Process for producing L-glutamic acid by fermentation

A process for producing L-glutamic acid by fermentation is disclosed, which process comprises culturing aerobically in a culture medium a mutant of the genus Brevibacterium or Corynebacterium which has an increased superoxide dismutase activity and is capable of producing L-glutamic acid in the culture medium and recovering the L-glutamic acid. The yield of L-glutamic acid can be increased by using the aforementioned mutants.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for producing L-glutamic acid by 
fermentation. 
2. Description of the Prior Art 
L-glutamic acid, in the form of the monosodium salt, has been used as a 
seasoning and has previously been produced by a fermentation process in 
which wild strains or artificial mutants of L-glutamic acid-producing 
bacteria, especially of the genus Brevibacterium or Corynebacterium, are 
used. 
Currently, various artifical mutants of the genus Brevibacterium or 
Corynebacterium capable of producing L-glutamic acid are known. Examples 
of such artificial mutants are mutants requiring L-arginine, L-histidine, 
pyrimidine, hypoxanthine, glycerol, chemical compounds having a disufide 
linkage, or an unsaturated fatty acid such as oleic acid (as described in 
Japanese Published Examined patent application Nos. 507/1967, 508/1967, 
509/1967, 27390/1970, 27391/1970, 19632/1975, 33997/1976, 2998/1977, 
6233/1978, 6234/1978, and 8798/1978); mutants resistant to 
chloramphenicol, streptomycin, chlortetracycline, S-(2-aminoethyl)cystein, 
monofluoracetic acid, fluorocitric acid, ketomalonic acid, 
.alpha.-amino-.beta.-hydroxyvaleric acid, DL-threonine hydroxamate, 
2-amino-3-phosphopropionic acid, 5-aminolevulinic acid, glutamic acid 
analogues, benzopyrone, naphthoquinone, or 2,6-pyridinedicarboxylic acid 
or to inhibitors of the bacterial respiratory system such as malonic acid, 
NaN.sub.3, KCN, sodium arsenite, 2,4-dinitrophenol, hydroxyamine, and 
guanidine (as described in Japanese Published Unexamined patent 
application Nos. 4398/1966, 126877/1975, 38088/1977, 89085/1979, 
21763/1980, 21764/1980, 124492/1980, 1889/1981, 35981/1981, 39778/1981, 
and 48890/1981); mutants sensitive to N-palmitoyl glutamic acid, lysozyme, 
or to a temperature of more than 34.degree. C. (as described in Japanese 
Published Unexamined patent application Nos. 64486/1975, 32193/1978, 
66687/1977, 122794/1979, and 114293/1980); and a mutant having reduced 
activity with respect to pyruvic acid dehydrogenase (as described in 
Japanese Published Unexamined patent application No. 21762/1980). However, 
because of the continued desirability of increased L-glutamic acid 
production, processes for the increased production of L-glutamic continue 
to be sought. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a method of increasing 
the production of L-glutamic acid. 
This and other objects of the invention as will hereinafter become more 
readily apparent have been accomplished by providing a process for 
producing L-glutamic acid by fermentation, which process comprises 
culturing aerobically in a culture medium a mutant of the species 
Brevibacterium or Cornyebacterium which has increased superoxide dismutase 
activity over that present in the parent strain from which said mutant is 
derived and which is capable of producing L-glutamic acid in the culture 
medium, and recovering the L-glutamic acid from the culture medium. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
It has now been found (and this discovery forms the basis of the present 
invention) that the production of L-glutamic acid can be increased when 
the superoxide dismutase activity of an L-glutamic acid-producing 
microorganic strain is reinforced or increased over that present normally, 
and that the mutants having increased superoxide dismutase activity can be 
obtained from among the artificial mutants resistant to a superoxide 
propagator, i.e., a chemical agent which produces a superoxide. 
The microorganisms employed in the process of the present invention are 
mutants which belong to the genus Brevibacterium or Corynebacterium, have 
an increased superoxide dismutase activity, and have an ability to produce 
L-glutamic acid. Preferably the mutant employed in the process of the 
present invention belongs to the species Brevibacterium lactofermentum, 
Brevibacterium flavum, Corynebacterium glutamicum or Corynebacterium 
acetoacidophilum. Preferable examples of such mutants of the present 
invention include the following: 
Brevibacterium lactofermentum: AJ 11796 FERM-P 6402, FERM BP-251, 
(DM.sup.r, SOD increased) 
Corynebacterium glutamicum: AJ 11801 FERM-P 6398, FERM BP-250, (MV.sup.r, 
SOD increased) 
where the stated terms have the following meanings: 
SOD increased: having an increased superoxide dismutase activity over that 
present in the unmutated parent strain 
DM.sup.r : resistance to Daunomycin, and 
MV.sup.r : resistance to Methylviologen. 
The AJ numbers are internal reference numbers of the present inventors. 
The mutants identified above by FERM-BP numbers were originally deposited 
on Mar. 1, 1982, at the Fermentation Research Institute, Agency of 
Industrial Science and Technology, Ministry of International Trade and 
Industry (FRI), 1-3 Higashi 1-chome, Yatabe-machi, Tsukuba-gun, 
Ibaragi-ken 305, Japan, at which time they received the indicated FERM-P 
numbers, and these deposits were converted to the FERM-BP deposit number 
under the Budapest Treaty on Feb. 3, 1983. FRI acquired the status of an 
International Depositary Authority as of May 1, 1981. 
The mutants indicated above were induced from parent strains of the genus 
Brevibacterium or Corynebacterium by a conventional method. The first step 
of the induction is to mutate the parent strain with a suitable chemical 
mutagen such as N-methyl-N'-nitro-N-nitrosoguanidine (hereinafter referred 
to as NG) or nitrous acid or by irradiation with ultraviolet light. 
The second step is to select a mutant resistant to a superoxide propagator 
by picking up the colonies grown on plates of nutrient agar medium 
containing an amount of a superoxide propagator which inhibits the growth 
of the parent strain. This amount can be easily be determined by simple 
experimentation. Finally, the mutants are evaluated for L-glutamic acid 
production according to a standard method which measures L-glutamic acid 
present in the culture medium. 
The mutants FERM BP-251 and FERM BP-250 have the same general 
characteristics as their parent strain, i.e., ATCC 13869 and ATCC 13032, 
and additionally have increased superoxide dismutase activity and 
resistance to Daunomycin or Methylviologen, respectively. 
As the parent strains, any wild strain capable of producing L-glutamic acid 
of the genus Brevibacterium or Corynebacterium can be employed. The 
preferred wild strains are coryne-form glutamic acid-producing bacteria 
and include the following examples: 
Brevibacterium lactofermentum: ATCC 13869 
Brevibacterium flavum: ATCC 14067 
Brevibacterium divaricatum: ATCC 14020 
Brevibacterium saccharoliticum: ATCC 14066 
Corynebacterium glutamicum: ATCC 13032 
Corynebacterium acetoacidophilum: ATCC 13870 
As another type of the parent strain, mutants of the genus Brevibacterium 
or Corynebacterium which are induced from wild strains as stated above and 
have biological characteristics known to be effective for production of 
L-glutamic acid, such as resistance to fluoromalonic acid, fluorocitric 
acid, ketomalonic acid, and 2,6-puridinedicarboxylic acid and sensitivity 
to lysozyme and N-palmitoylglutamate, are preferably used. 
These characteristics, useful for the production of L-glutamic acid, can be 
imparted prior to or after giving to the wild strains the resistance to 
the antibiotics. 
The superoxide radical is one of the active forms of oxygens and is 
represented by O.sub.2 --, .O.sub.2 -- or .O.sub.2 H. This radical is 
generated in vivo when oxygen (O.sub.2) is reduced in an oxidation 
reaction catalysed by Xanthineoxidase (EC 1,2,3,2) or in an autooxidation 
reaction of a cell's components. It is well known that the superoxide 
radical reacts with unsaturated fatty acids to produce a superoxide of the 
fatty acid. The superoxide thus produced is harmful to living cells and 
causes an undesirable drop in the various biochemical activities of the 
living cells. 
Similarly, a superoxide of an unsaturated fatty acid is generated in cells 
of L-glutamic acid-producing microorganisms which have been cultured 
aerobically, i.e., under oxidative condition, and which are producing 
L-glutamic acid in the culture medium. Thereby, the activity of the cells, 
such as their ability to produce L-glutamic acid, gradually drops with 
time when the cells are cultured for a long time. 
A superoxide propagator is a chemical agent which produces a superoxide 
harmful to living cells and usually inhibits the growth of microorganisms. 
Superoxide propagators include the following: Methylvioloben, 
Nitrofurantoin, Vitamin K, Morphine, Adriamycin, Mitomuycin C. Daunomycin, 
and Bleomycin. 
Superoxide dismutase (EC 1,15,1,1) can catalyze the following reaction: 
EQU 2 O.sub.2 --.+2 H.sup.+ .fwdarw.H.sub.2 O.sub.2 +O.sub.2 
It is known that a superoxide dismutase may defend a living body from 
disruption caused by an active oxygen such as a superoxide (Fridovich, 
Ann. Rev. Biochem., 44, 147-159 (1975)). 
Accordingly, the mutants of the present invention having an increased 
superoxide dismutase activity will maintain a cell's activity under 
oxidative condition for a longer time, and thereby can produce a larger 
amount of L-glutamic acid than their parent strains. 
Conveniently the process of the present invention also includes recovering 
the L-glutamic acid which accumulates in the culture medium.

The method by which the mutants of the present invention can be induced, 
the typical degree of resistance to the superoxide propagator obtained, 
and typical activities of the superoxide dismutase are exemplified in the 
following Experiments 1, 2 and 3, which are not intended to be limiting of 
the present invention unless otherwise specified. 
EXPERIMENT 1 
Cells of the known microorganic strain 
Brevibacterium lactofermentum ATCC 13869 grown on a slant of bouillion agar 
medium were scraped off and suspended in sterilized water containing 150 
.mu.g/ml NG, and the suspension was allowed to stand at 30.degree. C. for 
20 minutes. The microbal cells thus treated were washed with phosphate 
buffer solution and then inoculated on agar plates, the composition of 
which is given in Table 1, further containing 3 .mu.g/ml Daunomycin. 
TABLE 1 
______________________________________ 
Composition of the agar medium (pH 7.0) 
Component Conc. 
______________________________________ 
Yeast extract 1.0 g/dl 
Peptone 1.0 g/dl 
NaCl 0.5 g/dl 
Agar 2.0 g/dl 
______________________________________ 
The plates were then incubated at 30.degree. C. for 2 to 4 days until 
colonies developed on the plates. The colonies were collected as the 
Daunomycin-resistant mutants and were evaluated for producing L-glutamic 
acid according to a standard method. 
It was found that mutants which have an increased activity with respect to 
superoxide dismutase and provide greater production of L-glutamic acid 
than the parent strain were obtained with high frequency. 
From among these mutants, B. lactofermentum AJ 11796 FERM BP-251, which can 
produce more L-glutamic acid than any other mutant, was selected. The 
mutants resistant to Methylviologen of the present invention were obtained 
in a similar manner to that described above. 
EXPERIMENT 2 
Four-milliliter portions of an aqueous GM medium having the composition 
specified in Table 2, some of which contained the definite amount of the 
superoxide propagator specified in Table 3, were poured into small test 
tubes and heated for sterilization. 
TABLE 2 
______________________________________ 
Composition of GM medium 
Component Conc. Component Conc. 
______________________________________ 
Glucose 0.5 g/dl CaCl.sub.2 
0.1 mg/dl 
Ammonium 0.15 g/dl MnCl.sub.2.4H.sub.2 O 
0.36 mg/dl 
Sulfate 
Urea 0.15 g/dl Na.sub.2 B.sub.4 O.sub.7.10H.sub.2 O 
0.44 mg/dl 
K.sub.2 HPO.sub.4 
0.1 g/dl CuSO.sub.4.5H.sub.2 O 
1.95 mg/dl 
KH.sub.2 PO.sub.4 
0.3 g/dl ZnSO.sub.4.7H.sub.2 O 
44 mg/dl 
MgSO.sub.4.7H.sub.2 O 
0.01 g/dl Biotin 3 .mu.g/ml 
FeCl.sub.3.6H.sub.2 O 
4.85 g/dl Thiamine HCl 
10 .mu.g/ml 
(NH.sub.4)Mo.sub.7 O.sub.24.4H.sub.2 O 
0.18 g/dl 
______________________________________ 
Each strain to be tested was washed with GM medium and suspended in GM 
medium to prepare the cell suspension of which the optical density (at 
26-times dilution) at 562 nm was 0.1. A sample (0.1 ml) of cell suspension 
was then transferred from each batch of GM medium and placed in the test 
tubes. Cultivation was carried out at 30.degree. C. for 30 hours with 
shaking. After the cultivation, the degree of growth of each strain was 
determined by measuring optical density at 562 nm of a 26-times diluted 
solution of the resultant culture broth, and the results obtained are 
shown in Table 3. 
TABLE 3 
______________________________________ 
Degree of resistance 
Superoxide 
Conc. ATCC AJ ATCC AJ 
propagator 
(.mu.g/ml) 
13969 11796 13032 11801 
______________________________________ 
Daunomycin 
0.0 100 100 
0.5 50 80 
1.0 30 60 
2.0 0 30 
3.0 0 10 
Methyl- 0 100 100 
viologen 50 59 85 
100 20 63 
150 10 45 
200 0 25 
______________________________________ 
In Table 3, the degree of resistance to the superoxide propagator is 
represented by the relative values of the growth to the control. 
EXPERIMENT 3 
The activity of the superoxide dismutase of each test strain was determined 
according to the Nitroblue tetrazolium method (V. Ponti et al, Chem.-Biol. 
Interaction, 23, 281-291 (1978)). 
Thus, twenty ml portions of an aqueous medium, pH 7.0, which contain (per 
deciliter) 0.5 g yeast extract, 1.0 g peptone, 0.5 g bouillion and 0.5 g 
NaCl, were poured into 500 ml flasks and heated for sterilization. 
One loopful inoculm of each of the microorganisms listed in Table 4 below 
was transferred into each batch of the culture medium, and cultivation was 
carried out at 30.degree. C. for 24 hours with shaking. The microbial 
cells which accumulated in the culture broth were collected by 
centrifugation and washed twice with 0.1 M phosphate buffer, pH 7.0. The 
cells were then suspended in 20 ml of the same buffer, and the cell 
suspensions were subjected to sonication for 5 minutes (with cooling) to 
rupture the cells, and then centrifuged at 10,000 rpm for 10 minutes to 
obtain supernatant solutions. 
0.1 ml of each supernatant solution (sample solution) thus obtained was 
mixed with 2.4 ml of 0.05 M sodium carbonate buffer, pH 10.2; 0.1 ml nM 
Xanthine; 0.1 ml of 3 mM EDTA; 0.1 ml of 0.15% bovine albumin; and 0.1 ml 
of 0.75 mM nitro blue tetrazolium. Then the mixture was kept at 25.degree. 
C. for 10 minutes to activate enzyme activity. After the preincubation as 
above, 0.1 ml of Xanthin oxidase solution, which is 2 M ammonium sulfate 
solution containing 2.1.times.10.sup.-7 M Xanthine oxidase, was added to 
the preincubated mixture solution. Then the solution was allowed to stand 
at 25.degree. C. for 10 minutes followed by addition of 0.1 ml of 6 mM 
CuCl.sub.2 to stop the reaction. Thereafter, the optical density of the 
reaction solution at 560 nm was determined. As the control a similar 
reaction was performed using distilled water instead of the sample 
solution. 
1.0 unit of the enzyme activity of the superoxide dismutase is defined as 
the enzyme activity which inhibits 50% of the Xanthine oxidase reaction 
performed under the condition as above. 
The enzyme activity of the superoxide dismutase thus determined are shown 
in the following Table 4, in which the activity is represented by the 
relative value of the enzyme activity to the control. 
TABLE 4 
______________________________________ 
Superoxide dismutase activity 
Strain (%) 
______________________________________ 
ATCC 13869 
100 
AJ 11796 151 
ATCC 13032 
100 
AJ 11801 205 
______________________________________ 
The mutants are cultured aerobically in a conventional culture medium 
containing carbon sources, nitrogen sources and inorganic ions, and minor 
nutrients when required. 
As the carbon sources, saccharides such as glucose, sucrose, molasses and 
hydrolyzed starch, organic acids such as acetic acid and propionic acid, 
and alcohols such as ethanol can be used preferably. Nitrogen sources are, 
for example, ammonium sulfate, gaseous ammonia and urea. As the inorganic 
ions, K.sup.+, Na.sup.+, Ca.sup.++, Fe.sup.++, Mn.sup.++ Mg.sup.++, 
Zn.sup.++, So.sub.4.sup.--, Cl.sup.- and PO.sub.4.sup.-- are added to the 
culture medium when required. 
When a carbon source which does not contain biotin, such as hydrolyzed 
starch, is employed, biotin is added to the culture medium. Biotin 
concentratin in the medium has to be controlled to less than the maximum 
amount allowable for the growth of the mutant. 
On the other hand, when there is used a raw carbon source such as cane 
molasses containing more biotin than the proper amount for the growth of 
the mutant, an anti-biotin agent such as Penicillin, a higher fatty acid, 
or a surface-active agent is added to the medium. 
Cultivation is normally carried out under aerobic conditions for from 20 to 
80 hours at a temperature ranging from 30 to 38.degree. C. The pH of the 
culture medium is controlled between 6.0 to 8.0 with the addition of an 
organic or inorganic acid or an alkali. For this purpose, urea, CaCO.sub.3 
or gaseous ammonia is preferably used. 
L-Glutamic acid which accumulates in the culture broth can be recovered by 
any conventional recovery method. 
Having now generally described this invention, the same will be better 
understood by reference to certain specific examples which are included 
herein for purposes of illustration only and are not intended to be 
limiting of the invention or any embodiment thereof, unless specified. 
EXAMPLE 1 
Twenty-milliliter portions of an aqueous culture medium, of which the 
composition was as given in Table 5, were poured into 500 ml flasks and 
heated at 115.degree. C. for 10 minutes for sterilization. 
TABLE 5 
______________________________________ 
Composition of culture medium 
Component Conc. Component Conc. 
______________________________________ 
Glucose 36 mg/ml MnSO.sub.4.4H.sub.2 O 
8 .mu.g/ml 
Urea 2 mg/ml Thiamine.HCl 
10 .mu.g/dl 
KH.sub.2 PO.sub.4 
1 mg/ml Biotin 0.25 .mu.g/l 
MgSO.sub.4.7H.sub.2 O 
0.4 mg/ml Soy Protein 
5 .mu.l/ml 
FeSO.sub.4.7H.sub.2 O 
10 .mu.g/ml 
hydrolyzed 
______________________________________ 
Each strain to be tested, as listed in Table 5 and grown on bouillon agar 
medium, was inoculated into the medium and cultured at 31.5.degree. C. 
with shaking. During the cultivation, a small amount of an aqueous 
solution containing 450 mg/ml of urea was fed into the medium so as to 
maintain the pH of the culture medium in the range from 6.5 to 8.0. After 
30 hours of cultivation, the amount of L-glutamic acid which had 
accumulated in the culture broth was determined and the results obtained 
are shown in Table 6. 
TABLE 6 
______________________________________ 
L-glutamic acid 
Yield 
Strain No. accumulated (g/l) 
(%) 
______________________________________ 
ATCC 13869 16.2 45.0 
AJ 11896 18.5 51.4 
(FERM BP-251) 
ATCC 13032 15.5 43.0 
AJ 11801 17.7 49.2 
(FERM BP-250) 
______________________________________ 
EXAMPLE 2 
A culture medium (pH 7.0) containing, per milliliter, 100 mg sugar (cane 
molasses), 1 mg KH.sub.2 Po.sub.4, 1 .mu.g thiamine HCl, 1 mg MgSO.sub.4 
and 1 mg MgSO.sub.4.7H.sub.2 O was prepared. Thirty-ml portions of the 
medium were poured into 500-ml flasks and heated for sterilization. Then 
each strain to be tested, as listed in Table 7, was inoculated into the 
medium and cultured at 31.5.degree. C. with shaking. During the 
cultivation, a small amount of an aqueous solution of urea (400 mg/ml) was 
fed into the medium so as to maintain the pH of the medium in the range 
from 6.5 to 8.0. Polyethylene sorbitan monopalmitate was added to the 
medium when the optical density of a 26-times diluted solution of the 
culture medium came up to 0.300. 
After 36 hours of cultivation, the amount of L-glutamic acid which had 
accumulated in the culture broth was determined. The results are as shown 
in Table 7. 
TABLE 7 
______________________________________ 
L-glutamic acid 
Yield 
Strain No. accumulated (g/l) 
(%) 
______________________________________ 
ATCC 13869 49.5 49.5 
AJ 11796 52.5 52.5 
(FERM BP-251) 
ATCC 13032 49.3 49.3 
AJ 11801 51.5 51.5 
(FERM BP-250) 
______________________________________ 
The yield of L-glutamic acid can be increased by more than 2.0 percent by 
using the mutant of the present invention, and the increase in yield can 
produce a good increase in profit because more than 600,000 tons of 
L-glutamic acid have been produced by fermentation processes. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the invention as set 
forth herein.