JPH0815436B2 - Method for producing L-tryptophans - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing optically active substance L-tryptophan or L-5-oxytryptophan using an enzymatic function.

(Conventional Technology and Problems) L-Tryptophan is one of essential amino acids, and is an amino acid that is important in nutrition and physiologically. Currently, L-tryptophan is difficult to be mass-produced, so that its use is mainly limited to pharmaceuticals. However, if cheap manufacturing technology is established, new products such as foods, feed additives, polymer materials,
It is expected that a large market will be created.

L-5-oxytryptophan is an amino acid whose physiological action is mainly focused on, but currently, an effective production method has not been sufficiently established, and it is desired to develop an industrial production method that can be produced at low cost. .

As a conventional method for producing both of these amino acids, there has been known a method of using an ammonium ion and pyruvic acid, oxaloacetic acid, or L-malic acid in indole or 5-oxindole (Japanese Patent Publication No. 49-46917).

However, among these raw materials, especially pyruvic acid,
Oxaloacetic acid or L-malic acid is very expensive and is not suitable for the industrial production method which is the object of the present invention. Here, as a result of diligent research, the present inventors have been able to produce L-tryptophan from fumaric acid and ammonium ions and indole, and L-5-oxytryptophan from fumaric acid and ammonium ions and 5-oxindole. Therefore, the present invention has been completed.

(Structure and Effect of the Invention) The gist of the present invention is the following method (1).

(1) A: A microbial cell of a microorganism belonging to the genus Brevibacterium containing an enzyme capable of producing L-malic acid from fumaric acid.

B: A microbial cell belonging to the genus Escherichia, Proteus, or Erwinia containing an enzyme capable of producing oxaloacetate from L-malic acid.

C: A microbial cell belonging to the genus Escherichia, Proteus or Erwinia containing tryptophanase.

In the presence of the bacterial cells consisting of A, B, and C described above, fumaric acid, ammonium ion and the general formula (However, R represents a hydrogen atom or a hydroxyl group.) A corresponding L-tryptophan is produced from an indole represented by the following: A process for producing L-tryptophan.

The basis of the present invention is to use fumaric acid as one of the raw materials, and as the source of the enzyme system used in the reaction system,
The method is not limited to the method of using an enzyme existing in the same microbial cell, and naturally, a method of using a plurality of types of microbial cells or processed products thereof is also applicable.

When L-tryptophan is produced from indole and fumaric acid or a salt thereof and ammonium ion using the same microbial cell and L-5-oxytryptophan is similarly produced by using 5-oxyindole instead of indole In contrast, the culture cost is low because the microbial cell preparation can be performed in a single culture as compared with a plurality of types of microorganism-utilizing reactions, but each enzyme activity involved in the reaction is limited.

On the other hand, when a plurality of types of microbial cells are used, it is necessary to prepare the cells for each microorganism, but on the other hand, the reaction can be performed at the maximum rate in each reaction step, resulting in L-tryptophan. Alternatively, a high production rate of L-5-oxytryptophan can be obtained.

In any of the above cases, the method is either indole or 5-
The present invention provides a novel process for producing L-tryptophan or L-5-oxytryptophan from oxindole and fumaric acid or a salt thereof and ammonium ion.

This process consists of a reaction that can generate L-malic acid from fumaric acid, a reaction that can generate oxaloacetic acid from L-malic acid, and a reaction that can generate pyruvic acid from oxaloacetic acid and a tryptophanase reaction. , Which is a non-enzymatic decarboxylation reaction and is easily converted to pyruvic acid in an aqueous solution.

The strain used in each of the above reactions is not particularly limited, but for example, the strain used in the reaction IRONI is Escherichia coli K-1.
2 strain ATCC 27325, Escherichia coli
coli) K-12 strains (FERM BP-1733 and FERM BP
-1734), Proteus morgani
i) IFO−3848, Erwinia herbicola
cola) ATCC-21433, etc.

The strains used in the above-mentioned reaction a and reaction b include Brevibacterium flavu
m) MJ-233 (FERM BP-1497), Brevibacterium
Flavum (Brevibacterium flavum) MJ-233-AB-41
(FERM BP-1498) and the like.

In the present invention, the strain used in the above-mentioned reaction (a) and reaction (b) is preferably a bacterial cell of a microorganism belonging to the genus Brevibacterium or a processed product thereof, and particularly Brevibacterium flavum MJ-233 (FERM BP- 1497), Brevibacterium flavum MJ-233-AB-41 (FERM B
It is preferable to use bacterial cells such as P-1498).

When the above-mentioned microorganism belonging to the genus Brevibacterium is used, the enzyme activity is not particularly lowered, and the microbial cells used in the reaction can be reused.

Both of the above reactions are, for example, 0.
It is usually carried out at a temperature of about 20 to about 50 ° C., preferably about 30 to about 40 ° C. for about 1 to about 72 hours in an aqueous solution having a pH adjusted to 6 to 9 with 1M phosphate buffer or water. Indole or 5-
The amount of oxindole used in the reaction is not particularly limited, but it is generally suitable to use it in a concentration range of 0.1 to 20% (wt / vol). The fumaric acid or its salt and the ammonium ion concentration are not particularly limited, but generally 0.1 to 20% (wt / vol) is appropriately used. The amount of the bacterial cells or treated product thereof used in the reaction is generally
It can be used at a concentration of 0.1-10% (wt / vol).

The culture of the above strain can be carried out using a commonly used synthetic medium or natural medium. However, as the carbon source, carbohydrates such as glucose, glycerol, fructose, sucrose and molasses can be used in the case of Escherichia, Proteus and Erwinia. On the other hand, in the case of Brevibacterium, ethanol can be used as a carbon source in addition to the above carbohydrates. As a nitrogen source, tryptone, yeast extract,
Many of the natural organic nitrogen sources such as corn steep liquor and casein hydrolyzate can be carbon sources as well as nitrogen sources. The culture can be performed under aerobic conditions such as shaking culture or aeration-agitation tank culture. Culture temperature is generally 20-50
It is desirable to maintain the pH of the medium in the vicinity of neutral or slightly alkaline. Culture period is usually about 5
Time to about 3 days.

Using the cells obtained by the above-mentioned culture method, indole or 5-oxindole and fumaric acid or its salt and ammonium ion were used. When indole was used, L-tryptophan and 5-oxindole were used. In this case, L-5-oxytryptophan can be produced.

Separation and purification of L-tryptophan and the like formed in the reaction solution can be carried out by a known method such as adsorption and desorption treatment with an ion exchange resin, activated carbon and the like.

 Hereinafter, the present invention will be specifically described with reference to examples.

Reference Example-1: Preparation of Brevibacterium flavum bacterial cells Two sets of 100 ml medium having the composition shown in Table 1 were separately dispensed into two 500 ml Erlenmeyer flasks, and each was sterilized by heating at 120 ° C for 15 minutes. Aseptically add 2% by volume of ethanol to
These include Brevibacterium flavum
um flavum) MJ-233 (FERM BP-1497) or Brevibacterium flavum MJ-
233-AB-41 (FERM BP-1498) was inoculated into each platinum loop and cultured at 30 ° C for 24 hours.

20 ml of these two cultures were separately inoculated into the medium 1 having the composition shown in Table 2 in a 2-volume jar fermenter, and the mixture was stirred under the conditions of 33 ° C., pH 7.6, and aeration of 1 vvm, and the ethanol concentration was increased. Ethanol was added intermittently so that the concentration was kept at 1.0-1.5% by volume. After culturing for 30 hours, the culture solution was centrifuged (6,000 rpm, 15 minutes) to obtain each of the cells as a test cell.

Table 1 Urea 4.0g Ammonium sulphate 14.0g KH ₂ PO ₄ 0.5g K ₂ HPO ₄ 0.5g MgSO ₄・ 7H ₂ O 0.5g FeSO ₄・ 7H ₂ O 6.0mg MnSO ₄・ 4-6H ₂ O 6.0mg Yeast extract 1.0 g casamino acids 1.0g biotin 200μg thiamine hydrochloride 100μg distilled water 1000ml table 2 ammonium sulfate _{23.0g KH 2 PO 4 0.5g K 2} HPO 4 0.5g MgSO 4 · 7H 2 O 0.5g FeSO 4 · 7H 2 O 20 mg MnSO 4・ 4 to 6H ₂ O 20 mg Yeast extract 3 g Casamino acid 3 g Biotin 200 μg Thiamine hydrochloride 100 μg Distilled water 1000 ml Reference example-2: Preparation of Escherichia coli coli cells 50 ml of a medium having the composition shown in Table 3 in a volume of 500 ml Escherichia coli K-12 strain (ATC) was dispensed into an Erlenmeyer flask and sterilized at 120 ° C for 15 minutes.
C 27325 and FERM BP-1733 and FERM BP-1734), which were cultivated with shaking at 37 ° C. for 1 day, were similarly sterilized to prepare L-tryptophan at a concentration of 200 μg / ml and 1000 ml of L medium. Then, 20 ml was inoculated, and the mixture was cultured at 37 ° C. for 8 hours with shaking. Centrifuge the culture-finished solution (6000 rpm, 15 minutes, 4
C.) was used as the test cell.

Table 3 L medium Tryptone 10g Yeast extract 5g NaCl 5g Glucose 1g Distilled water 1 (pH 7.2) Reference example-3: Preparation of Proteus Morgany cells 50 ml of the medium having the composition shown in Table 4 was divided into 500 ml Erlenmeyer flasks. Injection, Proteus morganii IFO-3848 was inoculated into the sterilized at 120 ℃ for 15 minutes, shake cultured at 37 ℃ for 24 hours, the same sterilized L-tryptophan 200μg / 20 ml was inoculated into 1000 ml of the medium shown in Table 4 containing a concentration of ml, and the mixture was similarly cultivated with shaking at 37 ° C. for 8 hours. The cells obtained by centrifugation (6000 rpm, 15 minutes, 4 ° C.) of the culture-finished solution were used as test cells.

Table 4 _{KH 2 PO 4 0.5g / K 2} HPO 4 0.5g / MgSO 4 · 7H 2 O 0.5g / FeSO 4 · 7H 2 O 6ppm MnSO 4 · nH 2 O 6ppm yeast extract 10 g / casamino acid 5 g / (pH 7.5) Reference Example-4: Preparation of Erwinia herbicola cells As a strain, Erwinia herbicola (Erwinia herbicola)
ola) ATCC-21433 was used, and shaking culture was carried out at 37 ° C. for 8 hours in the same medium and operation as in Reference Example-3. The cells obtained by centrifugation (6000 rpm, 15 minutes, 4 ° C.) of the culture-finished solution were used as test cells.

Example 1 50 ml of the reaction solution having the composition shown in Table 5 was dispensed into a 500 ml Erlenmeyer flask, and then the reaction solution was mixed with Brevibacterium flavum prepared in Reference Example-1.
um) MJ-233 (FERM BP-1497) culture medium 40 ml obtained by centrifugation (6000 rpm, 15 minutes, 4 ° C.), and Escherichia col (Escherichia col) prepared in Reference Example-2.
i) The bacterial cells obtained by centrifugation (6000 rpm, 15 minutes, 4 ° C.) from 40 ml of the culture solution of K-12 strain ATCC 27325 were added, and 37
The shaking reaction was carried out at ℃ for 24 hours. After completion of the reaction, the cells were removed by centrifugation (4000 rpm, 15 minutes, room temperature), and L-tryptophan in the supernatant was quantified, and production of 800 mg / was found.

Escherichia coli (Escherichi coli) was added to the above reaction solution.
a coli) K-12 strain ATCC 27325 containing only cells collected from a culture solution of 40 ml and subjected to a shaking reaction at 37 ° C. for 24 hours, the production amount of L-tryptophan is 450 mg /
Met.

Table 5 Indole 20mM Sodium fumarate 50mM Ammonium chloride 300mM Nicotinamide adenine dinucleotide (NAD) 20mM Phosphate buffer (pH8.0) 100mM Example-2 50ml reaction solution similar to Example-1 Reference Example-1 Brevibacterium flavum prepared in
avum) MJ-233 AB-41 (FERM BP-1498) culture medium 40m
1 and the Escherichia coli K-12 strain (FERM BP) prepared in Reference Example-2.
-1733) and the bacterial cells from 40 ml of the culture solution were added, and
The reaction was shaken for 15 hours, and L-tryptophan in the supernatant after the reaction was quantified to be 3200 mg /.

After 40 ml of the reaction supernatant was passed through a column of ammonia type strongly acidic ion exchange resin (Diaion SK-IB, manufactured by Mitsubishi Kasei) to adsorb L-tryptophan, it was eluted with an alkaline solution and then concentrated to give crude L-tryptophan crystals. Was deposited. This was washed with acetone and dried to obtain 95 mg of L-tryptophan crystals.

In addition, as in Example-1, Escherichia coli (Esch.
erichia coli) K-12 strain (FERM BP-1733) culture medium obtained by using only cells obtained from 40 ml, L
-The amount of tryptophan produced was 460 mg /.

Example-3 In 50 ml of the same reaction solution as in Example-1, the Brevibacterium flavum prepared in Reference Example-1 (Brevibacterium fl
avum) MJ-233-AB-41 (FERM BP-1498) culture medium 40m
1 and the Escherichia coli K-12 strain (FERM BP) prepared in Reference Example-2.
-1734) and the bacterial cells obtained from 40 ml of the culture broth were added, and the mixture was shaken at 37 ° C for 15 hours to give L- in the supernatant after the reaction.
The quantification of tryptophan was 3100 mg /.

After 40 ml of the reaction supernatant was passed through a column of ammonia type strongly acidic ion exchange resin (Diaion SK-IB, manufactured by Mitsubishi Kasei) to adsorb L-tryptophan, it was eluted with an alkaline solution and then concentrated to give crude L-tryptophan crystals. Was deposited. This was washed with acetone and dried to obtain 87 mg of L-tryptophan crystals.

In addition, as in Example-1, Escherichia coli (Esch.
The amount of L-tryptophan produced was 430 mg / when only the bacterial cells obtained from 40 ml of the culture solution of E. coli K-12 strain (FERM BP-1734) were subjected to the reaction.

Example-4 50 ml of the same reaction solution as in Example-1 was added to Brevibacterium flavum prepared in Reference Example-1.
avum) MJ-233-AB-41 (FERM BP-1498) culture medium 40m
and the proteus prepared in Reference Example-3.
Culture medium of Morgany (Proteus morganii) IFO-3848 80m
The cells obtained from 1 were added and the mixture was subjected to a shaking reaction at 37 ° C. for 24 hours, and L-tryptophan in the supernatant after the reaction was quantified to be 1050 mg /.

When L-tryptophan was recovered from 40 ml of the reaction supernatant in the same manner as in Example-2, 32 mg of crystals were obtained.

When only the cells obtained from 80 ml of the Proteus morganii IFO-3848 culture solution were subjected to the reaction as in Example 1, the production of L-tryptophan was 430 mg /.

Example-5 As a tryptophanase-carrying strain, instead of Proteus morgany, Erwinia.
The reaction was carried out at 37 ° C. for 24 hours in the same manner as in Example 4 except that Erwinia herbicola ATCC-21433 was used. When L-tryptophan in the supernatant after the reaction was quantified, production of 710 mg / was found.

When only the cells obtained from 80 ml of the culture solution of Erwinia herbicola ATCC-21433 were subjected to the reaction as in Example 1, the amount of L-tryptophan produced was 320 mg /.

Example-6 After 50 ml of the reaction solution having the composition shown in Table 6 was dispensed into a 500 ml Erlenmeyer flask, the reaction was carried out in the same manner as in Example-2, and L in the supernatant after the reaction was completed. The quantification of -5-oxytryptophan was 2500 mg /.

40 ml of the reaction supernatant was passed through a column of an ammonia type strongly acidic ion exchange resin (Diaion SK-IB, manufactured by Mitsubishi Kasei) to adsorb L-5-oxytryptophan, then eluted with an alkaline solution and concentrated to L-5. -Crude crystals of oxytryptophan were precipitated. This was washed with acetone and dried to obtain 72 mg of L-5-oxytryptophan crystals.

In addition, as in Example-1, Escherichia coli (Esch.
erichia coli) K-12 strain (FERM BP-1733) culture medium obtained by using only cells obtained from 40 ml, L
The production amount of -5-oxytryptophan was 420 mg /.

Table 6 5-Oxindole 20 mM Sodium fumarate 50 mM Ammonium chloride 300 mM Nicotinamide adenine dinucleotide (NAD) 20 mM Phosphate buffer (pH8.0) 100 mM Example-7 Escherichia coli K-12 strain Instead of (FERM BP-1733), Escherichia coli K-12 strain (FERM BP-173)
The reaction was performed in the same manner as in Example 6 except that 4) was used, and the amount of L-5-oxytryptophan in the supernatant after the reaction was quantified to be 2560 mg /.

40 ml of the reaction supernatant was passed through a column of an ammonia type strongly acidic ion exchange resin (Diaion SK-IB, manufactured by Mitsubishi Kasei) to adsorb L-5-oxytryptophan, then eluted with an alkaline solution and concentrated to L-5. -Crude crystals of oxytryptophan were precipitated. This was washed with acetone and dried to obtain 70 mg of L-5-oxytryptophan crystals.

In addition, as in Example-1, Escherichia coli (Esch.
erichia coli) K-12 strain (FERM BP-1734) culture medium obtained from only 40 ml of culture medium
The production amount of -5-oxytryptophan was 420 mg /.

Example-8 After 50 ml of the reaction solution having the composition shown in Table 6 was dispensed into a 500 ml Erlenmeyer flask, the reaction was carried out in the same manner as in Example-4, and L in the supernatant after the reaction was completed. When -5-oxytryptophan was quantified, it was 850 mg /.

40 ml of the reaction supernatant was passed through a column of an ammonia type strongly acidic ion exchange resin (Diaion SK-IB, manufactured by Mitsubishi Kasei) to adsorb L-5-oxytryptophan, then eluted with an alkaline solution and concentrated to L-5. -Crude crystals of oxytryptophan were precipitated. This was washed with acetone and dried to obtain 27 mg of L-5-oxytryptophan crystals.

Incidentally, when only cells obtained from 80 ml of the culture solution of Proteus morganii (IFO-3848) were subjected to the reaction as in Example-1, the amount of L-5-oxytryptophan produced was 400 mg / Met.

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Claims (1)

[Claims] 1. A: A microbial cell belonging to the genus Brevibacterium containing an enzyme capable of producing L-malic acid from fumaric acid. B: A microbial cell belonging to the genus Escherichia, Proteus, or Erwinia containing an enzyme capable of producing oxaloacetate from L-malic acid. C: A microbial cell belonging to the genus Escherichia, Proteus or Erwinia containing tryptophanase. In the presence of the bacterial cells consisting of A, B, and C described above, fumaric acid, ammonium ion and the general formula (However, R represents a hydrogen atom or a hydroxyl group.) A corresponding L-tryptophan is produced from an indole represented by the following: A process for producing L-tryptophan.