Glutamine production in high concentrations with energy transfer employing glutamine synthetase from Micrococcus glutamicus.

Abstract

Glutamine was produced by coupling the glutamine synthetase from Micrococcus glutamicus with sugar fermentation of baker's yeast as an energy-generating system. With 429 units/ml of glutamine synthetase and 40 mg/ml of yeast cells, 120 mM of glutamate was converted completely to glutamine in 3-4 hr, and 160-170 mM of glutamine (approximately 23-25 g/l) was formed in 5 hr from 350 mM of the substrates glutamate and ammonium chloride with a yield of about 40%, based on the energy released by the yeast during sugar fermentation. The activity ratio of sugar fermentation and glutamine synthetase was again confirmed to be important for achievement of energy-coupling in systems with high concentrations of glutamate and ammonium chloride. Glutamine formation was partially stimulated by the addition of Mg2+ in concentrations at which yeast fermentation of sugar showed no response to the cation, but was greatly stimulated by the addition of a small amount of Cott This same Co2+ effect was also observed in the reaction with cell-free extracts or toluol-treated cells of M. glutamicus.We have established processes for the production of various substances, employing dried yeast cells as an enzyme source (1-9). The processes are driven by the energy released by the alcoholic fermentation of sugar by the yeast. We have also designed a new process in which the yeast fermentation of sugar is combined with an endergonic reaction catalyzed by an enzyme from a different microorganism (4, 10). The results suggest that the process offers the possibility of producing many compounds in high yield by using various biosynthetic reactions and high concentrations of substrates. We introduced the term "coupled fermentation 1 Amino acids are L-isomer unless otherwise stated .

Theanine Production by Coupled Fermentation with Energy Transfer EmployingPseudomonas taetrolensY-30 Glutamine Synthetase and Baker’s Yeast Cells

“…These results are similar to those observed in glutamine production, 12,13,21) and indicate that the ratio of the activities of sugar fermentation and theanine formation is important for effective theanine production by coupled fermentation with energy transfer.…”

“…A temporary reaction mixture for coupled fermentation with energy transfer 20) was used as an initial mixture, and was defined according to the previous findings. 11,12) The mixture (1 ml in a test tube) contained (1) 200 mM sodium glutamate and 300 mM ethylamine . HCl (substrates for theanine synthesis by GS), (2) 200 mM glucose and 50 mM potassium phosphate buffer, pH 7.0 (substrates for yeast fermentation of sugar), (3) 25 mM MgCl 2 and 5 mM MnCl 2 (cofactor for GS and several enzymes in yeast sugar fermentation), (4) 5 mM AMP (ATP source), and (5) enzyme preparations (GS and dried yeast cells; their concentration is indicated in the legend to each experiment).…”

“…Glutamine production was achieved by the use of GS from Corynebacterium glutamicum (Micrococcus glutamicus) or Brevibacterium flavum. 12,13) During the course of the investigation, formation of a small amount of theanine was observed when ammonia in the reaction mixture was replaced by ethylamine, and this depended on the reactivity of the GS toward ethylamine (the right side in Fig. 1).…”

“…11) This is an application of glutamine production from glutamic acid and ammonia by the ''coupled fermentation with energy transfer'', 12,13) which has been developed through work on fermentative production of nucleic acid-related compounds, sugar-phosphates, etc.…”

Production of .GAMMA.-glutamylmethylamide and .GAMMA.-glutamylethylamide by coupling of baker's yeast preparations and bacterial glutamine synthetase.

“…We have reported on glutamine production by coupling the reaction of bacterial glutamine synthetase with sugar fermentation of baker's yeast as an energy-supplying or an ATP-regenerating system (1)(2)(3). This paper describes the production of y-glutamylmethylamide (y-GMA) and y-glutamylethylamide (theanine) depending on the reactivity toward methylamine and ethylamine of glutamine synthetase from Micrococcus glutamicus ATCC 13032 (4).…”