FERMENTATIVE PRODUCTION OF L-GLUTAMIC ACID FROM α-KETOGLUTARIC ACID AND AMMONIUM SALT

Abstract

1. Searches for the microorganism which are able to grow in the medium, containing α-ketoglutaric acid and ammonium chloride, and do accumulate L-glutamic acid in the medium were carried out. The accumulation of L-glutamic acid was detected in the cultures of the microorganisms belonging to the following genera;
Agrobacterium, Aerobacter, Aeromonas, Bacillus, Bacterium, Erwinia, Escherichia, Micrococcus, Serratia, Pseudomonas, Xanthomonas; Debaryomyces, Hansenula, Mycotorula, Pseudosaccharomyces, Saccharomyces, Willia, Aspergillus, Penicillium and Rhizopus.
A strain of Pseudomonas ovalis showed the highest level of L-glutamic acid accumulation. A description of the morphological and physiological properties of this strain were presented. This strain was used in the subsequent experiments.
2. A test of the effects of nitrogen sources on L-glutamic acid formation revealed that ammonium chloride was the best nitrogen source for the L-glutamic acid formation.
3. The aerobic condition was favorable for the growth of this organism, but not for the accumulation of L-glutamic acid.
4. The accumulation process of L-glutamic acid was separated from the growth phase of the organism. After the organism was grown in the medium A3 on the shaker at 30°C for 17 to 18hrs., the cells were harvested by centrifugation and washed thrice. The washed cells were added to the fresh medium A3, and incubated at 30°C. The initial concentration of the cells were 30mg cells (wet weight) per ml. The highest yield of L-glutamic acid was obtained when the pH of the medium was maintained neutral or slightly alkaline and the incubation was carried out anaerobically. Within 24 to 30hrs., about 98% of the added α-ketoglutaric acid was consumed and 30μM L-glutamic acid per ml, an amount corresponding to 60% of the consumed α-ketoglutaric acid, were accumulated in the medium.
5. When the cell suspension was incubated aerobically or the pH of the medium was not controlled, the accumulation of L-glutamic acid remained at a lower level.
6. The enzymological studies suggested that L-glutamic acid was formed through either one, or both, of the following pathways;
1. α-ketoglutarate+TPNH2+NH3-L-glutamic dehydrogenase→ L-glutamate+TPN+H2O
2. α-ketoglutarate-TCA cycle→fumarate
fumarate+NH3-aspartate→L-aspartate
L-aspartate+α-ketoglutarate-aspartic-α-ketoglutaric transaminase →oxalacetate+L-glutamate
The pathway 1 seemed to be mainly working in our organism used.
7. The problems derived from this work and left in the future studies were pointed out.