JPS62215564A - Purification of tryptophan - Google Patents

Abstract

PURPOSE:To obtain the titled component in high purity, by passing a reaction liquid produced from indole by enzymatic action through a porous cation- exchange resin and separately eluting the titled compound and the unreacted raw material using an aqueous solution of an alkali or acid and a water- containing organic solvent. CONSTITUTION:Tryptophan useful as a pharmaceutical, health food, feed, etc., is produced from indole by enzymatic action. The reaction liquid containing unreacted raw material is passed through a porous cation exchange resin layer. The objective compound adsorbed to the ion-exchange group of the resin is eluted with an aqueous solution of an alkali or an acid, preferably with ammonia water. After the above elution, indole adsorbed to the porous part of the resin is eluted with a water-containing organic solvent miscible with water (preferably an aqueous solution of a low alcohol). The content of the unreacted raw material in the objective compound can be suppressed to <=10ppm and the objective compound containing little unreacted material can be produced with the above simple procedure.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to separating unreacted indole from 1-ributophane obtained by an enzymatic method using indole as a raw material, and reducing the indole content in tryptophan to 10 ppm.
The present invention relates to a method for purifying tryptophan that is suppressed within the scope of the present invention.

1-Lipdoane, especially L-1-lipdoane, is one of the essential amino acids and is a useful compound used in medicines, health foods, feeds, and the like.

In addition to the fermentation method using glucose as a raw material and the semi-fermentation method using anthranilic acid as a raw material, L-1-lipdoane can be produced by enzymatic condensation reaction with L-serine using indole as a raw material. how to get tryptophan,
Alternatively, a method is known in which L-1-rib1-fan is obtained by allowing enzymes to act on indole, pyruvic acid, and ammonia.

[Prior art and problems to be solved by the invention] The problem with the method of obtaining tryp-I-ributophane by enzymatic action using indole as a raw material is that the amount of unreacted indole in the obtained I-ributophane enzymatic reaction solution is It is separation.

Usually, the reaction solution contains 100 ppm or more of indole, and even a trace amount of indole gives off a characteristic bad odor. Therefore, the content in the final product should be at least 10 ppm, especially for feed use, 3 ppm.
It is necessary to make it up to about m.

However, indole and tribut-1-fan both have an indole skeleton and their chemical 11ζ structure is similar, so indole has a strong adhesion to tryptophan, and cannot be removed to an acceptable amount by means such as final crystal washing.

Furthermore, indole is a raw material with a high unit price, and if it cannot be recovered, it will lead to varnish 1-up.

Therefore, in the production of tryptophan using indole, in tryptophan purification step 1, which includes unreacted indole after the reaction, there is no method that can efficiently separate and recover indole and tryptophan while simultaneously completely removing other impurities. must not.

Normally, the enzymatic method for producing 1-Lift Fan involves reacting, for example, indole and serine in a near-neutral aqueous medium in the presence of an enzyme or bacterial cells that have the action of 1-lip 1-fan synthase. However, it is necessary to remove the bacterial cells and enzymes contained in the resulting reaction solution.
These bacterial cells and enzymes can be removed by adsorption separation using a solid material such as activated carbon or adsorbent silica gel in accordance with the usual amino acid treatment method, by sedimentation separation using a centrifuge, or by using ion exchange resin or other methods. A method of purifying amino acids from which these impurities have been removed using a non-polar porous resin has been adopted.

For example, JP-A-56-73050 describes a method for purifying and isolating L-1-lipdoane using a macroporous strongly acidic ion exchange resin.

However, in these methods, indole and trib I
~ Fan separation is not possible.

Indole and 1-lift fan are poorly soluble in water. For example, the slurry after the reaction is diluted with a large amount of water during the purification process to make 1-lift fan into an aqueous solution, and unreacted indole is treated with activated carbon. If you try to remove it by adsorption, a large amount of tryptophan will also be adsorbed along with indole.

In addition, when aromatic or heteroaromatic amino acids are adsorbed using a normal gel-type cation exchange resin as in the method described in the above publication, it is relatively difficult to elute, so porous cations that are easy to elute are When tryptophan is adsorbed and eluted using an exchange resin, it is naturally possible that indole is also contained in the eluent at the same time. In this case, in order to separate indole, it is necessary to add an organic solvent that is immiscible with water, such as benzene or toluene, to the eluted trib 1 to fan aqueous solution to extract and separate indole to the organic solvent side. Collection operations become complicated, and indole-containing jlt
It is difficult to reduce the amount to an acceptable range of about ppm in IC.

[Means for solving problems]

The present inventors have been intensively investigating a method for purifying tryptophan that is simple and contains almost no indole.
When the reaction solution of Tridi 1 to Fufu 2 containing indole is passed through a porous cation exchange resin layer, indole has an affinity for the resin, and surprisingly, indole is selectively adsorbed only in the porous portion. Furthermore, it has been found that even when tryptophan adsorbed on the ion exchange part is eluted, indole is hardly contained in the eluent. Furthermore, since a water-containing organic solvent is used to elute indole, it was found that the resin could be easily regenerated by simply washing the resin with water after indole elution, and that it could be reused for the separation of the 1-lift fan, leading to the present invention. This is what I did.

That is, the method of the present invention provides 1-lip 1- containing unreacted indole obtained by enzymatic action using indole as a raw material.
The fan reaction solution was passed through the porous cation exchange tree WI, and the tryptophan adsorbed on the ion exchange part of the resin was eluted using an aqueous alkali or acid solution. l characterized in that the indole is eluted using a water-containing organic solvent that is mixed with water.
- A method for refining lift fans.

In the present invention, the 1-Liftfan reaction solution used is, for example, a reaction solution obtained by enzymatically condensing indole and L-serine in the presence of 1-Liftfan synthase in an aqueous medium, or a reaction solution obtained by enzymatically condensing indole and L-serine, or indole and pyruvin. It is a reaction solution in which acid and ammonia are reacted in the presence of truptophanase, and of course it may be one that has been subjected to pretreatment such as removal of bacterial protein. In addition, if the purpose is to separate the active ingredients L-1-lipdoane and indole, it is of course possible to apply it to the liquid discharged during the purification process, such as the washing liquid, even if it is not the reaction liquid. .

It is preferable to remove solids that may cause clogging of the resin column from the liquids containing active ingredients, such as the reaction liquid and the furnace washing liquid, before actually using them.

In the method of the present invention, the ion exchange resin used is:
Any porous cation exchange resin may be used, but the
tSl)-112>, Amberl
ite 200C), Diaion P
A macroporous type with a strongly acidic cation exchange tree top such as K-220) is preferable.
However, the gel type cannot be used in the method of the present invention.

The amount of resin used is based on the total amount of 1,000 ions in the solution to be treated, that is, the total molar proportion of impurities such as amino acids, ammonium ions, sodium ions, etc. contained in normal reaction solutions in addition to L-)-lipidan. The amount should be within the total exchange capacity of the resin.

In the present invention, preferred embodiments for treating the reaction solution containing indole after the I-ribdoane reaction are as follows.

The reaction liquid p containing unreacted indole is passed through a porous cation exchange resin layer.

P)I of the liquid to be treated during liquid passage is preferably neutral to acidic, the temperature is 90°C or less, and the liquid passage is performed at 5V=1 to 10.

The adsorption and elution of tryptophan in the ion exchange part and the elution of indole adsorbed to the porous part may be performed alternately, but there may be a large difference in the concentration ratio of l-lipdoane and indole contained in the reaction liquid to be treated. and the adsorption mechanism is different, so the elution of the adsorbed indole is
After repeating the separation process of adsorption, elution, and regeneration using the l-lift fan many times, it is preferable to carry out the separation process when the adsorption capacity of indole is almost reached.

Otherwise, subsequent attempts to isolate and recover the indole from the eluate will be difficult. Therefore, it is usually preferable to elute indole after repeating the adsorption, elution, and regeneration of 1-lip 1 to 7 am at least five times or more, depending on the content ratio of indole. Tryptophan is completely adsorbed in one pass, and while the separation process is repeated, indole is adsorbed to the porous parts of the resin and stored h'! Therefore, when the concentration reaches a certain level, the indole solution can be passed through to make the solution 4E. By carrying out such a treatment, an indole solution with a high tlB If is obtained, and from this, highly pure indole can be easily recovered by separating it according to conventional methods such as concentration and crystallization.

Moreover, indole is hardly contained in the eluate from 1 to Ributofan during this period.

In the present invention, elution of 1 to lyptophans is carried out by passing water in a conventional manner each time after completion of adsorption and passing, and eluating the tree 1r6 after thorough washing. As the eluent, a commonly used alkali or acid aqueous solution is used, but ammonia water is preferable.
30wt% ammonia water from the top of the column at 5V = 1~
Either method may be used, such as eluting at a pH of 10 or circulating water in the column and blowing gaseous ammonia into it. Further, although L-1 to L-Fan are almost completely eluted by this operation, it is preferable to further extrude with water in order to recover the residual content in the column.

Through such normal repeated operations, 95 parts of the 1-lift fan contained in the liquid to be treated are recovered into the liquid to be treated. In addition, trace amounts of indole contained in the liquid to be treated (several ppp)
m is leaked into the processing solution.

If further purification is required, the L-1-ribdoane eluate can be purified by known isolation methods, i.e., unit operations such as concentration, crystallization, solid-liquid separation, drying, etc., to obtain a high-quality product containing less than the desired indole tolerance. L-1-rib 1-fan can be isolated.

1 - The resin after lipoan elution is thoroughly washed with water, and the resin is regenerated with hydrochloric acid, sulfuric acid, etc. in a conventional manner and used repeatedly.

In addition, in the present invention, when indole is eluted,
A water-containing organic solvent is used as the eluent.

Organic solvents alone have poor affinity for indole and cannot be completely eluted. Preferred organic solvents are lower aliphatic alcohols, such as methanol, ethanol, n-propatool, and isopropatool, with isopropatool being particularly preferred. An aqueous alcohol solution containing 10 liters or more, preferably 70 liters or more of these is prepared, and the solution is eluted from the top of the column at a rate of 5V-1 to 10 using at least 1 times, preferably about 2 times, the amount of resin.

The obtained indole eluate was concentrated in accordance with a conventional method, and after recovering the solvent, it was separated into a solid by a conventional method such as back extraction with a non-aqueous organic solvent such as 1-toluene or hexane, followed by cooling and crystallization. It can be collected by

The column after indole elution is thoroughly washed with water to remove the solvent, and then reused for the separation of tryptophan.

Reference examples and working examples are shown below, but we are sorry about the weight.

[Example-1] Contains L-1-rib l-7an obtained by condensing indole and L-serine in an aqueous medium in the presence of the enzyme TOP1-fan synthase produced by culturing E. coli. Activated carbon and water were added to the reaction solution, and P)I4 was added to 9 with sulfuric acid.
After heating at 5 to 100° C. for 1 hour to flocculate the bacterial cells, the bacterial cells adsorbed on the activated carbon were directly removed by filtration.

L-)-lift fan 38 (0.19M) obtained by pretreatment in this way, 0.1 yen of indole, L-
0.2% (0.02M) of serine, 0.8% (0.44M) of ammonium ions, and 0.8% (0.44M) of sodium to ions.
2'! (o, n 9 M) aqueous solution containing 2580
g (total molar equivalents of amino acids and cations: 19) was added to Revatit 5P-112 (Bayer product, exchange volume fi 1.9).
A column packed with 1.01 (mol/l) was subjected to conditioning regeneration, and the liquid was passed from the top at 5V=5.

L-1-lyptophan was detected by high performance liquid chromatography in the distilled liquid at the end of the liquid passage, but no L-1-lyptophan was detected.

Next, after washing with water 21% 5v = 10, 10% NH3 water 60
Elute at 0f, 5V=2, 1oo of water at 6.

Extrusion was performed at g to obtain eluent 160079.

In the eluent, L-1-rib 1-fan 5.9 (94
1g equivalent, recovery rate of 96.0 for L-1-rib 1 to fan in the adsorption stock solution), indole t ppm or less (
The indole removal rate was nearly 100%).

The eluate was concentrated under reduced pressure to approximately 4,700 ml and heated at 5°C. IHI crystallization a row nai 1'/:
F-Z 1m Upper A 1!4 $ After passing the stone, add 1 water
00g to obtain 136.8g of wet cake, which was dried to give 80.4g of yellow-white tuna flake-like crystals. Obtained IF @ total yield 82.0%/adsorption stock solution, product quality purity 996
-313° optical rotation, indole content 1.2 ppm
It contained almost no indole, and no indole odor was detected at all. Of course, this quality sufficiently satisfies the domestic feed additive standards.

In addition, the resin after elution with L-)-ributophane is further
After washing with 1 water, 80% isopropyl alcohol water 20
At 00.9, the indole eluate 2000.9 was obtained by flowing out from the top of the column at 5V=2□.

The eluent contained 0.12% indole (recovery rate of 92.0% based on indole in the adsorption stock solution).

[Example-2] In the same manner as in Example-1, only adsorption, elution, and regeneration of L-1-ributophane were repeated eight times without elution of indole. During this period, the indole content in the flowing liquid during L-1-rib 1-fan elution was checked, and no indole was detected.

A small amount of indole was detected in the 9th L-tryptophan eluate, so after washing with water after the 9th L-tryptophan elution, 2000 I of 80% isopropyl alcohol water was added from the top of the column at 5 V = 2. Elute indole with indole eluent 2000.9.
I got it.

The eluent contained 3.2% of indole. After concentrating the solution 111i to 500 g, n.
- Add 200 g of hexane, mix and extract, separate the layers, cool the n-hexane layer, crystallize at 5°C/2 hours, filter through Nutsche, dry the obtained weL cake, and indole 39. 0.1F was obtained. (Recovery rate 61.0%, gas chromatography purity 100φ) The recovered indole is
Although it was reused as a raw material for the L-1-ributophane enzymatic reaction, it was confirmed that the reaction had no effect at all.

In addition, after completing the elution of indole in this way, after washing with water and regenerating it again, adsorption and elution of L-1-rib 1-fan are repeated, and indole is detected in the L-1 ~ ribtofan eluent. Even if the elution and recovery of indole with isopropyl alcohol water was repeated, 5.5. −
- Even after the operation, the product obtained was of odorless quality that fully satisfied the domestic additive standards for feed L1.

Claims (1)

[Claims] 1. A tryptophan reaction solution containing unreacted indole obtained by enzymatic action using indole as a raw material is passed through a porous cation exchange resin layer and adsorbed on the ion exchange part of the resin. A method for purifying tryptophan, which comprises eluting tryptophan using an aqueous alkali or acid solution, and then eluting indole adsorbed to the porous part of the resin using a water-containing organic solvent that mixes with water. 2. The method according to claim 1, wherein the alkaline aqueous solution used as the tryptophan eluent is aqueous ammonia. 3. The method according to claim 1, wherein the water-containing organic solvent mixed with water used to elute indole is an aqueous solution of a lower aliphatic alcohol.