FR2611743A1 - Process for producing L-amino acids or 5'-inosinic acid by fermentation - Google Patents

Abstract

The subject of the invention is a process for the manufacture of an L-amino acid or 5'-inosinic acid. The process according to the invention consists in culturing an L-amino-acid-producing bacterium or a 5'-inosinic-acid-producing bacterium in a medium containing sugar cane molasses, sucrose or glucose as principal carbon source and containing at least one substance chosen from N-methylglycine, N,N-dimethylglycine, N,N,N-trimethylglycine and ( beta -hydroxyethyl)trimethylammonium hydroxide in order to produce an L-amino acid or 5'-inosinic acid and recover the L-amino acid or 5'-inosinic acid.

Description

The present invention relates to L-amino acids such as L-glutamic acid, L-lysine, L-glutamine, L-arginine,
L-phenylalanine, L-threonine, L-isoleucine, L-histidine, L-valine,
L-serine, L-ornithine, L-citrulline, L-tyrosine, L-tryptophan,
L-leucine, etc. and 5'-inosinic acid which have been widely used for seasonings, food additives, drugs, etc.

L-amino acids such as L-glutamic acid,
L-lysine, L-glutamine, L-arginine, L-phenylalanine, L-threonine,
L-isoleucine, L-histidine, L-valine, L-serine, L-ornithine, Lcitrulline, L-tyrosine, L-tryptophan, L-leucine, etc. and 5'-inosinic acid have so far been produced on an industrial scale by fermentation using microorganisms belonging to the genus Brevibacterium, to the genus Corynebacterium, to the genus
Bacillus or the genus Escherichia.

 The problem to be solved by the present invention is to develop a process for the production of L-amino acids or 5'-inosinic acid by fermentation on an industrial scale with low production costs.

Following extensive research to improve the yield of the above L-amino acids or 5'-inosinic acid by a fermentation process, the Applicant has found that the yield of desired L-amino acids or nucleic acid can be markedly improved by fermentation of media added with a defined quantity of at least one substance chosen from N-methylglycine (hereinafter referred to as MG), N, N-dimethylglycine (hereinafter
DMG), N, N, N-trimethylglycine (hereinafter TMG) and choline or (ss-hydroxyethyl) -trimethylammonium hydroxide (hereinafter HETMA).

In order to develop a process for the low cost industrial production of L-amino acids or 5'-inosinic acid, the present invention has been made on the basis of the above finding.

In other words, the present invention relates to a process for the production of an L-amino acid or 5'-inosinic acid which consists in cultivating a bacterium producing L-amino acids or a bacterium producing inosinic acid in a medium containing at least a substance chosen from MG, DMG, TMG and HETMA to produce an L-amino acid or 5'-inosinic acid and to collect L-amino acid or 5'-inosinic acid.

 The discovery was not known that by incorporating MG, DMG, TMG or HETMA into media containing sugars and other major sources of carbon, the yield of L-amino acids or 5'- acid could be improved. inosinique in an industrial scale fermentation process. We simply know the production of L-serine by fermentation from L-betaine using the genus Pseudomonas or the genus Escherichia (Japanese unexamined published patent application No. 49-134896) and the production of L-amino acids by fermentation using choline as the main source of carbon, using the genus Pseudomonas.

 However, in the production of L-serine by fermentation from betaine, betaine is specified as a precursor of serine from which an improved industrial scale yield of L- production cannot be expected. amino acids by fermentation. Furthermore, also in the production of L-amino acids by fermentation using choline as the main source of carbon, choline is used only as the main source of carbon and the yield of L-amino acids is limited to traces.

 On the other hand, it is known that the addition to the media in a defined amount of waste or Stephen residue, formed as a by-product in the stages of production of beet sugar, can clearly improve the yield of L-amino acids or 5 ′ acid -inosinics of a fermentation process (Japanese patent application unexamined published no. 59-216593). However, the discovery was not known that even in media supplemented with Stephen residue, the yield of L could be significantly improved. -amino acids or 5'-inosinic acid of a fermentation process by further adding MG, DMG, TMG or HETMA. The present invention is based on the new discovery which allows a large increase in yield of L-amino acids or of 5′-inosinic acid from a fermentation process, which is also extremely advantageous from the industrial point of view.

The term "L-amino acid" used in the present invention designates L-amino acids such as L-glutamic acid, L-lysine,
L-glutamine, L-arginine, L-phenylalanine, L-threonine, L-isoleucine,
L-tyrosine, L-tryptophan and L-leucine, etc. and although there are other L-amino acids than those described herein, the process of the present invention is applicable as long as they are produced by fermentation.

The L-amino acid producing bacteria used in the present invention can be any microorganism as long as they are capable of producing the
L-amino acids mentioned above.

In particular, the following bacteria can be used: 1. Bacteria producing L-glutamic acid
Brevibacterium divaricatum ATCC 14020
Brevibacterium flavum ATCC 14067
Brevibacterium lactofermentum ATCC 13869
Brevibacterium lactofermentum FERM-P 5012 (AJ 11360)
Corynebacterium acetoacidophilum ATCC 13870
Corynebacterium glutamicum ATCC 13032 2. L-lysine producing bacteria
Brevibacterium fLavum FERM-P 1708 (AJ 3419)
Corynebacterium glutamicum FERM-P 1709 (AJ 3420)
Brevibacterium lactofermentum FERM-P 1712 (A 3425)
Brevibacterium lactofermentum FERM-P 1711 (AJ 3424)
Brevibacterium flavum ATCC 21475
Corynebacterium glutamicum ATCC 12416
Brevibacterium lactofermentum ATCC 11470
Corynebacterium acetoglutamicum ATCC 11472 (AJ 11094) 3. L-glutamine producing bacteria:
Brevibacterium flavum FERM-P 4272
Corynebacterium acetoacidophilum ATCC 13870
Microbacterium flavum FERM-BP 664 (AJ 3684)
Brevibacterium flavum FERM-BP 662 (AJ 3409)
Corynebacterium acetoglutamicum ATCC 13870
Corynebacterium glutamicum FERM-BP 663 (AJ 3682) 4. L-arginine producing bacteria
Brevibacterium flavum FERM-P 4948
Corynebacterium glutamicum FERM-P 7274 (AJ 12093)
Brevibacterium flavum NRRL 12235 (AJ 11337)
Corynebacterium acetoglutamicum NRRL 12239 (AJ 11342) 5.Bacteria producing L-phenylalanine:
Brevibacterium lactofermentum FERM-P 5417
Brevibacterium flavum FERM-P 1916 (AJ 3439)
Corynebacterium glutamicum ATCC 21670
Arthrobacter citreus ATCC 21422
Brevibacterium lactofermentum ATCC 21420
Corynebacterium acetoacidophilum ATCC 21421
Brevibacterium flavum NRRL 12269 (AJ 11476)
Corynebacterium glutamicum ATCC 21670 6. L-threonine producing bacteria
Escherichia coli FERM-P 4900 (AJ 11334) Corynebacterium
glutamicum FERM-P 5835 (AJ 11654)
Brevibacterium flavum FERM-P 4164 (AJ 11172)
Brevibacterium lactofermentum FERM-P 4180 (AJ 11178)
Brevibacterium flavum ATCC 21269
Corynebacterium acetoacidophilum ATCC 21270 7. L-isoleucine producing bacteria
Brevibacterium flavum FERM-P 3672
Brevibacterium lactofermentum FERM-P 4192 (AJ 11190)
Corynebacterium glutamicum FERM-P 756 (AJ 12150)
Corynebacterium glutamicum FERM-P 757 (AJ 12151)
Corynebacterium glutamicum FERM-P 758 (AJ 12153)
Brevibacterium flavum FERM-P 759 (AJ 12149)
Brevibacterium flavum FERM-P 760 (AJ 12152) 8. L-histidine producing bacteria
Brevibacterium flavum FERM-P 2317
Brevibacterium lactofermentum FERM-P 1565 (AJ 3386)
Corynebacterium glutamicum ATCC 14297
Brevibacterium lactofermentum ATCC 21086
Corynebacterium acetoacidophilum ATCC 21407
Brevitacterium flavum ATCC 21406 (AJ 3225)
Bacillus subtilis FERM-BP 217 (AJ 11733)
Arthrobacter citreus ATCC 21412 9. L-valine producing bacteria:
Brevibacterium lactofermentum FERM-P 1845
Brevibacterium flavum FERM-P 512 (AJ 3276)
Corynebacterium lactofermentum FERM-P 1968 (AJ 3455)
Escherichia coli NRRL 12285 (AJ 11470) 10. L-serine producing bacteria:
Corynebacterium glycinophilum FERM-P 1687
Brevitacterium Lactofermentum FERM-P 1371 (AJ 3360)
Pseudomonas megarubessens FERM-P 4532 (AJ 11262) 11. L-çrinithine producing bacteria
Brevibacterium lactofermentum FERM-P 5936 (AJ 11678)
Corynebacterium glutamicum FERM-P 5644 (AJ 11589)
Arthrobacter citreus ATCC 21040
Corynebacterium glutamicum ATCC 13232 12. L-citrulline producing bacteria
Corynebacterium glutamicum FERM-P 5643 (AJ 1158 &
Brevibacterium flavum FERM-P 1645 (AJ 3408) 13. Bacteria producing L-tyrosine:
Corynebacterium glutamicum FERM-P 5836 (AJ 11655)
Brevibacterium flavum FERM-P 7914 (AJ 12180)
Bacillus subtilis FERM-P 6758 (AJ 11968) 14. L-tryptopha e-producing bacteria
Bacillus subtilis FERM-P 5286 (AJ 11483)
Brevibacterium lactofermentum FERM-P 7127 (AJ 12044)
Brevibacterium flavum FERM-P 2551
Corynebacterium glutamicum FERM-P 7128 (AJ 12052)
Brevibacterium flavum ATCC 21427
Brevibacterium flavum FERM-BP 114 (AJ 11667)
Bacillus subtilis FERM-BP 208 (AJ 11713)
Brevibacterium flavum FERM-BP 475 (AJ 12022)
Corynebacterium glutamicum FERM-BP 478 (AJ 12118)
Bacillus subtilis FERM-BP 200 (AJ 11709) 15. L-leucine producing bacteria
Brevibacterium lactofermentum FERM-P 1769 (AJ 3427)
Brevibacterium flavum FERM-P 420 (AJ 3226)
Corynebacterium glutamicum FERM-P 1966 (AJ 3453)
Brevibacterium flavum FERM-BP 755 (AJ 3686)
Corynebacterium glutamicum FERM-BP 756 (AJ 12150)
Corynebacterium glutamicum FERM-P 757 (AJ 12151)
Brevibacterium flavum FERM-BP 759 (AJ 12149) 16. Bacteria producing 5'-inosinic acid
Corynebacterium SP FERM-P 4973 (AJ 11352)
Corynebacterium equi FERM-P 4968 (AJ 11347)
Corynebacterium equi FERM-P 4971 (AJ 11350)
Corynebacterium equi FERM-P 5397 (AJ 11552)
Corynebacterium equi FERM-P 6261 (AJ 11749)
Corynebacterium equi FERM-P 6255 (AJ 11743)
Brevibacterium ammoniagenes FERM-P 7949 (AJ 12192)
Bacillus subtilis FERM-P 4120 (AJ 11158)
Bacillus subtilis FERM-P 6441 (AJ 11820), etc.

As the basic media for the production of L-amino acids or 5'-inosinic acid, conventional media known for the production of
L-amino acids suitable for bacteria producing Lamino acids and media suitable for the production of 5'-inosinic acid by fermentation.

 More particularly, sugars such as glucose, sucrose, fructose, maltose, sweet potato molasses, final sweet potato molasses, beet molasses, final beet molasses, raw sugar, sugar liquor, can be used as the main carbon sources. saccharified starch, saccharified cellulose liquor, saccharified pulp liquor, etc. ; fatty acids such as acetic acid, propionic acid, benzoic acid, etc. ; organic acids such as pyruvic acid, citric acid, succinic acid, fumaric acid, malic acid etc. ; alcohols such as ethyl alcohol, butyl alcohol, etc. ; individually or in mixtures with one another. In addition, it is also possible to use the precursors of the desired L-amino acids in the biosynthesis cycle, etc.

 As nitrogen sources, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc. can be used. ; urea, liquid ammonia, ammonia, in addition mixtures of amino acids which are organic nitrogen compounds, corn soaking liquor, casein acid hydrolyzate, Yeast extract, hydrolyzate soy, peptone, meat extract, etc. As inorganic salts, phosphates, magnesium salts, calcium salts, potassium salts, sodium salts, iron salts, manganese salts, zinc salts, copper and copper salts can be used. '' other metallic trace elements if necessary. In addition, if necessary, vitamins such as biotin, thiamin, etc. can also be used.

The culture conditions in the present invention are the same as in the ordinary production of amino acids by fermentation. Although the conditions vary a little depending on the
L-amino acids or the strain used, the culture temperature is preferably from 20 to 40 "C, in particular from 28 to 37" C. With regard to pH, good results are obtained when the pH is adjusted so that the medium is neutral. The culture is carried out under aerial conditions, for example culture in the air, culture with agitation, mixed culture, etc. The culture time is generally 20 to 7 days but the duration can also be extended by continuous culture, etc. To isolate each amino acid from each amino acid fermentation solution, treatment with an ion exchange resin can be used. and other known recovery techniques.

MG (CHJNHCH2COOH), DMG ((CH3) 2NCH2COOH),
TMG ((CH3) 3N CH2COO or (OH) (CH3) 3N CH2COOH) and HETMA ((OH) (CH3) 3 N + CH2CH2OH) and their salts are accessible not only as reactants, but also as industrial products in large low cost quantities. MG is known by the number of sarcosine and is found in nature in flower moss; MG is prepared by synthesis by condensation of methylamine and chloroacetic acid or by Strecker's synthesis of amino acids, etc. DMG is obtained by reaction of dimethylamine. TMG is known as glycine betaine and is found in large quantities in sugar beets, cotton seeds, etc.

and prepared by synthesis by reaction of chloroacetic acid with trimethylamine. HETMA is known as choline, found in large quantities in radish leaves, etc. and prepared by synthesis by reacting trimethylamine with a concentrated aqueous solution of ethylene oxide. Various other salts of these substances exist commercially; for example; MG sodium salt, DMG and TMG hydrochlorides and chloride, hydrogen tartrate, dihydrogen citrate, gluconate, etc. of HETMA are industrially produced and easily accessible.

 The MG, DMG, TMG or HETMA which are used in the present invention can be in the form of their salts or can be replaced by natural substances containing them.

 To use MG, DMG, TMG or HETMA in the present invention, they can be incorporated, alone or in combination of two or more, in the fermentation medium at a concentration of 0.01 to 5%, preferably from 0.05 to 2.0%. To incorporate them into the fermentation medium, they can be added beforehand to the fermentation medium, then subjected to incubation; or alternatively, they can be incorporated at once, in several portions or continuously during the incubation. In addition, they can be added to the inoculation culture which is then incorporated into the main fermentation.

 The following examples illustrate the invention without, however, limiting its scope.

Example 1
An inoculation medium containing 3 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.05 g / dl of MgSO4.7H20, 1 mg / dl of FeSO4.7H20, 1 is adjusted to pH 7.5. mg / dl MnSO4.4H20, 0.5 g / dl urea, 30 ug / l biotin, 100 pg / l thiamine hydrochloride and soy protein hydrolyzate containing 50 mg / dl nitrogen total and 50 ml of the inoculation medium are loaded into a 500 ml bottle and then sterilized by heating. The medium is inoculated with Brevibacterum lactofermentum ATCC 13869 and cultured in shaken culture for 16 h while maintaining the medium at 31.5 "C.

 On the other hand, a medium containing 8.0 g / dl of sugar cane molasses (in sugar), 0.2 g / dl of KH2PO4, 0.1 g / dl is prepared as the best production medium for glutamic acid. MgSO4.7H20, 2.0 mg / dL FeSO4.4H20, 200 pg / l thiamine hydrochloride, 30 mg / dl soy protein hydrolyzate (total nitrogen) and 200 mg / dl Stephen residue concentrated (total nitrogen concentration 2.0 g / dl) as total nitrogen concentration and a medium additionally containing 0.01 to 5 g / dl of TMG. After adjusting to pH 7.8, 285 ml of each medium are loaded into a small 1.0 I fermenter and then sterilized by heating at 115 "C for 10 min.

 Each medium is inoculated with 15 ml of the inoculation culture solution obtained by prior incubation and the culture is carried out with stirring in air at 31.5 "C. During the culture period, the pH of the solution is maintained culture at 7.8 with ammonia gas and at the same time sugar cane molasses is added beforehand sterilized to adjust the sugar concentration in the culture solution at 2-4 g / dl, calculated as sucrose; in this way, the culture is carried out. At a suitable time during the culture, polyoxyethylenesorbitan monopalmitate is added. When the sugar consumption rate is greatly reduced, the sugar supply is stopped and the fermentation is completed. 28 to 40 h. The quantity of L-glutamic acid accumulated in each culture solution is measured and the yield is determined relative to the sugar The results obtained are shown in Table 1 below.

TABLE 1
Amount of TMG Amount of acid Added yield added L-glutamic to sugar (o / dl) (a / dl) (%)
0 10.0 52.0
0.01 10.3 52.5
0.05 10.5 53.1
0.25 10.7 53.6
0.5 10.7 54.7
1.0 10.8 55.5
2.0 10.5 53.8
3.0 10.5 52.5
5.0 9.8 41.5
Example 2
An inoculation medium containing 5 g / dl of glucose, 0.2 g / dl of (NH4) 2SO4, 0.2 g / dl of urea, 0.15 g / dl of KH2PO4 is adjusted to pH 7.0. , 0.04 g / dl of MgS04.7H20, 1 mg / dl of FeS04.7H20, 100 ug / l of thiamine hydrochloride, 3 ug / l of biotin and 30 mg / dl of acid hydrolyzate of soy proteins as total nitrogen and 50 ml of the inoculation medium are loaded into a 500 ml bottle and sterilized by heating. The medium is inoculated with a glutamine-producing bacterium or Brevibacterium flavum
FERM-BP 662 and cultivated on a shaker for 12 h while maintaining the temperature at 31.50 C.

On the other hand, it is loaded into a 1.0 I fermenter and then sterilized 285 ml of a medium containing 13 g / dl of glucose, 0.25 g / dl of KH2PO4, 40 mg / dl of MgSO4.7H2O, 1.5 g / dl (NH4) 2SO4, 1.5 g / dl Na2SO4, 0.1 mg / dl FeS04.7H20, 3.5 llg / l biotin, 350 ug / l thiamine hydrochloride and 500 mg / dl of residue
Stephen concentrated, formed as a by-product in the steps of Stephen's process in the beet sugar manufacturing plant, as a total nitrogen concentration, and in addition 0.01 to 5 g / dl of HETMA, the medium being adjusted to pH 6.5. The culture is carried out at 31.5 "C with stirring in air while maintaining the pH between 6.5 and 6.0 with ammonia gas, until the remaining glucose reaches 0.5 g / dl.

 The yield of L-glutamine relative to the weight of sugar is shown in Table 2 below.

TABLE 2
Amount of HETMA added Yield of L-glutamine (/ dol) (%)
0 41.0
0.01 41.5
0.05 42.0
0.25 42.5
0.5 43.0
1.0 44.0
2.0 43.0
3.0 41.5
5.0 40.0
In the group with the addition of 0.01 to 3 g / dl of HETMA, the yield is improved compared to the control group. In the group added with 1.0 g / dl of HETMA, 1 l of the culture solution is obtained and the cells are separated by centrifugation. The supernatant liquid is passed through a strongly acidic ion exchange resin "DIA ION" (registered trademark) SK-1B (in the NH4 form).

After washing the resin with water, eluting with 2N NH4OH, the eluate is then concentrated and 35.9 g of crude crystals of L-glutamine are obtained from the concentrate.

Example 3
A medium containing 13 g / dl of glucose, 6 g / dl of (NH4) 2SO4, 0.1 g / dl of KH2PO4, 0.04 g / dl of MgSO4.7H20, 1 mg / is adjusted to pH 7.0 dl of FeS047.H20, 1 mg / dl of MnS04.4H20, 100 tig / dl of thiamine hydrochloride, 50 g / l of biotin, 500 mg / dl of concentrated Stephen residue, formed as a by-product in the stages of Stephen's process in the beet sugar manufacturing plant, as a total nitrogen concentration, with the addition of 0.01 to 3.0 g / dl of HETMA and 5 g / dl of calcium carbonate (sterilized separately), and 20 mg of the medium are loaded into a 500 ml flask and then sterilized by heating.

The medium is inoculated with Brevibacterum flavum
NRRL 12235 by means of a loop of platinum and cultivation on a shaker for 4 days while maintaining the temperature at 31.55C.

The yield of L-arginine relative to the weight of sugar is shown in Table 3 below.

TABLE 3
Amount of HETMA added Yield of L-arginine
(a / dl) (%)
0 30.5
0.01 31.0
0.05 31.3
0.25 32.0
0.5 33.0
1.0 32.0
2.0 31.0
3.0 29.5
In the group with the addition of 0.5 g / dl of HETMA, 1 l of the culture solution is obtained and the cells and other insoluble materials are separated therefrom by centrifugation. The supernatant liquid is passed through a weakly acidic ion exchange resin "AMBERLITE" (registered trademark) C-5O (NH4 form). After washing the resin with water, the L-arginine is eluted with NH4OH 2N, then the eluate is concentrated and 30.5 g of crude crystals of L-arginine are obtained from the concentrate.

Example 4
A medium is prepared for the production of L-isoleucine by adding 0.01 to 3 g / dl of DMG to a medium having the composition indicated in table 4 below, containing glucose as a carbon source. After having adjusted the pH to 7.2, 20 ml of each medium are loaded each time into a 500 ml shaker flask and then sterilized by heating at 115 ° C. for 10 min.

TABLE 4
Concentration component
Glucose 15.0 g / dl (NH4) 2S04 1.0 g / dl KH2PO4 0.15 g / dl
MgS04.7H20 0.04 g / dl FeS04.4H20 1.0 mg / dl MnSO4.4H20 1.0 mg / dl
Thiamine hydrochloride 300.0 ug / dl
Soy protein acid hydrolyzate (total nitrogen) 25.0 mg / dl
Biotin 5.0 ug / dl
Stephen residue (total nitrogen) 200 mg / dl (Calcium carbonate added after separate sterilization 5.0 g / dD
Each medium is inoculated with Brevibacterium flavum
FERM-BP 759 obtained previously by culture and cultured on a shaker at 31.5 "C until the glucose has disappeared.

 The yield of L-isoleucine based on the weight of sugar is indicated in Table 5 below.

TABLE 5
Amount of DMG added Yield of L-isoleucine
(a / dl) (%)
O 12.0
0.1 12.5
0.05 12.7
0.25 13.0
0.5 13.3
1.0 13.5
2.0 13.0
3.0 11.5
Example 5
A medium is prepared for the production of L-valine by adding 0.01 to 3 g / dl of TMG to a medium having the composition indicated in table 6 below and containing glucose as carbon source. After adjusting the pH to 7.0, 20 ml of each medium are added separately each time in a 500 ml flask and then sterilized by heating at 115 ° C. for 10 min.

TABLE 6
Concentration component
Glucose 15.0 g / dl (NH4) 2SO4 3.0 g / dl
KH2P04 0.1 g / dl
MgSO4.7H20 0.04 g / dl FeSO4.4H20 1.0 mg / dl MnSO4. 4H20 1.0 mg / dl
Thiamine hydrochloride 30.0 pg / dl
Soy protein acid hydrolyzate (total N) 80.0 mg / dl
Biotin 5.0 ug / dl
DI-methionine 60.0 mg / dl
Stephen residue (total nitrogen) 200 mg / dl (Calcium carbonate added after sterilization separates 5.0 g / dl)
Each medium is inoculated with Brevibacterium lactofermentum FERM-P1845 obtained previously by culture and cultured on a shaker at 31.5 "C until the glucose disappears.

 The yield of L-valine relative to the weight of sugar is indicated in Table 7 below.

TABLE 7
Amount of TMG added Yield of L-valine
(q / dl) (%)
O 12.0
0.01 12.5
0.05 13.0
0.25 13.5
0.5 14.0
1.0 13.5
2.0 12.5
3.0 11.5
EXAMPLE 6
Into 500 ml shaker flasks, 50 ml of inoculation medium having the composition indicated in Table 8 below are charged each time. After sterilization by heating at 110 ° C. for 5 min, it is inoculated with Brevibactérium flavum ATCC 21475 and cultured on a shaker at 31.5 ° C. for 20 h to give an inoculation culture solution. On the other hand, 20 ml of the medium are loaded separately each time for the production of L-lysine indicated in Table 4 in Sakaguchi vials and then sterilized. 1.5 g of calcium carbonate previously sterilized by heating is then added dry and inoculated each time with 1 ml of the inoculation medium described above and then cultured on a shaker at 31.50C. After 24 h of culture, TMG is added to the medium in the amount of O g / dl, 0.01 g / dl, 0.05 g / dl, 0.25 g / dl, 1.0 g / dl, 2 , 0 g / dl and 5.0 g / dl and the culture is continued.

The amount of L-lysine accumulated and the yield are indicated in Table 9 below.

TABLE 8
Composition of the environment
Middle Iron Medium
Prin mentation inoculation component
cipal
Glucose 3 g / dl
Sweet potato molasses - 15 g / dl KH2PO4 0.1 g / dl 0.1 g / dl
MgS04.7H20 0.04 g / dl 0.04 g / dl FeS04.7H20 1 mg / dl 1 mg / dl MnSO4 .4H20 1 mg / dl 1 mg / dl (NH4) 2S04 - 5.5 g / dl
Soy protein acid hydrolyzate (in total nitrogen) 100 mg / dl 100 mg / dl
Nicotinamide 0.5 mg / dl 1 mg / dl
DL-alanine 50 mg / dl 100 mg / dl
Biotin 100 pg / dl 200 zg / dl
Thiamine hydrochloride 200 pg / dl 200 g / dl
Urea 0.5 g / dl
Stephen residue (total nitrogen) - 200 mg / dl pH 7.0 7.0
TABLE 9
Amount of TMG Amount of L-lysine Accumulated added yield L-lysine
(c / dl) (q / dl) (%)
O 3.83 25.5
0.01 4.10 27.3
0.05 4.22 28.1
0.25 4.56 30.4
1.0 4.35 29.0
2.0 4.23 28.2
5.0 4.05 27.0
The fractions having completed the culture are collected in the group with the addition of 0.25 g / dl of TMG and the cells and the calcium salt are removed therefrom by centrifugation. 1 I of the supernatant liquid is passed through a strongly acidic ion exchange resin "AMBERLITE" (registered trademark) IR-120 type CH to adsorb the L-lysine there. The L-lysine adsorbed with 3% aqueous ammonia is then eluted and the eluate is concentrated under reduced pressure.

 After adding hydrochloric acid to the concentrate, the system is cooled to precipitate L-lysine in the form of L-lysine hydrochloride dihydrate and 36.5 g of crystals are obtained.

EXAMPLE 7
An inoculation medium containing 3 g / dl of glucose, 0.2 g / dl of (NH4) 2sO4, 0.2 g / dl of urea, 0.15 g / dl of KH2PO4 is adjusted to pH 7.0. , 0.04 g / dl of MgS04.7H20, 1 mg / dl of FeS04.7H20, 100 ug / l of thiamine hydrochloride, 300 ug / l of biotin and 140 mg / dl of acid hydrclysate of soy proteins, as total nitrogen and 50 ml of the inoculation medium are loaded into a 500 ml bottle and then sterilized by heating. The medium is inoculated with Brevibacterium flavum ATCC 21269 and Escherichia coli FERM-P 4900 (AJ 11334), respectively, and cultured on a shaker for 12 h while maintaining at 31.5 "C to give an inoculation culture solution.

 On the other hand, bottles of 500 ml each are loaded separately with 20 ml of a medium containing 13 g / dl of glucose, 0.25 g / dl of KH2PO4, 40 mg / dl of MgSO4.7H20, 2.0 g / dl of (NH4) 2SO4, 1 mg / dl of MnSO4.4H20, 1 mg / dl of FeS04.7H20, 40 mg / dl of L-isoleucire, 50 lag / l of biotin, 5 ug / l of hydrochloride thiamine, 32 mg / dl of Stephen residue, as total nitrogen, or of a medium additionally containing 0.01 to 5 g / dl of TMG, adjusted to pH 6.5, then sterilized by steam at 1100C for 10 minutes. To each medium is added 2 g of calcium carbonate previously sterilized by dry heating, respectively, and a further 1 ml of the inoculation culture solution described above is added thereto, then cultured at 31.50C. The yield of L-threonine obtained after the culture, relative to the weight of sugar, is indicated in Table 10 below.

TABLE 10
L-threonine yield
Amount of TMG Brevibactérium Escherichia
added flavum coli
C / dL) (%) (%)
0 12.0 25.0
0.01 12.5 26.0
0.05 12.8 27.5
0.25 13.5 28.5
0.5 14.0 30.0
1.0 14.5 31.0
2.0 13.0 28.0
5.0 12.5 26.0
From the group added with 0.25 g / dl of TMG, 1 l of the culture solution is obtained and the cells and the calcium carbonate are separated from the culture solution by centrifugation. The supernatant liquid, 500 ml, is passed through a column of highly acidic ion exchange resin "AMBERLITE" (registered trademark) IR-120 (form H +). Eluted with 3% aqueous ammonia and the amino acid fractions are collected, the salts are removed, discolored and concentrated under reduced pressure. Alcohol is added to the concentrate. After storage with cooling, the crystals formed are collected and dried to give 7.6 g of threonine crystals having a purity of 98% or more.

EXAMPLE 8
To a medium for the production of L-histidine having the composition indicated in Table 11 below, 0.01 to 3 g / dl of MG are added. After adjusting to pH 7.2, 20 ml of the medium are each loaded separately into a 500 ml shaker flask and then sterilized by heating at 115 ° C. for 10 min.

TABLE 11
Concentration component
Glucose 13.0 g / dl (NH4) 2S04 5.0 g / dl KH 2P04 0.1 g / dl
MgS04.7H20 0.04 g / dl FeS04.4H20 1.0 mg / dl
MnS04.4H20 1.0 mg / dl
Thiamine hydrochloride 50.0 wg / dl
Soy protein acid hydrolyzate (total nitrogen) 30.0 mg / dl
Biotin 50.0 llg / dl
Stephen residue (total nitrogen) 100 mg / dl (Calcium carbonate added after separate sterilization 5.0 g / dl)
Each medium is inoculated with Brevibacterium flavum
ATCC 21406 obtained by prior culture on an inclined broth and cultivation on a shaker at 31.5 "C until the glucose disappears.

 The yield of L-histidine relative to the weight of sugar is indicated in Table 12 below.

Table 12
Amount of MG Yield in
added (g / dl) L-histidine (^)
0 10.0
0.01 10.5
0.025 11.0
0.5 11.5
1.0 12.0
2.0 11.0
3.0 9.5
The cells and the calcium carbonate are removed from 1 l of the culture solution obtained in the group supplemented with 0.5 g / dl of MG and the supernatant is passed through a strongly acidic ion exchange resin "AMBERLITE "(registered trademark) IR-120 (H + form) to adsorb L-histidine. The L-histidine adsorbed with 3% aqueous ammonia is then eluted and the eluate is then concentrated under reduced pressure. The concentrate is cooled and allowed to stand to precipitate L-histidine crystals. The crystals are dried to give 7.1 g of L-histidine.

Example 9
A medium is prepared for the production of L-phenylalanine by adding 0.01 to 3.0 g / dl of DMG to a medium having the composition indicated in table 13 below containing sucrose as carbon source. After adjusting to pH 7.0, 20 ml are added separately each time to a 500 ml shaker flask and then sterilized by heating at 115 ° C. for 10 min.

TABLE 13
Concentration component
Sucrose 13.0 g / dl
KH2P04 1.5 g / dl (NH4) 2S04 1.0 g / dl MgS04.7H20 0.1 g / dl
MnS04.4H20 1.0 mg / dl FeS04.7H20 1.0 mg / dl
Thiamine hydrochloride 20.0 pg / dl
DL-methionine 50.0 mg / dl
L-tyrosine 40.0 mg / dl
Soy protein acid hydrolyzate (total nitrogen) 100.0 mg / dl
Fumaric acid 1.0 g / dl
Biotin 5.0 lig / dl
Acetic acid 0.3 mg / dl
Glutamic acid 0.2 g / dl
Stephen residue (total nitrogen) 100 mg / dl (Calcium carbonate added after separate sterilization 5.0 g / dl)
Each medium is inoculated with Brevibacterium lactofermentum ATCC 21420 obtained beforehand by culture on an inclined broth and cultured on a shaker at 31.50 C. until the glucose disappears. The yield of L-phenylalanine relative to the weight of sugar is shown in table 14 ci below.

TABLE 14
Quantity of DMG Yield in
added L-phenylalanine
(g / dl)
0 9.0
0.01 9.5
0.02 10.5
0.5 11.0
1.0 11.5
2.0 11.0
3.0 10.5
Example 10
A medium is prepared for the production of L-tryptophan by adding 0.01 to 5 g / dl of TMG to a medium having the composition indicated in table 15 below containing glucose as carbon source. After adjusting to pH 7.0, 20 ml of the medium are each loaded into a 500 ml shaker flask and then sterilized by heating at 1150C for 10 min.

TABLE 15
Concentration component
Glucose 13.0 g / dl (NH4) 2S04 4.0 g / dl KH2PO4 0.1 g / dl
MgS04.7H20 0.04 g / dl FeSO4. 7H20 1.0 mg / dl MnSO4. 4H20 1.0 mg / dl
Thiamine hydrochloride 200.0 pg / dl
Casein acid hydrolyzate 0.1 g / dl
Soy protein acid hydrolyzate (total nitrogen) 30.0 mg / dl
L-phenylalanine 20.0 mg / dl
Biotin 300.0 pg / dl
Stephen residue (total nitrogen) 100 mg / dl (Calcium carbonate added after separate sterilization 5.0 g / dl)
Each medium is inoculated with Bacillus subtilis
FERM-BP 208 obtained by prior culture and cultured on a shaker at 30 "C until the glucose disappears. The yield L-tryptophan relative to the weight of sugar is indicated in table 16 below.

TABLE 16
Quantity of TMG Yield in
added L-tryptophan
<g / dl) (%)
0 5.0
0.01 6.0
0.02 6.5
0.5 7.0
1.0 7.5
2.0 6.5
3.0 6.0
5.0 5.5
Example 11
A medium is prepared for the production of L-serine by adding 0.01 to 3 g / dl of DMG to a medium having the composition indicated in table 17 below containing glucose as carbon source. After adjusting to pH 7.0, 20 ml of medium are each loaded separately into a 500 ml shaker flask.

TABLE 17
Concentration component
Glucose 13.0 g / dl (NH4) 2SO4 0.2 g / dl KH2PO4 0.2 g / dl MgSO4.7H20 0.05 g / dl FeSO4. 4H20 2.0 mg / dl
Folic acid 1000 pg / dl
Thiamine hydrochloride 1000 pg / dl
Nicotinamide 2,500 ug / l
Soy protein acid hydrolyzate (total nitrogen) 60 mg / dl
Biotin 200 ug / l
Methionine 0.1 g / dl
L-leucine 20 mg / dl
Glycine 3.5 g / dl
DL-methionine 40 mg / dl
Stephen residue (total nitrogen) 100 mg / dl (Calcium carbonate added after separate sterilization 5.0 g / dl)
Each medium is inoculated with Corynebacterium glycinophilum FERM-P 1687 obtained by prior culture and cultured on a shaker at 34 "C.

 The yield of L-serine relative to the moles of glycine consumed is indicated in Table 18 below.

TABLE 18
Quantity of DMG Yield in
added L-serine
(g / dl) (%)
0 36.0
0.01 37.0
0.05 38.0
1.0 38.5
2.0 37.5
3.0 35.0
Example 12
0.1 to 5 g / dl of TMG are added to a medium having the composition indicated in table 19 below and 20 ml of the medium are each loaded separately in a 500 ml flask then sterilized by heating at 1200C for 10 minutes. Each cell is inoculated with three loops of platinum obtained by culturing Corynebacterium equi AJ 11749 (FERM-P 6262) at 31.5 "C for 18 h in an inclined medium having the following composition 20 g / l of glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l table salt and 20 g / l agar (pH 7.2). After culturing at 34 "C for 2 days, add 50 g / l of glucose and 10 g / l of inosine then cultivated on a shaker for another 3 days.

The amount of 5'-inosinic acid accumulated in the culture solution is indicated in Table 20 below.

TABLE 19
Concentration component
Glucose 13.0 g / dl
Yeast extract 1 g / dl
Ammonium sulfate 0.5 g / dl KH 2P04 2 g / dl
MgS04.7H20 0.6 g / dl
Calcium panthothenate 1 mg / dl
P-aminobenzoic acid 1 mg / dl
Adenine 8 mg / dl
Biotin 1000 pg / dl
Soy protein acid hydrolyzate (total nitrogen) 90 mg / dl
Glutamic acid 2 pg / dl
Stephen residue (total nitrogen) 100 mg / dl pH 6.5
TABLE 20
Amount of TMG Amount of acid
added 5'-inosinique
(g / dl) (5'-IMP Na2.7,5H20)
0 28
0.1 28.5
0.5 29
1.0 30
2.0 29.5
3.0 29
5.0 27
Example 13
An inoculation medium containing 3 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.04 g / dl of MgSO4.7H20, 0.001 g / dl of FeSO4.7H20, 0.001 is adjusted to pH 6.0. g / dl of MnSO4.4H2O, 0.3 g / dl of urea, acid hydrolyzate of soy proteins containing 60 mg / dl of total nitrogen, 30 pg / dl of thiamine hydrochloride and 20 pg / l of biotin and 50 ml of the inoculation medium are loaded into a 500 ml bottle and then sterilized by heating.

 The medium is inoculated with Brevibacterium lactofermentum FERM-P 5936 (AJ 11678) and cultured on a shaker for 13 h while maintaining at 31.5 "C.

On the other hand, 20 ml of a medium containing 13 g / dl of glucose 0.1 g / dl of KH2PO4, 0.04 g / dl of MgSO4.7H20, 0.001 g / is adjusted to pH 7.0 each time. dl FeSO4.7H20, 0.001 g / dl MnSO4.4H20, 4.0 g / dl ammonium sulfate, 65 mg / dl acid hydrolyzate of soy proteins, in total nitrogen, 20 pg / dl hydrochloride thiamine, 10 pg / dl of biotin and 78-mg / dl of
L-arginine or a medium additionally containing 0.01 to 3 g / dl of DMG, respectively, then sterilized by heating at 1150C for 10 min. To each medium is added 2 g of calcium carbonate previously sterilized by heating dry, respectively, and each time 1 ml of the inoculation culture solution described above is added thereto and then cultured at 31.50C. The yield in
Ornithine after the end of the culture, reported in sugar, is indicated in table 21 below.

TABLE 21
Quantity of DMG Yield in
added L-ornithine
(g / dl) (%)
0 24.5
0.01 26.0
0.05 27.5
1.0 29.0
2.0 27.0
3.0 23.0
Example 14
An inoculation medium containing 3 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.04 g / dl of MgSO4.7H20, 0.001 g / dl of FeSO4.7H20, 0.001 is adjusted to pH 6.0. g / dl of MnSO4.4H20, 0.3 g / dl of urea, 60 mg / dl of acid hydrolyzate of soy proteins, in total nitrogen, 30 ug / l of thiamine hydrochloride and 20 pg / l of biotin and 50 ml of the inoculation medium are loaded into a 500 ml bottle and then sterilized by heating.

The medium is inoculated with Corynebacterium glutamicum
ATCC 13232 and cultivated on a shaker for 13 h while maintaining at 31.5 "C.

 On the other hand, bottles of 500 ml each are loaded separately with 20 ml of a medium containing 13 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.04 g / dl of MgSO4.7H20, 0.001 g / dl of MnSO4.4H20, 4.0 g / dl of ammonium sulphate, 65 mg / dl of acid hydrolyzate of soy proteins in total nitrogen, 20 pg / l of thiamine hydrochloride, 10 pg / dl of biotin and 70 mg / dl of L-homoserine or of a medium additionally containing 0.01 to 3 g / dl of DMG, adjusted to pH 7.0, then sterilized by heating at 115 "C for 10 min. 2 g of calcium carbonate previously sterilized by dry heating is added to each medium, and 1 ml of the inoculation culture solution described above is added thereto each time, followed by culturing at 31.50 C.

The yield of L-ornithine after the end of the culture, relative to the sugar, is indicated in table 22 below.

TABLE 22
Quantity of DMG Yield in
added L-ornithine tg / dl) (%)
o 24.5
0.01 26.0
0.05 27.5
1.0 29.0
2.0 27.0
3.0 23.0
Example 15
An inoculation medium containing 3 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.04 g / dl of MgSO4.7H20, 1.0 mg / dl of FeSO4.7H20 is adjusted to pH 6.0. , 1.0 mg / dl of MnS04.4H20, 0.3 g / dl of urea, 60 mg / dl of hydrolyzate of soy protein (in total nitrogen), 30pg / dl of thiamine hydrochloride and 20pg / dl of biotin and 50 ml of the inoculation medium are loaded into a 500 ml bottle then sterilized by heating. The medium is inoculated with Corynebacterium glutamicum FERM-P 5643 (AJ 11588) and cultured on a shaker for 13 h. maintaining at 31.5 "C.

On the other hand, Example 14 is cultivated in a similar manner each time in a medium containing 13 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.04 g / dl of MgS04.7H20, 1.0 mg / dL of FeSD4. 7H20, 1.0 mg / dl of MnSO4.4H20, 3.0 g / dl of ammonium chloride, 65 g / dl of soy protein acid hydrolyzate (in total nitrogen), 20 g / dl of hydrochloride thiamine, lug / dl of biotin and 70 mg / dl of
L-arginine and a medium additionally containing 0.01 to 3 g / dl of G, adjusted to pH 7.0. The yield of L-citrulline, relative to the sugar after the end of the culture, is indicated in Table 23 below.

TABLE 23
Amount of MG Yield in
added L-citrulline
(g / dl) (%)
0 13.5
0.01 14.0
0.05 14.5
1.0 15.5
2.0 14.0
3.0 13.0
Example 16
This 500 ml is loaded separately into a shaking flask each time 50 ml of an inoculation medium having the c = -coition indicated in table 24 below. After sterilization by heating at 110 ° C. for 5 min, it is inoculated with Corynebacterium glutamicum FERM-P 5836 (AJ 11655) and cultured on a shaker at 31.5 ° C. for 12 h to give an inoculatic culture solution.

On the other hand, cultivation in a similar manner to Example 14 each time in a main fermentation medium of
L-tyrosine having the composition indicated in table 24 cs a medium additionally containing 0.01 to 3 g / dl of HETMA. The quantity e
L-tyrosine after the end of the culture is shown in Table 25 below.

TABLE 24
Composition of the environment
Middle of ino- Middle of fermen
Main culation component
Glucose 3 g / dl 13 g / dl
Urea 0.3 g / dl
Ammonium acetate 0.5 g / dl 1.0 g / dl
Ammonium sulfate - 3.0 g / dl KH2PO4 0.1 g / dl 0.2 g / dl MgS04.7H20 0.04 g / dl 0.1 g / dl MnSO4. 4H20 1 mg / dl 1 mg / dl
FeS04.7H20 1 mg / dl 1 mg / dl
Thiamine hydrochloride 300 pg / dl 50 pg / dl
Biotin 200 ug / dl 100 ug / dl
DL-phenylalanine 50 mg / dl
Dehydrated soy protein hydrolyzate (total nitrogen) 6.5 ml / l pH 7.0 7.0
TABLE 25
Quantity of HETMA Yield in
added L-tyrosine
(g / dl) (%)
0 6.0
0.01 6.5
0.03 8.0
0.5 9.0
1.0 8.2
2.0 7.5
3.0 6.0
Example 17
An inoculation medium containing 3 g / dl of glucose, 0.1 g / dl of KH2PO4, 0.3 g / dl of urea, 0.04 g / dl of MgSO4.7H20, 1 mg is adjusted to pH 6. / dl of FeS04.7H20, 1 mg / dl of MnS04.4H20, 40 mg / dl of DL-methionine, 10 pg / l of biotin, 200 ug / l of thiamine hydrochloride and 50 mg / dl of acid hydrolyzate soy protein (in total nitrogen) and 50 ml of the inoculation medium are loaded into a 500 ml flask then sterilized by heating at 1200C for 10 min. The medium is inoculated with Brevibacterium flavum FERM-BP 755 and cultured on a shaker for 20 h while maintaining at 31.5 "C.

 On the other hand, the culture is carried out in a similar manner to Example 14 each time in a medium containing 13 g / dl of beet molasses (calculated as glucose), 0.1 g / dl of KH2PO4, 2, 0 g / dl of CNH4) 2SO4, 0.04 g / dl of MgS04.7H20, 1 mg / dl of FeS04.7H20, 1 mt / dl of MnS04.4H20, 100 mg / dl of DL-methionine, 50 ug / l of biotin and 300 ug / l of thiamine hydrochloride and 50 mg / dl of acid hydrolyzate of defatted soy protein or medium additionally containing 0.01 to 3 g / dl of HETMA, adjusted to pH 7.0. The amount of L-leucine produced after the end of the culture is shown in Table 26 below.

TABLE 26
Quantity of HETMA Yield in
added L-leucine
(g / dl) (%)
0 12.0
0.01 13.2
0.5 14.2
1.0 15.0
2.0 14.0
3.0 12.5
It is understood that the invention is not limited to the preferred embodiments described above by way of illustration and that the person skilled in the art can make various modifications and various changes without however departing from the scope and the spirit of the invention.

Claims (1)

 CLAIM  Process for the manufacture of an L-amino acid or 5'-inosinic acid, characterized in that it consists in cultivating a bacterium producing L-amino acids or a bacterium producing 5'-inosinic acid in a medium containing sugar cane molasses, sucrose or glucose as the main carbon source and containing at least one substance chosen from N-methylglycine, N, N-dimethylgLycin, N, N, N-trimethylglycine and Hydroxide of (-hydroxyetbyl) trimethylammonium to produce an L-amino acid or 5'-inosinic acid and to be harvested L-amino acid or 5'-inosinic acid.