Purification and Characterization of Glutamine Synthetase ofPseudomonas taetrolensY-30: An Enzyme Usable for Production of Theanine by Coupling with the Alcoholic Fermentation System of Baker’s Yeast

Abstract

Concentrated cell-extract of Pseudomonas taetrolens Y-30, isolated as a methylamine-assimilating organism, formed gamma-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 30 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 Mg2+ -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.

Cloning and Expression ofMethylovorus maysNo. 9 Gene Encoding γ-Glutamylmethylamide Synthetase: An Enzyme Usable in Theanine Formation by Coupling with the Alcoholic Fermentation System of Baker’s Yeast

“…1C). 12) The results suggested that theanine production in high concentrations can be achieved by increasing the GS concentration in the reaction mixture. 13) This was unsuccessful, however, due to the low reactivity of P. taetrolens Y-30 GS to ethylamine, resulting in difficulty in dissolving large amounts of the enzyme.…”

“…10,11) In order to overcome these shortcomings, we have investigated theanine production from glutamic acid and ethylamine using the theanine synthetase-like enzyme, in which sugar fermentation of dried baker's yeast cells was coupled as an ATP-regenerating system (coupled fermentation with energy transfer). [12][13][14][15] In earlier reports, [12][13][14] we indicated the principle of coupled fermentation with energy transfer using the glutamine synthetase (GS; L-glutamate:ammonia ligase, ADP-forming, E.C. 6.3.1.2, Fig.…”

Cloning and Expression ofPseudomonas taetrolensY-30 Gene Encoding Glutamine Synthetase: An Enzyme Available for Theanine Production by Coupled Fermentation with Energy Transfer

“…Although several methods for preparing theanine have been reported, [13][14][15][16][17] they are usually complicated and show unsatisfactory yield. As described in previous papers, 18,19) theanine production by -glutamyl transfer reaction of bacterial glutaminase 20) or -glutamyl transpeptidase 21) has certain shortcomings with respect to supply of substrate (glutamine) and side reactions that lower product yield on glutamine.…”

“…18,19,22) In this method, a catalytic amount of another substrate ATP is regenerated by sugar fermentation of baker's yeast.…”

“…18,19) Mn 2þ as a cofactor and a high concentration of ethylamine as a substrate were effective for theanine production. In addition, it was found that increases in GS amounts in the mixture increased the yield of theanine, 19) indicating a need for an overexpression system of the P. taetrolens Y-30 GS for supplying large amounts of GS.…”