Purification and characterization of glutamine synthetase of Pseudomonas taetrolens Y-30: An enzyme usable for production of theanine by coupling with the alcoholic fermentation system of baker's yeast

Article abstract of DOI:10.1271/bbb.68.1888

Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed γ-glutamylethylamide (theanine) from glutamic acid and ethylamine in a mixture containing the alcoholic fermentation system of baker's yeast for ATP-regeneration. Glutamine synthetase (GS), probably responsible for theanine formation, was isolated from the extract of the organism grown on a medium containing 1% methylamine, 1% glycerol, 0.5% yeast extract, and 0.2% polypepton as carbon and nitrogen sources. The molecular mass was estimated to be 660 kDa by gel filtration and 55 kDa by SDS-polyacrylamide gel electrophoresis, suggesting that Ps. taetrolens Y-30 GS consists of 12 identical subunits. The enzyme required Mg²⁺ or Mn²⁺ for its activity. Under the standard reaction condition for glutamine formation (pH 8.0 with 3o mM Mg²⁺), GS showed 7% and 1% reactivity toward methylamine and ethylamine respectively of that to ammonia. Reactivity to the alkylamines varied with optimum pH of the reaction in response to divalent cation in the mixture: pH 11.0 was the optimum for the Mg ²⁺-dependent reaction with ethylamine, and pH 8.5 was the optimum for the Mn²⁺-dependent reaction. In a mixture of an optimum reaction condition with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn²⁺), reactivity increased up to 7% of the reactivity to ammonia in the standard reaction condition. The isolated GS formed theanine in the mixture with the yeast fermentation system.

Full text of DOI:10.1271/bbb.68.1888

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Purification and Characterization of Glutamine
Synthetase of Pseudomonas taetrolens Y-30: An
Enzyme Usable for Production of Theanine by
Coupling with the Alcoholic Fermentation System of
Baker’s Yeast
Sachiko YAMAMOTOa, Kousuke UCHIMURAa, Mamoru WAKAYAMAa & Takashi TACHIKIa
a Department of Bioscience and Biotechnology, Faculty of Science and Technology,
Ritsumeikan University
Published online: 22 May 2014.
To cite this article: Sachiko YAMAMOTO, Kousuke UCHIMURA, Mamoru WAKAYAMA & Takashi TACHIKI (2004) Purification
and Characterization of Glutamine Synthetase of Pseudomonas taetrolens Y-30: An Enzyme Usable for Production
of Theanine by Coupling with the Alcoholic Fermentation System of Baker’s Yeast, Bioscience, Biotechnology, and
Biochemistry, 68:9, 1888-1897, DOI: 10.1271/bbb.68.1888
To link to this article: http://dx.doi.org/10.1271/bbb.68.1888
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Biosci. Biotechnol. Biochem., 68 (9), 1888–1897, 2004
Purification and Characterization of Glutamine Synthetase of Pseudomonas
taetrolens Y-30: An Enzyme Usable for Production of Theanine by Coupling
with the Alcoholic Fermentation System of Baker’s Yeast
y
Sachiko YAMAMOTO, Kousuke UCHIMURA, Mamoru WAKAYAMA, and Takashi TACHIKI
Department of Bioscience and Biotechnology, Faculty of Science and Technology,
Ritsumeikan University, Shiga 525-8577, Japan
Received March 25, 2004; Accepted June 28, 2004
Concentrated cell-extract of Pseudomonas taetrolens
Y-30, isolated as a methylamine-assimilating organism,
formed ꢀ-glutamylethylamide (theanine) from glutamic
acid and ethylamine in a mixture containing the
alcoholic fermentation system of baker’s yeast for
ATP-regeneration. Glutamine synthetase (GS), proba-
bly responsible for theanine formation, was isolated
from the extract of the organism grown on a medium
containing 1% methylamine, 1% glycerol, 0.5% yeast
extract, and 0.2% polypepton as carbon and nitrogen
sources. The molecular mass was estimated to be
660 kDa by gel filtration and 55 kDa by SDS-polyacryl-
amide gel electrophoresis, suggesting that Ps. taetrolens
Y-30 GS consists of 12 identical subunits. The enzyme
required Mg2þ or Mn2þ for its activity. Under the
standard reaction condition for glutamine formation
(pH 8.0 with 30 mM Mg2þ), GS showed 7% and 1%
reactivity toward methylamine and ethylamine respec-
tively of that to ammonia. Reactivity to the alkylamines
varied with optimum pH of the reaction in response to
divalent cation in the mixture: pH 11.0 was the optimum
for the Mg2þ-dependent reaction with ethylamine, and
pH 8.5 was the optimum for the Mn2þ-dependent
reaction. In a mixture of an optimum reaction condition
with 1000 mM ethylamine (at pH 8.5 with 3 mM Mn2þ),
reactivity increased up to 7% of the reactivity to
ammonia in the standard reaction condition. The
isolated GS formed theanine in the mixture with the
yeast fermentation system.
Recently, various favorable physiological functions of
theanine have been reported,5–10) suggesting that de-
mand for theanine might become great not only as a
taste-enhancing additive but also as a supplement to
improve and/or maintain human health.
Several methods have been reported for producing
theanine including extraction of tea leaves, use of tea
callus,11–13) chemical synthesis,14,15) etc., but these
methods might be impractical due to complexity with
low yield and/or low purity.
Biochemical synthesis has also been investigated
using crude enzyme,16,17) which catalyzes a similar
reaction to that of theanine synthetase, but this was on an
experimental scale leaving out of consideration supply
of the enzyme and ATP. Recently, enzymatic production
was accomplished by ꢀ-glutamyl transfer reaction of
bacterial glutaminase18) or ꢀ-glutamyl transpeptidase19)
with glutamine and ethylamine as the substrates. The
method might be satisfactory to some extent, but the
process has certain shortcomings with respect to supply
of glutamine as well as side reactions lowering theanine
yield on glutamine: hydrolysis of glutamine and for-
mation of other ꢀ-glutamyl derivatives. These might be
resolved by the use of an enzyme similar to theanine
synthetase if ATP can be regenerated efficiently,
because glutamic acid is supplied more easily than
glutamine and the reaction is endergonic.
We have reported glutamine production using the
reaction of glutamine synthetase (GS; L-glutamate:am-
monia ligase, ADP-forming, E.C.6.3.1.2) of Corynebac-
terium glutamicum (Micrococcus glutamicus) or Brevi-
bacterium flavum,20,21) which was coupled with the sugar
fermentation reaction of dried baker’s yeast cells. This
process was an application of ‘‘coupled fermentation
with energy transfer,’’ in which the yeast fermentation of
glucose functioned as an ATP-regenerating system.22–25)
During the course of the investigation, we found that a
small amount of theanine was formed when ammonia,
one of the substrates of GS, was replaced by ethyl-
amine.26) But production of theanine in high concen-
Key words: theanine; glutamine synthetase; Pseudomo-
nas taetrolens Y-30; coupled fermentation
with energy transfer; baker’s yeast
Theanine, ꢀ-glutamylethylamide, is an amino acid
found by Sakato in Japanese green tea leaves (Camellia
sinensis).1) It is synthesized by theanine synthetase with
glutamic acid, ethylamine, and ATP as the substrates.2,3)
The content of theanine is 1.5–3% of the dry weight of
the tea leaves, and it renders the gracefully sweet taste.4)
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
Amino acid is L-isomer unless otherwise stated.

Ps. taetrolens Y-30 Glutamine Synthetase for Theanine Production
Table 1. Effect of Growth Conditions on GS Activity in Cells
1889
Exp. A
Medium
GS activity
SAꢃ3 TAꢃ4
Addition (%)
Growthꢃ2
1
2
1% methylamine
1% glucose
1% glycerol
0.3% YEꢃ1
0.3% YE
0.3% YE
0.8
1.0
0.8
2.7
2.9
2.0
2.2
0.7
4.7
5.5
5.9
7.0
0.015
0
1.2
0
3
0
0
4
1% methylamine
1% methylamine
2% methylamine
2% methylamine
1% methylamine
1% methylamine
1% methylamine
1% methylamine
1% methylamine
1% glycerol
2% glycerol
1% glycerol
2% glycerol
1% glycerol
1% glycerol
1% glycerol
1% glycerol
1% glycerol
0.3% YE
0.3% YE
0.3% YE
0.3% YE
0.1% YE
0.5% YE
1.0% YE
0.5% YE
0.5% YE
0.011
0.009
0.005
0.004
0.017
0.009
0.000
0.009
0.003
6.7
5.6
2.7
2.2
1.0
9.9
0.2
5
6
7
8
9
10
11
12
0.2% polypepton
0.4% polypepton
11
3.4
Exp. B
usable for theanine production by coupled fermentation
with energy transfer if it is stable. But the enzyme was
reported to be unstable.30)
GS activity
Cultivation
(d)
Growthꢃ2
SAꢃ3
TAꢃ4
1
2
3
4
5
6
3.5
5.5
5.7
5.5
5.1
3.6
0.000
0.003
0.008
0.009
0.015
0.010
0.4
5.0
7.1
8.7
15
We found that concentrated cell-free extract of a
bacterium, which was isolated as a methylamine-
assimilating organism, formed theanine from glutamic
acid and ethylamine in a reaction mixture of coupled
fermentation with energy transfer. An enzyme respon-
sible for theanine formation, tentatively indicated as GS,
was isolated from the cell-free extract and characterized
mainly in respect to reactivity for theanine synthesis.
10
Exp. A: Carbon and nitrogen sources in Medium B were changed as
indicated in the Table, and Ps. taetrolens Y-30 was grown for 3 d.
Exp. B: The organism was grown on an optimum medium for GS
.
production, which consisted of 1.0% methylamine HCl, 1.0% glycerol,
0.5% yeast extract, 0.2% polypepton, 1.0% NaCl, 0.02% KCl, 0.03%
MgSO4 7H2O, 0.005% KH2PO4, 0.005% K2HPO4, and 1 ꢀ 10ꢁ7% cyano-
.
Materials and Methods
cobaramine.
Cultivation was carried out at 30 ꢂC with 1 liter medium in a 2-liter
Sakaguchi flask. Cell-free extract was used to measure GS activity (ꢀ-
glutamylhydroxamate-forming reaction).
ꢃ1: yeast extract ꢃ2: turbidity at 610 nm ꢃ3: specific activity (unit/mg
protein) ꢃ4: total activity (units)
Isolation of methylamine-assimilating bacteria. Soil
samples were suspended in sterilized water and 0.1 ml of
the upper phase was inoculated into 5 ml of Medium A-1
or Medium A-2 in a test tube, which contained methyl-
amine as a carbon and a nitrogen source. Medium A-1
was designed according to the medium for a marine
methylotroph,29) with a small modification, and con-
trations was not successful due to the low reactivity of
the GS to ethylamine and to difficulty in controlling the
pH of the reaction mixture. The optimum pH for
theanine synthesis and the yeast fermentation was 10
and 7 respectively.27) Precise pH control of the mixture
was impossible due to large amounts of yeast cells in the
mixture.26) These findings suggested the need to search
for a new GS with high reactivity to ethylamine at
neutral pH range.
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 metabolism of methylamine.28,29) Syn-
thesis of GMA in the organisms is performed with
glutamic acid, methylamine, and ATP as the substrates
by GMA synthetase (GMAS; L-glutamate: methylamine
ligase, ADP-forming, EC 6.3.4.12). Differentiation of
GMAS from GS might be difficult because the reactions
catalyzed by the enzymes are very similar. But GMAS
has been reported to be active to methylamine rather
than to ammonia, and also to show high reactivity to
ethylamine,30) suggesting that the enzyme might be
.
tained 0.15% methylamine HCl, 2.5% NaCl, 0.02%
KCl, 0.03% MgSO4 7H2O, 0.001% CaCl2 2H2O,
.
.
.
0.001% FeSO4 7H2O, 0.005% KH2PO4, 0.005%
K2HPO4, 4 ꢀ 10ꢁ6% each of CuSO4 5H2O, ZnSO4
.
.
5H2O, NiSO4, Na2MoO4 2H2O, MnCl2 4H2O, and
.
.
CoSO4 7H2O, and 1 ꢀ 10ꢁ7% cyanocobalamine (pH
.
7). Medium A-2 was a simplified Medium A-1 with a
higher concentration of methylamine and a lower
.
concentration of NaCl: 1.0% methylamine HCl, 0.2%
NaCl, 0.02% KCl, 0.03% MgSO4 7H2O, 0.005%
.
KH2PO4, 0.005% K2HPO4, 0.03% yeast extracts, and
1 ꢀ 10ꢁ7% cyanocobalamine (pH 7). After 10–14 d of
cultivation at 30 ꢂC on a shaker (200 rpm), part of the
turbid culture was transferred into fresh Medium A-1 or
A-2 and cultivated further for 10–14 d. This procedure
was repeated 3–5 times, then the culture was streaked on
an agar plate containing 0.5% glucose, 1.0% polypepton,
0.5% yeast extract, 0.5% NaCl, and 2.0% agar. Bacterial
colonies that appeared after incubation at 30 ꢂC for 1–2 d
were purified and then isolated. The isolates were

1890
S. YAMAMOTO et al.
cultivated for 8 d in Medium A-1 and/or A-2, and the
methylamine-assimilating microorganisms, which gave
turbidity at 610 nm of more than 0.25, were submitted
for screening experiments.
The methylamine-assimilating organisms usually
started to grow 3–5 d after inoculation. Growth was
repressed (slightly or remarkably) by increasing the
concentration of methylamine to 1%, and the repressed
growth was recovered and stimulated by the addition of
0.05–0.1% yeast extract: early start as well as increase in
the growth. The addition of 1% glycerol further increas-
ed the growth of many organisms.
amine. The cells were collected by centrifugation
(12;000 ꢀ g, 4 ꢂC, 20 min), and washed twice with
10 mM potassium phosphate buffer (pH 6.0). The washed
cells were suspended in the same buffer and disrupted
with a sonicator (Nihonseiki, 20 khz for 20 min at 0–
15 ꢂC). The centrifugal supernatant was dialyzed against
10 mM potassium phosphate buffer (pH 6.0) and used to
measure GS activity.
In the second screening, the cell-free extract of the
organisms selected in the first screening was concen-
trated with ammonium sulfate (30–70% saturation; see
legend to Fig. 1), and examined in respect to theanine-
forming activity in a reaction mixture of the coupled
fermentation with energy transfer.
Selection of microorganism to form an enzyme
suitable for theanine production. The selection was
made with two screening experiments. The first was
dependent on the GS activity of the cell-free extract. The
isolated organism was cultivated for 3 d on 1 liter of
Medium B in a Sakaguchi flask. Medium B was defined
by several preliminary experiments to increase micro-
bial growth without remarkable decrease of GS activity
(see Table 1). The medium contained 1.0% methyl-
Assay of GS activity. The activities of two reactions
were measured.
Glutamine-forming reaction: Activity was determined
according to the previous method,31) with a small
modification: a reaction mixture consisting of 50 mM
sodium glutamate, 25 mM ammonium chloride, 7.5 mM
ATP, 30 mM MgCl2, 100 mM imidazole buffer (pH 8.0),
and a suitable amount of the final GS preparation (see
Table 2) was incubated at 30 ꢂC, and the amount of
inorganic phosphate formed was determined.
.
amine HCl, 1.0% glycerol, 0.3% yeast extract, 1.0%
NaCl, 0.02% KCl, 0.03% MgSO4 7H2O, 0.005%
.
KH2PO4, 0.005% K2HPO4, and 1 ꢀ 10ꢁ7% cyanocobal-
Fig. 1. Formation of Theanine by Coupling of Concentrated Cell-free Extract of Methylamine-assimilating Bacteria with Dried Yeast Cells.
Ammonium sulfate was added to the cell-free extract to 30% saturation with adjusting pH at 7, and then ammonium sulfate was added to the
centrifugal supernatant to 70% saturation. The precipitate was collected by centrifugation, dissolved in a small volume of 10 mM potassium
phosphate buffer (pH 6.0) and dialyzed against the same buffer. The concentrated extract, corresponding to 1 unit/ml of GS, was added to a
temporary reaction mixture for coupled fermentation with energy transfer (‘‘Materials and Methods’’). The reaction was carried out at 30 ꢂC for
24 h with shaking. Theanine was not formed in control mixtures (without yeast cells, data not shown; without GS preparation, see Fig. 4(A)).
(A), strain Y-30; (B), strain Z-10; (C), strain Z-76; (D), B. flavum. Symbols: , glucose; , theanine.

Ps. taetrolens Y-30 Glutamine Synthetase for Theanine Production
1891
Table 2. Purification of Ps. taetrolens Y-30 GS
The frozen cells were thawed and disrupted with the
sonicator (20 khz for 20 min at 0–15 ꢂC). The cell debris
was centrifuged off, and the supernatant was used as the
starting material.
Ammonium sulfate fractionation: To the cell-free
extract, ammonium sulfate was added to achieve 30%
saturation, with adjusting pH at 7 with 12% ammonium
hydroxide. The mixture was stirred for 30 min at 4 ꢂC
and the precipitate was centrifuged off. Ammonium
sulfate was added to the supernatant to 60% saturation.
After stirring for 30 min at 4 ꢂC, the precipitate was
collected by centrifugation and dissolved in the buffer.
The solution was dialyzed against the buffer.
DEAE-cellulofine column chromatography: The di-
alyzed solution was put on a DEAE-cellulofine column
(11:8 ꢀ 22 cm) equilibrated with the buffer. After
washing with the buffer, the column was developed
with the buffer containing 0.25 M, 0.45 M, and 1.0 M
NaCl. The enzyme was eluted with the buffer containing
0.45 M NaCl. The active fractions were combined, and
then concentrated with ammonium sulfate (70% satu-
ration). The precipitate formed was collected by cen-
trifugation and dissolved in a small volume of the buffer.
The solution was dialyzed against the buffer.
Butyl-Toyopearl 650M column chromatography: To
the dialyzed solution, ammonium sulfate was added to
achieve 30% saturation. The solution was put on a
Butyl-Toyopearl 650M column equilibrated with the
buffer containing 30% ammonium sulfate. After wash-
ing with the same buffer, the column was eluted by
decreasing the concentration of ammonium sulfate in the
buffer linearly. The active fractions, eluted at 20–10%
ammonium sulfate, were combined and concentrated
with ammonium sulfate (70% saturation). The precip-
itate formed was collected by centrifugation and
dissolved in a small volume of the buffer.
In this chromatography, GS was detected with a ꢀ-
glutamyl-transferring reaction because the ꢀ-glutamyl-
hydroxamate-forming reaction is interfered with ammo-
nium sulfate in the eluate. Ammonia is a substrate for
the glutamine-forming reaction. The mixture for the
transferring reaction contained 35 mM glutamine, 15 mM
hydroxylamine, 0.4 mM ADP, 1 mM MnCl2, 10 mM
arsenate, 100 mM imidazole–HCl buffer (pH 7.5), and
the enzyme solution. The reaction was carried out at
30 ꢂC and stopped, as was the ꢀ-glutamylhydroxamate-
forming reaction, followed by determination of ꢀ-
glutamylhydroxamate.
Sepharose CL-4B column chromatography: The con-
centrated enzyme solution was filtered through a column
of Sepharose CL-4B (2:8 ꢀ 130 cm) equilibrated with
the buffer. The active fractions were combined and used
as a final preparation.
Total protein Specific activity Total activity
(mg)
Purification step
(units/mg)
(units)
Cell-free extract
Ammonium sulfate
DEAE-cellulofine
Butyl-Toyopearl
Sepharose CL-4B
160,000
37,600
1,850
850
0.015
0.052
0.68
2,400
1,955
1,258
816
0.96
1.17
450
526
The mixture was the optimum for Ps. taetrolens GS
and it was used for general characterization of the
isolated enzyme. The condition is sometimes indicated
as ‘‘the standard reaction condition’’ in the text.
ꢀ-Glutamylhydroxamate-forming reaction: 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 GS preparation was
incubated at 30 ꢂC. After incubation, 2 volumes of a
.
mixture of 0.2 M FeCl3 6H2O, 0.12 M trichloroacetic
acid, 0.25 N HCl, and water (8:2:1:13) was added to stop
the reaction. The developed color of ꢀ-glutamylhydrox-
amate was determined spectrophotometrically at
540 nm. One unit of GS was defined as the amount of
enzyme forming 1 ꢁmol of ꢀ-glutamylhydroxamate per
min.
This method was used in the experiments for selection
of the organism, for estimation of the cultivation
conditions, and for purification of the enzyme.
Dried yeast cells and the mixture for coupled
fermentation with energy transfer. Baker’s yeast was
supplied by Oriental Yeast (Tokyo, Japan). It was air-
dried according to the previous method.24) A reaction
mixture for theanine production was defined temporarily
according to the mixture for production of glutamine21)
and GMA.26) It contained 200 mM glucose, 50 mM
potassium phosphate buffer (pH 7.0), 200 mM sodium
glutamate, 300 mM ethylamine, 25 mM MgCl2, 5 mM
MnCl2, 5 mM AMP, 40 mg/ml dried yeast cells, and
GS preparation. The mixture (1 ml in a test tube) was
incubated at 30 ꢂC with shaking (200 rpm). The reaction
was terminated by immersing the reaction tube in
boiling water for 3 min. The centrifugal supernatant of
the mixture (1000 rpm, 10 min) was submitted to the
assay.
Purification of GS. All operations were carried out at
0–10 ꢂC and 10 mM potassium phosphate buffer (pH
6.0), containing 10% glycerol, 1 mM MgCl2, and 1 mM
2-mercaptoethanol, was used unless otherwise specified.
Cell-free extract: The selected strain Y-30, tentatively
named Pseudomonas taetrolens Y-30, was grown at
30 ꢂC for 5 d on an optimum medium (1 liter in 2-liter
Sakaguchi flask), as described in ‘‘Results and Discus-
sion’’ (see Table 1).
N-terminal amino acid sequence analysis. The GS
protein on a gel of sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) was blotted onto
a poly-vinylidene difluoride (PVDF) membrane with a
The washed cells from 170-liter medium were
suspended in the buffer and stored at ꢁ20 ꢂC until use.

1892
S. YAMAMOTO et al.
semidry-blotting apparatus (AE-6670, Atto, Tokyo,
Japan). The protein on the PVDF membrane was then
stained with ponsew. The stained protein on the
membrane was cut off to analyze the N-terminal amino
acid sequence with a peptide sequencer (PPSQ-10S,
Shimadzu), according to the method of Edman.32) The
sequence was compared with the BLAST2 protein
database.
was formed inductively by methylamine. The addition
of 1% glycerol (medium 4) to the medium containing
1% methylamine and 0.3% yeast extract (medium 1)
was effective in increasing cell growth without signifi-
cant decrease of GS activity, bringing about a consid-
erable increase in total activity, but the addition of it at
higher concentrations was not as effective (medium 5).
The addition of 1% glucose repressed microbial growth
considerably (data not shown).
Assay. Protein was determined by Lowry’s method33)
with egg albumin as a standard. In column chromatog-
raphy, it was monitored with absorbance at 280 nm.
Glucose was assayed according to the method of
Somogyi,34) or enzymatically with glucose oxidase.
Inorganic phosphate was determined by the method of
Fiske and Subbarow.35) Theanine formed in the reaction
mixture was assayed by paper chromatography and
ninhydrin colorimetry according to the method describ-
ed previously.27)
Yeast extract significantly affected cell growth and
GS level in the cells (media 4, 8, 9, and 10). It stimulated
cell growth whereas it decreased the GS level, especially
at higher concentrations, and a concentration of 0.5%
was the most favorable for enzyme production. The
addition of 0.2% polypepton (media 9, 11, and 12)
increased cell-growth significantly with a certain in-
crease in enzyme production.
On the bases of the above results, an optimum
medium for GS production was defined. It is indicated in
the legend to Exp. B in Table 1. The cells under 5 d of
cultivation gave the highest GS activity.
Reagents. Glucose oxidase (glucose C II-test wako)
was supplied by Wako Pure Chemical. The theanine was
from Tokyo Kasei. The other reagents were highest-
grade commercial products.
Purification of GS
As summarized in Table 2, GS of Ps. taetrolens Y-30
was purified from the extract of cells grown on the
optimum culture medium (about 80-fold purification
with an overall yield of 22%). SDS-PAGE indicated that
the final preparation was homogeneous (Fig. 2).
The apparent molecular mass of the enzyme was
estimated to be 660 kDa by gel filtration with a Sepharose
CL-4B column standardized with catalase (232 kDa),
ferritin (440 kDa), and thyrogloblin (669 kDa), and to be
55 kDa by SDS-PAGE (Fig. 2). These results indicate
that the enzyme might consist of 12 identical subunits
(MW: 55 kDa), like the enzymes of several gram-
negative bacteria including Escherichia coli.36)
Results and Discussion
Selection of methylamine-assimilating bacteria
Among the methylamine-assimilating isolates that
grew on Medium A-1 or A-2 containing methylamine as
a carbon and nitrogen source, five strains were selected
in the first screening, depending on GS activity in the
cell-free extract (data not shown).
In the second screening, the theanine-forming activity
of the concentrated extract was examined in the mixture
of coupled fermentation with energy transfer. Figure 1
shows some of the results. These indicate that extracts of
strains Y-30, Z-10, and Z-76 formed theanine with a
decrease in glucose, an energy source for ATP-regen-
eration. The amounts were larger, especially that with
strain Y-30 extract, than the amount in the mixture with
the extract of B. flavum, which has been used for
glutamine production.21)
Strain Y-30 was chosen for further experiments. The
organism is a rod-shaped gram-negative bacterium, and
the other physiological properties suggested that it
belongs to Pseudomonas. The sequence of 16S riboso-
mal DNA showed 98% similarity to that of Pseudomo-
nas taetrolens. The organism is tentatively designated
Pseudomonas taetrolens Y-30 in this paper.
The N-terminal amino acid sequence was
SKSVQLIKDXDVKWI (X, not determined). This has
93% similarity to that of Pseudomonas syringae GS.
Stability
The GS of Ps. taetrolens Y-30 was stable at pH 6.0.
Activity was retained after 10 min of incubation at 55 ꢂC
and pH 6.0, and lowered to 60% at 65 ꢂC.
About 95% of the initial activity was maintained for
30 d at 5 ꢂC in a state of ammonium sulfate precipitate
(70% saturation), and full activity continued for 2
months at ꢁ20 ꢂC in a frozen state.
Substrate and divalent cation specificity
Table 3 shows the reactivity of Ps. taetrolens Y-30
GS to various substrates and that with several divalent
cations in the standard reaction condition.
Effect of cultivation conditions on GS level in
Ps. taetrolens Y-30
Experiment A of Table 1 summarizes the activities of
GS in cells grown under various nutritional conditions.
GS activity was barely detected when Ps. taetrolens Y-
30 was grown in the medium containing glucose or
glycerol (media 1, 2, and 3), indicating that the enzyme
The enzyme specifically required L-glutamic acid, and
utilized hydroxylamine, methylamine, and ethylamine
with 32, 7, and 1% reactivity of that to ammonia. GTP
and ITP were substituted for ATP with 26 and 18%
reactivity. The substrate specificity was not specific as

Ps. taetrolens Y-30 Glutamine Synthetase for Theanine Production
1893
ing to the increase in Mg2þ concentration with an
optimum of 15 mM, and did not vary at excess concen-
trations (data not shown). In the case of Mn2þ
,
maximum GS activity was obtained in the mixture with
3–4 mM, and decreased in the mixture with higher
concentrations of Mn2þ (data not shown). The optimum
concentration of divalent cation varied responding to the
concentration of ATP in the mixture (optimum ratios:
Mg2þ:ATP = 2:1; Mn2þ:ATP = 1:2). These are typical
properties observed with GS from various sour-
ces.31,37,38)
The low reactivity of the enzyme to methylamine and
ethylamine (Table 3) suggested that the Ps. taetrolens
Y-30 enzyme was GS but not GMAS, and indicated that
the enzyme is unusable for theanine production. But it
should be noted that the standard reaction condition was
the optimum for the glutamine-forming reaction with
30 mM Mg2þ. The reactivity of GS toward substrates,
especially ammonia analogs, frequently varied, respond-
ing to the divalent cation used and to the reaction
pH.27,36)
Effect of pH on the reactivity of GS to ammonia,
methylamine and ethylamine with Mg2þ or Mn2þ as a
divalent cation
Figure 3 compares the pH profiles of the GS reaction
with ammonia (A, B), methylamine (C, D), and ethyl-
amine (E, F) as a substrate in a mixture containing
30 mM Mg2þ (A, C, E) or 3 mM Mn2þ (B, D, F). The
concentration of Mg2þ and Mn2þ was optimum for the
glutamine-forming reaction under the standard reaction
condition.
Fig. 2. SDS-PAGE of Ps. taetrolens GS.
The gel was stained with Coomassie Brilliant Blue R-250. Lane 1,
molecular weight marker: myosin (Mr ¼ 200;000), ꢂ-galactosidase
(116,250), albumine (66,200), ovalbumine (45,000). Lane 2, GS.
With ammonia as a substrate, the optimum pH was
8.0 in the mixture containing 30 mM Mg2þ (A), and was
7.5 with 3 mM Mn2þ (B). Reactivity with Mg2þ was
about 10-fold higher than with Mn2þ, similarly to
findings with enzymes from other bacteria,36) but differ-
ently from findings with the C. glutamicum27) or
B. flavum enzyme.39)
The optimum pH for the reaction with methylamine
was 8.5 in the mixture with 30 mM Mg2þ (C), and 8.0
with 3 mM Mn2þ (D). The pH profiles were similar to
those for GS of C. glutamicum27) and GMAS of a
methylotroph, Methylophaga sp. AA-3030) (pH 8.5 and
pH 7.5–8.0 respectively).
In the mixture with ethylamine, the Mg2þ-dependent
reaction proceeded optimally at pH 11.0 (E) and the
Mn2þ-dependent reaction at pH 8.5 (F). The optimum
pH of 8.5 for the Mn2þ-dependent reaction was notice-
able for theanine production by coupling with sugar
fermentation of the yeast. The optimum pH of the
reaction by C. glutamicum GS was 10.0,27) and the
reaction could not be coupled with yeast fermentation.26)
Table 4 summarizes the Km values for the substrates
in the reactions with ammonia, methylamine, and
ethylamine under optimum pH conditions with 30 mM
Mg2þ or 3 mM Mn2þ, and indicates that the Km for the
substrates were variable except for ATP.
Table 3. Substrate Specificity of Ps. taetrolens GS
Relative
activity (%)
Divalent
cation (mM)
Relative
activity (%)
Substrate
L-glutamic acid
D-glutamic acid
L-aspartic acid
D-aspartic acid
Ammonia
Hydroxylamine
Methylamine
Ethylamine
ATP
100
0
Mg2þ (30)
Mg2þ (3)
Mn2þ (30)
Mn2þ (3)
Fe2þ (30)
Co2þ (30)
None
100
5
0
0
0
11
0
100
32
7
0
0
1
100
26
18
0
GTP
ITP
CTP
The reaction mixture described in ‘‘Materials and Methods’’ (the standard
reaction condition) was used with a 0.034 unit/ml enzyme preparation. One
of the substrates was replaced with a substrate analog, or 30 mM MgCl2 was
replaced with another divalent cation. The reaction was carried out at 30 ꢂC
for 10 min, and the amount of inorganic phosphate formed was determined.
The activity was expressed relatively to that in the standard reaction mixture:
100% ¼ 0:76 mM inorganic phosphate/10 min.
compared with those of GS from many sources.
As for the divalent cation, Mg2þ and Mn2þ were
effective for GS activity whereas the others were not
under the present condition. Activity increased respond-

1894
S. YAMAMOTO et al.
Fig. 3. Effects of pH and Divalent Cation on the GS Reaction with Ammonia, Methylamine or Ethylamine.
(A, B) The reaction mixture contained 50 mM sodium glutamate, 25 mM ammonium chloride, 7.5 mM ATP, 30 mM MgCl2 (A) or 3 mM MnCl2
(B), 100 mM buffer, and 0.034 units/ml GS. The mixture with 30 mM MgCl2 and imidazole buffer (pH 8.0) was the standard reaction condition.
(C, D) The reaction mixture contained 50 mM sodium glutamate, 500 mM methylamine, 7.5 mM ATP, 30 mM MgCl2 (C) or 3 mM MnCl2 (D),
100 mM buffer, and 0.034 units/ml GS. The concentration of 500 mM for methylamine was defined by a preliminary experiment on the effect of
its concentration to the activity. (E, F) The reaction mixture contained 50 mM sodium glutamate, 1000 mM ethylamine, 7.5 mM ATP, 30 mM
MgCl2 (E) or 3 mM MnCl2 (F), 100 mM buffer, and 0.034 units/ml GS. The concentration of 1000 mM for ethylamine was defined by a
preliminary experiment. The reaction was carried out at 30 ꢂC for 10 min and the amount of inorganic phosphate formed was determined.
Symbols: , acetate buffer; , imidazole–HCl buffer; , tris–HCl buffer; , borate–NaOH buffer.
The change in reaction velocity in response to the
concentration of glutamic acid was biphasic in all the
reactions. There was higher affinity for glutamic acid at
its lower concentrations. Similar observation was made
for the Mn2þ-dependent reactions of E. coli GS40) and
B. flavum GS.41) The value also varied in response to
divalent cation in the mixture.
The Km values for ammonia, methylamine, and
ethylamine varied in different ways responding to the
divalent cation in the mixture. The Km for ammonia was
not influenced, and those for methylamine and ethyl-
amine varied significantly with variations in optimum
pH. It was notable that the affinity of the enzyme toward
methylamine or ethylamine with 3 mM Mn2þ was higher
than that with 30 mM Mg2þ
.
Table 4 also compares the reactivity of the enzyme
under the conditions defined to be optimum on the bases
of the kinetic parameters. The reactivity to ethylamine
in the mixture containing 3 mM Mn2þ (at pH 8.5) was
7% of that to ammonia with 30 mM Mg2þ (at pH 8.0),
suggesting its participation in theanine synthesis in the
mixture of coupled fermentation with energy transfer.
Certain divalent cations have been reported to induce
unique structural changes of GS protein, both of

Ps. taetrolens Y-30 Glutamine Synthetase for Theanine Production
1895
Table 4. Km Values for Substrates and Relative Activities in the Reaction with Various Amines
Reaction conditions
Km value (mM) for
Glutamic acid
Lower Higher
Relative
Exp
Ammonia or
alkylamine
Divalent
cation
Ammonia
and amine
activity (%)
pH
ATP
conc.
conc.
A
B
C
D
E
F
Ammonia
Ammonia
30 mM Mg2þ
3 mM Mn2þ
30 mM Mg2þ
3 mM Mn2þ
30 mM Mg2þ
3 mM Mn2þ
8.0
7.5
0.65
1.4
2.5
5.6
2.0
2.5
4.0
1.1
2.0
1.5
1.4
1.4
1.3
1.5
0.56
0.59
100
100
11
45
14
18
7
Methylamine
Methylamine
Ethylamine
Ethylamine
8.5
8.0
0.5
0.5
20
700
11.0
8.5
0.67
0.22
500
The reaction mixture for each experiment contained:
A: 50 mM sodium glutamate, 15 mM ammonium chloride, 7.5 mM ATP, 30 mM Mg2þ, 100 mM imidazole buffer (pH, in the Table), and 0.068 unit/ml GS. This mixture is
for the glutamine-forming reaction, and was designated the standard reaction condition.
B: 50 mM sodium glutamate, 15 mM ammonium chloride, 7.5 mM ATP, 3 mM Mn2þ, 100 mM imidazole buffer (pH, in the Table), and 0.068 unit/ml GS.
C: 50 mM sodium glutamate, 500 mM methylamine, 7.5 mM ATP, 30 mM Mg2þ, 100 mM imidazole buffer (pH, in the Table), and 0.068 unit/ml GS.
D: 50 mM sodium glutamate, 500 mM methylamine, 7.5 mM ATP, 3 mM Mn2þ, 100 mM imidazole buffer (pH, in the Table), and 0.068 unit/ml GS.
E: 50 mM sodium glutamate, 1000 mM ethylamine, 7.5 mM ATP, 30 mM Mg2þ, 100 mM imidazole buffer (pH, in the Table), and 0.068 unit/ml GS.
F: 50 mM sodium glutamate, 1000 mM ethylamine, 7.5 mM ATP, 3 mM Mg2þ, 100 mM imidazole buffer (pH, in the Table), and 0.068 unit/ml GS.
Km values were obtained by varying the concentration of a substrate with fixed concentrations of the other substrates. Reactivities are expressed relative to that of
the glutamine-forming reaction (Exp. A).
Fig. 4. Formation of Theanine by Coupling of Dried Yeast Cells and Purified GS.
The temporary reaction mixture was described in ‘‘Materials and Methods.’’ The amounts of GS were null (A), 1 (B), and 10 (C) units/ml.
Incubation was done at 30 ꢂC with shaking (200 rpm). Symbols: , glucose; , theanine.
intrasubunit structure and subunit interaction. In addi-
tion, they brought about variation of GS-properties such
as the pH-activity profile, reactivity to the substrates, Km
values for substrates, etc. These changes in the structure
and properties of GS are perhaps related to one
another.36,42) The observations summarized in Fig. 3
and Table 4 might be caused by divalent cations
similarly to those described above, but detailed experi-
ments were not made in this study.
findings indicate that Ps. taetrolens Y-30 GS might be
usable for theanine production, and that production in
high concentrations can be achieved, as in the examples
of ‘‘coupled fermentation with energy transfer,’’20,25) by
improving the reaction mixture with larger amounts of
GS. This will be reported soon.
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