JPS5928398B2 - Method for producing L-isoleucine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing silicone inleucine by a fermentation method.

More specifically, ethanol-assimilating microorganisms to which DL-α-aminobutyric acid resistance has been actively imparted are aerobically cultured in a medium containing DL-α-aminobutyric acid and containing ethanol as the main carbon source, The purpose of the method for collecting L-inleucine from this culture solution is to industrially advantageously produce L-isoleucine using inexpensive raw materials.

L-inleucine is an amino acid that plays an important role in the nutrition of humans and animals as an essential amino acid, and its demand as a pharmaceutical, food, and feed fortifier has been rapidly increasing in recent years.

As for the industrial production method of L-inleucine, it is difficult to produce only L-inleucine by chemical synthesis method because stereoisomers exist like other amino acids, so fermentation method is the main method for producing L-inleucine. It is being done.

The fermentation method uses precursors of L-inleucine such as DL-α-aminobutyric acid and threonine (Japanese Patent Publication No. 4).
3-8709, Japanese Patent Publication No. 40-2880, etc.) and the so-called direct fermentation method in which no precursor is added (Special Publication No. 38-7091, etc.).
93586, 1973).

Regarding the carbon sources used in these fermentation methods,
In general, carbohydrates such as 1-glucose, fructose, mannose, caractose, sucrose, maltose, chrycerol, mannitol, starch hydrolyzate, and molasses can be used, and organic acids can also be used.

” (Special Publication Publication No. 38-7091, page 2, left column, lines 9-13)
In addition to glucose, fructose, sucrose, and maltose, used alone or in combination, starch, starch-saccharified sugar, molasses, organic acids, and alcohols are used.

(Japanese Patent Publication No. 40-2880, page 3, left column, lines 9 to 12), or 1- Sugars such as crucose, maltose, sucrose, starch decomposition products, blackstrap molasses, organic acids such as acetic acid, ethanol, and glycerin. Anything that can be assimilated by the bacteria used, such as alcohols such as
(Page 4, left column, lines 8 to 12) of the publication.

However, what is actually specifically disclosed is limited to glucose or starch saccharification solutions.

However, as an exception, methods have been proposed in which fatty hydrocarbons (Japanese Patent Publication No. 48-2355) and aromatic compounds (Japanese Patent Application Laid-Open No. 49-48890) are used as carbon sources other than carbohydrates.

The present inventor believes that it is cheaper than carbohydrates and can be supplied stably in the future, and is more advantageous in fermentation engineering because it is water-soluble compared to aliphatic hydrocarbons or aromatic compounds.
We are conducting intensive research with the goal of developing an industrial production method for L-isoleucine using ethanol as the main carbon source, and have already developed an ethanol-assimilating microorganism belonging to the genus Brevibacterium (Brevibacterium flavum MJ-233 microbial accession number microorganism). Koken Bacteria No. 3068 FERM-Pya 3068 is a representative strain) is cultured in a medium containing DL-α-aminobutyric acid and using ethanol as the main carbon source to produce L-isleucine industrially advantageously. Invented the method and published Japanese Patent Application Publication No. 51-1305.
The application has been filed as stated in Article 92.

In the method of JP-A-51-130592, naturally, the strain that produces and accumulates L-inleucine is DL-α
- Although it is thought that the microorganisms used have some degree of resistance (slight resistance) to aminobutyric acid, the inventor of the present application has conducted extensive research and has actively added DL-α-aminobutyric acid to the microorganisms used. It has been found that by imparting resistance, it is possible to significantly improve the amount of L-inleucine produced and accumulated.

As a result, an ethanol-assimilating microorganism belonging to the genus Brevibacterium and resistant to α-aminobutyric acid added a significant amount of L-isoleucine to a medium containing α-aminobutyric acid and using ethanol as the main carbon source. We succeeded in producing and accumulating L-inleucine, and completed a method for producing L-inleucine that is particularly advantageous industrially.

DL- which is an ethanol-assimilating microorganism that has been actively given resistance to DL-α-aminobutyric acid used in the present invention.
The α-aminobutyric acid resistant mutant strain can be obtained by the following procedure.

UV irradiation, or chemical agents (e.g. N-methyl-
The above L-inleucine producing strain (Brevibacterium
flavum MJ-233), this bacterial suspension was placed in a plate medium containing 10 m/m/m of α-aminobutyric acid (0.2% urea, 0.7% ammonium sulfate, KH2PO40,
05 Section, K2HPO40, 05 Section, MgSO4・7H
200.05%, NaC12mf/11CaC12.2
H202m/11 FeSO4・7H7H2O2/l
, Mn5O, ・4~6 H2O, ZnSO4・7H2
02 my/l, biotin 200 μg/l, thiamine hydrochloride 100 μg/l, DL-α-aminobutyric acid 1.
0, 2.0% agar, 3% ethanol by volume (added after sterilization) at 30°C for several days, and isolate the resulting large colony to obtain a resistant mutant strain.

A typical resistant mutant strain is Brevibacterium flavum MJ-233-AB-41 (hereinafter referred to as MJ-233-
AB-41), and this strain has been assigned the microbial accession number F''ERM-Pya 3812 to the Institute of Microbiology, Agency of Industrial Science and Technology, No. 3812 (December 16, 1978).
It has been entrusted as a contract.

This resistant mutant strain is Brevibacterium flavum MJ-
233 (Microorganism accession number: Microorganism Research Institute No. 3068 F
E RM-P A 3068 hereinafter referred to as MJ-233.

) was derived as an α-aminobutyric acid resistant strain.

The mycological properties of Brevibacterium flavum MJ-233 and the reasons for its taxonomic identification are as follows.

Mycological properties ■ Microscopic properties 06-0.8 X 1. OX 2.4μ short rods, usually in V-shaped pairs, palisade-like patterns also observed.

Durham positive, no spores, non-motile, no capsule, non-acid fast.

■Cultural properties 1, broth agar colonies are circular, the surface is smooth, and the colony ridges are flat or slightly ＼
The ridge, the periphery of the colony is wavy, yellow, opaque, and
Shiny.

2. Gravy agar slope Medium growth, filamentous, yellow.

3. Gravy Medium growth, sediment, no turbidity.

4 Meat juice agar puncture It grows well on the surface, and the internal growth is sparse and filamentous.

■Growth conditions■, Growth saturation: suitable vortex 30-37℃, range 15-40℃ 2, growth pH: optimal pH 7-8, range 5-103, oxygen requirement: aerobic 4, ammonium salt utilization : Yes 5, Availability of urea: Yes 6, Availability of nitrate: Yes ■ Physiological properties Other 1: Does not liquefy gelatin.

2. Lidmus milk does not change.

3. Reducibility of nitrate: positive 4, production of indole: negative 5, production of hydrogen sulfide: negative 6, hydrolysis of starch: negative 7, production of ammonia from peptone: positive 8, Voges-Gross Calaer reaction: negative 9. Methyl red test: Positive (weak) 10. Catalase: Positive 11. Urease: Positive 12. Production of acid from carbohydrates. Acid is produced from glucose, saccharose, trehalose, maltose, salicin, but no gas is produced.

arabinose, xylose, lactose, rhamnose,
raffinose, mannit, zulsit, inojit,
Does not produce acids and gases from atonite.

13. Vitamin requirement: Biotin requirement.

Based on the above-mentioned mycological properties, Bersey's Manual of Determinative Batteriology
gey's manual of determination
Differences with known bacterial groups were examined according to the classification criteria of the 7th edition of Bacteriology and the references listed in the 8th edition.

This strain is Durum-positive, non-sporulating, non-motile, and aerobic, and when observed under a microscope, all strains have an elliptic or short culm shape with V-shaped divisions.

Changes in Durham staining, morphological changes, and branched cells associated with the course of erect proliferation are not observed.

Comparing these properties with the above-mentioned Manual of Deterministic Bacteriology, 7th edition, this bacterium is Brevibacterium (Brevibacterium).
m) It is clear that it belongs to the genus, but there is no description of the same species.

Next, since this bacterium produces amino acids, we will compare it with known strains in the review article on glutamate-producing strains (Stratification No. 36).
Brevibacterium flavum (Vol. 2, No. 141-159, 1962).
ium flavum).

Therefore, this bacterium was identified as Brevibacterium flavum.

The relative growth rates of MJ-233-AB-41 (aminobutyric acid strain) and its parent strain MJ-233 against α-aminobutyric acid are shown in Table 1 below.

Note that the section indicates the overlapping section. (Note-1) The numerical values in the table are the growth rate when DL-α-aminobutyric acid is not added.
975)) is set as 100, and the relative growth rate is shown below.

(Note-2) Composition of medium used and culture method Urea 0.2 parts, ammonium sulfate 0.7 parts, KH2PO40.05 parts, K2HPO40.05 parts, MgSO4・7H200,
Section 05, yeast extract 0.01%, casamino acid 0.01
%, Fe SO4.7 H202mg/l, MnS
O4・4~6 H2O2mg//l, NaC12mg/
/l, CaCl2・2H2H2O2/l, ZnSO4・
A medium consisting of 202 mV/l of 7H, 200 μg/l of biotin, and 100 μg/l of thiamine hydrochloride (add the amount of DL-α-aminobutyric acid shown in Table 1).
Dispense into dog-shaped test tubes, sterilize at 120°C for 10 minutes, then MJ-
233-AB-41 strain was inoculated, 0.3 ml (3 volumes) of ethanol was added under aseptic conditions, and the mixture was incubated at 30°C.
A shaking culture was performed for 1 day.

In addition, in the present invention, DL, which is an ethanol-assimilating microorganism that has been actively given resistance to DL-α-aminobutyric acid,
-α-aminobutyric acid resistant mutant strain refers to the relative growth rate when DL-α-aminobutyric acid 2 was added to the medium of (Note-2) and cultured with shaking at 30°C for 3 days. -The growth rate when 2% α-AB was added. 0. D6□.

100 Growth rate - Growth rate without α-AB addition 0. D61° is 30
Defined as above.

(α-AB in the above formula is an abbreviation for DL-α-amino acid.

) The composition of the medium used for the culture of the present invention is not particularly limited as long as it contains ethanol as the main carbon source and DL-α-aminobutyric acid, and the nitrogen source inorganic salt is not particularly limited.

As the nitrogen source, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used alone or in combination.

As the inorganic salt, sodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used.

In addition, nutrients such as peptone, meat extract, yeast extract, cornstarch liquor, casamino acid, and various vitamins may be added to the medium if necessary for bacterial growth and L-inleucine production.

The culture is carried out under aerobic conditions such as aeration and shaking, and the culture temperature is 20 to 40°C, preferably 25 to 35°C.

The pH during the cultivation is kept at around 5-10, preferably around 7-8, and the pH in the culture solution is adjusted by adding acid or alkali.

The concentration of DL-α-aminobutyric acid added is 0.1 to 5 parts by weight, or 1 to 3 parts by weight.

Suitable ethanol concentration at the start of culture is 1 to 5% by volume, preferably 2 to 3% by volume.

The culture period requires 2-8 days, with an optimal period of 4-5 days.

L-inleucine can be easily recovered from the culture solution by centrifuging the culture solution, removing bacterial cells, etc., and then using known methods such as ion exchange resin treatment method or precipitation method. .

Examples are shown below.

The quality of L-inleucine was confirmed by the Rf value of paper chromatogram, the mobility of electrophoresis, and the biological activity value of microorganism quantification.

Determination Ha, Leuconostoc mesenteroides ATCC
A microbioassay method using 8042 was used.

Also, the term ``section'' refers to the overlapping section.

Example 1 Preculture medium (urea 0.4%, ammonium sulfate 1.4%
, KH2PO40,05, K2HPO40,05,
Mg504-7 H2O0.05%, CaCl2・2H
202ppm, FeSO4・7H202ppm,
Mn504H4~6H202ppr11, Zn5O・7
H7H2O2ppNaC12ppm1 Biotin 200
μfl/11. Thiamin-HCl 100μg/l, casamino acids 0.1%, yeast extract 0.1%) 10m/24
After dispensing into φ dog-shaped test tubes and sterilizing (pH 7.0 after sterilization), DL-α-aminobutyric acid resistant strain MJ-233-AB-4
1 (relative growth rate: 40), 0.3 ml of ethanol was added aseptically, and cultured with shaking at 30°C for 2 days.

Next, the main culture medium (0.4 part urea, 1 part ammonium sulfate.
4%, KH2P0.0.05%, K2HPO40.0
5%, Mg5O, -7H2O0.05%, CaCl2・
2H202ppm, FeSO4+ 7H202ppm
, Mn50444~6 H2O2ppm, ZnSO
4+7 H2O2ppm.

NaC 12ppm, biotin 200μg/11 thiamine/HCl 100μg/11, corn staple liquor 10ml/l, DL-a-aminobutyric acid 2%) 10
ml into a 24φ large test tube, and after sterilization (pH after sterilization)
7,0), inoculate 0.1 ml of the preculture solution, add 0.3 ml of ethanol aseptically, and culture with shaking at 30°C for 6 days.

Ethanol is added as it is consumed.

(At this time, do not exceed 3 volumes of ethanol.

The total ethanol consumption was 6 volumes.

) L-isoleucine was added per 14 days of culture for 5 days.
0g accumulated.

The filtrate from which bacterial cells and other impurities have been removed from the culture solution is passed through a column of strongly acidic cation exchange resin (H+ type) to adsorb L-isoleucine, washed with water, and eluted with 0.5N aqueous ammonia. The L-isoleucine fraction was concentrated, and crystals of L-inleucine were precipitated with cold ethanol to obtain 2.8 g of crude crystals per 11 culture solutions.

Furthermore, when using the parent strain (MJ-233) under the same conditions, the accumulation of L-inleucine in the medium was 3.0 per 11 culture solutions.
It was g.

Example 2 Pre-preculture medium (urea 0, 4, ammonium sulfate i,
4%, K2HPO40.05%, KH2PO40.05
MgSO4.7 H2O0.05%, Na Cl
2 ppm, CaCl2・2H202 ppm, ZnS
O4・7H202ppm, Fe SO4・7H2O
2ppm, MnSO4・4~66H2O2pp, biotin 200 ttg/l, thiamine HCl 100μg
/l, casamino acids 0.1%, yeast extract 0.1%) 10
Dispense m/ into a 24φ large test tube, sterilize it, and then add MJ-233.
- Inoculate AB-41 and add 0.3 ethanol aseptically.
ml and cultured with shaking at 30°C for 2 days.

Preculture medium (urea 0.4%, ammonium sulfate 1.4%
Related, KH2PO40.05%, K2HPO,0.05%, Mg504-7H200.05%, NaC12ppm
.

CaCl2-2H202ppm, ZnSO4#7H2
02ppm1FeS04・7H202ppm, Mn
SO4・4~66H2O2pp, Biothia 200t
4/l, thiamine HCl 100 μg/l, corn stapler 10 ml/l, DL-a-aminobutyric acid 1.
0%) 50m/ into a 500m/ Erlenmeyer flask,
After sterilization (pH 7.0 after sterilization), 10 m/ml of the pre-pre-culture solution was inoculated, 1.5 m/ml of ethanol was added aseptically, and cultured with shaking at 30°C for 1 day.

Main culture medium (urea 0.4 parts, ammonium sulfate 1.4 parts, KH2P 0.0.05%, K2HPO 40.05%,
Mg504-7 H2O0.05%, NaC12ppm
, CaCl2+ 2H202ppm, ZnSO4・7
H202ppm.

Fe SO4・7 H2O2ppm, Mn504H4
~6H6H2O2pp Biotin 200 ttjj/L
Thiamine/HCl 100 μal/11, corn stapler 10me/fDL-α-aminobutyric acid 2%)■
After sterilization (pH 7.0 after sterilization), inoculate with preculture solution 50771/, add 30 ml of ethanol aseptically, aerate IV VVM, stir 1.
Culture is carried out at 800 rprn, temperature of 30° C., and pH of 6.5 (adjusted with aqueous ammonia).

Ethanol is added as it is consumed and culture is continued.

11.0 g of L-isoleucine was produced and accumulated per 11 culture solutions in the pewter of the 5th culture (100 ml of total ethanol consumed).

Furthermore, when the parent strain (MJ-233) was cultured under the same conditions, 7.0 g of L-inleucine was produced and accumulated per 11 culture solutions.

Claims (1)

[Claims] 1. A DL-α-aminobutyric acid-resistant mutant strain, which is an ethanol-utilizing microorganism that belongs to the genus Brevibacterium and has been actively given resistance to DL-α-aminobutyric acid, was used to produce ethanol containing DL-α-aminobutyric acid. In a medium containing as the main carbon source,
A method for producing L-isoleucine by a fermentation method, which comprises carrying out aerobic culture to produce and accumulate L-isoleucine in a culture solution, and collecting inleucine from the culture solution.