JP2521095B2 - Method for producing L-isoleucine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing L-isoleucine by an enzymatic method. More specifically, a biotin-requiring microorganism belonging to the genus Brevibacterium or its immobilized product is subjected to an enzymatic reaction in a biotin-free aqueous solution containing ethanol and α-aminobutyric acid, The present invention relates to a method for producing L-isoleucine in high yield, which produces L-isoleucine.

(Prior art and its problem) L-isoleucine is an amino acid that plays an important role in human and animal nutrition as an essential amino acid, and the demand thereof as a pharmaceutical, food, and feed fortifier is rapidly increasing in recent years. As for the industrial production method of L-isoleucine, it is difficult to produce only the L-form by the chemical synthesis method because stereoisomers exist as in the case of other amino acids, and the production is mainly carried out by the fermentation method. ing.

As a fermentation method, a method using a precursor of L-isoleucine such as DL-α-aminobutyric acid and threonine (Japanese Patent Publication No.
-8709, Japanese Patent Publication No. 40-2880, etc.) and so-called direct fermentation method in which no precursor is added (Japanese Patent Publication No. 38-7091, Japanese Patent Publication No. 49-93586, etc.).

On the other hand, as an enzymatic method, L-isoleucine is produced from D-, L-, or DL-α-keto-β-methylvaleric acid in the presence of L- or DL-α-amino acid other than ammonium ion or isoleucine. Method (Japanese Patent Publication Sho 46-2
9789), ammonium ion or L other than isoleucine
-Or DL-amino acid, a method for producing L-isoleucine by acting on D-isoleucine or D-alloisoleucine alone or in a mixture, or an appropriate mixture of these and optical isomers thereof (JP-B-46- 29788),
A method for producing L-isoleucine from glucose and D-threonine using an immobilization product of a Serratia bacterium (Proceedings of the Japan Fermentation Engineering Society Conference p.47-48, 1977) and the like have been reported.

However, all of these methods have problems such as high raw material cost and low yield.

(Structure and Effect of the Invention) As a result of repeated studies to solve the above-mentioned problems, the present inventors have previously found that an ethanol-assimilating microorganism belonging to the genus Brevibacterium or a treated product thereof Invented was a method of enzymatically reacting an aqueous solution containing ethanol and α-aminobutyric acid in the presence of the immobilization product to produce L-isoleucine in the reaction solution.

Further, as a result of continuing the research on the present invention, the present inventors have found that in order to produce L-isoleucine with higher yield,
By using a completely synthetic medium containing no biotin, which is an essential component for the growth of the microorganism, as a reaction solution for the enzyme reaction,
It has been found to achieve that purpose. Further, the present inventors have found that, in order to enhance the effect of the invention and obtain a better yield, it is effective to carry out the reaction while maintaining the dissolved oxygen concentration of the reaction solution at 0.05 ppm or more. completed.

 The gist of the present invention is as described below.

"Biotin-requiring Brevibacterium
um) microorganisms belonging to the genus or its immobilization product, in an aqueous solution containing a carbon source and α-aminobutyric acid but not biotin, an enzyme reaction is carried out in the presence of dissolved oxygen to obtain L in the solution. -Produces isoleucine, from which L
-A method for producing L-isoleucine, which comprises collecting isoleucine. Heretofore, the production of L-isoleucine by the enzymatic method as in the present invention has not been reported or carried out, and the present invention is a novel method.

The microorganism used in the present invention may be one that belongs to the genus Brevibacterium that requires biotin, and is preferably ethanol-assimilating. Among these, L-isoleucine-producing bacteria are included. The microorganism is, for example, Brevibacterium flavum.
lavum) MJ-233 (FERM BP-1497), Brevibacterium flavum MJ-233-AB-41 (F
ERM BP-1498), Brevibacterium flavum (Brevi
bacterium flavum) MJ-233-ABT-11 (FERM BP-1500) and Brevibacterium flavum
vum) MJ-233-ABD-21 (FERM BP-1499) and the like, which are preferably used in the present invention.

The above-mentioned (FERM BP-1498) is an ethanol-assimilating microorganism positively endowed with DL-α-aminobutyric acid resistance using (FERM BP-1497) as a parent strain (Japanese Patent Publication No. 59-28398). ~ Column 4). (FERM BP-1500) is (FERM BP-14
It is a highly active mutant strain of L-α-aminobutyric acid transaminase whose parent strain is 97) (Japanese Patent Application No. 60-190609, specification 3 to
(See page 5). Also, (FERM BP-1499) is (FERM BP-149
It is a mutant strain of D-α-aminobutyric acid deaminase having a high activity, which is obtained by using 7) as a parent strain (see Japanese Patent Application No. 60-017501, pages 5 to 7).

The method for producing L-isoleucine of the present invention will be specifically described below.

The composition of the medium used for preparing the bacterial cells of the present invention preferably uses ethanol as a main carbon source, but is not particularly limited and may be that used for general microorganisms. As the nitrogen source, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea or the like can be used alone or in combination.

As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition to this, if necessary for the growth of the bacteria and the production of L-isoleucine,
Nutrients such as peptone, meat extract, yeast extract, corn stay liquor, casanomic acid and various vitamins are added to the medium and used.

Culturing is carried out under aerobic conditions such as aeration and stirring, and the cultivation 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 culture is preferably 1 to 5% by volume, more preferably 2 to 3% by volume. The culture period is 2 to 9 days, and the optimum period is 4 to 7 days.

The bacterial cells are collected from the thus obtained culture and washed with an appropriate buffer, and the washed bacterial cells are used for the enzymatic reaction of the present invention.

In the method of the present invention, DL-α-aminobutyric acid is added to an aqueous solution containing at least a carbon source (preferably ethanol) in the presence of the above-prepared microbial cells (including its immobilized product). To make the enzyme reaction, L
-Produce isoleucine. The concentration of ethanol added to the reaction solution is appropriately 1 to 20% by volume, preferably 2 to 10% by volume.

In addition, in the method of the present invention, an immobilized product of the bacterial cells can be used in the same manner as the above-mentioned bacterial cells.

The immobilization product of the bacterial cells of the present invention can be appropriately selected and adjusted from known immobilization methods such as an encapsulation method using acrylamide, alginate, carrageenan and the like, an ion binding method using DEAE-Sephadex, DEAE-cellulose and the like.

The aqueous solution contains water containing ethanol (pH:
7.0 to 9.0) or a buffer solution (pH 7.0 to 9.0) such as phosphoric acid or Tris hydrochloric acid can be used, but a completely synthetic medium containing ethanol is preferably used. Here, the completely synthetic medium is an aqueous solution containing an inorganic nitrogen source and an inorganic salt having a known chemical structure. Examples of the inorganic nitrogen source of the completely synthetic medium used in the present invention include ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate and the like, and inorganic salts include phosphate-potassium hydrogen, potassium dihydrogen phosphate, Examples include magnesium sulfate, manganese sulfate, iron sulfate and the like. These inorganic nitrogen sources and inorganic salts may be used alone or in admixture of two or more.

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

An example of a complete synthetic medium is (NH ₄ ) ₂ SO ₄ 2 g / l; KH ₂ PO _{4
0.5g / l; K 2 HPO 4} 0.5g / l; MgSO 4 · 7H 2 O0.5g / l; FeSO 4 · 7H 2
O 20 ppm; MnSO have pH7.6 aqueous solution containing ₄ · _{4~6H 2} O20ppm.

As mentioned above, the complete synthetic medium used in the present invention includes:
It does not contain biotin or natural products containing biotin. It is possible to add amino acids, vitamins, sugars, etc., of which the chemical structure is known to be free of biotin, which are known.

As the α-aminobutyric acid used in the method of the present invention, L- or DL-α-aminobutyric acid is used, and D-α
-Aminobutyric acid is used by being racemized to DL-α-aminobutyric acid.

The concentration of α-aminobutyric acid used in the enzymatic reaction in the method of the present invention is not particularly limited, but is generally 0.1 to 20.
% (Wt / vol), preferably in the concentration range of 2 to 10%. The amount of the microbial cells used is not particularly limited, but it can be used generally at a concentration of 1 to 50% (wt / vol), preferably 2 to 30%.

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

The above-mentioned enzymatic reaction is an aqueous solution containing no biotin used in the reaction, preferably an aqueous solution containing ethanol, and particularly preferably the dissolved oxygen in the above-mentioned ethanol-containing completely synthetic medium is preferably 0.05 ppm or more, but more than 8 ppm. Do so. When the dissolved oxygen in the reaction aqueous solution exceeds 8 ppm, enzyme reaction inhibition appears, which is not preferable.

To adjust the dissolved oxygen concentration of the reaction solution during the reaction, the dissolved oxygen of the reaction solution was measured with time using a dissolved oxygen measuring device (manufactured by Oriental Electric Co., Ltd.), and air or oxygen was fed to the reaction system. The amount of ventilation supplied continuously or intermittently should be adjusted.

In the enzymatic reaction of the present invention, it is advantageous and preferable to use a bacterial cell of a microorganism having an ability to racemize D-α-aminobutyric acid or its immobilized product without using L-isoleucine as a substrate. The reason is that L-α-aminobutyric acid is produced by racemizing D-α-aminobutyric acid, which remains as an unreacted product of DL-α-aminobutyric acid used as a raw material for this enzymatic reaction.

Examples of the microorganism having the ability to racemize D-α-aminobutyric acid without using L-isoleucine as a substrate include Pseudomonas Putida IFO 12
There are 996 etc.

The microbial cells having the ability to racemize the above D-α-aminobutyric acid may be added to the enzyme reaction system of the present invention from the beginning, but may also be added during the enzyme reaction.
It can also be used for the racemization reaction in a reaction system different from the enzyme reaction system.

The amount of the microbial cell having the ability to racemize D-α-aminobutyric acid or its immobilized product is 0.1 to 50% by weight, preferably about 1 to 30% by weight of the reaction solution.

The method for culturing this microorganism may be the same as the above-described method for culturing the microorganism originally used in the present invention (ethanol-utilizing microorganism belonging to the genus Brevibacterium that requires biotin). In addition, the method of treating the cells and the method of immobilization when used in the enzyme reaction are the same as those of the microorganism originally used in the present invention.

Separation and purification of L-isoleucine produced in the reaction solution obtained by the above reaction method can be easily carried out by an ion exchange resin treatment method, a precipitation method or the like.

Examples will be described below. The qualitative property of L-isoleucine was confirmed by the Rf value of a paper chromatograph, the mobility of the electrophoresis method, and the biological activity value of the microorganism quantification method. Quantitation is based on Leuconosto
micro bioassay using ATCC 8042 and high performance liquid chromatography (Shimadzu LC-5A)
Was done in combination with. Further, in the following 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 O 0.05%, CaCl 2 · 2
_{H 2 O 2ppm, FeSO 4 ·} 7H 2 O 2ppm, MnSO 4 · 4~6H 2 O2pp
_{m, ZnSO 4 · 7H 2 O} 2ppm, NaCl 2ppm, biotin 200 [mu] g /
l, thiamine / HCl 100 μg / l, casamino acid 0.1%, yeast extract 0.1%) 100 ml was dispensed into a 500 ml Erlenmeyer flask,
After sterilization (pH 7.0 after sterilization) Brevibacterium flavum MJ-233 (FERM BP-149
7) was inoculated, 2 ml of ethanol was aseptically added, and shaking culture was performed at 30 ° C. for 2 days.

After harvesting the cells by centrifugation from 100 ml of the culture, 0.5 g of ammonium sulfate, 0.5 g of DL-α-aminobutyric acid, and 1 ml of ethanol were suspended in 50 ml of 0.1 M phosphate buffer (pH 7.6) and shaken 30 times. The reaction was carried out at 48 ° C for 48 hours.

After the reaction was completed, 1.0 mg / ml of L-isoleucine was accumulated in the supernatant liquid that had been sterilized by centrifugation. 40 ml of the above supernatant,
After passing through a column of strong acid cation exchange resin (H ⁺ type) to adsorb L-isoleucine and washing with water and eluting with 0.5N ammonia water, the L-isoleucine fraction was concentrated and L-isoleucine was cooled with cold ethanol. Was precipitated to give 28 mg of crude crystals.

Example-2 Under the same conditions as Example-1, Brevibacterium flavum MJ-233-AB-41 (FERM
BP-1498) was cultured and reacted under the same conditions as in Example-1, and then L-isoleucine in the supernatant was quantified to be 1.3 mg / ml.

Furthermore, when L-isoleucine was recovered from 40 ml of the supernatant by the same operation as in Example-1, 36 mg of crude crystals were obtained.

Example-3 Under the same conditions as in Example-1, Brevibacterium flavum MJ-233-ABT-11 (FERM
BP-1500) was cultured and reacted under the same conditions as in Example-1, and L-isoleucine in the supernatant was quantified to be 1.4 mg / ml.

Furthermore, when L-isoleucine was recovered from 40 ml of the supernatant by the same procedure as in Example-1, 40 mg of crude crystals were obtained.

Example-4 Under the same conditions as in Example-1, Brevibacterium flavum MJ-233-ABD-21 (FERM
BP-1499) was cultured and reacted under the same conditions as in Example-1, and then L-isoleucine in the supernatant was quantified to be 1.1 mg / ml.

Furthermore, when L-isoleucine was recovered from 40 ml of the supernatant by the same procedure as in Example-1, 30 mg of crude crystals were obtained.

Example-5 Brevibacterium flavum
vum) MJ-233 (FERM BP-1497) was used, and shaking culture was performed in the same manner as described in the previous stage of Example-1.

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, biotin 200μg /
l, thiamine / HC 100 μg / l, casamino acid 0.3%, yeast extract 0.3%) 1000 ml into a 2 l aeration and agitation tank, and after sterilization (120 ° C., 20 minutes), 20 ml of ethanol and 20 ml of the preculture are added. After the addition, the culture was carried out at a rotation speed of 1000 rpm, an aeration rate of 1 vvm, a temperature of 33 ° C. and a pH of 7.6 for 48 hours.

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

After completion of the culture, after collecting the cells by centrifugation from 500 ml of the culture,
The bacterial cells washed twice with desalted distilled water were added to the reaction solution [ammonium sulfate 2 g / l,
_{KH 2 PO 4 0.5g / l,} K 2 HPO 4 0.5g / l, MgSO 4 · 7H 2 O 0.5g / l, F
eSO ₄・ 7H ₂ O 20ppm, MnSO ₄・ 4-6H ₂ O 20ppm, thiamine ・ HC 100μg / l, DL-α-aminobutyric acid 10g / l (pH 7.
6)] was suspended in 1000 ml, then the suspension was charged in a 2 l aeration and stirring tank, 20 ml of ethanol was added, and the rotation speed was 300 rpm, the aeration amount was 0.1 vvm, the temperature was 33 ° C., and the pH was 7.6 for 24 hours. The reaction was carried out.

After the reaction was completed, L-isoleucine was quantified in the supernatant liquid that had been sterilized by centrifugation (4000 rpm, 15 minutes, 4 ° C).

In addition, 500 ml of the culture solution after the reaction was passed through a column of a strongly acidic cation exchange resin (H ⁺ type) to adsorb L-isoleucine, washed with water and eluted with 0.5N ammonia water, and then L-isoleucine. Concentrate the fractions and add L with cold ethanol.
-Precipitated crystals of isoleucine. The results are shown in Table 1.

Example-6 Under the same conditions as in Example-5, Brevibacterium flavum MJ-233-AB-41 (FERM
BP-1498) was cultured, and L-isoleucine in the supernatant liquid reacted under the same conditions as in Example-5 was quantified. Also, crystals of L-isoleucine were precipitated in the same manner as in Example-5. The results are shown in Table 2.

Example-7 Under the same conditions as in Example-5, Brevibacterium flavum MJ-233-ABT-11 (FERM
BP-1500) was cultured, and L-isoleucine in the supernatant liquid reacted under the same conditions as in Example-5 was quantified. Also, crystals of L-isoleucine were precipitated in the same manner as in Example-5. The results are shown in Table 3.

Example-8 Under the same conditions as in Example-5, Brevibacterium flavum MJ-233-ABD-21 (FERM
BP-1499) was cultured and reacted under the same conditions as in Example-5, and L-isoleucine in the supernatant was quantified. Further, crystals of L-isoleucine were precipitated in the same manner as in Example-5. The results are shown in Table 4.

Example-9 Brevibacterium flavum
vum) MJ-233 (FERM BP-1497) was used for both pre-culture and main culture in the same manner as in Example-5.

After completion of the culture, after collecting the cells by centrifugation from 500 ml of the culture,
The bacterial cells washed twice with desalted distilled water were added to the reaction solution [ammonium sulfate 2 g / l,
_{KH 2 PO 4 0.5g / l,} K 2 HPO 4 0.5g / l, MgSO 4 · 7H 2 O 0.5g / l, F
_{eSO 4 · 7H 2 O 20ppm,} MnSO 4 · 4~6H 2 O 20ppm, thiamine HC100μg / l, DL-α- aminobutyric acid 10g / l (pH7.6)]
After suspending in 1000 ml of the above, the suspension was charged in a 2 l aeration stirring tank, 20 ml of ethanol was added, the rotation speed was 300 rpm, and the aeration rate was changed by changing the dissolved oxygen of the reaction solution as shown in Table 5.
The reaction was carried out at 33 ° C and pH 7.6 for 24 hours.

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. The results are shown in Table 5.

Example-10 Brevibacterium flavum
vum) Example using MJ-233-AB-41 (FERM BP-1498)-
After culturing in the same manner as in Example 9 and then reacting under the same conditions as in Example-9, L-isoleucine in the supernatant was quantified. The results are shown in Table 6.

Example 11 Brevibacterium flavum
vum) Example using MJ-233-ABT-11 (FERM BP-1500)-
After culturing in the same manner as in Example 9 and then reacting under the same conditions as in Example-9, L-isoleucine in the supernatant was quantified. The results are shown in Table 7.

Example-12 Brevibacterium flavum
vum) Example using MJ-233-ABD-21 (FERM BP-1499) −
After culturing in the same manner as in Example 9 and then reacting under the same conditions as in Example-9, L-isoleucine in the supernatant was quantified. The results are shown in Table 8.

Front page continuation (72) Inventor Makiko Fukushima 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Sanryo Petrochemical Co., Ltd. Central Research Laboratory (72) Hideaki Yukawa 8-3- Chuo, Ami-cho, Inashiki-gun, Ibaraki 1 Central Research Laboratory, Sanryo Yuka Co., Ltd.

Claims (5)

(57) [Claims] 1. A Brevibacterium which requires biotin.
E. coli in the presence of microorganisms belonging to the genus Evibacterium) or its immobilized product, an enzyme reaction is carried out in the presence of dissolved oxygen in an aqueous solution containing α-aminobutyric acid and a carbon source but not biotin, and L is added to the solution. -Producing isoleucine,
A method for producing L-isoleucine, which comprises collecting L-isoleucine from this. 2. A Brevibacterium which requires biotin.
The production method according to claim 1, wherein the enzyme reaction is carried out in the presence of bacterial cells obtained by culturing an ethanol-assimilating microorganism belonging to the genus evibacterium) in a medium containing ethanol as a main carbon source, or in the presence of this immobilized product. 3. An aqueous solution containing α-aminobutyric acid and a carbon source, but not biotin, which is a completely synthetic medium containing α-aminobutyric acid and a carbon source. The manufacturing method described. 4. The enzyme reaction at a dissolved oxygen concentration of 0.05 ppm or more and 8 ppm or less, claim 1, claim 2 or claim 3
The manufacturing method described in the item. 5. A microorganism belonging to the genus Brevibacterium is Brevibacterium flavum (Br
evibacterium flavum) MJ-233 (FERM BP-1497), Brevibacterium flavum M
J-233-AB-41 (FERM BP-1498), Brevibacterium
Brevibacterium flavum MJ-233-ABT-11 (FE
RM BP-1500) or Brevibacterium flavum (Brev
ibacterium flavum) MJ-233-ABD-21 (FERM BP-1499), The production method according to claim 1, claim 2, claim 3, or claim 4.