JPS582678B2 - Biseibutuniyoru L- Barin no Seizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides nutritional support for L-valine-producing bacteria that belong to the genus Corynebacterium or the genus Plevibacterium and have S-2-aminoethyl-L-cysteine (hereinafter referred to as thiarisin) replenishment. L- produced and accumulated in the medium by culturing in the medium
This method involves the production of L-valine using microorganisms, which is characterized by the collection of valine.The purpose of this method is to collect valine, which is important nutritionally and medicinally as an essential amino acid, and which has been used in seasonings and feed in recent years. The object of the present invention is to industrially advantageously produce L-valine.

Conventionally, methods for producing valine using microorganisms include methods using bacterial strains that require at least leucine for growth (Japanese Patent Publication No. 37-12448), and methods using bacterial strains that are sensitive to 2-thiazolealanine. The strain contains leucine,
A method using a strain imparted with isoleucine or threonine auxotrophy (Japanese Patent Publication No. 48-44877;
9-116293).

As a result of searching for strains of various microorganisms that have the ability to produce L-valine, the present inventor found that strains belonging to the genus Corynebacterium or Previbacterium, which are resistant to thiarisin, contained significant amounts of L-valine in the culture medium. He discovered that it could be produced and completed this invention.

The microorganism used in this invention belongs to the genus Corynebacterium or the genus Previbacterium, and is a mutant strain that is resistant to thiarisin.

Typical of these are Corynebacterium
Corynebacterium glutamicum KY1062 mutated from the parent strain Corynebacterium glutamicum ATCC13761
8 (Feiko Kenkyoku No. 2924) and Previbacterium lactofermentum KY10614 (Feiko Kenkyoku No. 2925), which was mutated using Previbacterium lactofermentum ATCC13869 as a parent strain.

These microorganisms are strains that have thiarisin resistance induced by ordinary mutagenesis operations, and their mycological properties, other than the above-mentioned compatibility and L-valine production ability, are based on the mycological properties of the species and genus to which the parent strain belongs. The properties are almost the same as those of

Examples of the above mutation induction methods include methods using ultraviolet irradiation, X-ray irradiation, and chemical agents (eg, nitrosoguanidine, methyl ethanesulfonate).

The degree of microorganism θ)i'' properties used in this invention is as follows.

The above-mentioned KY10628 (Feikoken Bibori No. 2924) and its parent strain ATCC13761 strain and KY
1 0 6 1 4 (Feikoken Bacteria Serial No. 2925) and its parent strain ATCC13869 were incubated at 28°C for 24 hours in a slant of medium A having the following composition.
After culturing for a period of time, the bacterial cells were scraped off and suspended in a small amount of basic medium B having the composition shown below.

1 rnl of this suspension was inoculated into 8 ml of basic medium B containing thiarisin in the amount shown in the table below, and cultured with shaking at 30°C for 24 hours.

The growth status of each strain at the end of culture is determined by optical density (OD
) The results measured were as shown in the table below.

≧? Medium A: yeast extract 5 g/l, peptone 10
El/l, NaCl 2.59/l, meat extract 5 9/
l, agar 2 5 9/1 Basic medium B: glucose 1 09/l, NH, NO31
El/t, NH, Ct5El/t, KOH19/t, monopotassium phosphate3. 4 &/LNa 2 SO4 2 g
/t. Mg SOc・7H200.2&/t, biotin 50γ/t, cystine 20m9/7, thiamine・HCt5 da/t, nicotinic acid 125 ~/L, calcium pantothenate 12.5TVLL-7 leonine 1 g/t It is clear from the table above Nagoto<KY10628 and:KY10614 are
Parent strains (ATCC13761 and ATCC13869)
Compared to thiarisin, it had a negative effect on growth inhibition.

As the fermentation medium used in this invention, carbon sources, nitrogen sources, inorganic substances, and other growth-promoting substances used in ordinary fermentation media can be used.

As carbon sources, glucose, fructose, sucrose, maltose, starch, molasses, blackstrap molasses, and various other hydrous carbons, organic acids such as acetic acid, citric acid, and propionic acid, and alcohols such as ethanol, glycerin, and alcohol are used. It will be done.

As nitrogen sources, the usual inorganic or organic nitrogen sources, such as ammonia, ammonium chloride, ammonium sulfate,
Ammonium phosphate, urea, yeast extract, corn steep liquor, peptone, etc. are used.

Inorganic substances include phosphates and sulfates such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, and other magnesium sulfates. Uum salts, calcium salts, iron salts, manganese salts and trace metal salts are used.

As the growth promoting substance, amino acids, vitamins, pantothenic acid, casein hydrolyzate, etc. are used.

If the pH decreases during culture, it is desirable to adjust the pH to around neutrality using a suitable neutralizing agent such as ammonia gas, aqueous ammonia, sodium hydroxide, ammonium carbonate, calcium carbonate, etc.

Furthermore, if the pH increases because an organic acid is used as the main carbon source, it is desirable to adjust the pH using the organic acid or mineral acid.

Isolation of L-valine from the fermentation process may be carried out by conventional methods, such as an ion exchange resin method, a concentration method, or a combination of other known methods.

Example 1 Corynebacterium glutamicum KY10628 and its parent strain Corynebacterium glutamicum A grown in the above medium A slant at 28°C for 24 hours
Separately, TCC13761 was inoculated into a seed culture medium with the following composition, and 28
°C, 220 r. p. m. Culture for 24 hours.

Seed culture medium; Glucose 509/t, Pep 1/n 11/t, Yeast extract 10 El/l, NaCl 2.5 9/t
t, urea 3g/t, biotin 50g/t, corn steeper 5g/l, (pH 7.2).

Next, the two kinds of seed culture media mentioned above were added at a ratio of 10% to the main fermentation medium having the following composition, and each was placed in a separate 250 WL flask at 28°C and 220 r.m. p. m. Culture for 72 hours.

Main fermentation medium: Molasses 180.9/4 (1oo.9/4 as glucose,
! )KH2804 29/L MnS04- 4H20
0.01g/l,. MgSO, ・7 H20
0. 5 9/l, FeS04・7 H20 0.
0 1 g/t, (NH4)2S0420g/L biotin 50γ/t, protein acid hydrolyzate 5g/LCaC03
30 &/l, (pH 6.8).

Among the two types of fermentation finished liquids mentioned above, the one using Corynebacterium glutamicum KY10628 has 2 to 3 valine.
However, the parent strain Corynebacterium glutamicum ATCC13761 did not produce valine.

A supernatant liquid was obtained from 250 liters of the former valine-containing fermentation liquid by centrifugation, and L-valine was separated from it by a conventional method using an acidic ion exchange resin and purified to obtain L-valine crystals 2. 1g was obtained.

Example 2. Previbacterium lactofermencum KY106
Example 1.14 and its parent strain Previbacterium lactofermentum ATCC13869 were used. Cultivate in the same way.

The fermentation liquid using the former strain contains valine at a concentration of 30.59/
However, valine was not produced in the fermentation liquid using the latter strain.

Example 1: 250 ml of the former valine-containing fermented i'ffl
．． The same procedure as above was carried out to obtain 2.89 L-valine crystals.

Example 3. Previbacterium lactofermentum KY106 grown in the above medium A slant at 28°C for 24 hours
14 and its parent strain Previbacterium lactofermentum ATCC 13869 were separately inoculated into a seed culture medium with the following composition, and incubated at 30°C with an aeration rate of 3 1
/mvL. Stirring 600r. p. m. Incubate for 18 hours.

During cultivation, the pH was adjusted to 6 with a 60% (W/V) acetic acid solution.
．． Adjust to 8.

Seed culture medium; acetic acid 7&/LKi{2P0.2 g/LMgSO4.7
H20 0.5.9/LFeSO, ・7 H20
0.0 1. ? /AMnSO4・4 H20 0.0
19/L (NH4)2 8042 6.6 9/
L-pyotine 50γ/t, Cohn Stibli force -2 0 1
171, (pH 7.4).

Next, the above two types of seed culture media were separately added to the main fermentation medium condensed below at a ratio of 20%, and using a 5t jar fermenter, the aeration rate was 3t/mvl, and the mixture was stirred for 7 hours.
0 0 r. p. m. Culture for 48 hours.

During cultivation, the pH was adjusted to 7.5 with 60% (W/V) acetic acid solution.
While adjusting the acetic acid concentration in the medium to 2 to 6 El/
Feed ammonium acetate so that the amount becomes t.

Main fermentation medium: Acetic acid 79/LKH2P0,2g/LMgS04・7H2
0 o. 5 g/LF e S O4 ・7 H2 0
0-0 1 9/LMn S 0 4 ・4H20
0.01g//. NH, C7 8 m/t biotin 5
0γ/L cone stripe-10. ! 9/L (pH 6.
8).

Among the two types of main fermentation media mentioned above, the one using Previbacterium lactofermentum KY10614 accumulated 30 E//t of valine;
No valine was produced when Lactofermentum ATCC13869 was used.

One ton of the valine-containing fermentation liquid was treated in the same manner as in Example 1 to obtain 10.9& of L-valine.

Claims (1)

[Claims] 1 Belongs to the genus Corynebacterium or the genus Previbacterium, and is a substitute for S-2-aminoethyl-L-cysteine.
1. A method for producing L-valine using a microorganism, which comprises culturing a valine-producing bacterium having E. in a nutrient medium and collecting L-valine produced and accumulated in the medium.