JPS63160592A - Production of l-valine - Google Patents

Abstract

PURPOSE:To efficiently obtain L-valine useful as a medicine, food or feed enriching agent by a fermentation method, by aerobically cultivating a microorganism, belonging to the genus Brevibacterium and having resistance to DL-alpha- aminobutyric acid in a nutrient culture medium. CONSTITUTION:Brevibacterium flavum MJ-233 (FERM-P No.3068) is subjected to ultraviolet light irradiation or chemical agent treatment to provide Brevibacterium flavum MJ-233-AB-41 (FERM-P No. 3812) having resistance to DL-alpha-aminobutyric acid. The resultant strain is the aerobically cultivated in a culture medium prepared by adding 0.1-5wt%, preferably 1-3wt% DL-alpha- aminobutyric acid to a culture medium containing 1-5vol%, preferably 2-3vol% carbon source, e.g. glucose, etc., as well as a nitrogen source, inorganic salt, if necessary, vitamin, etc., under aerobic condition at pH 5-10, preferably 7-8 and 20-40 deg.C, preferably 25-35 deg.C for 2-8 days, optimally 4-5 days to afford the aimed L-valine.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a method for producing L-valine. More specifically, it relates to a method of aerobically cultivating a microorganism belonging to the genus Prepibacterium and having resistance to DL-α-aminobutyric acid, and obtaining L-valine from the culture solution.

According to the method of the present invention, the amount of L-valine produced and accumulated can be greatly improved, and L-valine can be produced in high yield.

L-valine is an essential amino acid that plays an important role in the nutrition of humans and animals, and the demand for it as an ant, medicine, food, and feed fortifier has been rapidly increasing in recent years.

Prior art As for the industrial production method of L-valine, chemical synthesis method is used because stereoisomers exist as in the case of other amino acids.
Since it is difficult to produce only one L, fermentation is mainly used to produce it.

Conventionally, methods for manufacturing L-valine by fermentation have been carried out in various ways, including a method using an auxotrophic mutant strain as a fermentation bacterium (
Special Publications No. 37-1692, No. 37-12448, No. 42-
513, 43-11756, 51-33996, 52-116, 53-25034, 58-3259
4, JP-A-49-116293, etc.), a method of adding metal ions to the culture medium (Japanese Patent Publication No. 38-2528, etc.)
4, 38-25286, 40-4040, 42-
513 publications, etc.), the method of adding purine or pyrimidine derivatives (Japanese Patent Publication No. 40-1991), the method of adding carboxylic acid ester C (Japanese Patent Publication No. 11754-1975), barbylic acid, 2-thiobarbital acid (Japanese Patent Publication No. 43-11754), A method of adding a surfactant (Japanese Patent Publication No. 43-11755, etc.), a method of adding a surfactant (Japanese Patent Publication No. 41-21752, etc.), a method of adding acrylic acid (Japanese Patent Publication No. 43-13678), a method of adding an organic acid ( 1!!! Kosho 47-23035,
51-46836 and JP-A-49-50185).

However, since there is a limit to the accumulation of L-valine in various auxotrophic mutant strains that are known direct valine-fermenting bacteria, there is a need to provide a method for producing and accumulating significant amounts of L-valine from a new perspective. was.

SUMMARY OF THE INVENTION The present invention involves aerobically cultivating a microorganism belonging to the genus Prepibacterium and having resistance to DL-α-aminobutyric acid in a nutrient medium to produce and accumulate L-valine in the culture solution. The present invention provides a method for producing L-valine by fermentation, in which L-valine is collected from a culture solution.

Effects of the Invention According to the method of the present invention, an unprecedented amount of L-valine can be produced and accumulated in a nutrient medium. For this reason, L-valine can be produced with an inexpensive raw material in a high yield which is industrially advantageous.

DETAILED DESCRIPTION OF THE INVENTION Conventionally, a method for producing L-valine based on a fermentation method using a mutant strain having α-aminobutyric acid resistance has been proposed using Serratia marsecens (4? Kosho +5-24274), Proteus letgeri spp. Although there is a method based on a bacterial strain (Japanese Unexamined Patent Publication No. 143595/1983), no such findings are known regarding microorganisms belonging to the genus Prepibacterium.

The microorganism belonging to the genus Prepibacterium used in the present invention and having resistance to DL-α-aminobutyric acid can be made into a resistant mutant strain by the following operation.

That is, ultraviolet irradiation, chemical agents (such as 1fN-
After inducing mutations in the 9-valine producing bacterial strain by treatment (mef-N/-nitro-N-nitrosoquanidine, etc.), this bacterial suspension was transferred to a plate medium containing α-aminobutyric acid 101+9/117 [ Urea 0.2%, ammonium sulfate 0.7%, KH2
PO40.05%, KH2O40.05%, Mg5Oa
・7H200,05%, N1-2”F/Lx
Caαz・2HzO2'l/t, FeSO44? H
zO2't/ LNMnSO4・4-6H20, ZnS
O4・7H202q/L, biotin 200μV/L,
Thiamine hydrochloride 100μV/! , DL-α-aminobutyric acid 1.0%, agar 2.0%, ethanol 2% by volume (added after sterilization)] at 30°C for several days, and the resulting large colony was isolated. Obtain resistant mutants.

A typical resistant mutant strain is Prepibacterium flavum MJ-233-AB-41 (Feikoken Bacterium 3812
No., Special Publication No. 59-28398. Below MJ-233
-AB-41), and this resistant mutant strain was derived from Brevibacterium flavum MJ-233 (Feikoken Bacterium No. 3068, hereinafter referred to as MJ-233) using the above method. be.

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

Table 1 Note: The values in the table are the growth rate 0.0% without the addition of DL-α-aminobutyric acid. D, ato (Absorbance measurement 2 Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Volume 1, p.

212 Asakura Shoten, 1978)
The relative growth rate is shown as o.

Note) Composition of the medium used and culture method Urea 0.2%, ammonium 0.7%, KH2PO 40.05%
, K2HPO40.05%, MgSO4・7H200,
05%, yeast extract 0.01%, casamino acid 0.01%
, FeSO4・7Hz02 Wt/L, Mn5
O4a 4-'6HzO2q/z, NaCJ 2
'9/ L, Ca (Jz, 2H202N!/ L,
ZnSO4・7H202yy/t.

Biotin 200μy/l, thiamine hydrochloride 100μy/l
Dispense 10 x 1 of a medium consisting of 1 ml (DL-α-aminobutyric acid is added in the amount shown in Table 1) into 24 g3 large test tubes, and after sterilizing at 120°C for 10 minutes, MJ-233-
AB-41 strain and MJ-233 strain were each inoculated, and 0.2 Kt (2% by volume) of ethanol was added under aseptic conditions, followed by shaking culture at 30° C. for 3 days.

In addition, in the present invention, the DL-α-aminobutyric acid resistant strain or the microorganism resistant to DL-α-aminobutyric acid refers to DL
- In the above medium supplemented with 2% α-aminobutyric acid, 30
When cultured with shaking at ℃ for 3 days, the growth rate when 2% α-AB was added was 0. D 610 relative growth w X 100α-AB
Growth rate without additives: 0. Or+.

is defined as 30 or more. (α-AB in the above formula is D
It is an abbreviation for L-α-aminobutyric acid. ) The composition of the medium used for culture in the L-valine production method of the present invention is not particularly limited in terms of carbon source and nitrogen source inorganic salts. Generally, glucose is mainly used as the carbon source, and ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea can be used alone or in combination as the nitrogen source.

As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. are used. In addition, if necessary for the growth of bacteria and L-valine, Pelton, meat extract, yeast extract, cornstarch liquor, etc.
Nutrients such as casamino acids and various pressed vitamins can be added to the culture medium.

Culture is performed under aerobic conditions such as aeration, stirring, and shaking.
! ! The temperature is 20-40°C, preferably 25-35°C. The pH during the cultivation is maintained at around 5 to 10, preferably around 7 to 8, and the pH in the culture solution is adjusted by adding acid or alkali. The concentration of DL-α-aminobutyric acid added is preferably 0.1 to 5% by weight, and 1 to 3% by weight.

A suitable glucose concentration at the start of culture is 1 to 5 t% by volume, preferably 2 to 3 t% by volume. The culture period is 2-8 days, the optimal period is 4-5 days.

Recovery of L-valine from the culture solution can be easily carried out by centrifuging the culture solution to remove bacterial cells, and then using a known method such as an ion exchange resin treatment method or a precipitation method.

Experimental Examples In the following experimental examples, the quality of L-valine was confirmed by the Rf value of paper chromatogram, the mobility of electrophoresis, and the biological activity value by microorganisms.

The fixed Europe is Leuconostoc mesenteroides ATC,
The test was carried out using a microbioassay method using C8042. Moreover, the expression "shi" means weight %.

Example-1 Preculture medium (urea 0.4%, ammonium sulfate 1.4%
, K2HPO40.05%, KHzPOa 0.05%
, Mg5Oae7Hzo 0.05%, CaC2z 2H202ppm %FeSO4・7H202ppmS
MnSO4・4-6H20229m%Zn5Oi・7H
202ppmN Na (J2ppm, biotin 200μy/l, thiamin.) ICtl 00μ? /
11 Casamino acids 0.1%, yeast extract 0.1%) 10
Dispense d into 24β large test tubes and sterilize (pH 7.0 after sterilization)
)did. After adding 0.2 ml of a 50% glucose solution prepared by further sterilization (120°C, 15 minutes), Prepibacterium flavum (Brevib
acterium Flavum) MJ-23
3-A B-41 (FERM P-3812) was inoculated and cultured with shaking at 30° C. for 2 days while adjusting the pH to 7 to 7.5.

Next, use this culture medium (urea 0.4%, ammonium sulfate 1.
4%, KH2PO40.05%, K2HPO40.05
%, MgSO4・7H200,05%, CaC1z @
2H202m” %FeSO417HsO2ppmS
MnSO4.4-6I (202 ppm.

ZnSO4・7H202ppm, NaCt2ppm
, Biotin 200 fit/l, Thiamine” Hc
tl 00 pf/Z% corn staple liquor 10
d/1) 50- was dispensed into a 500d Erlenmeyer flask and sterilized (pH 7 after sterilization).

50% of this was further sterilized (120℃, 15 minutes)
After adding 2 d of glucose solution, 1 ml of the preculture was inoculated and cultured with shaking at 30° C. for 3 days while adjusting the pH to 7 to 7.5. On the third day of culture, L-valine added culture solution 1 to 92. Of accumulated.

Solution F, which has been removed from the culture solution by bacterial cells and other impurities, is passed through a column of strongly acidic cation exchange resin (H+ type) to adsorb L-valine, washed with water, and eluted with 0.5N ammonia water. Concentrate the L-valine fraction and add L-valine to cold ethanol.
Crystals of valine were precipitated. Thus, 1 culture solution per 1
．． A crude crystal of 3f was obtained.

Note that when the parent strain MJ-233 was used under the same conditions, the accumulation of L-valine in the medium was 50 or less per 1 culture solution.

Claims (1)

[Claims] (1) A microorganism belonging to the genus Prepibacterium and resistant to DL-α-aminobutyric acid is cultured aerobically in a nutrient medium, L-valine is produced and accumulated in the culture solution, and L-valine is produced and accumulated in the culture solution. -L by fermentation method, which is to collect valine
- Method for producing valine.