JP2582805B2 - Method for producing L-threonine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing L-threonine.

According to the present invention, L-threonine can be efficiently produced from L- or DL-aspartic acid or a salt thereof.

L-threonine is an amino acid that plays an important role in human and animal nutrition as an essential amino acid, and its demand as a medical, food and feed enhancer has been rapidly increasing in recent years.

Prior Art As an industrial method for producing L-threonine, it is difficult to produce only the L-form in chemical synthesis because of the presence of stereoisomers as in the case of other amino acids, and production is mainly performed by fermentation. Have been done. As a method of producing by a fermentation method, a method using an amino acid-requiring strain (Japanese Patent Publication No. 46-3319,
Nos. 34193 and 46-34194), and as a production method using a precursor fermentation method, a production method using homoserine as a precursor (Japanese Patent Publication Nos. 36-2896, 38-6590, and 43-8715). Gazettes).

Such a fermentation method requires complicated operations such as sterilization of a culture medium, is extremely difficult to control production, and has problems of by-products, and thus cannot be said to be an industrially advantageous method.

In addition, a production method using a fermentation method in which fixed costs and the like are lower than in a fermentation method and production control is easier is a method using glycine and acetaldehyde as precursors (Japanese Patent Laid-Open Nos.
However, these methods are not practical methods due to the by-product of allo-form threonine.

In addition to this, in the production method by the enzymatic method, L.
-Has been reported to produce threonine (Amin
o Acids, No. 1, P71-74 (1959)). However,
In these known methods, the amount of L-threonine produced was not yet sufficient.

SUMMARY OF THE INVENTION The present inventors have conducted intensive studies on a method for producing L-threonine by enzymatic production using L- or DL-aspartic acid or a salt thereof as a main raw material in order to solve the above problems, and completed the present invention. .

That is, the present invention, in the presence of microbial cells, L- or DL-
Enzymatic reaction of aspartic acid or a salt thereof in an aqueous solution,
L-threonine was formed in the solution, and L-threonine was
In a method for collecting threonine, the present invention provides a method for producing L-threonine, wherein the microorganism cells belong to the genus Brevibacterium requiring biotin.

Effects of the Invention According to the method of the present invention, L-threonine can be produced in high yield. When a reaction solution containing ethanol and / or glucose is used as an aqueous solution for the enzyme reaction, L
-Threonine can be produced in a high yield, no complicated operations such as sterilization of the culture medium in the fermentation method are required, and production control becomes extremely easy.

DETAILED DESCRIPTION OF THE INVENTION Examples of the microbial cells belonging to the genus Brevibacterium requiring biotin used in the present invention include the following, including L-threonine-producing bacteria.

Ethanol-assimilating ones, such as Brevibacterium flavum MJ-233 (FERM)
BP-1497), Brevibacterium flavum
terium flavum) MJ-233-AB-41 (FERM BP-1498),
Brevibacterium flavu
m) MJ-233-ABT-11 (FERM BP-1500) and Brevibacterium flavum MJ-2
33-ABD-21 (FERM BP-149) and the like. In addition to these, Brevibacterium ammoniagenes (Brevibacte
rium ammoniagenes) ATCC 6871, ATCC 13745, ATCC 13746,
Brevibacterium debaricatam
ivaricatum) ATCC 14020 and the like.

Among these microbial cells, those utilizing ethanol are preferable, and in particular, Brevibacterium flavum MJ-
233, Brevibacterium flavum MJ-233-AB-4
1, Brevibacterium flavum MJ-233-ABT-1
1, Brevibacterium flavum MJ-233-ABD-21
Is preferred.

The above (FERM BP-1498) was replaced with (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)
It is an L-α-aminobutyric acid transaminase highly active mutant using ERM BP-1497 as a parent strain (Japanese Patent Application No. 60-190609).
No. 3-5 pages). Further, (FERM BP-1499) is a D-α-aminobutyric acid deaminase highly active mutant strain (FERM BP-1497, see Japanese Patent Publication No. 57-26755) as a parent strain (Japanese Patent Application Laid-Open No. 61-176607). reference).

The "microbial cell" in the present invention includes the immobilized product thereof. Here, the `` immobilization '' can be performed by appropriately selecting the microorganism cells from a known solidification method, for example, an inclusive method using acrylamide, alginate, carrageenan, etc., an ion binding method using DEAE-Sephadex, DEAE-cellulose, etc. .

The method of the present invention is an enzymatic reaction under conditions that do not cause growth of microorganisms, and is carried out in an aqueous solution. As the aqueous solution, water or a buffer solution such as phosphoric acid or Tris-HCl is used, and more preferably, a reaction solution containing ethanol and / or glucose is used.

The reaction solution may further contain a biotin-free nitrogen source, an inorganic salt or the like. In this case, examples of the nitrogen source include ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea, and examples of the inorganic salt include potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, iron sulfate, and manganese sulfate. it can.

The concentration of ethanol and / or glucose added to the above reaction solution is 1 to 20% by volume for ethanol and 0.5 to 20% by weight for glucose. When ethanol and glucose are used in combination, they are used within the above ranges.

The concentration of L- or DL-aspartic acid or a salt thereof during the reaction is generally suitable to be used in a concentration range of 0.1 to 20% (wt / vol). Although the amount of the cells used is not particularly limited, they can generally be used at a concentration of 1 to 50% (wt / vol).

The enzyme reaction is carried out at about 20 to about 50 ° C, preferably about 30 to about 40 ° C.
At a temperature of about 10 to about 72 hours.

The separation and purification of L-threonine generated in the reaction solution obtained by the above reaction method can be easily performed by an ion exchange resin treatment method or a precipitation method.

A method for preparing a microorganism used in the present invention, that is, a microorganism having an ability to produce L-threonine from aspartic acid will be described below.

As a carbon source, for example, glucose, ethanol, methanol, molasses, etc., as a nitrogen source, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate,
Urea and the like can be used alone or in combination.

As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, various vitamins such as casamino acid and biotin can be added to the medium and used.

Culture is carried out under aerobic conditions such as aeration, shaking, and the like.
The cultivation temperature is 20 to 40 ° C, preferably 25 to 35 ° C. The pH during the culturing is adjusted to 5 to 10, preferably around 7 to 8. The pH during the culturing can be 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 the washed cells are used in the enzyme reaction of the present invention.

Experimental Examples In the following examples, the qualitative determination of L-threonine was confirmed by the Rf value of paper chromatography, the mobility of electrophoresis, and the biological activity value by microbial quantification. Quantification was performed using Leuconostoc meseloides.
nteroides) A microbioassay method using ATCC 8042 and high performance liquid chromatography (Shimadzu LC-5A) were used in combination. In the following examples, “%” means “% by weight”.

Example 1 Medium (urea 0.4%, ammonium sulfate 1.4%, 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 100 μg /, casamino acid 0.1%, yeast extract 0.1%) 100 ml was dispensed into a 500 ml Erlenmeyer flask, sterilized (pH 7.0 after sterilization), and then Brevibacterium flavum MJ-233 ( FERM BP-149
7) was inoculated, and 2 ml of ethanol was aseptically added thereto, followed by shaking culture 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 O 0.05%, FeSO ₄・
7H ₂ O 20ppm, MnSO ₄・ nH ₂ O 20ppm, biotin 200μg /,
1000 ml of thiamine / HCl 100 μg /, casamino acid 0.3%, yeast extract 0.3%) was charged into a two-volume aeration stirred tank, sterilized (120 ° C., 20 minutes), and 20 ml of ethanol and 20 ml of the preculture were added. Then, culture was performed for 48 hours at a rotation speed of 1000 rpm, an aeration rate of 1 vvm, and a temperature of 33 ° C. and a pH of 7.6.

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

After completion of the culture, after collecting the cells by centrifugation from 300 ml of the culture,
The cells washed twice with demineralized distilled water are suspended in 1000 ml of a reaction solution (DL-aspartic acid 2 mg, pyridoxal phosphate 5 μg, phosphate buffer 100 μmoles, ethanol 10 mg, pH 7.6 in 1 ml of the reaction solution). After turbidity, the suspension was charged into a two-volume agitating tank, and ethanol (20 ml) was added thereto.
The reaction was carried out for 10 hours at a ventilation rate of 0.1 vvm, a temperature of 33 ° C. and pH 7.6.

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

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

Example 2 Under the same conditions as in Example 1, Brevibacterium flavum MJ-233-AB-41 (FE
RM BP-1498) was cultured and reacted under the same conditions as in Example 1, and then L-threonine in the supernatant was quantified. In the same manner as in Example 1, crystals of L-threonine were precipitated and purified. The results are shown in Table 2.

Example 3 Under the same conditions as in Example 1, Brevibacterium flavum MJ-233-ABT-11 (F
ERM BP-1500) was cultured and reacted under the same conditions as in Example 1, and then L-threonine in the supernatant was quantified. In the same manner as in Example 1, crystals of L-threonine were precipitated and purified. The results are shown in Table 3.

Example-4 Under the same conditions as in Example-1, Brevibacterium flavum MJ-233-ABD-21 (F
After culturing ERM BP-1499) and reacting it under the same conditions as in Example 1, L-threonine in the supernatant was quantified. In the same manner as in Example 1, crystals of L-threonine were precipitated and purified. The results are shown in Table 4.

Example-5 The same operation as in Example-1 was performed, except that ethanol added during the reaction in Example-1 was changed to glucose.
The glucose concentration was 2% by weight. Table 5 shows the amount of L-threonine produced and the amount of purification in the supernatant after the reaction was completed.

Example-6 The reaction solution used in the reaction in Example-1 was (NH ₄ ) ₂ SO ₄ 2
g /; KH ₂ PO ₄ 0.5g /; KH ₂ PO ₄ 0.5g /; MgSO ₄・ 7H ₂ O
_{0.5g /; FeSO 4 · 7H 2} O 20ppm; MnSO 4 · 4~6H 2 O 20ppm;
Thiamine-HCl 100 μg /; DL-aspartic acid 2 g /
(PH 7.6) except that the operation was the same as in Example 1.

Table 6 shows the amount of L-threonine produced and the amount of purification in the supernatant after the reaction was completed.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Sachie Sato 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. In the Central Research Laboratory of Mitsubishi Yuka Co., Ltd. (72) Inventor Mitsunobu Shimazu 8 Ami-cho, Inashiki-gun, Ibaraki Central R & D Center, Mitsubishi Yuka Co., Ltd. (72) Inventor Hideaki Yukawa 8-3-1, Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref.

Claims (4)

(57) [Claims] 1. A method for enzymatically reacting L- or DL-aspartic acid or a salt thereof in an aqueous solution in the presence of a microbial cell, producing L-threonine in the solution, and collecting L-threonine therefrom. A method for producing L-threonine, wherein the microbial cells belong to the genus Brevibacterium requiring biotin. 2. The method according to claim 1, wherein the aqueous solution contains ethanol and / or glucose. 3. A biotin-requiring Brevibacterium (Br).
3. The method according to claim 1, wherein the microorganisms belonging to the genus Evibacterium are ethanol-assimilating. 4. A biotin-requiring Previbacterium flavum MJ-233, a Brevibacterium flavum MJ-233-AB-41, a Brevibacterium flavum MJ-233-ABT-11, a Brevibacterium. 3. The method according to claim 1 or 2, wherein the method is selected from Um Flavam MJ-233-ABD-21.