Theanine production by coupled fermentation with energy transfer using gamma-glutamylmethylamide synthetase of Methylovorus mays No. 9.
Gamma-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 Mn(2+) to the reaction mixture, whereas Mg(2+) 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 MnCl(2), 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).
Yamamoto
Sachiko
S
Department of Chemistry, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.
Morihara
Yosuke
Y
Wakayama
Mamoru
M
Tachiki
Takashi
T
D019731:Carbon-Nitrogen Ligases / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D004735:Energy Transfer; D005021:Ethylamines / Q000378:metabolism / Q000494:pharmacology; D005285:Fermentation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D005971:Glutamates / Q000096:biosynthesis*; D042762:Methylophilaceae / Q000145:classification / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D012970:Sodium Glutamate / Q000378:metabolism / Q000494:pharmacology
D002413:Cations, Divalent; D005021:Ethylamines; D005971:Glutamates; D011994:Recombinant Proteins; C026166:theanine; D019731:Carbon-Nitrogen Ligases; C020661:gamma-glutamylmethylamide synthetase; D005947:Glucose; D012970:Sodium Glutamate; C041564:ethylamine