To establish a practical method for the fermentative production of l -glutamine, cultural conditions for the accumulation of a large amounts of l -glutamine were investigated by using Flavobacterium rigense 703, which was previously reported by us as a l -glutamine-producing mutant. As a result, a yield of 25 mg of l -glutamine per ml was obtained after a 48-h cultivation in a medium containing glucose, yeast extract, (NH 4 ) 2 -fumarate, KH 2 PO 4 , K 2 HPO 4 , MgSO 4 .7H 2 O, and CaCO 3 (pH 6.4). Accumulation of l -glutamine was dependent upon the concentration of (NH 4 ) 2 -fumarate, and a suboptimum growth at a relatively high concentration of (NH 4 ) 2 -fumarate was essential for the maximum production of l -glutamine. At the optimum conditions, glutamic acid was formed as a by-product at a concentration of less than 1 mg/ml, but accumulation of the other amino acids was negligible. The product was isolated from the culture broth and readily purified by anion-exchange chromatography. The pure crystals of l -glutamine obtained in an 80% yield were optically and chromatographically pure.
The nutritional conditions for the production of l -glutamine by Flavobacterium rigense strain 703 were investigated. The optimum concentration of ammonia for achieving the highest yield of l -glutamine (25 mg/ml of broth) was relatively broad, from 0.9 to 1.6%, whereas fumaric acid had a narrow optimum range, near 5.5%. High concentration of inorganic ions such as chloride or sulfate ion clearly inhibited cell growth. Therefore, ammonium salts other than (NH 4 ) 2 -fumarate were unsuitable for the highest production. The optimum concentration of (NH 4 ) 2 -fumarate was 7%. To reduce the concentration of fumaric acid in the medium, many substances were evaluated as substitutes. The fumaric acid concentration required for highest l -glutamine yield could not be replaced by any one of the compounds tested. However, part of fumaric acid could be replaced with succinic acid and cupric ion; 4% (NH 4 ) 2 -fumarate plus 2.5% succinic acid or 5% (NH 4 ) 2 -fumarate plus 1 mM cupric ion produced results similar to 7% (NH 4 ) 2 -fumarate in the fermentation medium.
A penicillin-resistant mutant of Flavobacterium rigense designated as strain 703, FERM-P no. 3628, was obtained after ultraviolet treatment of F. rigense FERM-P no. 3556. The parent strain produces 0-2-hydroxypropylhomoserine from 1,2-propanediol. The mutant was found to be a good producer ofL-glutamine. The physiological characteristics of strain 703 were different from the general group of L-glutamic acid-producing bacteria. Strain 703 required L-tryptophan and thiamine but not biotin for its growth. L-Glutamine formation on a specific basis, however, was independent of L-tryptophan and thiamine. Biotin and penicillin were also not effective. Only ammonium fumarate acted as an effective factor on L-glutamine formation. Accumulation of L-glutamine by strain 703 was 10 mg/ml at 300C for 48 h in a chemically defined medium containing 3% diammonium fumarate.
During an investigation of microorganisms utilizing petrochemicals, a strain identified as Flavobacterium rigense was found to accumulate a new amino acid in a medium containing 1,2-propanediol as the sole carbon source. Cultural conditions for the accumulation of the product were investigated, and, as a result, the yield was increased to 2.8 mg/ml after a 5-day incubation in a medium containing 8% 1,2-propanediol. The pure amino acid was isolated, and its structure was investigated. Elemental analysis and infrared, nuclear magnetic resonance, and mass spectral analyses indicated that the amino acid is 0-2-hydroxypropylhomoserine.
Conditions for the production of tryptophanase from Achromobacter liquidum and for the conversion of L-serine and indole to L-tryptophan were studied. The enzyme could be produced in amnounts as great as 0.750 U/ml (degradation) and 0.294 U/ml (synthesis) by shaking cultures at 30°C in a medium containing dextrin, yeast extract, L-tryptophan, and L-glutamic acid. L-Tryptophan was produced most efficiently by shaking the cells at 37°C in a reaction mixture containing 60 mg of L-serine per ml, 60 mg of indole per ml, and 0.5 mM pyridoxal phosphate. After 3 days, 96 mg of L-tryptophan per ml was formed, and L-tryptophan was easily isolated to 85.4% yield by concentration of the reaction mixture.
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