JPS61234789A - Recovery of indole from triptophane - Google Patents

Abstract

PURPOSE:A triptophane reaction mixture containing indole is passed through a layer of cation-exchange resin and the indole adsorbed is eluted with an aqueous organic solvent to enable easy isolation of high-purity indole. CONSTITUTION:Indole is allowed to accumulate in the porous part by passing unreacting indole in the aqueous medium through an ion exchange resin and by repeating the operations. When the concentration reaches a certain level, an eluent is passed through to give an indole solution of high concentration. Then, the solution is subjected to concentration and crystallization to isolate indole.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for separating and recovering indole from tryptophan obtained by an enzymatic method using indole as a raw material.

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

Methods for producing L-tryptophan include a fermentation method using glucose as a raw material, a semi-fermentation method using anthranilic acid as a raw material, and an enzymatic condensation reaction with L-serine using indole as a raw material. how to get ribtophane,
Alternatively, a method is known in which L-tributophane is obtained by allowing enzymes to act on indole, pyruvic acid, and ammonia.

Problems to be Solved by the Prior Art and the Invention The problem with the method of obtaining tryptophan from indole as a raw material is that indole emits a characteristic bad odor even in small amounts, so it is necessary to reduce the content in the final product to at least 10 ppm. There is a need to.

However, since both indole and tryptophan have indole skeletons and their chemical structures are similar, indole has a strong adhesion to tryptophan, and cannot be removed to an acceptable amount by means such as final crystal cleaning.

Furthermore, indole is a raw material with a high unit price, and if it cannot be recovered, costs will increase.

Therefore, in the production of tryptophan using indole, it is necessary to separate unreacted indole and tryptophan after the reaction is completed, and also to separate indole and tryptophan when recovering tryptophan from the product p liquid in the purification process. The method must be able to completely remove impurities and at the same time efficiently separate and recover indole and tryptophan.

Normally, tryptophan is produced by an enzymatic method, in which indole and serine, for example, are reacted in a neutral aqueous medium in the presence of enzymes and microbial cells that have the action of tryptophan synthase. It is necessary to remove the bacteria and enzymes contained in the reaction solution, and to remove these bacteria and enzymes, adsorption and separation on solid materials such as activated carbon and adsorbent silica gel is performed in accordance with the usual amino acid treatment method. Methods such as sedimentation using a centrifugal separator, separation and purification using ion exchange resins or non-polar porous resins have been adopted.

However, separation of indole and tryptophan is not possible with these methods.

Indole and tryptophan are poorly soluble in water. For example, if you try to dilute the slurry after the reaction with a large amount of water during the purification process to make tryptophan into an aqueous solution, and then use activated carbon treatment to adsorb and remove unreacted indole. A large amount of tryptophan is also adsorbed along with indole, and its recovery is impossible. At this time, it is also possible to adsorb only tryptophan on an ion exchange resin, bring indole into contact with an organic solvent that is immiscible with water, such as benzene or toluene, and extract it to the organic solvent side. is required, and the solvent recovery operation becomes complicated.

Means for Solving the Problems The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, have arrived at the method of the present invention.

That is, the method of the present invention is a method for separating and recovering unreacted indole from tryptophan obtained by enzymatic action using indole as a raw material, in which a tryptophan reaction solution containing indole is passed through a porous cation exchange resin. After repeated adsorption, elution, and regeneration of tryptophan in the resin ion exchange section, indole adsorbed on the resin porous section is eluted using a water-containing organic solvent that mixes with water, and is constantly removed from the eluent. This is a method for separating and recovering indole from 1-tributophane, which is characterized by isolating indole according to a method.

In the present invention, unreacted indole in the aqueous medium after one reaction is removed by tryptophan separation operations such as tryptophan passing through a porous Sumiki ion exchange resin layer, adsorption, elution, and regeneration in the tryptophan ion exchange section. During repeated treatments, it is adsorbed and accumulated in the porous parts of the resin, so when a certain concentration is reached, a highly concentrated indole solution can be obtained by passing the indole eluent through and eluting it. If this is separated according to conventional methods such as concentration and crystallization, high purity indole can be easily isolated at low cost in the separation process. Moreover, it was found that the tryptophan eluent treated with the porous cation exchange resin contained almost no indole.

The tryptophan reaction solution used in the present invention is
For example, a reaction solution obtained by enzymatically condensing indole and L-serine in the presence of tryptophan synthase in an aqueous medium, or a reaction solution obtained by reacting indole with pyruvic acid and ammonia in the presence of tryptophanase. , even those that have been subjected to pretreatment such as removal of bacterial protein are acceptable. In addition, if the purpose is to recover the active ingredients L-)-ributophane and indole, a liquid discharged during the purification process, even if it is not a reaction liquid,
For example, it may be applied to furnace liquid, etc.

It is preferable to remove solid substances that may cause clogging of the resin tower from liquids containing active ingredients such as reaction liquids and furnace liquids before they are actually used.

In the method of the present invention, the ion exchange resin used is:
Any porous cation exchange resin may be used, but for example, Lewa t it t3p-1
12), Amberlite 20Q
C) Segroporous type of strongly acidic cation exchange resin such as Dtai□n pK-220 is preferable; gel type of strongly acidic cation exchange resin cannot be used in the method of the present invention. The amount of resin used depends on the amount to be treated. Make sure that the total amount of thione in the solution, that is, the total molar equivalent of contaminants such as amino acids, ammonium ions, and sodium ions contained in normal reaction solutions in addition to L-tryptophan, is within the total exchange capacity of the resin. .

It is preferable to use the resin by filling it in a column.

The PR of the liquid to be treated during liquid passing is preferably neutral to acidic, and the temperature is 90° C. or lower, and the liquid passing is performed at SV=1 to 10.

After passing through the resin, water is passed through the resin to thoroughly wash the resin, and then tryptophan is eluted according to a conventional method. Aqueous ammonia is preferable as the eluent, and when eluting with aqueous ammonia, 5 to 3 o wt % aqueous ammonia is eluted from the top of the column at 5V = 1 to 10, or the water in the column is circulated to the top of the column. Any method such as blowing gaseous ammonia may be used, and the pH inside the column should be at least 10.
It is better to set it to the above. Although L-)-lyptophan is almost completely eluted by this operation, it is preferable to further extrude with water in order to recover the residual content in the column.

In this way, 95% of the tryptophan normally contained in the liquid to be treated is recovered into the treatment liquid. In addition, less than 5% of indole contained in the liquid to be treated leaks into the treatment liquid, but L-
The treatment liquid containing tryptophan is obtained by known isolation methods, i.e., unit operations such as concentration, crystallization, solid-liquid separation, and drying, to obtain an odorless, high-quality L-) containing indole below the allowable level.
- Liptophan can be isolated.

After elution of tryptophan, the resin is thoroughly washed with water, and the resin is regenerated with hydrochloric acid, sulfuric acid, etc. in a conventional manner and used repeatedly.

As described above, tryptophan adsorption, elution, and resin regeneration operations are repeated, but in the present invention, there is a large difference in the concentration ratio of tryptophan and indole contained in the liquid to be treated, and the adsorption mechanism is different. Elution of the adsorbed indole must be carried out when the adsorption capacity of indole is almost reached after repeating the tryptophan separation process. Otherwise the subsequent eluent from India-
It will be difficult. Therefore, indole is usually eluted after adsorption, elution, and regeneration of tryptophan are repeated at least five times, depending on the content ratio of indole.

A water-containing organic solvent is used as the eluent. Organic solvents alone have a poor affinity ratio and cannot perform complete elution. Preferred organic solvents are lower aliphatic alcohols, such as methanol, ethanol, n-propatool, and isopropatool, with isopropatool being particularly preferred. Adjust these to an alcohol aqueous solution containing 10% or more, preferably 70% or more, and elute by passing the solution 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 indole eluate is concentrated and the solvent is recovered in accordance with a conventional method, followed by back extraction with a non-aqueous organic solvent such as toluene or hexane, followed by liquid separation followed by cooling crystallization to form a solid.

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 examples are shown below, and the numbers are weight %.

Reference Example Activated carbon and water were added to a reaction solution containing L-tryptophan obtained by condensing indole and L-serine in an aqueous medium in the presence of tryptophan synthase, an enzyme produced by culturing E. coli. PH4 is 95-10
After heating at 0° C. for 1 hour to flocculate the bacterial cells, the bacterial cells adsorbed on the activated carbon were directly removed by filtration. To 3.8% (0.19M) of L-tryptophan obtained by pretreatment in this way, 0.1% (0.1%) of indole, 0.2% (0.02M) of L-serine, and 0.0% (0.02M) of ammonium ion. 8
% (0,44M), sodium ion 0.2qb (0
An aqueous solution containing zsso, 9 (amino acid and l) was reconditioned and passed from the top at 5 V = 5. The distillate at the end of the passing was analyzed by high performance liquid chromatography to determine L - Detection of tryptophan was performed, but no tryptophan was detected.

Further, after washing with water 21.5 V = 10, 10% NH3 water 50
Elute with 0J, 5V=2, and extrude with 1000I of water, eluent 16oo! I got i+.

The eluent contained L-tryptophan 5.9t16 (94
, IF equivalent, recovery rate of 96.0% with respect to I, -tryptophan in the adsorption stock solution), and indole ippm or less (indole removal rate was almost 100%).

The eluate was concentrated under reduced pressure to about 630 g, 200 J of isopropyl alcohol was added, crystallization was carried out at 5°C for 2 hours, and after vacuum passing through a Nutsche filter, 30% isopropyl alcohol water (lOO!) was added. ! Wash it with wet, cake 106.61! This was dried to obtain 66.7N of white crystalline crystals.

NH (transmittance at 430 nm was measured as a 2% J aqueous solution), etc., could satisfy the Japanese Pharmacopoeia.

In addition, the resin after L-tryptophan elution was further reduced to 21
After washing with water, 80% isobrobyl alcohol water 200%
Of! i+ to flow out from the top of the column at 5V=2 to obtain 200ON of indole eluent.

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

Example 1 In the same manner as in Reference Example, only adsorption, elution, and regeneration of L-tryptophan were repeated 23 times without elution of m-indole. During this time, the distilled solution at the end of the flow was checked for detection of L-tryptophan using high-performance liquid chromatography, and after the detection of L-tryptophan was confirmed, the flow of the reaction solution was stopped and
(l) was flowed from the top of the column at 5V=2 to obtain an indole eluate of 2ooo,p.The eluent contained 3.2% indole.After concentrating the eluate to 5009, After mixing and extraction by adding 200 I of n-hexane at °C and separating the layers, the n-hexane layer was cooled to 5 °C/2 H.
The resulting wet cake was dried to obtain indole 39.01. (Recovery rate 61
．． The recovered indole was reused as a raw material for the L-tryptophan enzymatic reaction, but it was confirmed that it had no effect on the reaction at all.

Claims (1)

[Claims] 1. In a method for separating and recovering unreacted indole from tryptophan obtained by enzymatic action using indole as a raw material, a tryptophan reaction solution containing indole is passed through a porous cation exchange resin layer. adsorption and elution of tryptophan in the ion exchange part of the resin.
After repeated regeneration treatments, the indole adsorbed on the porous part of the resin is eluted using a water-containing organic solvent that mixes with water, and the indole is isolated from the eluent. Separation and recovery method. 2. The method according to claim 1, wherein the water-containing organic solvent to be mixed with water is an aqueous solution of a lower aliphatic alcohol.