JP2942995B2 - Method for producing L-alanine - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing L-alanine by an enzymatic method. According to the present invention, L-alanine can be efficiently produced with high yield.

As is well known, L-alanine is an important amino acid as a raw material for medicine, food or chemical industry, and its demand has been rapidly increasing in recent years.

(Prior art and problems) As an industrial production method of L-alanine, a method of producing L-alanine by enzymatic decarboxylation of L-aspartic acid (Japanese Patent Publication No.
53-27792) or a method of producing from fumaric acid and ammonia by reacting aspartase (EC. 4.3.1.1) and aspartate β-decarboxylase (EC.4.1.1.12) (JP-A-56-35991). Publication). However, in the former case, the production cost of alanine is high because L-aspartic acid as a raw material is relatively expensive, and in the latter case, since the optimal conditions under which both enzymes work are different, the reaction tank is separated, or When both enzymes are allowed to act at the same time, the reaction is performed at a relatively low temperature, so that the reaction rate is low.

The present inventors have found that in a single reaction vessel in the presence of a microbial cell containing aspartase or a processed product thereof and a microbial cell containing aspartic acid β-decarboxylase or a processed product thereof in a single reaction tank. A method for producing L-alanine from an acid or a salt thereof and ammonium or ammonium ion has been proposed (Japanese Patent Application No. 63-232570). The present inventors have further conducted intensive studies to improve the reaction rate of L-alanine production. As a result, in the aspartic acid β-decarboxylase reaction, the reaction temperature was increased by adding at least α-keto acid to the reaction solution. They found that they could react at 5050 ° C., and completed the present invention.

(Constitution and Effect of the Invention) The present invention provides a single reaction tank in the presence of a microbial cell containing aspartase or a processed product thereof and a microbial cell containing aspartic acid β-decarboxylase or a processed product thereof. In producing L-alanine from fumaric acid or a salt thereof and ammonia or ammonium ion at a reaction temperature of 40 ° C. with an aqueous solution containing at least α-keto acid in the reaction solution.
It is intended to provide a method for producing L-alanine, which is maintained at 〜50 ° C. According to the present invention, the reaction can be performed at a high reaction rate by maintaining the reaction temperature at 40 to 50 ° C. in the reaction solution containing α-keto acid, and L-alanine can be efficiently produced.

The α-keto acid used in the present invention is preferably pyruvic acid or a salt thereof, or α-ketobutyric acid or a salt thereof.

As the microorganism containing aspartase used in the present invention, any strain having the enzyme activity and belonging to a coryneform bacterium can be used. For example, Brevibacterium flavum can be used. ) M
J-233 (Jikken No. 1497), Brevibacterium flavum MJ-233-AB-41
(Microtechnical Lab. No. 1498), Brevibacterium ammoniagenes ATCC 6
872, Corynebacterium glutamicum
rium glutamicum) ATCC 31830 etc.
These bacteria are preferably used.

On the other hand, as the microbial cells containing aspartic acid β-decarboxylase, any strains having the enzyme activity and belonging to the genus Pseudomonas can be used. For example, Pseudomonas dacunha (Pseudomonas dacunha) can be used.
e) IAM 1152, ATCC 21192, Pseudomonas putida ATCC 21812, IAM 1506, Pseudomonas fluorescein
ns) IFO 3081, Pseudomonas aeruginosa (Pseu
domonas aeruginosa) IAM 1054 and the like, and these cells are preferably used.

The above-mentioned microbial cells used in the present invention can be used as they are, or can be used as a processed product thereof, that is, a destruction product of the cells. Destruction of the cells can be performed using a method known per se, for example, sonication, squeezing or the like.

The medium used for preparing the above-mentioned microbial cells used in the method of the present invention is not particularly limited, and may be one used for general microorganisms.

The carbon source of the medium used for the preparation of the microbial cells containing aspartase is not particularly limited. For example, glucose, ethanol, organic acids such as acetic acid and fumaric acid, and the like can be used.

As the nitrogen source of the medium, 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, nutrients such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and various vitamins are added to the medium if necessary for the growth of bacteria and L-aspartic acid production.

Cultivation of the microbial cells containing aspartase is performed under aerobic conditions such as aeration stirring and shaking, and the culture temperature is 20 to 40.
C., preferably at 25-35.degree. PH during culture is 5-1
The reaction is carried out at 0, preferably around 7 to 8, and the pH is adjusted during culture by adding an acid or an alkali. The cultivation time is 2 to 9 days, and the optimal period is 4 to 7 days.

On the other hand, the carbon source of the medium used for preparing the microbial cells containing aspartic acid β-decarboxylase is not particularly limited, and examples thereof include fumaric acid, succinic acid, and aspartic acid. Among them, fumaric acid is preferably used. As the nitrogen source of the medium, inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, and urea can be used, and organic nutrients such as peptone, yeast extract, constellite liquor, and casamino acid can also be used. it can. As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used.

Culture of the cells containing aspartic acid β-decarboxylase is performed under aerobic conditions such as aeration and agitation and shaking, and the culture is performed at a temperature of 20 to 40 ° C, preferably 28 to 32 ° C. During culturing
The pH is 5-10, preferably around 7-8.
Is adjusted by adding an acid alkali. The concentration of fumaric acid at the start of the culture is preferably 0.1 to 5% by weight, more preferably 0.5 to 2% by weight. The culture period is 10 hours to 4 days, and the optimal period is 1 to 3 days.

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

In the method of the present invention, an enzymatic reaction is carried out in an aqueous solution containing at least fumaric acid or a salt thereof, ammonia or ammonium ion and α-keto acid in the presence of the microbial cells prepared above or a processed product thereof. Here, the concentration of fumaric acid or a salt thereof as a reaction raw material for producing L-alanine added to the aqueous solution is 0.5 to 30% by weight, preferably 5 to 15% by weight. The concentration of ammonia or ammonium ions added is 0.1 to 5 mol, preferably 0.5 to 5 mol.
3.5 moles.

As the α-keto acid added to the reaction solution, pyruvic acid or a salt thereof, or α-ketobutyric acid or a salt thereof is suitably used. The addition concentration is 0.0001-0.5% by weight,
Preferably 0.001 to 0.2% by weight is used.

Examples of fumaric acid salts that can be added to the above aqueous reaction solution include ammonium salts, sodium salts,
Potassium salts, calcium salts and the like can be mentioned. Examples of pyruvic acid or α-ketobutyric acid salts include sodium salts and potassium salts. Further, as an ammonium ion source, ammonium chloride, ammonium sulfate and the like can be added.

The aqueous solution further contains pyridoxal 5 'phosphoric acid at 0.
0005 to 0.05% by weight, preferably 0.001 to 0.01% by weight. If necessary, a nonionic surfactant such as polyoxyethylene (9) octyl phenyl ether (Triton X-100), polyoxyethylene (20) sorbitan monolaurate (Tween 20)
And the like can be used by adding 0.01 to 0.5% by weight, preferably 0.03 to 0.2% by weight. In the present invention, the pH during the enzymatic reaction is 6.0 to 10.0, preferably pH 7.0 to 8.5,
The reaction temperature is about 40 to about 50 ° C, preferably about 42 to about 47 ° C, and the reaction is usually performed for about 5 to about 72 hours.

The separation and purification of L-alanine generated in the reaction solution obtained by the above reaction method can be performed by a known ion exchange resin treatment or the like.

Experimental Examples In the following experimental examples, the qualitative determination of L-alanine was confirmed by the Rf value of a paper chromatograph, the retention time of a high performance liquid chromatograph, and the specific rotation of the purified product. The quantification was performed using high performance liquid chromatography (Shimadzu LC-5A). In the following experimental examples, “%” means “% by weight”.

Experimental Example-1 Preparation of Aspartase-Containing Bacteria (1) A) Culture medium of Brevibacterium flavum MJ-233 cells (0.4% urea, 1.4% ammonium sulfate, KH ₂ PO ₄₀ .
_{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 2p
_{pm, ZnSO 4 · 7H 2 O} 2ppm, NaCl · 2ppm, biotin 200 [mu] g /
, Thiamine / HCl 100 μg /, casamino acid 0.1%, yeast extract 0.1%) was dispensed into a 500 ml Erlenmeyer flask, sterilized (pH 7.0 after sterilization), and then Brevibacterium flavum MJ-233 (fine). Koken Joyo No.
No. 1497) and aseptically add 2 ml of ethanol,
Shaking culture was performed at 2 ° 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 · 4~6H 2 O 20ppm, biotin
200 μg /, thiamine / HCl 100 μg /, casamino acid 0.3
%, Yeast extract 0.3%) in a 2-volume aeration and stirring tank, sterilized (120 ° C., 20 minutes), added with 20 ml of ethanol and 20 ml of the above culture, and rotated at 1000 rpm and aerated at 1 rpm.
The cells were cultured at vvm, a temperature of 33 ° C. and pH 7.6 for 48 hours.

In addition, ethanol was added intermittently about every 1 to 2 hours so that the concentration of the culture value during the culture did not exceed 2% by volume, and finally, up to 100 ml.

After completion of the culture, the cells were collected by centrifugation from 1000 ml of the culture.

B) Removal treatment of fumarase activity Since the side reaction enzyme fumarase in addition to aspartase is present in the microbial cells prepared in section A) above, there is a problem that fumaric acid as a raw material is partially converted to malic acid. Therefore, the fumarase activity was removed in advance.

The cells prepared in the above section A) were mixed with a reaction mixture [100 g of L-aspartic acid, ammonia (aqueous solution containing 28% ammonia)] 1
40 ml, 1 g of CaCl ₂ .2H ₂ O, polyoxyethylene sorbitan monolaurate 0.8; contained in distilled water 1], and then heat-treated at 45 ° C. for 5 hours. The treated product was collected by centrifugation, and the cells were used as aspartase-containing cells.

Experimental Example-2 Preparation of Aspartase-Containing Bacteria (2) A) Brevibacterium ammoniagenes ATCC 6872
Cultivation of bacterial cells 100 ml of a medium for preparing the aspartase-containing bacterial cells used in Experimental Example 1 was dispensed into a 500 ml Erlenmeyer flask and sterilized (pH after sterilization).
7) After that, Brevibacterium ammoniagenes (B
(revibacterium ammoniagenes) ATCC 6872 was inoculated, 2 ml of a 50% glucose solution was added aseptically, and shaking culture was performed at 30 ° C. for 24 hours.

Next, 1000 ml of the main culture medium of Experimental Example 1 was charged into a 2-volume agitating tank, sterilized (120 ° C., 20 minutes), and 40 ml of a 50% glucose solution and 20 ml of the culture were added. Culture was performed at 1,000 rpm, aeration rate of 1 vvm, temperature of 33 ° C. and pH 7.6 for 24 hours.

In addition, glucose was added by 5 g every about 1 to 2 hours, and finally added to 70 g. After completion of culture, culture
The cells were collected by centrifugation from 00 ml.

B) Removal treatment of fumarase activity The cells prepared in the above A) were suspended in the reaction solution 1 used in B) of Experimental Example 1, and then heated at 45 ° C for 2 hours. The treated product was collected by centrifugation, and the cells were used as aspartase-containing cells.

Experimental Example-3 Preparation of aspartase-containing cells (3) A) Culture of Corynebacterium glutamicum ATCC 31830 cells Preparation medium for aspartase-containing cells of Experimental Example-1 100 m
is dispensed into a 500 ml Erlenmeyer flask, sterilized (pH 7 after sterilization), and then Corynebacterium glutamicum (Corynebact
erium glutamicum) ATCC 31830 and aseptically inoculate 50
Then, 2 ml of a% glucose solution was added, and shaking culture was performed at 30 ° C. for 24 hours.

Next, 1000 ml of the main culture medium of Experimental Example 1 was charged into a 2-volume agitating tank, sterilized (120 ° C., 20 minutes), and 40 ml of a 50% glucose solution and 20 ml of the culture were added. Culture was performed at 1000 rpm, aeration rate of 1 vvm, temperature of 33 ° C. and pH 7.6 for 24 hours.

Note that glucose was added in an amount of 5 g every about 1 to 2 hours, and finally, 70 g was added. After culture, culture 1000m
and collected by centrifugation.

B) Removal treatment of fumarase activity The cells prepared in the above section A) were suspended in the reaction solution 1 used in the section B) of Experimental Example 1, and then heated at 45 ° C for 2 hours. The treated product was collected by centrifugation, and the cells were used as aspartase-containing cells.

Experimental Example-4 Preparation of Aspartic Acid β-Decarboxylase-Containing Bacteria A) Culture Medium of Pseudomonas dacne IAM 1152 Bacteria (Sodium Fumarate 0.5%, Ammonium Fumarate 1.0%, Yeast Extract 0.5%, Monopotassium Phosphate) 0.05
%, MgSO ₄ .7H ₂ O 0.05%, pH 7.0) was dispensed into a 500 ml Erlenmeyer flask, sterilized, inoculated with Pseudomonas dacunhae IAM1152, and cultured at 30 ° C. for 1 day with shaking. Performed (pre-culture). Next, a medium 1 similar to the above medium was charged into a two-volume agitated tank, and sterilized (12
0 ° C, 20 minutes), add 20 ml of the preculture, and rotate at 1 rpm.
Culture was performed at 000 rpm, aeration rate of 1 vvm, temperature of 30 ° C. and pH 7.3 for 1 day.

After completion of the culture, the cells were collected by centrifugation from 1000 ml of the culture.

B) Removal treatment of fumarase activity In the microbial cells prepared in the above A), the raw material and fumaric acid are partially converted to malic acid because the side reaction enzyme fumarase exists in addition to aspartate β-decarboxylase. Therefore, the fumarase activity was removed in advance.

The cells prepared in the above section A) were suspended in 1 of the reaction solution (0.11 g of sodium pyruvate, 10 mg of pyridoxal 5'-phosphoric acid; contained in 1 of distilled water), and then heat-treated at 50 ° C. for 2 hours. Was done. The treated product was collected by centrifugation, and the cells were used as aspartic acid β-decarboxylase-containing cells.

Example-1 Each microbial cell collected by centrifugation from 200 ml of the cell suspension prepared in section B) of Experimental Example-1 and section B) of Experimental Example-4 was combined, and a reaction solution [ Ammonium fumarate 1
Mol, sodium pyruvate 5 mmol, pyridoxal 5'-phosphate 0.04 mmol, polyoxyethylene (2
0) Sorbitan monolaurate 0.05%, pH 7.5 (prepared with 28% ammonia water)], and put into a 1-volume agitating tank. The reaction was performed at several 300 rpm for 20 hours.

After the completion of the reaction, alanine produced in the reaction solution was quantified. The results are shown in Table 1. 100 ml of the reaction solution was adjusted to pH 4.0, filtered by boiling, and the filtrate was filtered using Amberlite IRC-50 (H ⁺
After washing with water, wash with water and elute with 4.5% aqueous ammonia. After concentrating this eluate under reduced pressure, crystals were precipitated with cold ethanol. Table 1 shows the amount of L-alanine recovered.
All was measured specific rotation for further recovery alanine ▲ [alpha] was ²³ _D ▼ 14.3 ° (C = 10,6N-HCl).

As a comparative example, the amount of L-alanine produced when pyruvic acid was not added to the reaction solution is shown in Table 1.

Example-2 The sodium pyruvate in the reaction solution of Example-1 was changed to α-
The same experiment as in Example 1 was conducted except that ketobutyric acid was changed to 5 mmol. The results are shown in Table 2.

The specific rotation of the recovered L-alanine is ▲ [α]
²⁵ _D ▼ = + 14.3 was ° (C = 10,6N-HCl).

Example 3 In the same manner as in Example 1, using each of the microbial cells collected from 200 ml of the cell suspension prepared in paragraphs B) of Experimental Example-2 and B) of Experimental Example-4. Reaction and purification. The results are shown in Table 3. Further, the specific rotation of the recovered alanine was measured. [[Α] ²⁵ _D ▼ = + 14.4 ゜ (C = 10, 6N-HCl)
Met.

Example-4 Sodium pyruvate in the reaction solution of Example-3 was changed to α-
Example-except that sodium ketobutyrate was changed to 5 mmol
The same experiment as in No. 3 was performed. The results are shown in Table 4.

The specific rotation of the recovered L-alanine is ▲ [α]
²⁵ _D ▼ = + 14.4 was ° (C = 10,6N-HCl).

Example-5 In the same manner as in Example-1, using each microbial cell collected from 200 ml of the cell suspension prepared in paragraph B) of Experimental Example-3 and B) of Experimental Example-4, Reaction and purification. The results are shown in Table 5. Further, the specific rotation of the recovered alanine was measured. [[Α] ²⁵ _D ▼ = + 14.4 ゜ (C = 10, 6N-HCl)
Met.

Example-6 The sodium pyruvate in the reaction solution of Example-5 was converted to α-
Example-except that sodium ketobutyrate was changed to 5 mmol
The same experiment as in Example 5 was performed. The results are shown in Table 6.

The specific rotation of the recovered L-alanine is ▲ [α]
²⁵ _D ▼ = + 14.3 was ° (C = 10,6N-HCl).

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Koichi Uchida 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Prefecture Inside the Tsukuba Research Laboratory, Mitsubishi Yuka Corporation (72) Inventor Hideaki Yukawa Ami-cho, Inashiki-gun, Ibaraki Prefecture 8-3-1, Chuo Mitsubishi Tsukasa Co., Ltd. Tsukuba Research Laboratory (58) Field surveyed (Int. Cl. ⁶ , DB name) C12P 13/06 WPI (DIALOG) BIOSIS (DIALOG)

Claims (1)

(57) [Claims] 1. Fumaric acid or a salt thereof in a water-soluble solvent in the presence of a microbial cell containing aspartase or a processed product thereof and a microbial cell containing aspartic acid β-decarboxylase or a processed product thereof. And ammonia or ammonium ions in a single reaction vessel to produce L-alanine in the reaction solution.
A process for producing L-alanine, which comprises adding keto acid and reacting at a reaction temperature of 40 to 50 ° C.