JPS63192394A - Production of l-isoleucine - Google Patents

Abstract

PURPOSE:To obtain the titled compound useful as a medicine, etc., in high yield, by keeping the dissolved oxygen concentration in an alpha-aminobutyric acid-containing culture medium within a specific range in aerobically cultivating an ethanol-assimilating microorganism of the genus Brevibacterium in the culture medium. CONSTITUTION:An ethanol-assimilating microorganism, preferably Brevibacterium flavum MJ-233-AB-41 (FERM-P No. 3812), etc., belonging to the genus Brevibacterium is aerobically cultivated in an alpha-aminobutyric acid- containing culture medium while keeping the dissolved oxygen concentration in the culture medium at 0.5-8ppm to afford the aimed compound. Furthermore, the cultivation is preferably carried out at 25-35 deg.C cultivation temperature and pH 7-8 during the cultivation for 4-7 days.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing L-isoleucine by a fermentation method. More specifically, Brevibacterium (1) Brevibacterium Ii
When culturing a microorganism that belongs to E and has the ability to assimilate I-9/-7L/anaerobically in a medium containing α-aminobutyric acid, the dissolved oxygen concentration of the medium should be set to 0.5 ppm or more at 8F.
) It relates to a method for producing L-isoleucine, characterized in that the length is maintained at 9 m or less.

(Background of the Invention) L-isoleucine 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 medical, food, and feed enrichment agent has been increasing rapidly in recent years. As for the industrial production method of L-isoleucine, it is difficult to produce only the L-isomer by chemical synthesis method because stereoisomers exist like other amino acids, so it is mainly produced by fermentation method. ing.

(Prior Art and Problems) Regarding the production of L-isoleucine by fermentation, various studies have been made on culture medium composition, culture conditions, etc., but little is known about the influence of dissolved oxygen concentration.

The present inventors have completed the present invention by intensively studying the optimal culture method for producing L-isoleucine from α-aminobutyric acid.

(Structure and Effects of the Invention) The present inventors have discovered that when a microorganism belonging to the genus Brevibacterium and capable of assimilating ethanol is aerobically cultured in a medium containing α-aminobutyric acid, the dissolved oxygen concentration of the medium is By intermittently or continuously aerating the medium with a gas such as pure oxygen or a mixture of pure oxygen and air to maintain the concentration of o, 5 ppm or more and 13 ppm or less, L-isoleucine increases in the medium. It was discovered that the yield is N product, and the present invention was achieved. Conventionally, in the fermentation production of L-isoleucine, it is known that the dissolved oxygen concentration decreases as the bacterial cells multiply and reaches zero during the culture, but it is possible to reduce the concentration by actively adding pure oxygen etc. However, there is no report that it improves dissolved oxygen concentration (the present invention is a new method).

(Specific method of the invention) The microorganism used in the present invention is Brevibacterium (Br.
Any substance belonging to the genus evibacterium and capable of assimilating ethanol may be used. This includes L-isoleucine producing bacteria. The producing bacterium is, for example, Brevibacterium flavum.
avu+w) M J-233 (Feikoken Bacteria 30th
No. 68), Brevibacterium flavum (Brevi
bacterium flavum) MJ-233
-41 (Feikoken Bacteria No. 3812, Brevibacterium flavum (Brevibacterium fl)
avum) M J -233-A B T
-11 (Feikoken Bacteria No. 8423) and Brevibacterium flavum (Brevibacterium flavum)
flavu-) MJ-233-ABD-21 (Feikoken Bibori No. 8055), etc., and are suitably used in the present invention.

In addition, the above-mentioned (Feikoken Bokuyori No. 3812) is an ethanol-assimilating microorganism that has been actively imparted with DL-α-aminobutyric acid resistance using (Feikoken Bikyouri No. 3068) as its parent strain ( (See columns 3 and 4 of Japanese Patent Publication No. 59-28398),
(Feikoken Bikori No. 8423) is (Feikoku Kenbiyori No. 3)
068) as the parent strain and is a mutant strain with high activity of α-aminobutyric acid transaminase (Patent application No. 19060-1980).
(Refer to pages 3 to 5 of the specification of No. 9)
No. 55) is a D-α-aminobutyric acid deaminase high activity mutant (
See pages 5 to 7 of the specification of Japanese Patent Application No. 60-017501! ! 4)
.

The method for producing L-isoleucine of the present invention will be explained in more detail below.

The medium composition used for the preparation of microbial cells of the present invention preferably includes ethanol as the main carbon source, but the carbon source is not particularly limited and may be any one used for general microorganisms.The nitrogen sources include ammonia, ammonium sulfate, Inorganic salts that can be used alone or in combination such as ammonium chloride, ammonium nitrate, and urea include potassium phosphate-hydrogen,
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-isoleucine production.

The amount of DL-α-aminobutyric acid, which is a precursor of L-isoleucine, is 0.1 to 5% by weight, preferably 0.25% by weight.
~3% by weight.

Cultivation is carried out under aerobic conditions such as aeration agitation and shaking, and the culture temperature is 20 to 40°C, preferably 25 to 35°C.
The pH during the cultivation is maintained at around 5 to 10, preferably around 7 to 8, and the pH during the cultivation is adjusted by adding acid or alkali.

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

The culture period is 1-9 days, with an optimal period of 4-7 days.

The dissolved M concentration in the medium was measured over time using a dissolved oxygen needle (manufactured by Oriental Electric Co., Ltd.), and the dissolved oxygen concentration was measured at 0.5p.
Pure f! ! A mixed gas of oxygen or pure oxygen and air is added intermittently or continuously. Note that the upper limit of the dissolved oxygen concentration is set at 8 ppm in view of oxygen inhibition of bacterial growth.

The separation and purification of L-isoleucine produced in the culture solution obtained by the above culture method can be performed by an ion exchange resin treatment method, a precipitation method, or the like.

(Example) An example will be shown below. The quality of L-isoleucine was confirmed by the Rf value of paper chromatography, the mobility of electrophoresis, and the biological activity value of microorganism quantification. Quantification was performed using Leuconostoc mesenteroides (Leuco
NosLoc mesenteroides) was used in combination. Moreover, in the following examples, % means weight %.

Example-1 Medium (urea 0.4%, ammonium sulfate 1.4%, KH
2PO40.05%, K, HPo, 0°05%, Mg5
O+ ・7H200,05%, CaC1,・2Hz
0.2ppm, Fe5Oa HIHz 0.2ppm
, Mn5o4 -4~6Hz O2ppm, Zn5O
*・7H202ppm.

NaC 12ppm-Biotin 200pg/I! - Thiamine/HCjl 100 μg/l, Casamino acid 0.1%
, yeast extract 0.1%) 100mj! 500mlml
After dispensing Brevibacterium flavum into a flask and sterilizing it (pH 7.0 after sterilization),
ium flavus+) MJ-233 (Feikoken Bibori No. 3068) was inoculated, 2 mj of ethanol was added aseptically, and shaking culture was performed at 30 t for 2 days (preculture).

Next, the main culture medium (ammonium sulfate 2.3%, KH2
PO20.05%, K2 HPO40.05%, Mg5
Oa ・IHz OO, 05%, Fe5Q, -782
020 ppm, MnSO4.nH, 020 ppm, biotin 200 μg/j.

Thiami7・HCj l OQpg/It, Casamino acids 0
．． 3%, yeast extract 0.3%, DL-α-aminobutyric acid 0
．． 5%) was charged into a 21 volume aerated stirring tank.
After sterilization (120 °C, 20 minutes), 20 ml of ethanol
and 20 mj of the above preculture, and the rotation speed was 1100.
Culture was carried out for 24 hours at Qrp, aeration volume of 1 vvm, temperature of 33° C., and pH of 7.6.

Note that ethanol was added intermittently so that the concentration in the medium did not exceed 2% by volume during culture. Further, pure oxygen was added intermittently or continuously so that the dissolved acid and elemental concentrations were kept within the categories shown in Table 1.

After completion of the culture, L-isoleucine in the supernatant liquid was sterilized by centrifugation (4000 rpm, 15 minutes, room temperature) and quantified. In addition, 500 mJ of the culture solution was passed through a column of strongly acidic cation exchange resin (H+ type) to adsorb L-isoleucine, and after washing with water, it was eluted with 0°5N ammonia water, and the L-isoleucine fraction was concentrated. and L- with cold ethanol.
Crystals of isoleucine were precipitated. The results are shown in Table 1. In addition, L- when pure oxygen is not added (only air is added)
The amount of isoleucine produced was used as a comparative example.

Table 1 Note 1: 16 hours after the start of main culture, the concentration becomes 0.2 ppm or less.

Note 2 Amount obtained as two crystals Example-2 Brevibacterium flavum was grown under the same conditions as Example-1.
) M J -233-AB-41 (Microtechnology Research Institute 3rd
No. 812) was cultured and purified in the same manner as in Example-1.

The results are shown in Table 2 below.

Example-3 Brevibacterium flavum (1) was grown under the same conditions as Example-1.
rium flavus+) M J -233-A
Cultivate BT-11 (Feikoken Bacteria No. 8423゜),
Purification was then carried out in the same manner as in Example-1. The results are shown in Table 3 below.

Example-4 Brevibacterium flavum (1) was grown under the same conditions as Example-1.
rium flavum) M J -233-AB
D-21 (Feiko Kenbokuyori No. 8055) was cultured and purified in the same manner as in Example-1. The results are listed in the 4th section below.
Shown in the table.

Table 2 Note 2: Quantity obtained as crystals Table 3 Note 1: 0.2 ppm or less after 16 hours from the start of main culture.

Note 2 = Amount obtained as crystals Table 4 Note 1: 0.2 ppm or less after 16 hours from the start of main culture.

Note 2: Amount obtained as crystals

Claims (3)

[Claims] (1) Brevibacterium
When producing L-isoleucine by aerobically culturing ethanol-assimilating microorganisms belonging to the genus um) in a medium containing α-aminobutyric acid, the dissolved oxygen concentration of the medium is set to 0.5 pp.
A method for producing L-isoleucine, which comprises culturing while maintaining the concentration of L-isoleucine at a concentration of m or more and 8 ppm or less. (2) The manufacturing method according to claim 1, wherein pure oxygen is added to maintain dissolved oxygen in the medium at 0.5 ppm or more. (3) Brevibacterium
A microorganism that belongs to the genus um) and has the ability to assimilate ethanol is
Brevibacterium flavum
rium flavum) MJ-233, Brevibacterium flavum MJ-233-AB-41, Brevibacterium flavum MJ-233-ABT-11, Brevibacterium flavum MJ-233-ABD-2
1. The manufacturing method according to claim 1 or 2, which is claim 1.