1210
S. YAMAMOTO et al.
A
B
70
60
50
40
30
20
10
0
700
600
500
400
300
200
100
0
300
250
200
150
100
50
0
0
10 20 30 40 50
0
10 20 30 40 50
Time (h)
Time (h)
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.,
Enzymatic production of theanine, an ‘‘umami’’ compo-
nent of tea, from glutamine and ethylamine with
bacterial ꢀ-glutamyltranspeptidase. Enzyme Microb.
Technol., 31, 884–889 (2002).
12) Yamamoto, S., Uchimura, K., Wakayama, M., and
Tachiki, T., Purification and characterization of gluta-
mine synthetase of Pseudomonas taetrolens Y-30: an
enzyme usable for production of theanine by coupling
with the alcoholic fermentation system of baker’s yeast.
Biosci. Biotechnol. Biochem., 68, 1888–1889 (2004).
13) Yamamoto, S., Wakayama, M., and Tachiki, T., The-
anine production by coupled fermentation with energy
transfer employing Pseudomonas taetrolens Y-30 gluta-
mine synthetase and baker’s yeast cells. Biosci.
Biotechnol. Biochem., 69, 784–789 (2005).
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.
References
1) Sakato, Y., The chemical constituents of tea: III. A new
amide theanine. Nippon No¯geikagaku Kaishi (in Japa-
nese), 23, 262–267 (1949).
2) Sasaoka, K., Kito, M., and Inagaki, H., Studies on the
biosynthesis of theanine in tea seedlings: synthesis of
theanine by the homogenate of tea seedlings. Agric. Biol.
Chem., 27, 467–468 (1963).
3) Juneja, L. R., Chu, D. C., Okubo, T., Nagato, Y., and
Yokogoshi, H., L-Theanine-a unique amino acid of green
tea and its relaxation effect in humans. Food Sci.
Technol., 10, 199–204 (1999).
4) Yokogoshi, H., Kato, Y., Sagesaka, Y., Matsuura, T.,
Kakuda, T., and Takeuchi, N., Reduction effect of
theanine of blood pressure and brain 5-hydroxyindoles in
spontaneously hypertensive rats. Biosci. Biotechnol.
Biochem., 59, 615–618 (1995).
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) Ito, K., Nagato, Y., Aoi, N., Juneja, L. R., Kim, M.,
Yamamoto, T., and Sugimoto, S., Effect of L-theanine on
the release of ꢁ-brain waves in human volunteers.
Nippon No¯geikagaku Kaishi (in Japanese), 72, 153–157
14) Yamamoto, S., Wakayama, M., and Tachiki, T., Cloning
and expression of Pseudomonas taetrolens Y-30 gene
encoding glutamine synthetase: an enzyme available
for theanine production by coupled fermentation with
energy transfer. Biosci. Biotechnol. Biochem., 70, 500–
507 (2006).
15) Yamamoto, S., Wakayama, M., and Tachiki, T., Char-
acterization of theanine-forming enzyme from Methyl-
ovorus mays No. 9 in respect to utilization of theanine
production. Biosci. Biotechnol. Biochem., 71, 545–552
(2007).