Method for production of L-arginine by fermentation

Abstract

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.

Description

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:

______________________________________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, andCorynebacterium glutamicum ATCC 13032.______________________________________

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:

______________________________________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.)______________________________________ 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______________________________________Composition of agar-medium (pH: 7.2)Component Conc. Component Conc.______________________________________Glucose 2.0 g/dl FeSO.sub.4.7H.sub.2 O 10 mg/dlUrea 0.3 g/dl MnSO.sub.4.4H.sub.2 O 1.0 mg/dlAmmonium sulfate 1.0 g/dl Biotin 10 mg/dlKH.sub.2 PO.sub.4 0.1 g/dl Thiamine.HCl 100 .mu.g/lMgSO.sub.4.7H.sub.2 O 0.04 g/dl______________________________________

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______________________________________Degree of ResistanceChemical Strains Concentration (g/dl)agent tested 0 1 2 4 5 10______________________________________Keto-malonic AJ 3277 100 59 8 5acid AJ 11595 " 100 100 20 AJ 3278 " 44 5 0 AJ 11598 " 100 95 25Fluoro-malonic AJ 3277 " 54 5 0acid AJ 11596 " 103 102 60Monofluoro- AJ 3277 " 45 0 0 60acetic 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______________________________________ TABLE 3______________________________________Degree of ResistanceChemical Strains Concentration (g/dl)agent tested 0 0.1 0.5 1.0______________________________________.beta.-Aspartate AJ 3277 100 50 18 8hydrazide 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 83 132,6-Diamino AJ 3277 100 55 22 10succinate AJ 11338 100 92 65 25 AJ 3278 100 43 19 5 AJ 11342 100 91 55 23Hadacidin AJ 3277 100 55 16 9 AJ 11339 100 85 65 23 AJ 11344 100 92 79 17 AJ 11193 100 41 8 4______________________________________ 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______________________________________Composition of Culture Medium MediumComponent A B C D______________________________________Glucose (g/dl) 10.0 3.0 -- --Ethanol (g/dl) -- -- 1.0 1.5Ammonium sulfate " 6.0 2.0 0.5 --Ammonium acetate " -- 0.5 -- 0.3Urea " -- 0.2 0.2 --KH.sub.2 PO.sub.4 " 0.1 0.1 0.1 0.1MgSO.sub.4 " 0.04 0.04 0.04 0.04FeSO.sub.4 (mg/dl) 1.0 1.0 1.0 1.0MnSO.sub.4 " 1.0 1.0 1.0 1.0Bitotin (.mu.g/l) 50 50 50 50Thiamine.HCl " 20 20 50 200Soy proteinhydrolyzate (7.0%) (ml/dl) 1.0 2.5 2.5 1.5CaCO.sub.3 (g/dl) 5.0 -- -- --pH 7.0 7.5 7.5 7.5______________________________________ 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______________________________________Strain L-arginine accumulated______________________________________AJ 11600 3.6 g/dlAJ 11343 3.5 g/dl______________________________________ 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______________________________________Strain L-arginine accumulated (g/dl)______________________________________AJ 3277 1.80AJ 11595 1.92AJ 11596 1.84AJ 11597 1.90AJ 11337 2.00AJ 11338 1.90AJ 11339 1.85AJ 3278 1.70AJ 11598 1.86AJ 11599 1.91AJ 11341 1.90AJ 11342 1.85AJ 11193 3.30AJ 11600 3.60AJ 11344 3.45______________________________________

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.

Claims

1. A method for the production of L-arginine by fermentation which comprises:

(1) culturing aerobically in a culture medium a mutant capable of producing L-arginine of the genus Brevibacterium or Corynebacterium, and

(2) recovering the L-arginine accumulated in the culture medium; said mutant being resistant to Keto-malonic acid, fluoro-malonic acid, monofluoro-acetic acid or aspartate-antagonist,

wherein said mutant belongs to the species Brevibacterium flavum or Corynebacterium acetoacidophilum selected from the class consisting of

______________________________________Brevibacterium flavum NRRL B-12235Brevibacterium flavum NRRL B-12236Brevibacterium flavum NRRL B-12237Brevibacterium flavum NRRL B-12240Brevibacterium flavum NRRL B-12242Brevibacterium flavum NRRL B-12243Brevibacterium flavum NRRL B-12244Brevibacterium flavum NRRL B-12241Brevibacterium flavum NRRL B-12247Corynebacterium acetoacidophilum NRRL B-12238Corynebacterium acetoacidophilum NRRL B-12239Corynebacterium acetoacidophilum NRRL B-12245Corynebacterium acetoacidophilum NRRL B-12246______________________________________

2. The method according to claim 1 where the mutant resistant to keto-malonic acid is Brevibacterium flavum NRRL B-12242 or Brevibacterium flavum NRRL B-12245.

3. The method according to claim 1 where the mutant resistant to fluoro-malonic acid is Brevibacterium flavum NRRL B-12243.

4. The method according to claim 1 where the mutant resistant to monofluoro-acetic acid is Brevibacterium flavum NRRL B-12244 or Brevibacterium flavum NRRL B-12247, or Corynebacterium acetoacidophilum NRRL B-12246.

5. The method according to claim 1 where the mutant resistant to aspartate-antagonist is Brevibacterium flavum NRRL B-12235 or Corynebacterium acetoacidophilum NRRL B-12238 or Brevibacterium flavum NRRL B-12240.