JP2582810B2 - Method for producing L-isoleucine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing L-isoleucine by an enzymatic method.

According to the present invention, L-isoleucine can be efficiently produced with high yield.

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 pharmaceutical, food, feed additive or the like has been rapidly increasing in recent years.

2. Description of the Related Art As an industrial method for producing L-isoleucine, it is difficult to produce only the L-isomer by a chemical synthesis method because stereoisomers exist as in the case of other amino acids. Is being done. As a fermentation method, a method using a precursor of L-isoleucine such as DL-α-aminobutyric acid and threonine (Japanese Patent Publication Nos. 43-8709 and 40-2880),
A so-called direct fermentation method in which no precursor is particularly added (Japanese Patent Publication No. 38-70)
No. 91, JP-A-49-93586, etc.). On the other hand, as an enzymatic method, D-, L-, and L- or DL-amino acids other than ammonium ion or isoleucine are used.
Or DL-α-keto-β-methylvaleric acid to L-
Method for producing isoleucine (Japanese Patent Publication No. 46-29789), ammonium ion or L other than isoleucine
-Or D- isoleucine or D-alloisoleucine alone or in the presence of a DL-amino acid,
Alternatively, a method for producing L-isoleucine by acting on an appropriate mixture of these and optical isomers thereof (JP-B-46-29788)
Publication), a method for producing L-isoleucine from glucose and D-threonine using an immobilized product of a Serratia genus bacterium (Abstracts of the Annual Meeting of the Fermentation Engineering Society of Japan, p.47-
48, 1977). However, these methods have a problem that the raw material cost increases and the yield is low.

SUMMARY OF THE INVENTION The present invention provides a biotin-requiring Brevibacterium (Br
(A) α-ketobutyric acid or a salt thereof, (B) L- or DL-α-aminobutyric acid or a salt thereof, or (C) L- Or DL-α-hydroxybutyric acid or a salt thereof is subjected to an enzymatic reaction in an aqueous solution containing at least acetic acid or a salt thereof to produce L-isoleucine in the aqueous solution, from which L-isoleucine is collected. And a method for producing L-isoleucine.

According to the method of the present invention, (A) α-ketobutyric acid or a salt thereof, (B) L- or DL-α-aminobutyric acid or a salt thereof, or (C) L- or DL-α-hydroxy L-Isoleucine can be efficiently produced in high yield from butyric acid or a salt thereof, and acetic acid or a salt thereof which is relatively inexpensive as a reaction raw material.

The method of the present invention is an enzymatic reaction, and the reaction solution at that time is an aqueous solution containing at least acetic acid, and does not require complicated operations such as sterilization of the medium used in the case of the fermentation method. is there.

In the method of the present invention, the production of L-isoleucine by such an enzymatic method has not been reported or practiced, and the present invention is a novel method.

DETAILED DESCRIPTION OF THE INVENTION The microorganism used in the present invention belongs to the genus Brevibacterium that requires biotin, and is preferably one that can utilize ethanol. These include L-isoleucine producing bacteria. Examples of the microbial cells used in the present invention include Brevibacterium flavum MJ-233 (FERM).
BP-1497), Brevibacterium flavum (Breviba)
cterium flavum) MJ-233-AB-41 (FERM BP-1498),
Brevibacterium flavu
m) MJ-233-ABT-11 (FERM BP-1500) and Brevibacterium flavum MJ-233
-ABD-21 (FERM BP-1499), and such bacteria are suitably used in the present invention.

Note that the above (FERM BP-1498) is the same as (FERM BP-149)
7) is an ethanol-assimilating microorganism positively imparted with DL-α-aminobutyric acid resistance using the parent strain as a parent strain (JP-B-59-28).
No. 398, columns 3-4). (FERM BP-1500)
RM BP-1497) as a parent strain, which is a mutant with high activity of L-α-aminobutyric acid transaminase (see JP-A-65-21998). (FERM BP-1499) is replaced with (FERM BP-14
97) is a mutant strain having a high activity of L-α-aminobutyric acid deaminase having the parent strain (see JP-A-61-177993).

In addition to these microbial cells, Brevibacterium ammoniagenes ATCC 6
871, ATCC 13745, ATCC 13746, Brevibacterium divaricatum AT
CC 14020 or the like can also be used.

The microbial cells belonging to the genus Brevibacterium requiring biotin for use in the present invention can be used as they are, or immobilized products obtained by immobilizing them by a known method can be used. Examples of the immobilization method include immobilizing the cells using a polymerizable monomer such as acrylamide, or immobilizing the cells on a suitable carrier such as alginate or carrageenan.

The culture medium used for preparing the above-mentioned biotin-requiring microorganism belonging to the genus Brevibacterium used in the method of the present invention is not particularly limited, and may be any medium used for general microorganisms. Among them, a medium containing ethanol as a main carbonic acid source is preferable.

Ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used alone or in combination as a nitrogen source of a medium used for preparing the microbial cells used in the present invention.

As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. If necessary for the growth of other bacteria and L-isoleucine production,
Nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and various vitamins are added to the medium and used.

The cultivation is performed under aerobic conditions such as aeration stirring and shaking, and the culturing temperature is 20 to 40 ° C, preferably 25 to 35 ° C. The pH during the culturing is 5 to 10, preferably around 7 to 8. The pH during the culturing is adjusted by adding an acid or an alkali.

The ethanol concentration at the start of the culture is preferably 1 to 5% by volume, more preferably 2 to 3% by volume. The culturing period is 2 to 9 days, and the optimal period is 4 to 7 days.

The cells are collected from the culture thus obtained, washed with water or a suitable buffer, and used for the enzymatic reaction of the method of the present invention.

In the method of the present invention, in the presence of the microbial cells prepared above (including the immobilized product thereof),
(A) α-ketobutyric acid or a salt thereof, (B) L- or DL-α
-Aminobutyric acid or a salt thereof, or (C) L- or DL-
α-Hydroxybutyric acid or a salt thereof is subjected to an enzymatic reaction in an aqueous solution containing at least acetic acid or a salt thereof.

Here, the concentration of acetic acid or a salt thereof added to the aqueous solution is
It is 0.5 to 5.0% by weight, preferably 1 to 3.0% by weight.
(A) α-ketobutyric acid or a salt thereof, (B) L- or DL-α
-Aminobutyric acid or a salt thereof, or (C) L- or DL-
The concentration used during the reaction of α-hydroxybutyric acid or a salt thereof,
Generally, 0.1 to 20% by weight is suitably used.

In the method of the present invention, the aqueous solution used for the enzyme reaction may be water containing acetic acid or a salt thereof, or a buffer solution such as phosphoric acid or tris-hydrochloric acid. And an aqueous solution containing inorganic salts and the like.

As the above-mentioned nitrogen source and inorganic salt, known nitrogen sources and inorganic salts used in a medium used for a usual culture medium of microbial cells can be used. Specifically, the nitrogen source is ammonia,
Examples of inorganic nitrogen sources such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium phosphate,
Examples of the inorganic salt include potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, and iron sulfate. These inorganic nitrogen sources and inorganic salts can be used alone or in combination of two or more.

Amino acids, vitamins, saccharides, and the like having a known chemical structure that clearly does not contain biotin other than the above-mentioned nitrogen source and inorganic salt can be added to the enzyme reaction of the method of the present invention.

It is necessary that biotin is not present in the enzyme reaction system of the method of the present invention. If biotin is present, microbial cells used for the enzymatic reaction multiply, and the separation / purification of fermentation products other than the target and the target becomes complicated, which is not preferable.

The concentration of the inorganic nitrogen source and / or the inorganic salt or the like as an aqueous solution may be in the same range as a medium used for culturing ordinary microbial cells, and is not particularly limited.

In the method of the present invention, the amount of the microbial cells used for the enzyme reaction is not particularly limited, but can be generally used at a concentration of 1 to 50% (wt / vol).

In the present invention, the enzymatic reaction is carried out at a temperature of about 20 to about 50C, preferably about 30 to about 40C, usually for about 10 to about 72 hours.

The enzyme reaction, the concentration of dissolved enzyme in an aqueous solution containing at least acetic acid or a salt thereof used in the reaction is 0.05 ppm
As described above, it is preferable to supply air or oxygen to the reaction system continuously or intermittently so as to be 8 ppm or less.

Separation and purification of L-isoleucine produced in the reaction solution obtained by the above reaction method can be performed by a known ion exchange resin treatment method or a precipitation method.

Experimental Examples In the following experimental examples, the qualitative value of L-isoleucine was
Rf value of paper chromatograph, mobility of electrophoresis method,
It was confirmed by the biological activity value by the microorganism determination method. Quantification was performed using Leuconostoc mesenroides
senteroides) ATCC 8024 microbioassay method and high performance liquid chromatography (Shimadzu LC-5A). In the following experimental examples, “%” means “% by weight”.

Example 1 Medium (urea 0.4%, ammonium sulfate 1.4%, KH ₂ PO ₄₀ .
_{05%, K 2 HPO 4 0.05} %, MgSO 4 · 7H 2 O0.05%, CaCl 2 · 2H 2 O
_{2ppm, FeSO 4 · 7H 2 O2ppm} , MnSO 4 · 4~6H 2 O2ppm, ZnSO 4
· 7H ₂ O2ppm, NaCl2ppm, biotin 200 [mu] g /, thiamine · HCl100μg /, 0.1% casamino acid, 0.1% yeast extract)
Dispense 100 ml into a 500 ml Erlenmeyer flask and sterilize (pH 7.
0) followed by Brevibactium flavum
erium flavum) MJ-233 (FERM BP-1497) or the same MJ-233
233-AB-41 (FERM BP-1498) or MJ-233-ABT-
11 (FERM BP-1500) or MJ-233-ABD-21 (FERM BP-1500)
BP-1499), and aseptically add 2m of ethanol
l, and shaking culture was performed at 30 ° C. for 2 days.

Next, the main culture medium (ammonium sulfate 2.3%, KH ₂ PO
_{4 0.05%, K 2 HPO 4} 0.05%, MgSO 4 · 7H 2 O0.05%, FeSO 4 · 7
_{H 2 O20ppm, MnSO 4 · nH} 2 O20ppm, biotin 200 [mu] g /, thiamine · HCl100μg /, 0.3% casamino acid, yeast extract
(0.3%) was charged into a two-volume aeration stirred tank, and four identical ones were prepared. Sterilize each (120 ° C, 2
After 0 min), 20 ml of ethanol and 20 ml of the preculture obtained above were added, respectively, and the number of rotations was 1,000 rpm and the aeration rate was 1
Culture was performed for 48 hours at 33.degree.

In addition, ethanol was added intermittently about every 1 to 2 hours so that the concentration in the medium during the culture did not exceed 2% by volume.

After completion of the culture, the cells were collected by centrifugation from 500 ml of each culture. The cells obtained by washing twice with these demineralized distilled water were respectively reacted with [(NH ₄ ) ₂ SO ₄ 2 g /
_{; KH 2 PO 4 0.5g /;} KH 2 PO 4 0.5g /; MgSO 4 · 7H 2 O0.5g /;
_{FeSO 4 · 7H 2 O, 20ppm} ; MnSO 4 · 4~6H 2 O20ppm; Thiamine -
Hydrochloric acid 100 μg / (pH 7.6)] and suspended in 1000 ml, and the suspension was charged into a 2-volume agitated tank.
The reaction was carried out at a rotation speed of 300 rpm, a temperature of 33 ° C. and a pH of 7.6 by adding 30 g and each of the reaction raw materials shown in Table 1 at 10 g / addition. At this time, the dissolved oxygen concentration in all the reaction solutions is 0.1 ppm
Was kept.

The reaction time was 24 hours when DL-α-aminobutyric acid or DL-α-hydroxybutyric acid was used as a raw material, and 15 hours when sodium α-ketobutyrate was used as a raw material.

After the completion of the reaction, L-isoleucine in the supernatant liquid was removed by centrifugation (4000 rpm, 15 minutes, 4 ° C.), and the amount was determined.

After the reaction was completed, 500 ml of each reaction solution was passed through a column of a strongly acidic cation exchange resin (H ⁺ type) to adsorb L-isoleucine, washed with water, and eluted with 0.5 N aqueous ammonia. The L-isoleucine fraction was concentrated, and crystals of L-isoleucine were precipitated with cold ethanol and purified. The results are shown in Table 1.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Shoichi Nara 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Inside the Central Research Laboratory, Mitsubishi Yuka Co., Ltd. 8-3-1, Mitsubishi Yuka Central Research Laboratory (72) Inventor Hideaki Yukawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Mitsubishi Yuka Central Research Laboratory

Claims (1)

(57) [Claims] 1. A biotin-requiring Brevibacterium (Br)
(A) α-ketobutyric acid or a salt thereof, (B) L- or DL-α-aminobutyric acid or a salt thereof, or (C) L- Or DL-α-hydroxybutyric acid or a salt thereof is subjected to an enzymatic reaction in an aqueous solution containing at least acetic acid or a salt thereof to produce L-isoleucine in the aqueous solution, from which L-isoleucine is collected. A method for producing L-isoleucine.