A method for producing l-glutamic acid by fermentation, which comprises culturing a microorganism having l-glutamic acid-producing ability in a liquid medium of which ph is adjusted to a condition under which l-glutamic acid produced by the microorganism is allowed to be precipitated, to allow l-glutamic acid to be produced and accumulated with precipitation of l-glutamic acid accompanied, wherein an operation causing existence of l-glutamic acid crystals in the medium is performed when a concentration of l-glutamic acid in the medium is lower than the concentration at which spontaneous crystallization occurs.

Patent
   RE41800
Priority
Feb 20 2002
Filed
Jun 16 2009
Issued
Oct 05 2010
Expiry
Feb 20 2022
Assg.orig
Entity
Large
0
64
all paid
1. A method for producing l-glutamic acid by fermentation, which comprises culturing a microorganism having l-glutamic acid-producing ability in a liquid medium of which ph is adjusted within the range of 3 to 5 to allow l-glutamic acid to be produced and accumulated with precipitation of l-glutamic acid accompanied, wherein an operation causing existence of l-glutamic acid crystals in the medium is performed after the fermentation begins and when a concentration of l-glutamic acid in the medium is lower than the concentration at which spontaneous crystallization occurs,
wherein the operation causing existence of l-glutamic acid crystals in the medium is addition of l-glutamic acid crystals, and
wherein l-glutamic acid crystals are not added to the medium before the fermentation.
2. The method according to claim 1, wherein the crystals are α-crystals.
3. The method according to claim 2, wherein an amount of the crystals added to the medium is 0.2 g/l or more.
4. The method according to claim 1, wherein the microorganism belongs to the genus Enterobacter Pantoea.
5. The method according to claim 2, wherein the microorganism belongs to the genus Enterobacter Pantoea.
6. The method according to claim 3, wherein the microorganism belongs to the genus Enterobacter Pantoea.
7. The method according to claim 4, wherein the microorganism is Enterobacter agglomerans Pantoea ananatis.
8. The method according to claim 5, wherein the microorganism is Enterobacter agglomerans Pantoea ananatis.
9. The method according to claim 6, wherein the microorganism is Enterobacter agglomerans Pantoea ananatis.
10. The method according to claim 1, wherein the microorganism can metabolize a carbon source in a liquid medium contacting l-glutamic acid at a saturation concentration and the carbon source, the liquid medium having a ph within the range of 3 to 5 and has an ability to accumulate l glutamic acid in an amount exceeding the saturation concentration of l-glutamic acid in the liquid medium at the ph.
11. The method according to claim 2, wherein the microorganism can metabolize a carbon source in a liquid medium containing l-glutamic acid at a saturation concentration and the carbon source, the liquid medium having a ph within the range of 3 to 5 and has an ability to accumulate l-glutamic acid in an amount exceeding the saturation concentration of l-glutamic acid in the liquid medium at the ph.
12. The method according to claim 3, wherein the microorganism can metabolize a carbon source in a liquid medium containing l-glutamic acid at a saturation concentration and the carbon source, the liquid medium having a ph within the range of 3 to 5 and has an ability to accumulate l glutamic acid in an amount exceeding the saturation concentration of l-glutamic acid in the liquid medium at the ph.
13. The method according to claim 4, wherein the microorganism can metabolize a carbon source in a liquid medium containing l-glutamic acid at a saturation concentration and the carbon source, the liquid medium having a ph within the range of 3 to 5 and has an ability to accumulate l-glutamic acid in an amount exceeding the saturation concentration of l-glutamic acid in the liquid medium at the ph.
14. The method according to claim 5, wherein the microorganism can metabolize a carbon source in a liquid medium containing l-glutamic acid at a saturation concentration and the carbon source, the liquid medium having a ph within the range of 3 to 5 and has an ability to accumulate l-glutamic acid in an amount exceeding the saturation concentration of l-glutamic acid in the liquid medium at the ph.
0. 15. The method according to claim 7, wherein said microorganism is Pantoea ananatis AJ13355 strain.
0. 16. The method according to claim 8, wherein said microorganism is Pantoea ananatis AJ13355 strain.
0. 17. The method according to claim 9, wherein said microorganism is Pantoea ananatis AJ13355 strain.

This application is a continuation of U.S. application Ser. No. 10/077,999, filed on Feb. 20, 2002, now abandoned.

The present invention relates to a method for producing L-glutamic acid by fermentation. L-Glutamic acid is widely used as a raw material of seasonings and so forth.

L-Glutamic acid is produced mainly by fermentation utilizing so-called L-glutamic acid-producing coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium or mutant strains thereof (Amino Acid Fermentation, Gakkai Shuppan Center, pp. 195-215, 1986). As methods for producing L-glutamic acid by fermentation by using other bacterial strains, there are known a method using a microorganism belonging to the genus Bacillus, Streptomyces, Penicillium or the like (U.S. Pat. No. 3,220,929), a method using a microorganism belonging to the genus Pseudomonas, Arthrobacter, Serratia, Candida or the like (U.S. Pat. No. 3,563,857), a method using a microorganism belonging to the genus Bacillus, Pseudomonas, Serratia, Aerobacter aerogenes (currently referred to as Enterobacter aerogenes) or the like (Japanese Patent Publication (Kokoku) No. 32-9393), a method using a mutant strain of Escherichia coli (Japanese Patent Application Laid-open (Kokai) No. 5-244970) and so forth. In addition, the inventors of the present invention proposed a method for producing L-glutamic acid by using a microorganism belonging to the genus Klebsiella, Erwinia or Pantoea (Japanese Patent Application Laid-open No. 2000-106869).

Further, there have been disclosed various techniques for improving L-glutamic acid-producing ability by enhancing activities of L-glutamic acid biosynthetic enzymes through use of recombinant DNA techniques. For example, it was reported that introduction of a gene coding for citrate synthase derived from Escherichia coli or Corynebacterium glutamicum was effective for enhancement of L-glutamic acid-producing ability in Corynebacterium or Brevibacterium bacteria (Japanese Patent Publication (Kokoku) No. 7-121228). In addition, Japanese Patent Application Laid-open No. 61-268185 discloses a cell harboring recombinant DNA containing a glutamate dehydrogenase gene derived from Corynebacterium bacteria. Further, Japanese Patent Application Laid-open No. 63-214189 discloses a technique for increasing L-glutamic acid-producing ability by amplifying a glutamate dehydrogenase gene, an isocitrate dehydrogenase gene, an aconitate hydratase gene and a citrate synthase gene.

Although L-glutamic acid productivity has been considerably increased by the aforementioned breeding of microorganisms or improvement of production methods, development of methods for more efficiently producing L-glutamic acid at a lower cost is required to meet to further increase of the demand in future.

There is known a method wherein fermentation is performed as L-amino acid accumulated in culture is crystallized (Japanese Patent Application Laid-open No. 62-288). In this method, the L-amino acid concentration in the culture is maintained below a certain level by precipitating the accumulated L-amino acid in the culture. Specifically, L-tryptophan, L-tyrosine or L-leucine is precipitated during fermentation by adjusting temperature and pH of the culture or adding a surfactant to a medium.

While a method of carrying out fermentation with precipitation of L-amino acid accompanied is known as described above, amino acids suitable for this method are those showing a relatively low water solubility, and no example of applying the method to highly water-soluble amino acids such as L-glutamic acid is known. In addition, the medium must have low pH to precipitate L-glutamic acid. However, L-glutamic acid-producing bacteria such as those mentioned above cannot grow under an acidic condition, and therefore L-glutamic acid fermentation is performed under neutral conditions (U.S. Pat. Nos. 3,220,929 and 3,032,474; K. C. Chao & J. W. Foster, J. Bacteriol., 77, pp. 715-725 (1959)). Thus, production of L-glutamic acid by fermentation accompanied by precipitation is not known. Furthermore, it is known that growth of most acidophile bacteria is inhibited by organic acids such as acetic acid, lactic acid and succinic acid (Yasuro Oshima Ed., “Extreme Environment Microorganism Handbook”, p. 231, Science Forum; R. M. Borichewski, J. Bacteriol., 93, pp. 597-599 (1967) etc.). Therefore, it is considered that many microorganisms are susceptible to L-glutamic acid, which is also an organic acid, under acidic conditions, and there has been no report that search of microorganisms showing L-glutamic acid-producing ability under acidic conditions was attempted.

Under the circumstances as described above, an object of the present invention is to provide a method for producing L-glutamic acid by fermentation, with precipitation of L-glutamic acid accompanied.

The inventors of the present invention found that, in a method for producing L-glutamic acid by fermentation, which comprising culturing a microorganism having L-glutamic acid-producing ability, to allow L-glutamic acid to be produced and accumulated in a medium with precipitation of L-glutamic acid accompanied, a yield of L-glutamic acid when existence of crystals is artificially caused before spontaneous crystallization occurs, is better that that when spontaneous crystallization of L-glutamic acid produced by the microorganism is allowed to occur. Thus, they accomplished the present invention.

The present invention provides the followings.