Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
2002-02-20
2003-11-25
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing alpha or beta amino acid or substituted amino acid...
Reexamination Certificate
active
06653110
ABSTRACT:
BACKGROUND OF THE INVENTION
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.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for producing L-glutamic acid by fermentation, which enables efficient L-glutamic acid production even when a material containing an organic acid that inhibits growth of a microorganism at a low pH, is used as a sugar source.
The inventors of the present invention found that, at a neutral pH, an L-glutamic acid-producing bacterium consumed an organic acid that inhibited growth of the L-glutamic acid-producing bacterium at a low pH, and that based on this property, L-glutamic acid could be efficiently produced by using a material containing an organic acid that inhibited growth of a microorganism at a low pH as a sugar source. Thus, they accomplished the present invention.
The present invention provides the followings.
(1) A method for producing L-glutamic acid by fermentation, which comprises culturing a microorganism having L-glutamic acid-producing ability in a medium having pH of 5.0 or less, wherein a total content of an organic acid that inhibits growth of the microorganism at the pH is one at which the growth of the microorganism is not inhibited.
(2) The method according to (1), wherein L-glutamic acid is produced and accumulated in the medium with precipitation of the L-glutamic acid accompanied, during the culture in the medium.
(3) The method according to (1) or (2), wherein the total content of the organic acid is 0.4 g/L or less.
(4) The method according to any one of (1) to (3), wherein the organic acid is an organic acid having carbon number of 1 to 3.
(5) The method according to any one of (1) to (4), wherein the microorganism belongs to the genus Enterobacter.
(6) The method according to (5), wherein the microorganism is Enterobacter agglomerans.
(7) The method according to any one of (1) to (6), wherein the microorganism can metabolize a carbon source in a liquid medium containing L-glutamic acid at a saturation concentration and the carbon source, at a specific pH, 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.
(8) The method according to (7), wherein the specific pH is 5.0 or less.
(9) A method for producing L-glutamic acid by fermentation, which comprises culturing a microorganism having L-glutamic acid-producing ability at a first pH at which growth of the microorganism is not inhibited by an organic acid in a medium, and then culturing the microorganism at a second pH that is suitable for L-glutamic acid production by the microorganism and is lower than the first pH.
(10) The method according to (9), wherein the organic acid is an organic acid having carbon number of 1 to 3.
(11) The method according to (9) or (10), wherein the second pH is 3.0 to 5.0.
(12) The method according to any one of (9) to (11), wherein the culture at the first pH is performed while pH of the medium is maintained to be the first pH by adding an alkalizing substance to the medium.
(13) The method according to (12), which comprising lowering pH of the medium by controlling the addition amount of the alkalizing substance after the culture at the first pH.
(14) The method according to any one of (9) to (13), wherein the culture at the first pH is continued until the organic acid in the medium is depleted.
(15) The method according to any one of (9) to (14), wherein the microorganism belongs to the genus Enterobacter.
(16) The method according to (15), wherein the microorganism is
Enterobact
Akiyoshi Naoki
Sato Masakazu
Ajinomoto Co. Inc.
Lilling Herbert J.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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