Method for production of L-arginine by fermentation

Mutants of the genus Bervibacterium or Corynebacterium are given resistance to keto-malonic acid, fluoro-malonic acid, monofluoro-acetic acid or aspartate antagonist and used to produce L-arginine by aerobic fermentation.

BACKGROUND OF THE INVENTION 
This invention relates to a method for producing L-arginine by 
fermentation. 
It has been known that L-arginine is produced by a fermentation process, in 
which mutants of the genus Brevibacterium, Coryne-bacterium resistant to a 
sulfa drug or arginine antagonist are used (Japanese Published Unexamined 
Patent Application No. 48189/1975). 
SUMMARY OF THE INVENTION 
It has now been found that the productivity of L-arginine is significantly 
increased when a resistance to keto-malonic acid, fluoro-malonic acid, 
monofluoro acetic acid, or aspartate-antagonist is given to the known 
mutants which belong to the genus Brevibacterium or Corynebacterium and 
are capable of producing a L-arginine. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The microorganisms employed according to the present invention are the 
mutants which belong to the genus Brevibacterium or Corynebacterium, are 
resistant to keto-malonic acid, fluoro-malonic acid, monofluoroacetic acid 
or aspartate-antagonists and have the ability to produce L-arginine. 
The mutants as stated above may be induced from parents strains of the 
genus Brevibacterium or Corynebacterium by conventional mutation methods 
such as an irradiation with UV-ray or exposure to 
N-methyl-N'-nitro-N-nitroso guanidine, and thereafter picking up the 
colonies which are formed on the nutrient agar-medium containing the 
amount of the chemical agents inhibitive to the growth of the parent 
strain. 
As the parent strains, mutants capable of producing L-arginine or wild 
strains of the genus Brevibacterium or Corynebacterium are employed. When 
the wild strains are used as the parent, L-arginine productivity is given 
to the wild strains prior to giving the resistance to the chemicals of 
this invention, or after giving to the wild strains the resistance, 
L-arginine productivity is given. 
In order to give the L-arginine productivity to the microorganisms of the 
genus Brevibacterium or Corynebacterium, as is known, resistance to 
arginine-antagonists such as 2-thiazole alanine and arginine hydroxamate, 
or to sulfa-drug is given to the microorganisms. The arginine antagonists 
are such chemicals as those which inhibit the growth of the microorganisms 
of the genus Brevibacterium and Corynebacterium and the inhibition is 
suppressed when L-arginine coexists in the medium. 
The preferred wild strains belonging to the genus Brevibacterium or 
Corynebacterium and are coryne-form L-glutamic acid producing bacteria and 
the examples are: 
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Brevibacterium divaricatum 
ATCC 14020, 
Brevibacterium flavum 
ATCC 14067, 
Brevibacterium lactofermentum 
ATCC 13869, 
Brevibacterium saccharolyticum 
ATCC 14066, 
Brevibacterium roseum 
ATCC 13825, 
Corynebacterium acetoacidophilum 
ATCC 13870, 
Corynebacterium lilium 
ATTC 15990, and 
Corynebacterium glutamicum 
ATCC 13032. 
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Aspartate-antagonists of this invention inhibit the growth of the 
microorganisms of genus Brevibacterium and Corynebacterium and the 
inhibition is suppressed partly or completely when L-aspartate coexists in 
the medium, and are, for instance, .beta.-aspartylhydrazide, 
diamino-succinic acid and hadacidin. 
Specimens of the mutants of this invention are: 
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Brevibacterium flavum 
AJ 11337, FERM-P 4940, 
NRRL B-12235 
(SD.sup..gamma., AspHd.sup..gamma.) 
Brevibacterium flavum 
AJ 11338, FERM-P 4941, 
NRRL B-12236 
(SD.sup..gamma., AS.sup..gamma.) 
Brevibacterium flavum 
AJ 11339, FERM-P 4942, 
NRRL B-12237 
(SD.sup..gamma., HD.sup..gamma.) 
Corynebacterium acetoacidophilum 
AJ 11341, FERM-P 4944, 
NRRL B-12238 
(SD.sup..gamma., AspHd.sup..gamma.) 
Corynebacterium acetoacidophilum 
AJ 11342, FERM-P 4945, 
NRRL B-12239 
(SD.sup..gamma., AS.sup..gamma.) 
Brevibacterium flavum 
AJ 11343, FERM-P 4946, 
NRRL B-12240 
(2TA.sup..gamma., SG.sup..gamma., His.sup.-, 
HD.sup..gamma.) 
Brevibacterium flavum 
AJ 11595, FERM-P 5637, 
NRRL B-12242 
(SD.sup..gamma., KM.sup..gamma.) 
Brevibacterium flavum 
AJ 11596, FERM-P 5638, 
NRRL B-12243 
(SD.sup..gamma., FM.sup..gamma.) 
Brevibacterium flavum 
AJ 11597, FERM-P 5639, 
NRRL B-12244 
(SD.sup..gamma., FA.sup..gamma.) 
Corynebacterium acetoacidophilum 
AJ 11598, FERM-P 5640, 
NRRL B-12245 
(SD.sup..gamma., KM.sup..gamma. ) 
Brevibacterium flavum 
AJ 11344, FERM-P 4947, 
NRRL B-12241 
(2TA.sup..gamma., SG.sup..gamma., His.sup.-, 
HD.sup..gamma.) 
Corynebacterium acetoacidophilum 
AJ 11599, FERM-P 5641, 
NRRL B-12246 
(SD.sup..gamma., FA.sup..gamma.) 
Brevibacterium flavum 
AJ 11600, FERM-P 5642, 
NRRL B-12247 
(2TA.sup..gamma., SG.sup..gamma., His.sup.-, 
FA.sup..gamma.) 
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SD.sup..gamma. : resistance to sulfadiazine 
2TA.sup..gamma. : resistance to 2thiazolealanine 
SG.sup..gamma. : resistance to sulfaguanidine 
His.sup.- : requirement of histidine for growth 
Asp Hd.sup..gamma. : resistance to aspartylhydrazide 
AS.sup..gamma. : resistance to diaminosuccinic acid 
HD.sup..gamma. : resistance to hadacidin 
KM.sup..gamma. : resistance to ketomalonic acid 
FM.sup..gamma. :resistance to fluoromalonic acid 
FA.sup..gamma. : resistance to monofluoroacetic acid 
The method by which the mutants of the present invention were induced are 
shown below: 
Experiment 1 
Brevibacterium flavum AJ 3277 (SD.sup..gamma.) which was derived from ATCC 
14067 was treated with 250 .mu.g/ml N-methyl-N'-nitro-N-nitro-soguanidine 
at 30.degree. C. for 30 minutes. Then, the microbial cells were spread on 
an agar medium containing the amount of keto-malonic acid which inhibit 
the growth of the parent strain. 
After the cultivation, colonies which turned up on the agar medium were 
picked up and their productivities of L-arginine were examined. 
Among the mutants thus obtained, B. flavum AJ 11595 which can produce more 
increased amount of L-arginine than any other mutants was selected. 
The other mutants of this invention were obtained by the analogous manner. 
The degree of the resistance of the mutants of this invention to the 
chemical agents were determined by the following experiment. 
Experiment 2 
Each testing strain was washed with the aqueous culture medium shown in 
Table 1, the cells were suspended in the same medium (the optical density 
at 562 mm of 26 times dilute of the suspension was 0.3 to 0.33), and 0.1 
ml of the suspension was put into 40 ml of the same medium which further 
contains the amount of chemical agents shown in Table 2 and 3 and was 
placed in a small-size test tube. 
TABLE 1 
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Composition of agar-medium (pH: 7.2) 
Component Conc. Component Conc. 
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Glucose 2.0 g/dl FeSO.sub.4.7H.sub.2 O 
10 mg/dl 
Urea 0.3 g/dl MnSO.sub.4.4H.sub.2 O 
1.0 mg/dl 
Ammonium sulfate 
1.0 g/dl Biotin 10 mg/dl 
KH.sub.2 PO.sub.4 
0.1 g/dl Thiamine.HCl 
100 .mu.g/l 
MgSO.sub.4.7H.sub.2 O 
0.04 g/dl 
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A cultivation was carried out at 31.5.degree. C. for 48 hours with shaking. 
Then, the growth of each strain was determined by measuring the optical 
density at 562 mm of the resulted broths and the results are shown in 
Table 2 and 3. In Table 2 and 3 the degree of the resistance is 
represented by the relative values of the growth to the control. 
TABLE 2 
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Degree of Resistance 
Chemical Strains Concentration (g/dl) 
agent tested 0 1 2 4 5 10 
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Keto-malonic 
AJ 3277 100 59 8 5 
acid AJ 11595 " 100 100 20 
AJ 3278 " 44 5 0 
AJ 11598 " 100 95 25 
Fluoro-malonic 
AJ 3277 " 54 5 0 
acid AJ 11596 " 103 102 60 
Monofluoro- 
AJ 3277 " 45 0 0 60 
acetic acid 
AJ 11597 " 110 100 100 
AJ 11193 " 53 4 0 
AJ 11600 " 110 100 90 
AJ 3278 " 47 0 0 
AJ 11599 " 110 90 90 
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TABLE 3 
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Degree of Resistance 
Chemical Strains Concentration (g/dl) 
agent tested 0 0.1 0.5 1.0 
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.beta.-Aspartate 
AJ 3277 100 50 18 8 
hydrazide AJ 11337 100 90 63 27 
AJ 3278 100 45 23 8 
AJ 11341 100 93 51 19 
AJ 11193 100 48 12 7 
AJ 11343 100 91 83 13 
2,6-Diamino 
AJ 3277 100 55 22 10 
succinate AJ 11338 100 92 65 25 
AJ 3278 100 43 19 5 
AJ 11342 100 91 55 23 
Hadacidin AJ 3277 100 55 16 9 
AJ 11339 100 85 65 23 
AJ 11344 100 92 79 17 
AJ 11193 100 41 8 4 
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The mutant are cultured aerobically in a conventional culture medium 
containing carbon sources, nitrogen sources, and inorganic ions, and when 
required minor nutrients. 
As the carbon sources saccharides such as glucose, fructose and sucrose, 
and molasses and starch hydrolyzed containing those saccharides, organic 
acids such as acetic acid and propionic acid, and alcohols can be used 
preferably. Nitrogen source are, for example, ammonia sulfate, gaseous 
ammonia and urea. 
Cultivation is carried out preferably under aerobic condition for 2 to 7 
days and the temperature of the culture medium is controlled in the range 
from 24.degree. to 37.degree. C., preferably adjusting the pH of the 
medium at 5.0 to 9.0 with organic or inorganic acid or alkali. For this 
purpose, urea, CaCO.sub.3 or gaseous ammonia may also be used. 
L-arginine accumulated in the culture broth may be recovered by an entirely 
conventional recovering method such as using an ion exchanging resine.

The invention will now be illustrated by the following Examples. 
EXAMPLE 1 
Twenty five ml portions of Culture medium (A) of which the composition is 
given in Table 4 were put into 500 ml-flasks, and heated at 110.degree. C. 
for 5 minutes for sterilization. Then each flask was supplemented with 1.0 
g CaCO.sub.3 separately sterilized. 
TABLE 4 
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Composition of Culture Medium 
Medium 
Component A B C D 
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Glucose (g/dl) 10.0 3.0 -- -- 
Ethanol (g/dl) -- -- 1.0 1.5 
Ammonium sulfate 
" 6.0 2.0 0.5 -- 
Ammonium acetate 
" -- 0.5 -- 0.3 
Urea " -- 0.2 0.2 -- 
KH.sub.2 PO.sub.4 
" 0.1 0.1 0.1 0.1 
MgSO.sub.4 " 0.04 0.04 0.04 0.04 
FeSO.sub.4 (mg/dl) 1.0 1.0 1.0 1.0 
MnSO.sub.4 " 1.0 1.0 1.0 1.0 
Bitotin (.mu.g/l) 
50 50 50 50 
Thiamine.HCl 
" 20 20 50 200 
Soy protein 
hydrolyzate (7.0%) 
(ml/dl) 1.0 2.5 2.5 1.5 
CaCO.sub.3 (g/dl) 5.0 -- -- -- 
pH 7.0 7.5 7.5 7.5 
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Brevibacterium flavum AJ 11600 and AJ 11343 previously cultured on bovillon 
agar slants were inoculated into the each bach of culture medium, and 
cultured with shaking at 31.degree. C. for 72 hours. After 72 hours 
cultivation, determination of L-arginine accumulated in the resultant 
culture broth were carried out colorimetrically and the results are shown 
in Table 5: 
TABLE 5 
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Strain L-arginine accumulated 
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AJ 11600 3.6 g/dl 
AJ 11343 3.5 g/dl 
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One liter of culture broth of AJ 11600 prepared by the same manner as above 
were collected and centrifuged to remove microbial cells and solid 
CaCO.sub.3. One liter supernatant solution thus obtained was passed 
through the column of "Amberlite C-50" in the form of NH.sub.4.sup.+, 
thereby L-arginine was adsorbed on the resine, and it was eluted with 2 N 
ammonia water. The elute was evaporated and the concentrated solution was 
cooled to a low temperature enough to crystalize L-arginine. After the 
completion of the crystallization, 23.3 g crystalline L-arginine was 
separated from the mother liquor. 
In a similar manner as above, 22.7 g crystalline L-arginine was obtained 
from the culture broth of AJ 11343. 
EXAMPLE 2 
Each of the strains listed in Table 5 previously cultured on a bovillon 
agar slant was cultured in the same manner as described in Example 1 and 
the amount of L-arginine accumulated in the cultured broth was determined. 
The result is given in the table below. 
TABLE 5 
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Strain L-arginine accumulated (g/dl) 
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AJ 3277 1.80 
AJ 11595 1.92 
AJ 11596 1.84 
AJ 11597 1.90 
AJ 11337 2.00 
AJ 11338 1.90 
AJ 11339 1.85 
AJ 3278 1.70 
AJ 11598 1.86 
AJ 11599 1.91 
AJ 11341 1.90 
AJ 11342 1.85 
AJ 11193 3.30 
AJ 11600 3.60 
AJ 11344 3.45 
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EXAMPLE 3 
Three hundred ml of Medium (C) shown in Table 4 was placed in 1.0 
liter-fermentation vessel and heated at 110.degree. C. for 5 minutes for 
sterilization. Then it was inoculated with 15 ml seed culture broth of 
Brevibacterium flavum AJ 11599 which had been previously culturing in 
Medium (D) aerobically at 31.degree. C. with agitation and aeration. 
During the cultivation, the pH of the medium was maintained in the range 
from 7.2 to 8.0 with addition of acetic acid and acetic acid solution. 
After 55 hours of the cultivation, 4.24 g/dl L-arginine was accumulated in 
the culture broth. The volume of acetic acid solution used during the 
cultivation was 20% to the initial volume of the culture medium. From 300 
ml culture broth thus prepared, 9.20 g crystalline L-arginine was obtained 
in the same manner as described in Example 1. 
EXAMPLE 4 
Brevibacterium flavum AJ 11343 were cultured in Medium (D) shown in Table 4 
at 31.degree.0 C. for 18 hours with agitation and aeration to prepare seed 
culture broth. 
Thereafter 300 ml of Medium (C) was placed in 1.0 liter-fermentation 
vessel, sterilized at 110.degree. C. for 5 minutes, was inoculated with 15 
ml of the seed culture broth and held at a temperature of 31.degree. C. 
with agitation and aeration. During the cultivation, the pH of the medium 
was maintained at a pH ranging from 7.2 to 7.8 with gaseous ammonia. The 
concentration of ethanol in the medium was determined by 
gas-chromatography and small portions of ethanol were fed to the medium 
when the ethanol concentration became about 0.3%. After 48 hours of the 
cultivation, 48 g of ethanol was consumed and 2.57 g/dl of L-arginine was 
found in the culture broth. By the manner shown in Example 1, 4.75 g of 
L-arginine was recovered.