JPS63130580A - Purification of tryptophan - Google Patents

Abstract

PURPOSE:To crystallize and purify the titled compound useful as a medicine, in high purity, by passing an aqueous solution of tryptophan containing impurities through a nonpolar highly porous polymer resin, eluting with ammonia water and concentrating the eluate with a reverse osmosis membrane. CONSTITUTION:An aqueous solution of tryptophan containing impurities is passed through a nonpolar highly porous polymer resin to effect the adsorption of tryptophan and the tryptophan adsorbed to the resin is eluted with ammonia water (the 1st step). The eluate obtained by the 1st step is concentrated with a reverse osmosis membrane (the 2nd step). The objective compound is crystallized by removing ammonia from the concentrated liquid obtained by the 2nd step (the 3rd step). The amount of the resin used in the 1st step is usually 5-100%, preferably 5-30% of the aqueous solution of tryptophan and the concentration of the ammonia water used in the 1st step is preferably about 0.5-5N.

Description

DETAILED DESCRIPTION OF THE INVENTION Technical Field The present invention relates to a method for purifying tryptophan.

According to the method of the present invention, an efficient method for purifying tryptophan with high purity and high recovery rate is provided when producing a large amount of trit-7an useful for pharmaceuticals, feed additives, etc.

Prior art methods for producing tryptophan include a method of synthesizing serine and indole using citryptophan synthetase, a method of synthesizing pyruvate, ammonia and indole using tryptophanase, and an intermediate fermentation method from anthranilic acid. well known.

In either case, the reaction crude liquid contains a considerable amount of impurities. For example, in the synthesis method from serine and indole,
Unreacted D-serine and coexisting inorganic salts are present in the synthesis method from pyruvic acid, ammonia, and indole, and unreacted pyruvic acid, a large amount of ammonium salt, and other coexisting inorganic salts are present, and intermediates from anthranilic acid are present. In fermentation, there are large amounts of inorganic salts contained in the culture solution.

The conventional method for separating and purifying tryptophan from these impurities is to adsorb the crude reaction solution containing tryptophan onto a strongly acidic cation exchange resin, and then use an appropriate eluent to remove the tryptophan and desorb D-serine. There is a method of eluting coexisting amino acids such as, and then eluting tryptophan with aqueous ammonia.

However, in this method, a large amount of acid is required to regenerate the ion exchange resin, and when there are many coexisting inorganic salts, the exchange ability of the ion exchange resin is exchanged with the inorganic salt. However, there are problems such as the fact that it requires

Another known method for producing tryptophan is to adsorb an amino acid mixture containing tryptophan onto a non-ion exchange decolorizing resin, and elute the amino acids other than tryptophan, which have lower molecular weights, with water in that order.) (See Japanese Patent Publication No. 30-2982).

However, this method requires a large amount of water to elute all tryptophan, the resulting tricyto7an solution is not dilute, and it requires a large amount of energy to recover tryptophan from this tryptophan solution. There's a problem.

SUMMARY OF THE INVENTION The present invention has been made for the purpose of solving the problems of the prior art described above.

That is, the present invention provides a method for separating and purifying tryptophan from an aqueous tryptophan solution containing impurities, which includes the following steps: Step 1: The ice-water solution is passed through a non-polar high-porous polymer resin to adsorb tryptophan, and then ammonia water is added to the resin. Elute the adsorbed tryptophan. Step 2: Concentrate the eluate obtained in Step 1 using a reverse osmosis membrane. Step 3: Remove ammonia from the concentrated solution obtained in Step 2 and crystallize tryptophan. The present invention provides a method for purifying tryptophan, which is characterized by employing the following steps.

Effects of the Invention According to the method of the present invention, tryptophan can be purified with high purity and high recovery rate from an aqueous tryptophan solution containing impurities. Furthermore, by reusing the aqueous ammonia produced in Step 2 as the tryptophan eluate in Step 1, the consumption of auxiliary agents used in purification can be extremely reduced, resulting in such an economical d! Regardless of the manufacturing method, the target tryptophan can be purified with high purity and high recovery rate.

Detailed Description of the Invention Step 1 in the method of the present invention consists in passing an impurity-containing tryptophan aqueous solution through a non-transparent high porous polymer resin to adsorb tryptophan, and then eluting tryptophan with aqueous ammonia. .

By using aqueous ammonia as the eluent, it is possible to concentrate the eluate to several times the amount of the reaction solution passing through the tryptophan membrane, which reduces the burden on the next step of concentration using a reverse osmosis membrane. can be significantly reduced.

Step 2 consists in concentrating the ammonia aqueous solution of tryptophan obtained in the above step 1 using a reverse osmosis membrane while remaining as an ammonia aqueous solution.

) IJ f ) 7an has low solubility in water and also has a small temperature dependence of solubility, so the recovery rate can be kept low by commonly used cooling crystallization. A high #condensation degree can be obtained by concentrating the ammonia aqueous solution eluted from the ammonia using a reverse osmosis membrane.

Step 3 is to remove ammonia by evaporation or the like from the tricito-7 ammonium solution concentrated using reverse osmosis GTL in step 2, and to crystallize and recover tryptophan.

From tryptophan water bath solution concentrated using reverse osmosis membrane,
Just by removing ammonia, tryptophan crystals can be easily recovered with a high recovery rate.

In the method of the present invention, during the shrinkage of tryptophan using a reverse osmosis membrane in step 2, the ammonia water that permeates through the reverse osmosis membrane is directly absorbed into the tryptophan adsorbed on the non-polar high deluxe polymer resin in step 1. Preferably, it is used as an eluent.

The reverse osmosis membrane used in step 2 eliminates ammonia water.
Since it is not concentrated, ammonia (ammonium ions) supplied to the reverse osmosis membrane at the same time as tryptophan moves to the permeation side at the same concentration as the concentration side. By using this ammonia water to elute tryptophan from the non-polar highly porous polymer-based fat in Step 1, most of the ammonia water that has passed through the reverse osmosis membrane can be recovered without going through a recovery step. Of course, since the concentration side also carries ammonia water with the same concentration as the permeation side, the concentration of the permeated ammonia water or before the concentration step can be slightly adjusted by using concentrated ammonia water or ammonia gas.

Even if the ammonia water permeated through the reverse osmosis membrane produced in Step 2 is reused as is in Step 1, the purity and recovery rate of tryptophan in purification of the target tryptophan will not decrease, and the consumption of ammonia as an auxiliary agent will also be reduced. It has the advantage of being able to reduce

Nonpolar hyperporous used in the present invention? The remer-based resin is a non-polar resin that is a neutral vinyl chloride polymer or copolymer, such as styrene and divinylbenzene, which does not have ion polarity unlike ion exchange resins.
Moreover, these resins are highly porous polymer resins having a pore radius of 100 to 100OX.

Specific examples of the above-mentioned non-polar high porous remer resin include "Diaion HP-10",
1 Tsukasa HP-20', "Do HP-21", "Do HP-3"
0'', ``Same HP-40'', ``Same HP-50'', ``Sepapies 5P-206', ``Same 5P-207'', ``Same 5P''
-soo", "5P-9001 product name, manufactured by Mitsubishi Chemical Corporation), "Amberlite XAD-2',""X
This includes AD-4' (all trade names, manufactured by Rohm and Haas).

The tryptophan concentration in the impurity-containing tryptophan aqueous solution used in the present invention may be any concentration as long as the tryptophan is dissolved, but it is preferably a solution with a concentration of 5 to 15 Iilt. The impurities contained in the aqueous solution, which is the raw material, vary depending on the process through which the aqueous solution was obtained, but some examples are as follows.

For example, in the enzymatic production method using DL-serine and indole as raw materials, unreacted L-serine, D-serine that is not used in the reaction, unreacted indole, pyridoxal phosphate as a coenzyme, and its low chloride sodium,
This includes inorganic salts such as ammonium chloride and potassium dihydrogen phosphate.

In addition, in the enzymatic production method using indole, pyruvate and ammonium salt as raw materials, unreacted indole, pyruvate or its salt, ammonium salts such as ammonium acetate and ammonium chloride, the coenzyme pyridoxal phosphate and its This includes inorganic salts such as potassium dihydrogen phosphate, which are used for low pH adjustment. Intermediate Ii using anthranilic acid! In the production method using fermentation, unreacted anthranilic acid, glucose, ammonium sulfate,
This includes fermentation liquid components such as sodium hydrogen phosphate, potassium dihydrogen phosphate, and manganese sulfate.

The amount of the non-polar non-volatile polymer resin used in step 1 varies depending on the concentration of tryptophan in the impurity-containing tryptophan aqueous solution to be passed, but it is usually preferably 5 to 100% of the tryptophan aqueous solution. is 5-30%
capacity is sufficient. After passing the tryptophan aqueous solution,
You can immediately elute the tritophane with aqueous ammonia, but since the bottom of the column is filled with non-polar high porous polymer resin, stop passing the liquid just before the volume of tryptophan leaks out, and add a small amount of pure water. It is preferable to pass water through it in order to separate tryptophan from impurity amino acids, organic salts, and inorganic salts.

The degree of ammonia water used to elute tryptophan in step 2 of the present invention is not particularly limited, and it is sufficient to use a sufficient concentration to obtain a tryptophan aqueous solution from a non-polar high porous polymer resin, but from 0.5N to Aqueous ammonia of about 5N is preferable.

Even when a large amount of indole is mixed into the reaction crude solution, which is the tryptophan aqueous solution used as the raw material in step 1,
Since indole is not eluted by such an ammonia aqueous solution, tryptophan can be obtained with high purity, which is extremely convenient.

Indole adsorbed to high porous polymer resin,
As it accumulates as it is used, methanol,
Elution and recovery can be performed using alcohols such as ethanol and impronol, a mixture of such alcohol and water, acetone, and the like.

The reverse osmosis membrane used to concentrate the tryptophan eluted from the non-polar high porous polymer resin used in step 2 is a normally used reverse osmosis membrane, even if it is a so-called tight reverse osmosis membrane with a high salt rejection rate. Even a so-called loose reverse osmosis membrane with a low salt rejection rate may be used as long as it can withstand use with aqueous ammonia at a predetermined concentration.

The concentration ratio by the reverse osmosis membrane may be appropriately selected depending on the reverse osmosis membrane used, the concentration of ammonia water, and the elution concentration from the non-polar high porous polymer resin. Further, the reverse osmosis membrane module format is not limited to any of the so-called snot-irral type, tubular type, and holofiber type.

The tryptophan solution concentrated by the reverse osmosis membrane is then subjected to simple distillation or a conventional crystallizer to remove ammonia by evaporation, thereby easily precipitating and recovering highly pure tryptophan crystals.

According to the present invention, tryptophan can be selectively separated and purified from coexisting inorganic salts, unreacted raw materials, etc., and moreover, it is possible to easily reduce energy consumption and recover crystalline tryptophan with high purity and good yield.

Examples Example 1 Indole and DL using tryptophan synthase
- Serine was reacted to obtain a crude reaction liquid having the following composition.

Tryptophan Log/lD-serine 6.211/lL-serine
1.11 i/l pyridoxal-5-phosphate 1 da/l sodium chloride
2 i / 6 tons of the above reaction crude solution was mixed with non-polar high porous polymer resin "Diaion HP-20J (
5V3H in a column packed with 2t (product name, manufactured by Mitsubishi Kasei)
The liquid was passed through at r-1. Subsequently, after passing 1 t of demineralized water,
25 tons of 2N ammonia water and 2 tons of demineralized water were passed through the column, and 2-7 tons of the tryptophan fraction was collected while detecting the composition of the outlet liquid with a refractometer at the bottom of the column. The tryptophan concentration of this tritophane fraction was 229/l, which was 42 times that of the original solution, the purity was 97%, and the recovery rate was approximately 100%.

Subsequently, after adjusting the ammonia concentration of the above eluate to 2N using concentrated ammonia water, a tubular reverse osmosis membrane 1'-
It was concentrated using 9077/14 using "PBILTL-198 Mini Module" (part name: manufactured by Teijin Engineering). (The concentration ratio by the reverse osmosis membrane was 41 times.) The permeate was 2N ammonia water and did not contain tryptophan. The concentrated water had an ammonia concentration of 2N and a tryptophan recovery rate of 97N. The obtained a-condensed liquid 640 go,
Simple distillation was carried out at 40° C. and 60 −Hg, and ammonia and water were removed by evaporation until the − of the residual liquid became 9 or less. Tryptophan crystals were crystallized, and then filtered through a filter to obtain 47.8 g of crystalline tryptophan. Ta. The crystal recovery rate was 80%, and white crystals had a purity of 99.5%. The evaporation i- of water was 94.71 per gram of triglyceride.

Example 2 6 tons of the reaction solution obtained in exactly the same reaction as in Example 1 was treated with a non-polar high porous polymer resin [Sepapeas 5p-soo J (trade name: manufactured by Mitsubishi Kasei) 2 in the same manner as in Example 1.
5V3Hr-1 solution was passed through the column packed with T. Subsequently, after passing demineralized water Xt, 2t of the permeate from the reverse osmosis membrane used in Example 1 was passed through, and further 2t of demineralized water was passed through. As in Example 1, 2.71 tryptophan fractions were collected. The tryptophan concentration of this tritophane fraction is
The concentration was 21.5 g/l, 215 times that of the original solution, the purity was 97%, and the recovery rate was 97%.

Subsequently, in exactly the same manner as in Example 1, the eluate was adjusted to 2N, concentrated to 90 g/l using a reverse osmosis membrane, and subjected to simple distillation and filtration to obtain white tryptophan crystals with a concentration of 45.7 g/l.
I got a 9. The recovery rate was 76% and the purity was 99.5%. The concentration ratio by reverse osmosis membrane was 4.2 times. The amount of water evaporated by simple distillation was 4.7 g per 1 g of tryptophan.

Example 3 Indole, sodium pyruvate, and ammonium chloride were reacted using tryptophanase to obtain a crude reaction solution having the following composition.

Tryptophan 10gZt pyruvate sodium pyruvate s g7t indole
z g7t, ammonium chloride
231i/L Sodium Chloride 2.
99/l pyridoxal-5-phosphate 111
19/ was passed through a column filled with 2 tons of non-polar non-ibolus resin "Diaion HP-20j (trade name, manufactured by Mitsubishi Kasei) at 5V3Hr".

Subsequently, 1 t of demineralized water was passed through it, and then 2 t of the permeate from the reverse osmosis membrane used in Example 2 was passed through it, and further 2 t of demineralized water was passed through it. As in Example 1, 2.71 tryptophan fractions were collected. The tryptophan concentration of this tryptophan fraction was 21.51/l, concentrated 2.15 times that of the original solution, and its purity was 98%, with a recovery rate of 98%.

Subsequently, the bath solution was adjusted to 2N in the same manner as in Example 1, concentrated to 90g7t using a reverse osmosis membrane, and subjected to simple distillation and filtration to obtain 46.8 I of white crystals of tryptophan.
! I got it. The recovery rate was 78% and the purity was 99.7%. The concentration ratio with the reverse osmosis membrane was 4.2 times, and the amount of water evaporated by simple distillation was 4.7 g per 1 g of tryptophan.

Comparative Example 6 tons of crude reaction liquid obtained in exactly the same reaction as Example 1,
In the same manner as in Example 1, a column packed with 2 t of non-polar non-irborous polymer resin "HP-20J" was heated at 5V3Hr.
The solution was passed at -1. Subsequently, 30 tons of demineralized water was passed through. As in Example 1, 22 tons of the tryptophan fraction was collected while detecting the composition of the outlet liquid using a refractometer at the bottom of the tower. The tryptophan concentration of this tryptophan fraction was 2.3 g/l, which was 4.3 times diluted with respect to the crude reaction solution. Furthermore, the recovery rate of tryptophan was 85%, and the purity was 96%.

This tryptophan fraction was distilled into 2. ．． Concentrate to 2L, and then heat the concentrated solution at 5°C.
The mixture was cooled to 100 mL, left as it was for 5 hours to crystallize tryptophan crystals, and then filtered. The recovery rate of the tryptophan crystals obtained was 651, and the purity was 97.5%. The amount of water required for concentration is 1 g of tryptophan (F)59
'#Met.

Claims (1)

[Claims] (1) In a method for separating and purifying tryptophan from an aqueous tryptophan solution containing impurities, ￥Step 1￥: The aqueous solution is passed through a non-polar highly porous polymer resin to adsorb tryptophan, and then adsorbed to the resin with aqueous ammonia. Step 2: Concentrate the eluate obtained in Step 1 using a reverse osmosis membrane. Step 3: Remove ammonia from the concentrated solution obtained in Step 2 and crystallize tryptophan. A method for purifying tryptophan characterized by adopting a process consisting of.