GB2071646A - Method of Producing L- glutamic Acid by Fermentation - Google Patents

Abstract

An improved method for making L-glutamic acid by fermentation involves aerobic culture in an aqueous medium of a mutant induced from an L-glutamic acid-producing micro- organism of the genus Brevibacterium or Corynebacterium, the mutant being resistant to glutamic acid or glutamic acid analogues.

Description

SPECIFICATION Method for Producing L-Glutamic Acid by Fermentation This invention relates to a method of producing L-glutamic acid by fermentation. L-glutamic acid, of which mono-sodium salts are used as seasonings, has been produced by fermentation processes using microorganisms of the genus Brevibacterium or Corynebacterium.

We have successfully induced, from known L-glutamic acid-producing microorganisms of the genus Brevibacterium and Corynebacterium, mutants resistant to glutamic acid or glutamic acid analogues, which mutants have a higher productivity of L-glutamic acid than the known L-glutamic acid-producing microorganisms.

According to our invention, we provide a method of producing L-glutamic acid by fermentation, which comprises culturing aerobically in an aqueous medium a mutant induced from an L-glutamic acid-producing microorganism of the genus Brevibacterium or Corynebacterium, the mutant being resistant to glutamic acid or to glutamic acid-analogues; and recovering the. L-glutamic acid accumulated in the aqueous medium.

Specimens of the mutants used for practising the method of this invention are as follows: Brevibacterium lactofermentum AJ 1 1292, (FERM-P 4585, NRRLB-12035) (resistant to ammonium glutamate) Brevibacterium lactofermentum AJ 1 1293, (FERM-P 4586, NRRLB-12036) (resistant to glutamic acid-y-monohydroxamate) Brevibacterium flavum AJ 1 1294, (FERM-P 4587, NRRLB-12037) (resistant to ammonium glutamate) Brevibacterium flavum AJ 11295 (FERM-P 4588, NRRL-B 12038) (resistant to a-methylglutamic acid) Corynebacterium acetoacidophilum AJ 11296 (FERM-P 4581, NRRL-B 12039) (resistant to ammonium glutamate) Corynebacterium acetoacidophilum AJ 11297 (FERM-P 4582, NRRL-B 12040) (resistant to glutamic acid-y-monohydroxamate) Corynebacterium glutamicum AJ 11298 (FERM-P 4583, NPRL-B 12041) (resistant to ammonium glutamate) Corynebacterium glutamicum AJ 11299 (FERM-P 4584, NRRL-B 12042) (resistant to a-methylglutamic acid) FERM-P numbers are the accession numbers of the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, 3-go, 1-ban, 1-chome, Yatabecho-higashi, Tsukuba-gun, Ibaragi-ken, Japan. NRRL-numbers are the accession numbers of the Agricultural Research, North Central Region, Northern Regional Research Center, 1815 North University Street, Peoria Illinois 61604, U.S.A.

The morphological characteristics of the above 8 specific mutants are as follows: They have the same morphological characteristics of their respective parent strains on page 5 lines 1-4. The mutants additionally have the characteristics shown in the parentheses.

The characteristics of the parent ATCC Nos. 13869, 14067, 13870 (Corynebacterium Acetoacidophilum) and 13032 have been disclosed in J. Agr. Chem. Soc. Japan Vol 36, 141-159 (1962), U.S. Patent No. 3,1 17,915, and Bull. Agr. Chem. Soc. Japan Vol 22, 176-185 (1958), respectively.

Those mutants were derived from Brevibacterium lactofermentum ATCC 13869, Brevibacterium flavum ATCC 14067 Corynebacterium Acetoacidophilum ATCC 1387 and Corynebacterium glutamicum ATCC 13032, respectively. Brevibacterium divaricatum NRRLB-231 1, Brevibacterium snccharoliticum ATCC 14066, Corynebacterium acetoglutamicum ATCC 15806, Microbacterium ammoniaphilum ATCC 1 5354 and other so-called Coryne-form L-glutamic acid-producing bacteria can also be used as the parent strain to induce the mutants of this invention.

When other biological characteristics, which are known to be effective for the production of Lglutamic acid such as sensitivity to N-palmitoyl-glutamic acid, are also present in the mutant, the yield of L-glutamic acid can be increased still more.

The methods of mutation are conventional such as exposing the parent strain to ultra-violet light, or to X-rays or to chemical mutagents. For example, the parent strains are exposed to N-nitro-N'methyl-N-nitrosoguanidine at 300C for 20 minutes.

The screening of the mutant strains of this invention from the exposed parent strains can be effected by isolating the strains which can grow in a medium which contains the amounts of glutamic acid or glutamic acid or glutamic acid analogues inhibitive to the growth of the parent strains.

The glutamic acid analogues which inhibit the growth of the parent strains of this invention, the inhibition being suppressed by L-glutamic acid, are, for example, a-methylglutamic acid, P- hydroxyglutamic acid, glutamic acid-y-monohydroxamate, a-amino-4-phosphono-butyric acid, glutamic acid-y-methylester, glutamic acid-dimethylester, glutamic acid-y-ethylester, glutamic acid-dit-butylester and glutamic acid-diethylester.

The mutants resistant to glutamic acid or to glutamic acid analogues can grow in an agarmedium which contains an amount of glutamic acid analogues inhibitive to the growth of the parent strains.

The degrees of resistance of the mutants mentioned above to glutamic acid and to glutamic acid analogues are shown in Tables 1 and 2 below, as follows: Table 1

Ammonium glutamate ATCC AJ ATCC AJ ATCC AJ ATCC AJ gIdl(as glutamic acid 13869 11292 14067 11294 13870 11296 13032 11298 0 100 100 100 100 100 100 100 100 1 105 105 95 101 98 99 101 102 5 102 110 98 97 96 97 98 99 10 85 102 65 95 68 96 95 96 15 50 95 40 68 52 96 77 95 20 10 60 20 30 20 58 52 85 Table 2

L-glutamic acidy-mono ATCC AJ ATCC AJ ATCC AJ ATCC AJ hydroxamate yg/ml 13869 11293 14067 11295 13870 11297 13032 11299 0 100 100 100 100 100 100 100 100 10 99 99 72 98 95 99 96 98 20 85 97 52 95 90 98 92 97 50 70 96 45 93 72 95 72 95 100 55 60 30 65 54 88 55 88 1000 20 50 15 55 36 60 43 55 The resistance of the mutants was determined as follows: The microorganisms to be tested previously cultured on an agar slant containing 1/dl peptone, 1 g"dI yeast extract, 0.5 g/dl NaCI and 2 g/dl agar of pH 7.0, at 300C for 24 hours, were inoculated in aqueous media containing, per deciliter, 0.5 g glucose, 0.15 g urea, 0.15 g (NH4)2SO4, 0.3 g KH2PO4, 0.1 g K2HPO4, 0.01 g MgSO4.7H2O, 0.1 mg CaCI2.2H2O, 10 g thiamine.HCI, 3 y9 biotin, 0.44 mg Na2S4O7. 10H20,4.85 mg mg FeCI3.6H2O, 1.95 mg CuSO4.5H2O, 0.185 mg (NH4)6MowO24.4H2O, 44 mg ZnSO4.7H20:0.36 mg MnCI2.4H2O and the amount of ammonium-L-glutamate of L-glutamic acid-ymonhydroxamate shown in Tables 1 and 2 above.

After 24 hours' cultivation at 30 C, the growth was determined by measuring the optical density of the aqueous medium.

The fermentation media used in accordance with this invention are conventional media containing carbon sources, nitrogen sources, inorganic salts and, where required, other minor organic nutrients such as vitamins and aminoacids.

The carbon sources used for practising the invention are also conventional such as carbohydrates (sucrose, glucose, molasses or starch hydrolysate), acetic acid or ethanol.

The cultivation in the fermentation media is carried out in a conventional manner.

In the resulting fermentation broth, higher amounts of L-glutamic acid are accumulated than when various known L-glutamic acid-producing bacteria are used.

L-Glutamic acid in the fermentation broth can be recovered by various known methods such as precipitation at the isoelectric pH.

The invention is illustrated by the following Examples.

Example 1 Thirty milliliter batches of a fermentation medium containing, per milliliter, 100 mg sugar of cane molasses 1 mg KH2PO4. 1 mg MgSO4.7H2O, and thiamine.HCI, of pH 7.0, were placed in 500 ml shaking flasks, and heated.

The microorganisms listed in Table 3 below were inoculated in the fermentation media and cultured at 31 .50C with shaking. During the cultivation, a small portion of urea solution (containing 400 mg/ml urea) was fed to the fermentation media to maintain the pH of the fermentation media at from 6.5 to8.0.

When the optical density at 562 mu of a 26 times dilution of each fermentation medium reached 0.3, polyoxyethylenesorbitanmonopalmitate (PESP) (3 mg/ml) was added to the medium. After 36 hours' cultivation, the amounts of L-glutamic acid shown in Table 3 were accumulated.

Table 3

c Microorganism tested ATCC AJ ATCC AJ 138F9 11292 14067 11294 Yield of L-glutamic acid (weight percent) 55 58 49 52 ATCC AJ ATCC AJ Microorganism tested 13870 11296 13032 11298 Yield of L-glutamic acid {weight percent) 50 52 51 53 Example 2 An aqueous medium was prepared to contain, per milliliter, 50 mg (as sugar) of beet molasses 1.0 mg heH2PO4, 1.0 mg MgSO4.7H2O, 10 ,ug FeSO4.7H2O, 8 149 MnSO4. 4H2O, and 1 yg thiamine.HCI, adjusted to pH 7.0 and 300 ml batches of the medium were placed in a 11 fermenter and heated to sterilization point.

The microorganisms listed in Table 4 (below were inoculated in the media and cultured at 31 .50C with agitation and aeration. During the cultivation, gaseous ammonia was fed into the medium so as to maintain the pH of the medium at 7.8. When the optical density at 562 ml of a 26 times dilution of the culture medium reached 0.3, 0.5 mg/ml PESP was added to the medium.

After 24 hours' cultivation, L-glutamic acid arcumulated in the resulting culture liquid was determined. The results are. shown in Table 4, as follows: Table 4

Microorganism tested Yield of L-glutamic acid AJ11293 65% ATCC 13869 60% AJ 11297 54% ATCC 13870 50% Example 3 Twenty milliliter batches of an aqueous culture medium containing, per milliliter, 36 mg glucose, 2 mg urea, 1 mg KH2PO4, 0.4 mg MgS04.7H20, 10 ,ug FeS04.7H20, 8 g MnSO4.4H20, 5 u1 soyprotein-hydrolysate 0.1 fLg thiamine.HCI and 0.005 ,ug biotin were placed in 500 ml shaking flasks and heated at 11 50C for 10 minutes. Brevibacterium lactofermentum AJ 11292 or its parent ATCC 13869 was inoculated in the culture medium and cultured at 31 .50C with shaking.

During the cultivation, a small portion of urea solution (of 450 mg/ml) was fed to the medium to maintain the pH of the medium in the range from 6.5 to 8.0. When the optical density at 562 um of a 26 times dilution of the culture medium reached 0.3, PESP was added to the culture medium to contain 3 mg/ml.

AJ 11292 produced 19.8 mg/dl L-glutamic acid in the resulting culture liquid, and ATCC 13869 produced 18.7 mg/dl L-glutamic acid.

Example 4 Twenty milliliter batches of a culture medium containing, per milliliter, 100 mg raw sugar. 1 mg KH2PO4, 0.4 mg MgS04.7H20,10 y9 FeS04.7H20,8,ug MnSO4.4H20, 5 mg urea and 5 yl of soyprotein-acid-hydrolysate were placed in 500 ml shaking flasks, and each batch was heated to sterilization.

AJ 11299 or ATCC 13032 was inoculated in the culture medium and cultured at 31 C with shaking.

During the cultivation, a small portion of a urea solution containing 400 mg/ml urea was fed to maintain the pH of the medium at from 6.5 to 8.0. When the optical density at 562 m,u of a 26 times' dilution of the medium reached 0.30, and 3 mg/ml PESP were added to the medium.

After 30 hours' cultivation, AJ 11299 produced 50 mg/ml of ~L-glutamic acid in the culture medium, and ATCC 13032 produced 48 mg/ml L-glutamic acid.

Example 5 Brevibacterium lactofermentum AJ 11292 was cultured in 300 ml of the following seed culture medium placed in an 11-fermenter.

Seed culture medium: glucose 5.0 g/dl KH2PO4 0.1 g/dl MgS04.7HzO 40 mg/dl FeS04-7H20 1.0 mg/dl MnSO4.4H20 1.0 mg/dl Soy-protein hydrolysate 24 mg/dl (as total nitrogen) urea 0.2 g/dl biotin 30,ug/dl thiamine-HCI 20 ,ug/dl The following culture medium (300 ml) was placed in an 1 I-fermenter and heated to sterilization.

cane molasses 6.0 g/dl (as sugar) KH2PO4 0.2 g/dl MgS04.7HzO 80 mg/dl FeS04-7H20 2.0 mg/dl Soy-protein hydrolsate 6.8 mg/dl (as total nitrogen) thiamine.HCl 20,ug/dI The above seed culture broth (15 ml) was transferred into the 300 ml culture medium, and cultivation was carried out at 31.50C, the pH of the medium being adjusted to 7.8. When the optical density at 562 mu of 26 times' dilution of the culture medium became 0.45, and polyoxyethylenesorbitane-monopalmitate was added to contain 0.4 g/dl in the medium. The sugar concentration was maintained at 2~4g/dl by feeding cane molasses containing 45 g/dl sugar.

When the cultivation was continued for 33 hours, 1 70 ml of the cane molasses were fed and 20.1 g/dl sugar were used.

Brevibacterium lactofermentum ATCC 13869 was cultured by the same method as above.

The results are shown in Table 5, as follows: Table 5

L-glutamic acid accumulated Yieldfweightbasesl Brev. Iactofermentum AJ 1 1292 9.84 g/d 48.0% ATCC 13869 9.21 g/d 45.8%

Claims (3)

Claims

1. A method of producing L-glutamic acid by fermentation which comprises culturing aerobically in an aqueous medium a mutant induced from an L-glutamic acid-producing microorganism of the genus Brevibacterium or Corynebacterium, the mutant being resistant to glutamic acid or glutamic acid-analogues: and recovering the L-glutamic acid accumulated in the aqueous medium.

2. A method according to claim 1, wherein said L-glutamic acid-producing microorganism is selected from: A-A (see pages 2 and 3).

3. A method according to claim 1 substantially as herein described with reference to any of the specific examples.