JP2716470B2 - Method for producing L-isoleucine - Google Patents

Description

The present invention relates to a method for producing L-isoleucine, and more particularly, to a method for producing L-isoleucine in a high yield by suppressing the production of by-products by an enzymatic method or a fermentation method.

L-isoleucine is one of the essential amino acids, and is used in pharmaceuticals, foods, feed additives and the like as an amino acid that plays a major role in nutrition of humans and animals, and its demand has been rapidly increasing in recent years. .

Since L-isoleucine has a stereoisomer like other amino acids, it is generally difficult to chemically synthesize L-isoleucine, and industrially it is mainly produced by a fermentation method. For example, L- such as DL-α-aminobutyric acid and threonine
A method of fermenting in a precursor of isoleucine (see JP-B-43-8709, JP-B-40-2880, etc.); a method of so-called direct fermentation without using such a precursor (Japanese Patent Publication No. 38-7091) And Japanese Patent Publication No. 49-93586).

On the other hand, as an enzymatic method, for example, a method for producing L-isoleucine from D-, L- or DL-α-keto-β-methylbutyric acid in the presence of an ammonium ion or an L- or DL-amino acid other than isoleucine (Special Publication 46
-29789); in the presence of an ammonium ion or an L- or DL-amino acid other than isoleucine, acting on D-isoleucine or D-alloisoleucine alone or in a mixture or an appropriate mixture of these and an optical isomer thereof. (See Japanese Patent Publication No. 46-29788); a method for producing L-isoleucine from glucose and D-threonine using an immobilized Serratia bacterium (Japanese Society of Fermentation Engineering) Abstracts of lectures, pp. 47-48, 1977) have been reported.

However, these methods increase raw material costs,
There are problems such as a low yield, which is not satisfactory.

Further, the present inventors have previously described Brevibacterium (Brev
Microorganisms belonging to the genus ibacterium and having ethanol assimilation are aerobically cultured in a medium containing ethanol containing DL-α-aminobutyric acid as a main carbon source to produce and accumulate L-isoleucine in the culture broth. At least, L-isoleucine is collected from this culture solution, and L-isoleucine is obtained by a fermentation method.
A method for producing isoleucine (see Japanese Patent Publication No. 57-26755 and Japanese Patent Publication No. 59-28398); In the presence of L-isoleucine, an aqueous solution containing ethanol and α-aminobutyric acid is subjected to an enzymatic reaction to produce L-isoleucine in the solution, from which L-isoleucine is collected.
A method for producing isoleucine (see Japanese Patent Application No. 61-133773) has been proposed.

The present inventors have proposed the above-mentioned two methods,
In the course of conducting an improvement study to obtain L-isoleucine at a higher yield, O-ethylhomoserine is by-produced in the fermentation broth or the enzyme reaction solution, and this substance is produced from the culture broth or the reaction broth. It was clarified that the separation and purification of L-isoleucine was complicated and the recovery rate of L-isoleucine was reduced. Therefore, the present inventors believe that suppressing the by-product of O-ethylhomoserine in a fermentation broth or an enzyme reaction solution can improve the yield of L-isoleucine, and have studied diligently on a method for the suppression. This time,
If the production of L-isoleucine by a fermentation method or an enzymatic method using a biotin-requiring microorganism belonging to the genus Brevibacterium described above or a processed product thereof is carried out in the presence of L- or DL-methionine, O-ethylhomoserine can be obtained. It has been found that by-products are significantly reduced, separation and purification of L-isoleucine from a culture solution or an enzyme reaction solution are facilitated, and the yield of L-isoleucine can also be improved. Thus, the present invention has been completed. .

Thus, according to the present invention, Brevibacterium (Brev
L- or DL-α-amino-n-butyric acid or a salt thereof, α-ketobutyric acid or a salt thereof, and L- or DL- In producing L-isoleucine by subjecting a butyric acid derivative selected from α-hydroxybutyric acid or a salt thereof to an enzymatic reaction in an aqueous ethanol solution,
There is provided a method for producing L-isoleucine (hereinafter referred to as an enzymatic method), wherein the enzymatic reaction is carried out in the presence of L- or DL-methionine.

According to the present invention, further, a biotin-requiring microorganism belonging to the genus Brevibacterium can be produced by using L- or DL-α-amino-n-butyric acid or a salt thereof, α-ketobutyric acid or a salt thereof, and L- or DL-hydroxybutyric acid. Alternatively, aerobically culturing in a medium containing ethanol containing a butyric acid derivative selected from salts thereof as a main carbon source, and producing and accumulating L-isoleucine in the culture broth, the culture is treated with L- or DL-methionine. And a method for producing L-isoleucine [hereinafter referred to as a fermentation method].

 Hereinafter, the method of the present invention will be described in more detail.

The microorganism used in the method of the present invention is a biotin-requiring microorganism belonging to the genus Brevibacterium, and preferably has ethanol utilization. Among them, L-isoleucine producing bacteria are also included. Specific examples of such microorganisms include Brevibacterium flavu
m) MJ-233: (Microtechnical Laboratory No. 1497), Brevibacterium flavu
m) MJ-233-AB-41 (Jikken-Jikken No. 1498), Brevibacterium flavu
m) MJ-233-ABT-11 (Microtechnical Laboratory No. 1500), Brevibacterium flavu
m) MJ-233-ABD-21 (Microtechnical Research Institute No. 1499), and the like, and these bacteria are suitably used in the present invention.

In addition, the microbial strain No. 1498 strain is a ethanol-assimilating microorganism positively imparted with DL-α-aminobutyric acid resistance using the microbial strain No. 1497 strain as a parent strain. (See columns 3 and 4 of JP-B-59-28398). The strain of No. 1500 is a highly active mutant of L-α-aminobutyric acid transaminase using the strain of No. 1497 as a parent strain (Japanese Patent Application No. 60-190609). 3-5). Further, the strain of No. 1499 is a D-α-aminobutyric acid deaminase highly active mutant using the strain of No. 1497 as a parent strain (see Japanese Patent Application No. 61-177793). ).

In addition to these microorganisms, Brevibacterium ammoniagenes ATCC 687
1, ATCC 13745, ATCC 13746; Brevibacterium
Devaricatum (Brevibacterium divaricatum) ATCC 14
020 or the like can also be used.

The cultivation of the biotin-requiring microorganism belonging to the genus Brevibacterium as described above can be performed using a medium containing nutrients such as an ethanol main carbon source, a nitrogen source, and an inorganic salt, according to a conventional method. . When L-isoleucine is produced by fermentation, L-isoleucine is added to the medium.
-Or DL-α-amino-n-butyric acid or a salt thereof,
It contains a butyric acid derivative selected from α-ketobutyric acid or a salt thereof and L- or DL-hydroxybutyric acid or a salt thereof.

As the nitrogen source that can be contained in the medium, nitrogen-containing organic or inorganic substances that can be generally used in culturing microorganisms, such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea can be used alone or in combination. As the inorganic salt, for example, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like can be used. In addition, if necessary for growth of bacteria and production of L-isoleucine, 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.
The cultivation temperature can be generally in the range from 20 to 40 ° C, preferably from 25 to 35 ° C. The pH of the medium is usually in the range of 5 to 10, preferably around 7 to 8.
Can be adjusted by appropriately adding an acid or an alkali to the medium.

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

In the fermentation method, the concentration of the butyric acid derivative added to the medium can be generally in the range of 0.1 to 5% by weight, preferably 1 to 3% by weight.

When the method of the present invention is carried out by a fermentation method, the present invention has an essential feature in that culturing is performed in the presence of L- or DL-methionine. L- or DL to the medium
-The concentration of methionine can be changed depending on the composition of the medium to be used, the type of microorganism, and the like.
A suitable range is from 5 to 5% by weight, preferably from 0.05 to 2% by weight.

On the other hand, when the method of the present invention is carried out by the enzymatic method, the cells can be collected from the culture obtained by culturing as described above, washed with water or an appropriate buffer, and then used directly. Alternatively, the cells can be immobilized by a method known per se and used as an immobilized product, or the cells can be crushed by means of ultrasonication, squeezing, or the like, and the crushed product or a crushed product obtained by a method known per se Alternatively, an immobilized enzyme immobilized in step (1) can be used. Examples of the method for immobilizing the microbial cells or the crushed product thereof include a method using a polymerizable monomer such as acrylamide, and a method of insolubilizing using a suitable carrier such as alginate or carrageenan.

In the enzymatic method according to the present invention, the above-mentioned butyric acid derivative is reacted in an aqueous ethanol solution in the presence of the above-mentioned microbial cells or a processed product thereof and in the presence of L- or DL-methionine. The concentration of ethanol in the aqueous ethanol solution is generally 0.5 to 40% by volume, preferably 1%.
It can be in the range of ~ 20% by volume.

The aqueous solution may be water containing ethanol or a buffer solution such as phosphoric acid or Tris-HCl, but preferably an aqueous solution containing ethanol and further containing a nitrogen source and / or inorganic salts is used.

In the method of the present invention, the nitrogen source and the inorganic salts that can be added to the enzyme reaction system can be selected from those added to the medium used in the culture of ordinary microbial cells. However, in this case, it is desirable to use one that does not contain biotin.

Specific examples of the nitrogen source that can be contained in the aqueous solution include ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, inorganic nitrogen sources such as ammonium phosphate, and the like.
These hydrogen sources and inorganic salts can be used alone or as a mixture of two or more thereof. Examples thereof include potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, and iron sulfate.

The concentration of these nitrogen sources and / or inorganic salts in an aqueous solution can be in the same range as in a medium used for culturing ordinary microbial cells, and is not particularly limited.

An example of such a reaction solution is (NH ₄ ) ₂ SO ₄ 2 g /
,; KH ₂ PO ₄ 0.5 g /; K ₂ HPO ₄ 0.5 g /; MgSO ₄・ 7H ₂ O 0.5
_{g /; FeSO 4 · 7H 2} O 20ppm; aqueous solution of MnSO ₄ · 4~6H ₄ O 20ppm containing pH7.6 and the like.

Furthermore, amino acids, vitamins, saccharides and the like which do not contain bition can be added to the aqueous solution.

The concentration of the butyric acid derivative selected from the L- or DL-α-amino-n-butyric acid or a salt thereof, α-ketobutyric acid or a salt thereof and L- or DL-α-hydroxybutyric acid or a salt thereof in an aqueous solution is as follows: There is no particular limitation, but generally 0.1 to 20% (wt / vol), preferably 0.2 to 20% (wt / vol).
A range of 5% (wt / vol) is appropriate.

On the other hand, the concentration of the microbial cells or the processed product thereof which is present in the aqueous solution is not particularly limited, either.
Generally, 1 to 50% (wt / vol), preferably 2 to 20% (wt / vol)
/ vol).

The concentration of L- or DL-methione coexisting in the enzyme reaction system according to the method of the present invention at the time of the reaction is as follows:
It can vary depending on the type and form of the microbial cells used or the processed product thereof, the composition of the aqueous solution, etc., but is generally 0.01 to 5% by weight, preferably 0.05 to 2% by weight.

The enzymatic reaction according to the present invention is generally 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 separation and purification of L-isoleucine produced in the culture solution or the reaction solution by the fermentation method and the enzyme method described above from the culture solution or the reaction solution may be performed according to a method known per se, for example, an ion exchange resin treatment method, a precipitation method, or the like. It can be performed by appropriately combining methods and the like.

Next, the method of the present invention will be described more specifically with reference to examples. In the following examples, the qualities of O-ethylhomoserine and L-isoleucine were determined by paper chromatography.
The determination was performed based on the Rf value and the mobility of the electrophoresis method. The quantification was performed using high performance liquid chromatography (Shimadzu LC-5A). In the following examples,% means% by weight.

Example 1 Medium (0.4% urea, 1.4% ammonium sulfate, KH ₂ PO ₄
0.05%, K ₂ HPO ₄ 0.05%, MgSO ₄・ 7H ₂ O 0.05%, CaCl ₂・
_{2H 2 O 2ppm, FeSO 4 ·} 7H 2 O 2ppm, MnSO 4 · 4~6H 2 O 2pp
_{m, ZnSO 4 · 7H 2 O} 2ppm, NaCl 2ppm, biotin 200 [mu] g /
, Thiamine / HCl 100μg /, casamino acid 0.1%, yeast extract 0.1%) 100ml is dispensed into a 500ml Erlenmeyer flask,
After sterilization (pH 7.0 after sterilization), Brevibacterium flavum MJ-233 (Microtechnical Research Institute No. 1497) was inoculated, and 2 ml of ethanol was aseptically added.
Shaking culture was performed at 0 ° 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 O 0.05%, FeSO ₄・
7H ₂ O 20ppm, MnSO ₄・ nH ₂ O 20ppm, Bition 200μg /
, Thiamine / HCl 100μg /, casamino acid 0.3%, yeast extract 0.3%)
After sterilization (102 ° C., 20 minutes), 20 ml of ethanol and 20 ml of the preculture were added, and the rotation speed was 1000 rpm, and the aeration rate was 1 vv.
Culture was carried out at a temperature of 33 ° C. and a pH of 7.6 for 48 hours.

In addition, the concentration of the ethanol in the culture medium was 2% by volume.
Was added intermittently about every 1-2 hours so as not to exceed.

After completion of the culture, the cells were collected by centrifugation from 500 ml of the culture. This is washed twice with demineralized distilled water, and the obtained cells are
Reaction solution [(NH ₄ ) ₂ SO ₄ 2.3%, KH ₂ PO ₄ 0.05%, K ₂ HPO _{4
0.05%, MgSO 4 · 7H 2} O 0.05%, FeSO 4 · 7H 2 O 200ppm, Mn
SO ₄ · 4~6H ₂ O 200ppm, Thiamine - HCl 100 [mu] g / (pH
7.6)] After suspending in 1000 ml, the suspension was charged into a 2-volume agitating tank, and further added with 20 ml of ethanol, 0.5 g of DL-methionine and 1% of DL-α-aminobutyric acid or 0.5% of sodium α-ketobutyrate. Alternatively, 1% of sodium DL-α-hydroxybutyrate is added, and the number of rotations is 300 rpm, the temperature is 33 ° C., and the pH is 7.6 (25%).
% Adjusted with aqueous ammonia) for 15 hours.

After the completion of the reaction, O-ethylhomoserine and L-isoleucine in the supernatant liquid that had been sterilized by centrifugation (4000 rpm, 15 minutes, 4 ° C) were quantified.

As a comparative example, the same reaction was performed without adding DL-methionine. The results are shown in Table 1 below.

Example 2 Microorganisms were used for Brevibacterium flavum.
rium flavum) MJ-233-AB-41 (Microtechnical Laboratories No. 1498)
The procedure was performed in the same manner as in Example 1 except that

 The results are shown in Table 2 below.

Example 3 Microorganisms were used for Brevibacterium flavum.
rium flavum) MJ-233-ABT-11
) Was carried out in the same manner as in Example 1 except that

 The results are shown in Table 3 below.

Example 4 A microorganism was used for Brevibacterium flavum.
rium flavum) MJ-233-ABD-11
) Was carried out in the same manner as in Example 1 except that

 The results are shown in Table 4 below.

Example 5 50 ml of the main culture medium of Example 1 was dispensed into a 500 ml Erlenmeyer flask, sterilized, and then added with 0.05% of DL-methionine and DL.
-Α-aminobutyric acid 0.5% or α-sodium ketobutyrate 0.5
% Or 0.5% of DL-α-hydroxybutyric acid and 1 ml of ethanol
Was added, and 2 ml of the preculture of Example 1 was further added, followed by shaking culture at 220 rpm and a temperature of 33 ° C. for 48 hours.

After the cultivation, O-ethylhomoserine and L-isoleucine in the supernatant liquid removed by centrifugation (4000 rpm, 15 minutes, 4 ° C) were quantified. In addition, as a comparative example, the same culture was performed without adding DL-methionine. The results are shown in Table 5 below.

Example 6 A microorganism was used for Brevibacterium flavum.
rium flavum) MJ-233-AB-41 (Microtechnical Laboratories No. 1498)
Example 5 was carried out in the same manner as in Example 5, except that

 The results are shown in Table 6 below.

Example 7 The microorganism was used for Brevibacterium flavum.
rium flavum) MJ-233-ABT-11
Example 5 was carried out in the same manner as in Example 5 except that the above was changed.

 The results are shown in Table 7 below.

Example 8 Microorganisms were used for Brevibacterium flavum.
rium flavum) MJ-233-ABD-21
Example 5 was carried out in the same manner as in Example 5 except that the above was changed.

 The results are shown in Table 8 below.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Wado, 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Inside the Central Research Laboratory, Mitsubishi Yuka Co., Ltd. (72) Inventor Masato Terasawa, Ami-cho, Inashiki-gun, Ibaraki 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 (2)

(57) [Claims] (1) L- or DL-α-amino-n-butyric acid or a salt thereof, α-ketobutyric acid in the presence of a biotin-requiring microorganism belonging to the genus Brevibacterium or a processed product thereof. Alternatively, when enzymatically reacting a salt thereof and a butyric acid derivative selected from L- or DL-α-hydroxybutyric acid or a salt thereof in an aqueous ethanol solution to produce L-isoleucine,
-Or a method for producing L-isoleucine, which is carried out in the presence of DL-methionine. 2. A biotin-requiring microorganism belonging to the genus Brevibacterium, comprising L- or DL-amino-n-butyric acid or a salt thereof, α-ketobutyric acid or a salt thereof, and L- or DL-hydroxybutyric acid or a salt thereof. Aerobically cultured in a medium containing ethanol as a main carbon source containing the selected butyric acid derivative, and producing and accumulating L-isoleucine in the culture medium, the culture was carried out in the presence of L- or DL-methionine. A method for producing L-isoleucine.