JPS63105687A - Production of tryptophans - Google Patents

Abstract

PURPOSE:To produce L-tryptophan or L-5-hydroxytriptophan at a high production rate, by reacting fumaric acid with ammonium ions and indoles in the presence of plural enzymes. CONSTITUTION:Fumaric acid is reacted with ammonium ions and indole or 5-hydroxyindole in the presence of an enxyme capable of producing L-malic acid from fumaric acid or microbial cell, containing the above-mentioned enzyme and belonging to the genus Brevibacterium, etc., or treated microbial cell thereof, an enzyme capable of producing oxalacetic acid from L-malic acid or microbial cell, containing the above-mentioned enzyme and belonging to the Escherichia, etc., or treated microbial cell thereof and tryptophanase or microbial cell, containing the tryptophanase and belonging to the genus Escherichia, etc., or treated microbial cell thereof at pH6-9 and about 30-40 deg.C temperature for about 1-72hr.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing optically active shi-tryptophan or L-5-oxytryptophan using enzyme function.

(Prior Art and Problems) L-tryptophan is one type of essential amino acid, and is particularly important nutritionally and physiologically. Currently, tributophane is difficult to produce in large quantities, so its use is mainly limited to pharmaceutical use. However, if inexpensive manufacturing technology can be established, it is expected that new, large-scale markets for foods, feed additives, polymeric materials, etc. will be created.

L-5-oxytryptophan is an amino acid that has attracted attention mainly for its physiological effects, but currently no effective manufacturing method has been established, and there is a desire to develop an industrial manufacturing method that can be produced at low cost. .

Conventionally, methods for producing both of these amino acids have been known in which ammonium ions and pyruvic acid, oxaloacetic acid, or L-malic acid are used in indole or 5-oxindole (Japanese Patent Publication No. 46917/1985, etc.). .

However, among these raw materials, pyruvic acid, oxaloacetic acid, or L-IJ malic acid are very expensive;
It cannot be passed through the industrial manufacturing method that is the object of the present invention. As a result of intensive research, the present inventors have found that it is possible to produce L-5-oxytryptophan from fumaric acid, ammonium ions, and indole mustard, and L-5-oxytryptophan from fumaric acid, ammonium ions, and 5-oxindole. They found that this is the case, and completed the present invention.

(Structure and effects of the invention) The gist of the present invention is the following method (11).

[1) A: An enzyme capable of producing L-malic acid from fumaric acid, a microbial cell containing the enzyme, or a processed product thereof.

B: An enzyme capable of producing oxaloacetic acid from L-IJ malic acid, a microbial cell containing the enzyme, or a processed product thereof.

C: Tryptophanase, microbial cells containing tryptophanase, or processed products thereof.

Production of L-tryptophans, which is characterized in that the corresponding L-) liptraanes are produced from fumaric acid, ammonium ions, and indoles represented by the general formula in the presence of the enzyme systems A, B, and C described above. Law.

The basis of the present invention is to use fumaric acid as one of the raw materials, and the source of the enzyme system used in the reaction system is not limited to methods that utilize enzymes existing within the same microorganism. Naturally, methods that utilize multiple types of microbial cells or processed products thereof are also covered.

When indole, fumaric acid or its salt, and ammonium ion mustard-tryptophan are produced using the same type of microbial cells, and L-5-oxytryptophan is similarly produced using 5-oxindole instead of indole. Compared to reactions involving multiple types of microorganisms, this method requires only a single culture for bacterial cell preparation, resulting in lower culture costs; however, there are restrictions on the activity of each enzyme involved in the reaction.

On the other hand, when using multiple types of microbial cells, it is necessary to prepare cells for each microorganism, but on the other hand, it is possible to react at maximum speed in each reaction step.
As a result, a high production rate of L-tryptophan or L-5-oxytryptophan can be obtained.

In either case, the present method provides a novel process for producing monotryptophan or L-5-oxytryptophan from indole or 5-oxindole, fumaric acid or its salt, and ammonium ion.

This process consists of reaction (■) which can produce L-malic acid from fumaric acid, reaction (◎) which can produce oxaloacetic acid from L-malic acid, and reaction (O) which can produce pyruvic acid from oxaloacetate.
and tryptophanase reaction @, of which reaction θ is a non-enzymatic decarboxylation reaction and is easily converted to pyruvic acid in an aqueous solution.

Bacterial strains used in each of the above reactions are not particularly limited, but for example, Escherichia coli (Escherichia coli) strains used in reaction
colt) K-12 strain ATCC27325
, Escherichia coli
coli) K-12 strain (FERM P-884
4 and FERM P-8845), Proteus
morganii (Proteus morganii) I
FO-3848, Erwinia herbicola (Erwin
ia herbjcola) ATCC-21433
and so on.

The bacterial strain used in the above reaction and reaction ◎ is Brevibacterium flavum (Brevibacterium flavum).
erium flavum) M J -233 (F
ERM P-3068).

Brevibacterium flavum (Brevjbacte)
riumflavum) MJ-233-AB-41(
FERM P-3812).

In the present invention, the bacterial strains used in the reactions ◎ and ◎ are preferably cells of microorganisms belonging to the genus Brevibacterium or processed products thereof, particularly Brevibacterium flavum MJ-233 (FER?I). P-
3068), Brevibacterium flavum MJ-2
It is preferable to use cells such as 33-AB-41 (FERM P-3812).

When using the above-mentioned microorganisms belonging to Brevibacterium genus,
In particular, there is no decrease in enzyme activity, and the microbial cells used in the reaction can be reused.

The above C) shift reaction is similar to normal enzymatic reactions, such as O
, about 20 to about 50°C, preferably about 30°C, in an aqueous solution whose pH is adjusted to 6 to 9 with 1M phosphate buffer or water.
The process is typically carried out at a temperature of from about 1 to about 40°C for about 1 to about 72 hours. There is no particular restriction on the amount of indole or 5-oxindole used in the reaction, but it is generally 0.1-20% (wt/
It is appropriate to use it within the concentration range of vol). Also,
The concentration of fumaric acid or its salt and ammonium ion is not particularly limited, but is generally 0.1 to 20% (w
t/vol) is suitably used. The amount of bacterial cells or their processed material used in the reaction is generally 0.
．． It can be used at a concentration of 1-10% (wt/vow).

In addition, the above-mentioned bacterial strain can be cultured using a commonly used synthetic medium or natural medium. In the case of Escherichia, Proteus and Erwinia, carbohydrates such as glucose, glycerol, fructose, sucrose and molasses can be used as carbon sources.

On the other hand, in the case of Previbacterium, ethanol can be used as a carbon source in addition to the carbohydrates mentioned above. In addition, many natural organic nitrogen sources such as tryptone, yeast extract, corn steep liquor, and casein hydrolyzate can serve as a nitrogen source as well as a carbon source.

Cultivation can be carried out under aerobic conditions such as shaking culture or aerated agitated tank culture. Culture temperature is generally 20-50℃
℃, and it is desirable to maintain II)H of the medium in the vicinity of neutral or slightly alkaline. The culture period is usually about 5 hours to about 3 days.

Using the bacterial cells obtained by the above-mentioned culture method or a processed product thereof, indole or 5-oxindole, fumaric acid or its salt, and ammonium ion are used. If indole is used, L-tryptophan, 5-oxindole, When oxindole is used, L-5-oxytryptophan can be produced.

In the present invention, the term "processed product" includes a crushed product obtained by crushing microbial cells by ultrasonication or the like, and also includes a microbial cell or a crushed product fixed on a carrier or the like by a known method.

Separation and purification of the tryptophan and the like produced in the reaction solution can be carried out by known methods such as adsorption and desorption treatment using ion exchange resins, activated carbon 1, and the like.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Reference Example-1: Preparation of Brevibacterium flavum cells.
Dispense separately into two 2-volume Erlenmeyer flasks and heat at 120℃.
Heat sterilize for 15 minutes and add 2% ethanol by volume to each
Add aseptically and add Brevibacterium flavum to these.
M J-233 (FERM P-3068) or Brevibacterium flavum (Brevibacterium
ium flavum) M J-233-AB-4
1 (FERM P-3812) was inoculated in loopfuls and cultured at 30°C for 24 hours.

20mff1 of these two sets of culture solutions were separately inoculated into 1p of a medium with the composition shown in Table 2 in a 21-volume jar fermentor, and incubated at 33°C, pH 7.6, and aerated iJ-1v v.
The ethanol concentration was 1.0-1.
Ethanol was added intermittently to maintain a concentration of 5% by volume. After 30 hours of culture, the culture solution was centrifuged (6, OOO
rpm, 15 minutes) were used as test cells.

Table 1 Urea 4.0g Ammonium sulfate 14.0g Continuation of Table 1 KH, PO40, 5g K2HP○, 0.5gMg
5O,-7H200,5g Fe50. =IH206,0+ng Mn Sot ・4-6 H206, Orq yeast extract 1.0g Casamino acids
1.0g biotin
200μg thiamine hydrochloride
100μg distilled water
10100O Table 2 Ammonium sulfate 23.0gK H
2P 04 0, 5 g
K2HPO, 015g Mg SO4・7 H200,5g FeSO,・lH2O20try MnSO4・4~6H2020 ■ Yeast extract 3g Casamino acids 3g Table 2 continued Biotin 200 μg Thiamine hydrochloride 100 It, g
Distilled water 10100O Reference example-2 = 8 g of Escherichia coli cells Pour the medium 5 Q m A with the composition shown in Table 3 into a 500 ml Erlenmeyer flask, sterilize it at 120°C for 15 minutes, and add Escherichia coli cells. Ko'J (Esche-richia c
o order) K-12 strain (ATCC27325 and FE
RM P-8844 and FHRM P-8845)
was inoculated and cultured with shaking at 37°C for 1 day, and 200 μg/m of L-)ributophane prepared in the same manner was sterilized.
Contain 20 m in 1000 ml of culture medium at a concentration of 1.
1 was inoculated and cultured with shaking at 37°C for 8 hours. Centrifuge the cultured solution (6000 rpm, 15 minutes).

The cells obtained at 4° C. were used as test cells.

Table 3 - Tryptone 10g Yeast extract 5g Na Cj! 5 g glucose
1g Distilled water 1β (pH 7,2) Reference example-3 = Proteus morganii - Preparation of bacterial cells Soum the medium 5 with the composition shown in Table 4. Proteus morganii (Proteus +a) was dispensed into Erlenmeyer flasks and sterilized at 120°C for 15 minutes.
organii) IFO-3848 and 37°C
The cells were cultured with shaking at 37° C. for 24 hours, then sterilized in the same manner and cultured with shaking at 37° C. for 8 hours. The cells obtained by centrifuging the cultured solution (6000 rpm, 15 minutes, 4° C.) were used as test cells.

Table 4 KH2PO40.5g/I! 2HP0. 0.5g/j! ! Mg5O-,
・7H200,5g/j! FesOt' 7H20
6 ppm MnSO4/nH2C) 5 ppm yeast extract ° 10 g/l Casamino acids
5 g/12 (pH 7,
5) Reference Example-4: Erwinia herbicola (Erwiniaherb) as a bacterial strain for preparation of Erwinia herbicola
icola) ATCC-21433 was used, but shaking culture was performed at 37° C. for 8 hours using the same medium and procedure as in Reference Example-3. Centrifuge the cultured solution (600 orp
The microbial cells obtained at 4° C. for m and ts were used as the test microorganisms.

Example-1 50mA of reaction solution with the composition shown in Table 5! was dispensed into a 500 ml Erlenmeyer flask, and Brevibacterium flavum (Brevibacterium flavum) prepared in Reference Example-1 was added to the reaction solution.
bacterium flavum) MJ 23
3 (FERM P-3068) culture solution 4 Q
m7! Centrifugation (6000 rpm, 15 minutes, 4°C)
) and Escherichia coli (P, 5cherichia colt) prepared in Reference Example-2.
K-12 strain 8TCC27325 culture solution 40mj
The microbial cells obtained by centrifugation (6000 rpm, 15 minutes, 4°C) were added thereto, and a shaking reaction was performed at 37°C for 24 hours. After the reaction, centrifugation (4000 rpm, 1
The bacterial cells were removed at room temperature for 5 minutes, and tryptophan was quantified in the synovial fluid, and 800 μ/β production was observed.

In addition, Escherichia coli (Escherichia coli) was added to the above reaction solution.
richia colt) K-12 strain ATC
When only the bacterial cells collected from 40 ml of culture solution of C27325 were added and a shaking reaction was performed at 37° C. for 24 hours, the amount of L-)liptophan produced was 450 g/A.

Table 5 Indole 20mM Sodium Fumarate 50mM Ammonium Chloride 300mM Nicotinamide Adenine Dinucleotide (NAD) 20mM Phosphate Buffer (pH 8,0) 100mM Example-
2 Reaction solution 5 similar to Example-1 Reference Example-1 to Qmffi
Brevibacterium flavum (Brevi
bacterium flavun) MJ-233
AB-41 (PHI? M P-3812) culture solution 4
The bacterial cells obtained from Q m It and Escherichia coli prepared in Reference Example-2
) i) Bacterial cells from 40 ml of culture solution of L12 strain (FERM P-8844) were added and incubated at 37°C.
The shaking reaction was carried out for 5 hours, and the amount of L-tryptophan in the supernatant after the reaction was determined to be 3200 ■/j! Met.

40 mβ of the reaction supernatant was passed through a column of ammonia-type strongly acidic ion exchange resin (Diaion 5K-IB, manufactured by Mitsubishi Kasei Co., Ltd.) to adsorb L-) tryptophan, and then eluted with an alkaline solution and concentrated to obtain L-tryptophan. Crude crystals were precipitated. This was washed with acetone, cooled, and dried to obtain 95 μm of crystals of L-)liptophan.

In addition, as in Example-1, Escherichia coli (Esc
herichia colt) K-12 strain (
When only the bacterial cells obtained from culture solution 4 Qmff of FERM P-8844) were subjected to the reaction, the amount of L-tryptophan produced was 460 mg/R.

Example-3 Reaction solution 5 similar to Example-1 Reference example for Q m j2-
Brevibacterium flavum (Brev
ibacterium flavum) M J −
233A B-41 (FER?I P'-3812
) culture solution 4 Q m It and Escherichia coli (Es.
cherichia coli) K-12 strain (
FERM P-8845) culture solution 40mj! The L-tryptophan in the supernatant after the reaction was quantified and found to be 3100 mg/A.

40mj of reaction supernatant! After adsorbing L-1-lyptophan through a column of ammonia-type strongly acidic ion exchange resin (Diaion SK-I B, manufactured by Sani Kasei),
After elution with an alkaline solution, it was concentrated to precipitate crude crystals of L-tryptophan. Wash this with acetone and dry it.
- 87 mg of tryptophan crystals were obtained.

In addition, as in Example-1, Escherichia Co. IJ (Es
cherichia colt) K-12 strain
(['ERM P-8845) culture solution 49 m j
! When only the bacterial cells obtained from L-) were subjected to the reaction, the amount of L-ributophane produced was 430 mg/j! Met.

Example-4 Brevibacterium flavum (Breviba prepared in Reference Example-1) was added to 50mj+ of the same reaction solution as in Example-1.
cterium flavum) M J 233
AB 41 (FEI?MP-3812)
Bacteria and bodies obtained from 40 m of culture solution and Reference Example-3
Proteus morganii (Proteus morganii) prepared in
morganii) IFO-3848 culture solution 80
The microbial cells obtained from mIt were added thereto, and a shaking reaction was carried out at 37°C for 24 hours. After the reaction, L-tryptophan in the supernatant liquid was quantified and found to be 1050 mg/l.

Reaction supernatant liquid 4 Qmj! was prepared in the same manner as in Example-2. When L-tryptophan was collected from the sample, 32 mg of crystals were obtained.

In addition, as in Example-1, Proteus molga-1-(
Proteus morganii) IFO-384
When only the bacterial cells obtained from 80 m J of the culture solution of No. 8 were subjected to the reaction, the production of L-)ributophane was 430 mg/J.
! Met.

Example 5 Erwinia herbicola AT prepared in Reference Example 4 instead of Proteus morganii as a tryptophanase-bearing strain
The reaction was carried out at 37° C. for 24 hours in the same manner as in Example 4 except that CC-21433 was used. When the amount of tryptophan in the supernatant after the reaction was quantified, it was 710m.
g/j! The formation of was observed.

In addition, as in Example-1, Erwinia herbicola (E
rwinia herbicola) ATCC-2
When only the bacterial cells obtained from 8Q m 1 of the culture solution of 1433 were subjected to the reaction, the amount of L-tryptophan produced was 320
mg/! Met.

Example-6 50 m of the reaction solution having the composition shown in Table 6 was heated to 500 m
After dispensing into a 12-volume Erlenmeyer flask, the reaction was carried out in the same manner as in Example 2, and after the completion of the reaction, L-5 in the superior synovial fluid was
- Oxytryptophan was determined at 2500 m
g/β.

Reaction supernatant liquid 4 Q m j! Ammonia-type strongly acidic ion exchange resin (Diaion SK-I B, manufactured by Mitsubishi Kasei)
After L-5-oxytryptophan was adsorbed through the column, it was eluted with an alkaline solution and concentrated to precipitate crude crystals of L-5-oxytryptophan. This was washed with acetone and dried to obtain 72 mg of L-5-oxytryptophan crystals.

In addition, as in Example-1, Escherichia coli (Esc
herichia coli) K-12 strain (F
ERM P-8844) culture solution 4 Q m j! When only the bacterial cells obtained from L-5-oxytryptophan were subjected to the reaction, the amount of L-5-oxytryptophan produced was 420 mg/It.
Met.

Table 6 - Oxindole 20mM Sodium Fumarate 50mM Ammonium Chloride 300mM Nicotinamide Adenine Dinucleotide (NAD) 20mM G Phosphate
Canned solution (pH 8,0) 100mM Example-7 Escherichia coli (Escherichia c
olt) K-12 strain (FE[1M P-884
4) instead of Escherichia coli (Escheri
chia coli) K-12 strain (FERM
The reaction was carried out in the same manner as in Example 6, except that P-8845) was used, and the amount of 5-oxytryptophan in the supernatant after the reaction was determined to be 2560 mg/β.
Met.

40 ml of the reaction supernatant was passed through a column of ammonia-type strongly acidic ion exchange resin (Diaion SK-I B, manufactured by Mitsubishi Kasei) to adsorb L-5-oxytryptophan, eluted with an alkaline solution, and concentrated. Crude crystals of L-5-oxytryptophan were precipitated. This was washed with acetone and dried to obtain 7 Qmg of crystals of L-5-oxytryptophan.

In addition, as in Example-1, Escherichia coli (Esc
herichia colt) L12 strain (FE
RM P-8845) culture solution 40 mj! When only the bacterial cells obtained from the above were subjected to the reaction, the amount of L-5-oxytryptophan produced was 420 mg/n.

Example-8 50ml of the reaction solution having the composition shown in Table 6 was heated to 500mI! ,
After dispensing it into an Erlenmeyer flask, the reaction was carried out in the same manner as in Example 4, and the amount of L-5-oxytryptophan in the supernatant liquid after the reaction was determined to be 85' Om g.
/ It was 1.

40mj of reaction supernatant! After adsorbing L-5-oxytryptophan through a ram using only ammonia-type strongly acidic ion-exchange resin (Diaion 5K-I B, manufactured by Mitsubishi Kasei), it was eluted with an alkaline solution and concentrated to obtain L-5-oxytryptophan. Crude crystals were precipitated. This was washed with acetone and dried to obtain 27 mg of L-5-oxytryptophan crystals.

In addition, as in Example-1, Proteus morganii (P
roteus morganii) IFO-3848
When only the bacterial cells obtained from the culture solution 80 m j+ were subjected to the reaction, the amount of L-5-oxytryptophan produced was 400 mg/It.

Claims (2)

[Claims] (1) A: An enzyme capable of producing L-malic acid from fumaric acid, a microbial cell containing the enzyme, or a processed product thereof. B: An enzyme capable of producing oxaloacetate from L-malic acid, a microbial cell containing the enzyme, or a processed product thereof. C: Tryptophanase, microbial cells containing tryptophanase, or processed products thereof. In the presence of the enzyme system consisting of A, B and C above, fumaric acid, ammonium ion and general formula ▲ mathematical formula, chemical formula,
A method for producing L-tryptophans, which is characterized by producing the corresponding L-tryptophans from indoles represented by ▼ (where R represents a hydrogen atom or a hydroxyl group). (2) Claim 1, wherein A is a cell of a microorganism belonging to the genus Brevibacterium, and B and C are cells of a microorganism belonging to the genus Escherichia, Proteus, or Erwinia or a processed product thereof. the method of.