting material.
(ii) Ammonium sulfate fractionation: To the cell-free
extract, ammonium sulfate was added to achieve 33%
saturation, with pH adjusted to 6–7 with 12% ammo-
nium hydroxide. The mixture was stirred gently for
60 min, and the precipitate, collected by centrifugation,
was dissolved in buffer A. The solution was dialyzed
against the same buffer.
AMP, 40 mg dried yeast cells, and a suitable amount
of theanine-forming enzyme. The reaction was done
at 30 ꢂC with shaking (200 rpm), and terminated by
immersing the reaction tube in boiling water for 3 min.
The supernatant, obtained by centrifugation at 1;500 ꢀ g
for 10 min, was submitted to the assay.
Assay. Theanine, inorganic phosphate, protein, glu-
cose, and fructose 1,6-bisphosphate (FBP) were deter-
mined by methods described previously.12,13)
(iii) DEAE-cellulofine column chromatography: The
dialyzed solution was put on a DEAE-cellulofine (2 ꢀ
10 cm) column equilibrated with buffer A. After wash-
ing it with the buffer, the column was eluted by in-
creasing the concentration of NaCl in buffer A to 0.5 M
linearly, and the enzyme was eluted at 0.3–0.4 M NaCl
(see Fig. 2 under ‘‘Results and Discussion’’). The active
fractions were combined and dialyzed against buffer A.
(iv) Hydroxyapatite column chromatography: The
dialyzed solution was put on a hydroxyapatite column
(1 ꢀ 5 cm) equilibrated with buffer A. After washing it
with the same buffer, the column was eluted by in-
creasing the concentration of the potassium phosphate
buffer to 600 mM linearly. The active fractions, eluted
at 300–400 mM buffer, were combined and dialyzed
against buffer A. The dialysate was used as a final
preparation.
Reagents. Glucose oxidase (glucose CII-test wako)
was supplied by Wako Pure Chemical (Osaka, Japan),
and theanine was by Tokyo Kasei (Tokyo). The other
reagents were highest-grade commercial products.
Results and Discussion
Isolation of methylamine- and/or methanol-assimilat-
ing bacteria
Pseudomonas taetrolens Y-30, described in previous
papers,12–14) was selected by a complicated method. The
first step was isolation of methylamine-assimilating
bacteria, the second was dependent on the activity of the
GS reaction in cell-free extract, and the third was
dependent on the activity of the concentrated extract to
form theanine in a mixture of coupled fermentation with
energy transfer.12) The results indicated that the GS
activity in the extract did not always reflect the theanine-
forming activity, and the reactivity of P. taetrolens Y-30
GS to ethylamine was not very high, though the enzyme
could be used for theanine production.12,13) Taking this
into consideration, a fast and convenient two-step
procedure was designed to obtain an enzyme more
efficient than P. taetrolens Y-30 GS.
N-Terminal amino acid sequences. Determination of
the N-terminal amino acid sequence of the isolated
enzyme was done as described in a previous paper.12)
The sequence was compared with the FASTA program
of the DDBJ homepage (http://www.ddbj.nig.ac.jp/
search/fasta-j.html).
Dried yeast cells and coupled fermentation with
energy transfer. Baker’s yeast was supplied by Oriental
Yeast (Tokyo), and was air-dried according to the
previous method.19) The reaction mixture for coupled
fermentation with energy transfer12) (1 ml in a test tube)
contained 200 mM sodium glutamate, 300 mM ethyl-
In the first step, were isolated about 200 strains of
methylamine- and/or methanol-assimilating bacteria
using medium A (see ‘‘Materials and Methods’’). In
the second, the strains were examined as to the theanine-
forming activity of their permeated cells, and six strains
were selected (Table 1). The amount of theanine formed
by these isolates was much larger than that in the
mixture with the cells of E. coli AD494 (DE3) harboring
.
amine HCl, 200 mM glucose, 50 mM potassium phos-
phate buffer (pH 7.0), 30 mM MgCl2, 5 mM MnCl2, 5 mM

548
S. YAMAMOTO et al.
Table 2. Effect of Nutritional Conditions on Theanine-Forming Activity in Cells
Addition (%)
Theanine-forming
activity
Medium
Nitrogen source (%)
YEꢃ3 PPꢃ4
Growthꢃ5
Carbon source
(0.25%)
SAꢃ6
TAꢃ7
ꢃ2
1
2
MeOHꢃ1
MeNH2
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.4
1.0
2.0
0
0
0.23
0.25
0
2.6
0.0
3.0
0.0
MeOH
MeNH2
Glucose
Glycerol
Fructose
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
0
3
0
N.D.ꢃ8
N.D.
N.D.
N.D.
2.6
N.D.
N.D.
N.D.
N.D.
3.0
4
0
0
5
0
0
6
0
0
7
MeNH2
MeNH2
MeNH2
MeNH2
MeNH2
MeNH2
MeNH2
MeNH2
0
0.23
1.00
0.16
0.10
0.38
1.08
1.37
1.08
8
0
1.9
0.0
9.5
0.0
9
0
10
11
12
13
14
0
0.0
2.0
0.0
3.8
0.03
0.4
1.0
2.0
0
1.8
1.7
9.5
0
11.5
8.5
0
1.6
Carbon and nitrogen sources in medium A were changed as indicated in the Table. M. mays No. 9 was grown on 5-ml medium in a test tube at 30 ꢂC for 3 d. The cells
colleted from culture medium were used to measure the theanine-forming activity of permeated cells. The reaction was done at 30 ꢂC for 5 h.
ꢃ1Methanol; ꢃ2Methylamine; ꢃ3Yeast extract; ꢃ4Polypepton; ꢃ5Turbidity at 610 nm;
ꢃ6Specific activity = the amount of theanine formed (mM) ꢄ turbidity at 610 nm of the reaction mixture;
ꢃ7Total activity = specific activity ꢀ total turbidity (multiply turbidity at 610 nm of culture medium by culture volume (ml)); ꢃ8Not determined.
pET21aGS.14) As Table 1 indicates, Mg2þ was more
effective as a cofactor than Mn2þ. The divalent cation
dependence was different from that of P. taetrolens Y-
30 GS, which suggests that these organisms form an
enzyme distinct from the GS.
The cell growth and theanine-forming activity de-
creased with the addition of a high concentration of
yeast extract (media 7–10), whereas a high concentra-
tion of polypeptone increased cell growth while main-
taining a certain amount of theanine-forming activity
(media 11–14). The effects of yeast extract and that of
polypeptone were noticeable in the role of the enzyme
responsible for theanine formation in nitrogen metabo-
lism in M. mays No. 9. Detailed experiments on enzyme
formation under various nutritional conditions have not
been done yet.
According to the results of media 7–14, an experiment
was carried out again using a 2-l Sakaguchi flask and 1 l
of culture medium containing various concentrations of
polypepton with a fixed low level of yeast extract. The
results (data not shown) revealed that the addition of
0.5% polypepton with 0.03% yeast extract (medium B,
see legend to Fig. 1) was optimum in increasing cell
growth without a significant decrease in theanine-
forming activity in the cells.
All the selected organisms were rod-shaped and gram-
negative, and the sequences of their 16S ribosomal DNA
showed 98–100% similarity to those of the several
bacteria indicated in Table 1. These bacteria have been
reported to assimilate C1 compounds such as methanol
and/or methylamine.20,21) Methylovorus mays, which
had extremely high sequence similarity to isolate No. 9,
is an obligate methanol-assimilating organism.22) Strain
No. 9 also did not grow without methanol in the medium
(see Table 2), indicating that it was an obligate
methylotroph. The other organisms were facultative
methylotrophs that grew on glycerol or glucose as
carbon source (data not shown).
Strain No. 9 was chosen for subsequent experiments
on the basis of its high theanine-forming activity as well
as its unique assimilating ability as a carbon source. It is
referred to as Methylovorus mays No. 9 in this paper.
Figure 1 shows the growth of the organism on the
optimum medium, and changes in theanine-forming
activity of the cells during cultivation. The cells of 1 d of
cultivation gave the highest activity.
Effect of cultivation conditions on theanine-forming
activity in M. mays No. 9
Table 2 summarizes theanine-forming activity in the
cells of M. mays No. 9 grown under various nutritional
conditions. The organism did not grow without meth-
anol (media 3, 4, 5, and 6), which confirmed that
M. mays No. 9 was an obligate methylotroph. Theanine-
forming activity did not appear when methylamine was
removed from the medium (medium 2), suggesting that
the enzyme responsible for theanine formation was
induced by methylamine, and that it had certain role in
methylamine metabolism.
Purification of theanine-forming enzyme
Preliminary experiments (data not shown) indicated
that the thermal stability and pH stability of the
theanine-forming reaction in the cell-free extract of
M. mays No. 9 were similar to those of the GS reaction
in the extract. The behavior of both activities was almost
the same in the ammonium sulfate fractionation of the
cell-free extract (data not shown), and also on DEAE-
cellulofine column chromatography (Fig. 2A).
This suggests that one enzyme catalyzed both reac-

Characterization of Theanine-Forming Enzyme of M. mays No. 9
549
Table 3. Purification of Theanine-Forming Enzyme
2.0x103
1.5x103
1.0x103
2.0
1.5
1.0
0.5
0.0
0.8
0.6
0.4
0.2
0.0
Total protein Specific activity Total activity
(mg)
Purification step
(units/mg)
(units)
Cell-free extract
Ammonium sulfate
DEAE-cellulofine
Hydroxyapatite
913
143
17
0.15
0.79
4.67
3.72
136
113
79
9.5
35
The enzyme activity was determined by the ꢀ-glutamylhydroxamate-
forming reaction.
5.0x102
0.0
0
1
2
3
4
5
The apparent molecular mass of the enzyme was
estimated to be 410–470 kDa by gel filtration on a
Sepharose CL-6B column standardized with aldolase
(158 kDa), catalase (232 kDa), ferritin (440 kDa), and
thyrogloblin (669 kDa), and to be 51 kDa by analysis by
SDS–PAGE (Fig. 2B). The molecular weight of the
enzyme is different from that of GS of P. taetrolens Y-
30 and other gram-negative bacteria.23)
The N-terminal amino acid sequence of the enzyme
was MTSLEEAQKFLEDDHVVYVL, and this has
64.7% similarity to that of GS of Methylobacillus
flagellatus KT, which is a predicted enzyme by gene
analysis (see ‘‘Materials and Methods’’).
Cultivation (d)
Fig. 1. Variations in Theanine-Forming Activity during the Growth
of M. mays No. 9.
M. mays No. 9 was grown on an optimum medium B, which
.
consisted of 0.25% methylamine HCl, 0.25% methanol, 0.03%
yeast extract, 0.5% polypenton, 1.0% NaCl, 0.02% KCl, 0.03%
ꢁ7
.
MgSO4 7H2O, 0.005% KH2PO4, 0.005% K2HPO4, and 1 ꢀ 10
%
cyanocobalamine. Cultivation was carried out at 30 ꢂC with 1 l of
medium in a 2-l Sakaguchi flask on a reciprocal shaker (100 rpm).
The permeated cells were used to measure theanine-forming
activity. Specific and total activities were estimated as indicated in
Table 2. Symbols: , turbidity at 610 nm; , specific activity;
total activity.
,
Stability of theanine-forming enzyme
tions. GS activity was monitored for speedy purification
of the theanine-forming enzyme.
The isolated enzyme was stable between pH 5.5 and
pH 6.0 (data not shown). Activity was retained after
10 min of incubation at 35 ꢂC at pH 6.0, and fell to 60%
at 40 ꢂC (data not shown). Full activity was maintained
for at least 2 months at ꢁ20 ꢂC in a frozen state.
This indicates that the theanine-forming enzyme is
more stable than ꢀ-glutamylmethylamide synsthetase
(GMAS) of Methylophaga sp. AA-30.17)
As shown in Table 3, the enzyme was purified about
25-fold from the extract of M. mays No. 9 with an
overall yield of 26%. Analysis by sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS–PAGE)
indicated that the final preparation was homogeneous
(Fig. 2B).
1
2
A
B
0.4
0.3
0.2
0.1
0.0
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
7
6
5
4
3
2
1
0
0
10
20
30
40
Fraction No.
Fig. 2. Purification of Theanine-Forming Enzyme by DEAE-Cellulofine Column Chromatography (A) and SDS–PAGE Analysis (B) of the Isolated
Enzyme.
A, Ammonium sulfate fraction (0–33%) of the cell-free extract (143 mg protein) was chromatographed on a DEAE-cellulofine column
(2 ꢀ 10 cm), as described in ‘‘Materials and Methods.’’ Symbols: , theanine-forming activity (mM, concentration of theanine formed by 70 min
of incubation in 0.1 ml mixture containing 0.05 ml of the eluate); , GS activity (units/ml); broken line, protein concentration; solid line, NaCl
concentration in the buffer. B, Final preparation of theanine-forming enzyme was analyzed by SDS–PAGE. The gel was stained with coomassie
brilliant blue R-250. Lane 1, molecular weight marker: myosin (Mr = 200,000), ꢁ-galactosidase (121,000), bovine serum albumin (92,000),
glutamate dehydrogenase (62,000), obalvumin (47,500), and carbonic anhydrase (36,800). Lane 2, final enzyme preparation. The arrow indicates
purified enzyme.

550
S. YAMAMOTO et al.
Table 4. Reactivity of Theanine-Forming Enzyme to Various
Amines and Km Values for Substrates
A
0.08
0.06
0.04
0.02
0.00
Relative activity
(%)ꢃ1
Km
(mM)ꢃ2
Substrate
Ammonia
0.75
40
84
Hydroxylamine
Methylamine
Ethylamine
3.9
100
75
0.18
0.57
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
0
50
100
150
200
250
Glutamic acid
with methylamine
with ethylamine
1.4
1.3
Glutamic acid (mM)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
ATP
with methylamine
with ethylamine
1.3
0.63
0.08
0.06
0.04
0.02
0.00
B
C
The standard reaction condition, described in ‘‘Materials and Methods,’’ with
0.07 units/ml enzyme preparation, was incubated at 30 ꢂC for 10 min, and
the inorganic phosphate formed in the mixture was measured.
ꢃ1The concentration of methylamine in the mixture was lowered to 2 mM,
and methylamine was replaced with 2 mM substrate analog. The activity was
expressed relative to that in the reaction mixture with methylamine: 100% =
0.050 mM inorganic phosphate/min.
ꢃ2Methylamine in the standard reaction mixture was replaced with substrate
analog. Km values were obtained by varying the concentrations of amine or
ammonium chloride, glutamic acid, and ATP with fixed concentrations of
the other substrates. The values were calculated from double-reciprocal plots
of rate of inorganic phosphate formation versus substrate concentration.
0
5
10 15 20 25 30 35 40
ATP (mM)
100
80
60
40
20
0
Substrate specificity of theanine-forming enzyme
Table 4 compares reactivity and affinity to several
substrates. As it indicates, the enzyme was more highly
active to methylamine than to ammonia. This reactivity
was very similar to that of GMAS of Methylophaga sp.
AA-30.17) The isolated enzyme of M. mays No. 9 also
showed high reactivity to ethylamine, and the Km value
for ethylamine was about 1,000 times lower than that in
the case of P. taetrolens Y-30 GS,12) suggesting that the
enzyme is more effective for theanine production than
P. taetrolens Y-30 GS.
On the other hand, this experiment indicated that the
activity of the theanine-forming enzyme was repressed
by a high concentration of glutamic acid (Ki value,
160 mM with methylamine as a substrate, 110 mM with
ethylamine as a substrate) and ATP (Ki value, 31 mM
with methylamine as a substrate, 20 mM with ethyl-
amine) (Fig. 3A and B). In addition, a high concen-
tration of theanine in the mixture lowered the enzyme
activity (Fig. 3C; Ki value for theanine, 51 mM). Inhib-
ition by ATP might not occur in coupled fermentation
with energy transfer because ATP in low concentrations
is regenerated by sugar fermentation in the mixture.
However, the effects of glutamic acid and theanine
should be taken into consideration to achieve theanine
production at high concentrations, which requires high
concentrations of glutamic acid as a substrate.
0
50 100 150 200 250 300 350
Theanine (mM)
Fig. 3. Effect of Concentration of Glutamic Acid (A), ATP (B), and
Theanine (C) on ꢀ-Glutamylmethylamide- and Theanine-Forming
Activity.
A, B, Part of the experimental result to estimate the Km values for
glutamic acid and ATP respectively. Symbols: , with methylamine
as another substrate; , with ethylamine. C, Various concentrations
of theanine were added to the standard reaction mixture containing
ethylamine. Incubation was done with 0.075 units/ml theanin-
forming enzyme at 30 ꢂC for 10 min, and the inorganic phosphate
formed was measured. The activity was expressed relatively to
that in the reaction mixture without theanine: 100% = 0.058 mM
inorganic phosphate/min. The standard reaction mixture containing
ethylamine with 100 mM theanine was used for calculation of the Ki
value. The value was calculated from double-reciprocal plots of rate
of inorganic phosphate formation versus ethylamine concentration.
of theanine-forming enzyme of M. mays No. 9 was
different from those of the others: Mg2þ was effective
for M. mays No. 9 enzyme, whereas Mn2þ was effective
for the latter two enzymes.12,17)
Effect of pH on reactivity of theanine-forming enzyme
to ethylamine and theanine production by coupled
fermentation with energy transfer
Ore preceding papers12,13) reported that the theanine-
forming reaction in a neutral pH range is favorable for
theanine production by coupling with the sugar fermen-
tation system of baker’s yeast because the fermentation
proceeds optimally in a neutral pH range.24)
Divalent cation specificity of theanine-forming en-
zyme
The enzyme required divalent cation for its ꢀ-
glutamylmethylamide- and theanine-forming reactions,
as did GMAS of Methylophaga sp. AA-3017) and GS of
P. taetrolens Y-30.12) However, the cation dependence

Characterization of Theanine-Forming Enzyme of M. mays No. 9
551
A
B
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
25
20
15
10
5
100
80
60
40
20
0
200
150
100
50
0
0
0
5
10
15
20
25
4
5
6
7
8
9 10
Time (h)
pH
Fig. 4. pH-Profile of Theanine-Forming Reaction (A) and Theanine Production by Coupling the Reaction with Yeast Sugar Fermentation (B).
A, The standard reaction mixture containing ethylamine was incubated at 30 ꢂC for 10 min with 0.078 units/ml of theanine-forming enzyme.
The amount of inorganic phosphate formed was measured. Symbols: , acetate buffer; , imidazole buffer; , Tris–HCl buffer; , borate
buffer. B, The mixture and the conditions for the reaction are described in ‘‘Materials and Methods.’’ The reaction was done with 1.0 unit/ml
theanine-forming enzyme preparation. Symbols: , glucose; , FBP; , theanine.
Biochem., 59, 615–618 (1995).
As Fig. 4A indicates, the optimum pH for the
theanine-forming reaction of isolated enzyme from
M. mays No. 9 with 30 mM Mg2þ was 7.5–8.0, predict-
ing that the enzyme would form theanine in a reaction
mixture of the coupled fermentation with energy trans-
fer.
3) Kimura, R., and Murata, T., Influence of alkylamides of
glutamic acid and related compounds on the central
nervous system. I. Central depressant effect of theanine.
Chem. Pharm. Bull., 19, 1257–1261 (1971).
4) Kimura, R., and Murata, T., Influence of alkylamides of
glutamic acid and related compounds on the central
nervous system. II. Synthesis of amides of glutamic acid
and related compounds, and their effects on the central
nervous system. Chem. Pharm. Bull., 19, 1301–1307
(1971).
5) Kakuda, T., Nozawa, A., Unno, T., Okamura, N., and
Okai, O., Inhibiting effects of theanine on caffeine
stimulation evaluated by EEG in rat. Biosci. Biotechnol.
Biochem., 64, 287–293 (2000).
6) Kobayashi, K., Nagato, Y., Aoi, N., Juneja, L. R., Kim,
M., Yamamoto, T., and Sugimoto, S., Effect of L-
theanine on release of ꢂ-brain waves in human volun-
teers. Nippon No¯geikagaku Kaishi (in Japanese), 72,
153–157 (1998).
7) Matsuura, T., and Kakuda, T., Effects of precursor,
temperature, and illumination of theanine accumulation
in tea callus. Agric. Biol. Chem., 54, 2283–2286 (1990).
8) Lichtenstein, N., Preparation of ꢀ-alkylamides of gluta-
mic acid. J. Am. Chem. Soc., 64, 1021–1022 (1942).
9) Sasaoka, K., Kito, M., and Onishi, Y., Synthesis of
theanine by pea seed acetone powder extract. Agric.
Biol. Chem., 28, 318–324 (1964).
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).
As Fig. 4B indicates, the theanine-forming enzyme of
M. mays No. 9 formed 60 mM of theanine in 24 h by
coupled fermentation with energy transfer. Theanine
formation was achieved with a small amount of purified
enzyme (1 unit/ml), and it was about 20 times larger
than that by the same amount of P. taetrolens Y30
GS.12) This result indicates that theanine production in
high concentrations might be possible by increasing the
amount of the enzyme. A preliminary experiment on
coupled fermentation using various concentrations of
glutamic acid (data not shown) suggested that the rate of
theanine formation was not lowered by increasing the
glutamic acid concentration. This result perhaps implies
that glutamic acid in high concentrations is not so
inhibitory as it was the standard reaction condition for
isolated theanine-forming enzyme.
The present paper indicates that the reactivity and the
optimum pH of the theanine-forming enzyme of
M. mays No. 9 are favorable for theanine production
by coupled fermentation with energy transfer. Details of
theanine production in high concentrations using
M. mays No. 9 enzyme will be reported soon.
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