Abstract:
Mutants of the genus Brevibacterium or Coryne-bacterium resistant to a respiratory inhibitor or ADP phosphorylation inhibitor produced L-glutamic acid in a high yield, when they are cultured aerobically in an aqueous culture medium.

Description:
FIELD OF THE INVENTION 
     This invention relates to a method for producing L-glutamic acid by fermentation. 
     DESCRIPTION OF THE PRIOR ART 
     L-Glutamic acid has been produced by fermentation using a microorganism of the genus Brevibacterium or Corynebacterium. Various attempts have been done to improve the productivity of the known glutamic acid producing strains by artificial mutation techniques. Examples of such artificial mutants are mutants of Brevibacterium resistant to S-2-amino-ethyl-cysteine (Japanese Published Unexamined Patent Application No. 126877/1975, mutants of Brevibacterium and Corynebacterium resistant to fluorocitric acid, ketomalonic acid, α-amino-β-hydroxyvaleric acid, DL-threoninehydroxamate, 2-amino-3-phosphopropionic acid or 5-aminolevulinic acid, (Japanese Published Unexamined Patent Application No. 89045/1979), mutants of Brevibacterium and Corynebacterium sensitive to lysozyme (Japanese Published Unexamined Patent Application No. 122794/1979), mutant of Brevibacterium and Corynebacterium having reduced activity of pyruvic acid dehydrogenase (Japanese Published Unexamined Patent Application No. 21762/1980), mutants resistant to glutamic acid or glutamic acid-analogue of Brevibacterium or Corynebacterium (Japnese Published Unexamined Patent Application No. 21763/1980), and mutants of Brevibacteirum resistant to 2,6-pyridine-dicarboxylic acid (Japanese Published Unexamined Patent Application No. 21764/1980). 
     SUMMARY OF THE INVENTION 
     It is now found that mutants of the genus Brevibacterium or Corynebacterium resistant to a respiratory inhibitor or ADP phosphorylation inhibitor produce L-glutamic acid in an improved yield. 
     Now, it is provided a method for producing L-glutamic acid which comprises culturing in an aqueous culture medium a mutant of the genus Brevibacterium or Corynebacterium which is resistant to a resipiratory inhibitor or ADP phosphorylation inhibitor, and recovering L-glutamic acid accumulated in the resulted culture liquid. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The respiratory inhibitors of this invention inhibit, as is well known, respiration of the microorganisms, and then their growth. The examples of the respiratory inhibitors are malonic acid, potassium cyanid, sodium azide, sodium arsenite. 
     The ADP phosphorylation inhibitors inhibit the phosphorylation of ADP (adenosine diphosphate) of microorganisms, and production of ATP (adenosine triphosphate), and then their growth. ADP phosphorylation inhibitors include uncouplers, which inhibit coupling reaction in respiratory chain, such as 2,4-dinitrophenol, hydroxylamine and arsenic, and energy trasfer inhibitors, which inhibit energy transferring reaction such as guanidine. 
     Specimens of the mutants of this invention are: 
     Brevibacterium lactofermentum AJ 11426 (FERM-P 5123, NRRL B-12211), (malonic acid) 
     Brevibacterium flavum AJ 11427 (FERM-P 5124, NRRL B-12203), (malonic acid) 
     Corynebacterium glutamicum AJ 11441 (FERM-P 5138, NRRL B-12210), (malonic acid) 
     Brevibacterium lactofermentum AJ 11428 (FERM-P 5125, NRRL B-12212), (sodium azide) 
     Brevibacterium lactofermentum AJ 11429 (FERM-P 5126, NRRL B-12213), (potassium cyanid) 
     Brevibaterium flavum AJ 11430 (FERM-P 5127, NRRL B-12204), (potassium cyanid) 
     Corynebacterium glutamicum AJ 11431 (FERM-P 5128, NRRL B-12207), (potassium cyanid) 
     Brevibacterium lactofermentum AJ 11432 (FERM-P 5129, NRRL B-12214), (sodium arsenite) 
     Brevibacterium lactofermentum AJ 11433 (FERM-P 5130, NRRL B-12215), (2,4-dinitrophenol) 
     Brevibacterium flavum AJ 11434 (FERM-P 5131, NRRL B-12205), (2,4-dinitrophenol) 
     Brevibacterium lactofermentum AJ 11435 (FERM-P 5132, NRRL B-12216), (hydroxylamine hydrochloride) 
     Corynebacterium glutamicum AJ 11436 (FERM-P 5133, NRRL B-12208), (hydroxylamine hydrochloride) 
     Brevibacterium lactofermentum AJ 11437 (FERM-P 5134, NRRL B-12217), (arsenic) 
     Brevibacterium lactofermentum AJ 11438 (FERM-P 5135, NRRL B-12218), (guanidine) 
     Brevibacterium flavum AJ 11439 (FERM-P 5136, NRRL B-12206), (guanidine) 
     Corynebacterium glutamicum AJ 11440 (FERM-P 5137, NRRL B-12209), (guanidine) 
     The mutants of Brevibacterium flavum, Brevibacterium lactofermentum and Corynebacterium glutamicum were derived from the parent strains, Brevibacterium flavum ATCC 14067, Brevibacterium lactofermentum ATCC 13869 and Corynebacterium glutamicum ATCC 13032, respectively. Brevibacterium divaricatum ATCC 14020, Brevibacterium saccharoliticum ATCC 14066, Corynebacterium acetoacidophilum ATCC 13870, and other glutamic acid-producing bacteria of the genera Corynebacterium and Brevibacterium can be used as the parent strains. 
     The parent strains can be mutated to obtain the mutants of this invention by a conventional manner such as UV-radiation X-ray radiation, exposure to a mutagen. For instance, the parent strain is mutated by exposing to 250 μg/ml N-nitro-N&#39;-methyl-N-nitrosoguanidine at 30° C. fof 20 minutes. 
     Inhibitory action to the growth of the mutants of this invention and their parents in the presence of the respective inhibitors to which the mutants resist was tested and the results are shown in Tables 1, 2 and 3. As can be seen in Tables 1, 2 and 3, the growth of the mutants of this invention was less inhibited than that of their parents by the inhibitors. 
     The determination of growth was performed by the following way; the medium containing 1 g/dl yeast extract, 1 g/dl peptone, 0.5 g/dl sodium chloride and 2 g/dl agar of pH 7.0 was sterilized at 120° C. for 15 minutes, and was poured into plates. The plates were inoculated with the microorganisms, which had been cultured on meat extract agar slant for 24 hours and had been suspended into sterilized water. The inoculum size was 10 5  ˜10 6  cells per plate. A paper disk containing the amount of inhibitor shown in each Table was placed on each plate. Growth around the paper disk was observed after 16 to 48 hours incubation of the plates at 30° C. 
     In Tables &#34;++&#34;, &#34;+&#34; and &#34;-&#34; show that good growth, poor growth and scarce growth were observed around the disk, respectively. 
     
                       TABLE 1______________________________________inhibitor indisk(mg/ml)    Growth______________________________________    ATCC    AJ      ATCC  AJ    ATCC  AJmalonic acid    13869   11426   14067 11427 13032 11441______________________________________1        ++      ++      ++    ++    +     +3        ±    ++      ±  ++    -     +5        -       +       -     +     -     +10       -       +       -     -     -     -______________________________________    ATCC    AJ      ATCC  AJ    ATCC  AJKCN      13869   1429    14067 11430 13032 11431______________________________________0.1      +       ++      +     ++    +     +0.3      ±    +       +     + +   ±  +0.5      -       +       -     +     -     +0.8      -       -       -     -     -     -______________________________________    ATCC    AJNaN.sub.3    13869   11428______________________________________ 0.01    ±    + 0.05    -       +0.1      -       -0.5      -       -______________________________________    ATCC    AJNa.sub.3 AsO.sub.3    13869   11432______________________________________0.5      ++      +++1.0      -       ++5.0      -       +10       -       -______________________________________ 
    
     
                                           TABLE 2__________________________________________________________________________2,4-Di-nitroGrowth          Hydro- Growth               GrowthphenolATCC    AJ  ATCC            AJ  xylamine                       ATCC                           AJ  ATCC                                   AJ  Arsenic                                            ATCC                                                AJ(mg/ml)13869    11433        14067            11434                HCl(mg/ml)                       13869                           11435                               13032                                   11436                                       (mg/dl)                                            13869                                                11437__________________________________________________________________________0.1  ++  ++  ++  ++  0.5    ++  ++  ++  ++  1    ++  ++0.5  +   ++  ±            ++  1.0    -   ++  +   ++  3    +   ++0.8  -   +   +   +   3.0    -   +   -   +   5    -   +1.0  -   -   -   -   5.0    -   -   -   -   10   -   +__________________________________________________________________________ 
    
     
                       TABLE 3______________________________________GrowthGuanidine   ATCC    AJ      ATCC  AJ    ATCC  AJ(mg/ml) 13869   11438   14067 11439 13032 11440______________________________________1       ++      ++      ++    ++    +     +3       -       ++      -     ++    -     +5       -       +       -     +     -     -10      -       -       -     -     -     -______________________________________ 
    
     The effect of respiratory inhibitor on NADH oxidation of the mutants resistant to respiratory inhibitor and their parents was tested, and the results are shown in Table 4. 
     NADH oxidation activity was determined by the following manner: Ten ml batches of reaction mixture containing 500 μmoles of phosphate buffer (pH 7.5), 6 μmoles of NADH, a certain volume of cell extract of the test strain and the amount of respiratory inhibitor shown in Table 4 were kept airtight in vessels having oxygen sensor &#34;Beckman model 777&#34;, stirred, and held at 32° C. NADH oxidation activity was determined based on the decreasing rate of the partial pressure of oxygen, It was observed that the NADA oxidation activity of AJ 11429, AJ 11430 and AJ 11431 was less reduced by the inhibitors shown in Table 4 than that of their parents. 
     
                                           TABLE 4__________________________________________________________________________       Relative activity of NADH oxidationInhibitor  conc.       ATCC AJ  ATCC                    AJ   ATCC                             AJadded  (mg/ml)       13869            11429                14067                    11430                         13032                             11431__________________________________________________________________________malonic acid  5    40   70  35  75   20  75KCN    0.5  0    85  0   75   0   65NaN.sub.3  0.05 0    50  0   60   0   75Na.sub.3 AsO.sub.3  1.0  15   90  25  95   10  80none   --   100  100 100 100  100 100__________________________________________________________________________ 
    
     Respiration activity and ADP phosphorylation activity of the mutant resistant to ADP phosphorylation inhibitor and their parent were determined in the presence of uncoupler (Table 5) or energy transfer inhibitor (Table 6). 
     Determination of respiration activity and ADP phosphorylation activity was carried out as follows: 
     RESPIRATION ACTIVITY 
     Test strains were cultured for 20 hours in a medium containing, per milliliter, 36 mg glucose, 2 mg urea, 1 mg KH 2  PO 4 , 0.4 mg MgSO 4 .7H 2  O, 10 μg FeSO 4 .7H 2  O, 8 μg MnSO 4 .4H 2  O, 5 μl soybean acid-hydrolysate, 0.1 μg thiamine.HCl, and 0.0025 μg biotin, Cells in the resulted culture liquids were harvested, washed twice with 0.5% NaCl, and suspended in 1/15 M phosphate buffer of pH 7.5. The cell suspension was shaken at 28° C. for 3 hours, and 0.5 ml of the resting cell suspension was put into 2 ml of a reaction mixture containing 100 μmoles of phosphate buffer of pH 7.5, 100 μmoles of glucose and the amount of the inhibitor shown in Table 5 or 6. The reaction mixture was held at 32° C. for  3 hours, and oxygen uptake was measured with warburg manometer. 
     ADP PHOSPHORYLATION ACTIVITY 
     The resting cells mentioned above were dried at a room temperature and dried further in a phosphorus pentoxide desiccator under a vacuum overnight. Fifty mg of the dried cells were put into 2 ml of a reaction mixture containing 10 μmoles ADP, 200 μmoles glucose 250 μmoles phosphate buffer, 5 μmoles MgCl 2 .6H 2  O and the amount of the inhibitor shown in Table 5 or 6. The reaction mixture was held at 30° C. for 3 hours, and then heated in boiling water for 3 minutes. ATP formed in the reaction mixture was determined colorimetrically. 
     
                                           TABLE 5__________________________________________________________________________              Relative activityUnconpler conc.      Activity              ATCC                  AJ  ATCC                          AJ  ATCC                                  AJadded (mg/ml)      determined              13869                  11433                      14067                          11434                              13032                                  11436__________________________________________________________________________2,4-dini- 0.5  repiration              125 160 145 145 135 140trophenol  ADP phospho-              0   65  0   70  0   50      rylationhydroxyla- 1.0  respiration              130 175 120 150 135 160mine       ADP phospho-              0   50  0   65  0   45      rylationarsenic 5.0  respiration              130 160 145 160 140 160      ADP Phospho-              0   50  0   70  0   45      rylationnone  --   repiration              100 100 100 100 100 100      ADP phospho-              100 100 100 100 100 100      rylation__________________________________________________________________________ 
    
     
                                           TABLE 6__________________________________________________________________________Relative activityconc.     Activity             ATCC                 AJ  ATCC                         AJ  ATCC                                 AJInhibitor(mg/ml)     determined             13869                 11438                     14067                         11439                             13032                                 11440__________________________________________________________________________guanidine3    respiration             100 110 100 110 100 100     ADP phospho-              0   85  15  80  10  75     rylationnone --   respiration             100 100 100 100 100 100     ADP phospho-             100 100 100 100 100 100     rylation__________________________________________________________________________ 
    
     From these experiments, it was observed that ADP phosphorylation activity of both of the mutants resistant to uncoupler and mutant resistant to energy transfer inhibitor was less decreased by uncoupler or energy transfer inhibitor than that of their parents. 
     The mutants of this invention are cultured to produce L-glutamic acid by conventional manner. Namely, the cultivation is carried out under an aerobic condition at a temperature in the range from 30° C. to 40° C. The culture medium employed in this invention contains carbon sources, nitrogen sources and inorganic ions as in conventional medium. 
     Preferred carbon sources are carbohydrates (sucrose, glucose, fructose and crude materials containing the carbohydrates such as cane molasses beet molasses and starch hydrolyzate), acetic acid, and ethanol. Most preferred nitrogen sources are urea, gaseous ammonia and aqueous ammonia. 
     L-Glutamic acid thus accumulated in the resulted culture liquid can be recovered also by conventional manner. 
    
    
     EXAMPLE 1 
     Twenty milliliter batches of a culture medium containing, per milliliter, 36 mg glucose, 2 mg urea, 1 mg KH 2  PO 4 , 0.4 mg MgSO 4 .7H 2  O, 10 μg FeSO 4 .7H 2  O, 8 μg MnSO 4 .4H 2  O, 0.1 μg thiamine.HCl, 0.0025 μg biotin and 5 μl soy protein hydrolyzate, were placed in 500 ml shaking flasks, and sterilized at 115° C. for 10 minutes. 
     The microorganisms listed in Table 7 below were inoculated in the culture media and cultured at 31.5° C. with shaking. During the cultivation, a small portion of urea solution (containing 450 mg/ml urea) was fed to the fermentation media to maintain the pH of the fermentation media at from 6.5 to 8.0. 
     After 30 hours&#39; cultivation, the amounts of L-glutamic acid shown in Table 7 were accumulated. 
     EXAMPLE 2 
     An aqueous medium was prepared to contain, per milliliter, 100 mg (as sugar) of cane molasses, 1.0 mg KH 2  PO 4 , 1.0 mg MgSO 4 .7H 2  O, and 0.1 μg thiamine.HCl, adjust to pH 7.0, 30 ml batches of the medium were placed in 500 ml flasks and heated to sterilize. 
     The microorganisms listed in Table 8 were inoculated in the media and cultured at 31.5° C. with shaking. During the cultivation, an urea solution containing 400 mg/ml urea was fed in the medium so as to maintain the pH of the medium at 6.5˜8.0. When the optical density at 562 mμ of a 26 times dilution of the culture medium reached 0.3, 0.5 mg/ml polyoxyethylenesorbitanmonopalmitate were added to the medium. 
     After 36 hours&#39; of the cultivation, the amounts of L-glutamic acid shown in Table 8 were accumulated. 
     
                       TABLE 7______________________________________            L-Glutamic acidMicroorganism tested            accumulated (g/l)______________________________________ATCC 13869       16.2ATCC 14067       15.0ATCC 13032       15.5AJ 11426         17.5AJ 11427         17.0AJ 11428         17.8AJ 11429         17.8AJ 11430         18.2AJ 11431         17.5AJ 11432         18.0AJ 11433         17.5AJ 11434         17.0AJ 11435         18.0AJ 11436         17.2AJ 11437         16.9AJ 11438         18.0AJ 11439         18.0AJ 11440         17.5AJ 11441         17.8______________________________________ 
    
     
                       TABLE 8______________________________________            L-Glutamic acidmicroorganism tested            accumulated (g/l)______________________________________AJ 11426         52.0AJ 11427         49.5AJ 11429         51.5AJ 11430         50.3AJ 11431         51.0AJ 11433         50.9AJ 11434         50.3AJ 11435         51.5AJ 11436         50.8AJ 11438         52.9AJ 11439         52.0AJ 11440         52.5AJ 11441         50.8______________________________________