WO1989002923A1 - Process for preparing purified aqueous indole solution - Google Patents

Abstract

A process for preparing a substantially purified aqueous indole solution, which comprises dissolving crude indole containing organic impurities in a water-immiscible organic solvent wherein said impurities have a larger partition coefficient than said indole has between said solvent and water, and bringing the solution into contact with water or an aqueous reaction medium to form two liquid phases and transfer the indole to the aqueous phase by partition.

Description

 Saying Moon Itoda »

 Preparation method of purified indole aqueous solution [Technical field]

 The present invention relates to a method for preparing a purified aqueous solution of indole, and more particularly, to a method using indole as a raw material, a crude oil containing organic impurities for use in the reaction. The present invention relates to a method for obtaining a substantially purified aqueous solution of indole from indole.

 [Background Art]

 There are many known methods for synthesizing useful compounds by enzymatic reaction using indole as one substrate. For example, a method of synthesizing L-tributphan from indol and L-selin by tributyne synthase is a typical example. This is a typical example, and has already been realized as an industrial manufacturing method.

 In general, indole is produced by a chemical synthesis method, a fractionation method from coal tar, etc., but a substituted product such as ethylindole or other aromatic compound is used. Is contained as an impurity. Some of these impurities, when present in the enzyme reaction solution, not only inhibit the activity of certain enzymes but also cause inactivation. Absent. Therefore, in order to carry out the enzymatic reaction smoothly, these impurities should be prevented from accumulating in the reaction solution as well as in the enzyme reaction solution. There is a need .

—On the other hand, for many enzymes used in enzymatic reactions, Water acts as an ideal solvent. For this reason, enzymatic reactions are often performed in aqueous solutions. Also, many enzymes exhibit high activity near room temperature between 15 ° C and 50 ° C, so reactions are often performed at temperatures in this range.

 However, indole is poorly soluble in water, and its solubility near room temperature is at most 3 to 4 I £. Therefore, in an enzymatic reaction using indole as a raw material, it is not easy to prepare an indole aqueous solution, particularly to prepare a quantitative indole aqueous solution and subject it to the reaction. This is the situation.

 Until now, the method of preparing an indole aqueous solution has been to add indole in the form of solid powder to water or an aqueous solution containing and already dissolving indole, and dissolve by forced stirring and mixing. For this purpose, undissolved indole is collected by solid-liquid separation to obtain an aqueous solution of indole, or a solvent miscible with water, for example, methyl alcohol A method of dissolving indole in an aliphatic lower alcohol such as that described above to prepare a solution and mixing the resulting solution with an aqueous solution containing the indole has been carried out.

According to these methods, the quantitativeness of the indole concentration in the aqueous solution is ensured. However, if crude indole, which is industrially manufactured and available, is used, impurities contained therein will be dissolved together in the indole aqueous solution and the enzymatic reaction will occur. Will be supplied as is- That is, when crude indole is used, the above method does not purify the indole, which is not preferable for performing the enzymatic reaction. In addition, when a solvent that is miscible with water is used, the solvent itself often causes denaturation, activity inhibition, or inactivation of the enzyme, and its use should be restricted. .

 Conventionally, a method has been proposed in which an organic solvent is used in a reaction system as an enzymatic reaction using indole as one of reaction substrates.

 That is, in Japanese Patent Publication No. 56-45593, when performing an enzyme reaction in which the substrate in the enzyme reaction is almost insoluble in the enzyme-containing solution, the substrate is immiscible with water, A method for performing the reaction in the presence of a miscible organic solvent has been proposed. However, in this method, the function of the organic solvent is to ensure that the substrate is at a constant concentration in the aqueous phase where enzymatic conversion occurs, substantially equal to its saturated concentration. .

 Also, Japanese Patent Application Laid-Open No. Sho 59-111187 discloses that in a reaction method in which at least one kind of substrate deteriorates the activity of an enzyme, an organic compound that is not miscible with water but is miscible with a substrate is used. It describes a reaction method using an enzyme characterized in that the reaction is carried out by using a solvent to reduce the substrate concentration in the aqueous phase to substantially less than the activity inhibition concentration of the enzyme. In this method, particularly, a method relating to a method of synthesizing L-tributyphan by enzymatic reaction using indole as one of the substrates is disclosed.

In addition, Nong et al. For the production of L-tributane from L- and L- or DL-Serine, dissolve indol in a water-insoluble organic solvent, A method has been described for the use as a source of biosynthesis (Biotechnology and Bioengineering. Vol. XXV 999-1011, 1983).

 Performing enzymatic reactions using organic solvents that are immiscible with water is effective when substances that are almost insoluble in water, such as indole, are used as substrates. It is a way. However, none of these proposed methods takes into account any impurities contained in the indole, and targets substantially pure indole. It is what it is. Therefore, the role of the organic solvent in these methods is to store the indole and carry out the indole while maintaining a certain concentration in the aqueous phase when performing the enzymatic reaction. To supply.

However, when the raw indole for conducting the enzymatic reaction as described above is a crude indole containing organic impurities, care must be taken in selecting an organic solvent to be used in the reaction. It costs. According to the findings of the present inventors, for example, an enzyme, tributafuncinase, which synthesizes L-tributaphan from indole and L-serine O-Ethylaniline and 3-methylethyl in concentrations of about 200 ppra, and 2-ethylethyl indole up to 20 ppra. Inhibits even at about ppm. If other impurities are present at high concentrations of 1000 ppm or more, Some may inhibit.

 Therefore, even if an organic solvent that is immiscible with water can store the indole and maintain a constant indole concentration in the aqueous phase, it can also remove organic impurities at the same time. If the same distribution as indole is made in the aqueous phase, the impurities will coexist in the enzyme reaction system and will inhibit the enzyme. In addition, even if the organic solvent has a distribution ratio somewhat lower than that of indole, the substrate indole is consumed by the enzymatic reaction, while the impurity is not consumed. As a result, the accumulation of impurities results in inhibition of the enzyme. As described above, if an organic solvent is used in which the distribution ratio of impurities in the crude indole to the aqueous phase is not sufficiently small, inhibition of the enzymatic reaction and contamination of the enzymatic reaction product may occur. It is not easy to avoid, and it becomes more difficult to carry out the enzyme reaction smoothly.

However, the above-mentioned prior art using an organic solvent immiscible with water does not disclose any knowledge or method concerning the selection of an organic solvent from such a viewpoint. Simply using these methods does not allow the enzymatic reaction to proceed smoothly when using crude indole. For this reason, a method of purifying and isolating a crude indole, which is a raw material, and using this as a raw material can be considered. However, if an attempt is made to purify indole based on such an idea, a rectification operation or a recrystallization operation is applied as a method, but many impurities are contained. The physical properties such as the boiling point of the substituted product of indole are similar to those of indole. There are practical limits to improving the purity of indole in crops. Above all, purification by recrystallization is a method suitable for obtaining high-purity indole, but the recovery rate of indole is low, and the purification in the crystallization filtrate is difficult. There is a problem with the accumulation of impurities, etc., and the cost involved in purification is generally high. Therefore, in reality, there is a long-awaited need for a method of purification without going through recrystallization.

 [Disclosure of the Invention]

 An object of the present invention is to provide a method for preparing a substantially purified aqueous solution of indole that can be subjected to a reaction from crude indole containing organic impurities in a reaction using indole as a raw material. It is to provide

 Another object of the present invention is to provide, in an enzymatic reaction using indole as a raw material, a purified indole aqueous solution from which impurities that inhibit the reaction have been removed, in a reaction system of an aqueous medium containing the enzyme. An object of the present invention is to provide a method for industrially performing an enzyme reaction.

The purpose of the present application is to convert a crude indole containing an organic impurity into an organic solvent phase which is an organic solvent immiscible with water and between the organic solvent phase and the aqueous phase. After previously dissolving in an organic solvent having a distribution ratio larger than the distribution ratio of the indole to the organic solvent phase, the solution is brought into contact with water or an aqueous reaction medium for carrying out the reaction. A method for preparing a substantially purified aqueous solution of indole, which is characterized by forming a liquid phase and distributing and transferring the indole to an aqueous phase. Achieved by providing the law.

 According to the present invention, it is known that an indole produced by a chemical synthesis method contains an impurity which inhibits the activity of the enzyme, and the crude indole containing such an impurity is known. Instead of isolating the crude oil by the complicated purification method as described above and subjecting it to the reaction, a crude indole is used as a reaction raw material, and a reaction system containing an enzyme includes: A purified indole aqueous solution is prepared by a simple method and used as a purified indole to carry out the reaction.

 Conventionally, in an enzymatic reaction using indole as one substrate, in order to obtain a purified indole aqueous solution to be used for the reaction, the purified indole solution must be purified in advance by a rectification operation or a recrystallization operation. Obtained an indole and used it to prepare an aqueous solution. However, according to the method of the present invention, a relatively low-purity crude indole which is industrially available without using these purification operations can be used as a reaction raw material as it is. Not only can the enzyme reaction system be supplied as a high-purity aqueous solution of purified indole that does not inhibit enzyme activity and that is substantially free of impurities, and can easily produce an aqueous solution of a desired concentration. And it can be prepared quantitatively.

 That is, the method of the present invention is useful as a method for carrying out an enzymatic reaction using crude indole produced by a chemical synthesis method or the like as a raw material, and its industrial utility value. Is expensive.

It should be noted that the method of the present invention is not limited to organic reactions It is most effective when applied to a reaction in which a mixture of pure substances is particularly problematic, but it is not necessarily limited to enzymatic reactions, and dyes and other various substances derived from indole It can also be applied to reactions for producing

 [Best mode for carrying out the invention]

 The crude indole used in the present invention can be produced by a well-known method in this field, for example, a method for synthesizing anilines and ethylenic alcohols in the presence of various catalysts. Chemical synthesis from aniline ethanol and O-ditoluene by a catalytic reaction, or a synthesis by indolin dehydration reaction. The obtained indole and those obtained by the coal tar fractionation method are exemplified. Organic impurities contained in the purified indole obtained by these methods include, for example, 2—methylindole, 3—methylindole, N—methylindole, 2—Ethylindole, 3—Ethylindole, N—Indole-substituted product such as ethylindole, 2—Methylquinoline, N—Cyclic compound such as phenylvirol, a O-Ethylaniline, O-Ethylaniline, N-Ethylaniline and aniline-substituted derivatives and derivatives, such as acetoaldehyde, o-Triluidine, anilino Ethanol and the like can be mentioned.

The organic solvent that dissolves crude indole containing these organic impurities is one that forms two liquid phases without being mixed with water, and dissolves indole. In order not to interfere with the enzymatic reaction, solvents such as aliphatic or aromatic Preferred are those that have very low solubility in water, and include aliphatic saturated hydrocarbons such as benzene, n-hexane, heptane, isooctane, and nonane. Alicyclic hydrocarbons such as benzene, cyclohexane and methylcyclohexane; aromatic hydrocarbons such as benzene, toluene and xylene; butyl acetate; Esters such as butyl citrate and ethyl citrate and ketones such as methylisobutylketone and diisobutylketone are preferred. Among them, aliphatic solvents are more preferable than aromatic solvents because they have less denaturation and inhibition of enzymes when dissolved in a small amount in the enzyme reaction solution.

Further, in order to obtain a substantially purified aqueous solution of indole, an organic solvent having a difference in affinity between the indole and the organic impurity for the solvent is selected. That is, an organic solvent in which the distribution ratio of organic impurities between the organic solvent phase and the aqueous phase to the organic solvent phase is larger than that of the indole to the organic solvent phase is selected. Organic impurities contained in crude indole produced industrially by chemical synthesis and the like have a slight difference in melting point and boiling point from indole as described above. In order for the partition ratio between the organic solvent phase and the aqueous phase to be significantly different from that of indole, it must be at least an organic solvent with high solubility of the impurities. Solvents having a solubility parameter value of 7 to 9.5 at the temperature at which the reaction is carried out are more preferred. Also, depending on the organic impurities contained in the crude indole, The appropriate solvent can be selected. In other words, for the separation of impurities having a higher melting point and boiling point than indole, such as 2-methylindole and 3-methylindole, the solubility parameter ( A methylcyclohexane having an SP value of about 8 is more preferable, and a cyclohexane is more preferable.

For impurities with a lower melting point than indole, such as 2-ethylethyl or 3-ethylindole, n-hexanedioctane with an SP value of about 7 A sufficient purification effect can be obtained. A preferred solvent may be selected in an industrially implemented process such as whether or not the enzyme reaction to be applied is affected or the solvent itself is easily handled.

 Purification int for the reaction: To prepare an aqueous solution, the crude indole can be dissolved in the organic solvent described above and then contacted with water. The water to be used is not limited to water, but may be an aqueous solution containing all or a part of a substrate other than indole, an inorganic salt, and the like necessary for performing an enzyme reaction. You may. In addition, if there is no problem with the inhibition of the enzyme by the organic solvent, an enzyme source, that is, a chick enzyme, bacterial cells, and those obtained by immobilizing these on a suitable carrier may be present.

Dissolve the crude indole in the organic solvent as described above, and thoroughly contact and mix with water or an aqueous solution containing substances necessary for the reaction such as a substrate. Almost all impurities between the indole and the organic impurities remain in the organic solvent layer due to the difference in the partition coefficient between the solvent and water, and the aqueous phase The water phase is obtained as a substantially purified aqueous solution of indole from which the impurities have been removed, since it hardly moves. If necessary, the above-mentioned mixed solution may be subjected to an enzymatic reaction after being statically separated.

 The method for bringing the organic solvent phase and the aqueous phase into contact with each other is not limited, and a device usually used for liquid-liquid extraction such as a stirring tank and a static mixer may be used, and the moving speed between the two phases may be used. A device having a large liquid-liquid interface area is preferable in order to increase the liquid content. If necessary, the stationary separation after the two-phase contact may be performed using the same device as for ordinary liquid-liquid extraction.

 In addition, when the organic solvent phase and the aqueous phase are brought into contact, the concentration of the crude indole dissolved in the solvent, the volume ratio of both phases, the contact temperature, and the contact time are arbitrarily combined. Thus, one of the features of the present invention is that an aqueous solution of indole having a desired concentration can be quantitatively prepared.

 Also, it is not possible to adjust the concentration by adding new crude indole to the organic solvent phase after standing and separating, and then use it again for preparing the purified aqueous solution of indole. Needless to say. By repeating such reuse, organic impurities accumulate in the solvent phase, but if necessary, distillation and recrystallization may be performed on this to obtain the The recovered indole can be used again for the preparation of a purified indole aqueous solution according to the method disclosed in the present invention, as long as the recovered indole is recovered.

Hereinafter, the present invention will be described in more detail with reference to Examples, but it should be understood that the present invention is not limited to these Examples. Needless to say.

Example 1

2—Methylindole, 3—Methylindole, N—Ethylindole and 10 g of crude indole powder containing 98.5% by weight of impurities containing aniline as impurities. Le e emissions (SP value 8.9) 5 0 because Shi was dissolved in ral, this is the after distilled water 5 O ml were mixed shaken for 10 minutes at introduced 3 0 ^e C to a separatory b over Bok, It was allowed to stand and separated. Analysis of the concentrations of indole and various impurities in the aqueous phase showed that the indole concentration was 2.0! The proportion of indole in the total organic components in the aqueous phase was 99.9% by weight. The same crude indole was dissolved in 50 ml of tert-butyl alcohol (SP value 11-0) with the same weight in 50 ml, and the same operation was carried out. As a result, the indole concentration in the aqueous phase was 5.9. The ratio of g / β and indole was 98.9% by weight.

Example 2

16 g of crude indole containing the same impurities as in Example 1 and having a purity of 97.5% by weight is dissolved in 500 ml of n-hexane (SP value: 7.3) and contains 7.5 g of L-serine The mixture was contact-mixed with 500 ml of an aqueous solution at 30 and then allowed to stand still. The resulting aqueous phase had a concentration of 1 · 9 in the concentration of I 、, indole and impurities derived from indole. The ratio was 99.8% by weight to 0.2% by weight. Before and after this operation, no change was observed in the concentration of L-serine in the aqueous phase. The n-hexane concentration in the aqueous phase was 10 ppm. Example 3

Indole 99.95% by weight, 3—Methyl indole 15% perfume indole consisting of 0.05% by weight of perfume indole and 99.9% by weight of 3—methyl indole in a prescribed amount After mixing and adjusting to various purities, dissolve in 9 O ml of n-hexane, 2.8 g of L-serine, 1.0 g of glycin, and calcium chloride dihydrate 0.1 g, containing 2 rag of pyridoxal-5'-phosphate, contact-mixed with 210 ral of aqueous solution adjusted to pH 8.5 with ammonia water, and the two liquid phases were allowed to stand and separated The concentrations of indole and 3 -methylindole in the aqueous phase were analyzed by high-performance liquid chromatography. Before contact, the weight ratio of n — hexane dissolved in hexane to 3 — methylindole, and the indole and 3 — methylin in the aqueous phase after contact Table 1 shows the comparison with the weight ratio of the doles.

Table 1

Organic phase before contact Aqueous phase after contact Indole 3-Methylindole Indole 3-Methylindole

98.5 wt% 1.5 wt% 99.7 wt% 0.3 wt% 94.0 wt% 6.0 wt% 98.5 wt% 1.5 wt% 90.0 wt% 10.0 wt% 97.6 wt% 2.4 wt% 80.0 wt% 20.0 wt% 94.5 wt% 5.5 wt%

Reference example 1

 Table 2 shows Escherichia coli MT-10232 (FERM BP-19), a fungus producing fungus, in a 500 ml Sakaguchi flask. 100 ml of the medium having the composition was inoculated and cultured at 35 ° C for 24 hours. 200 ml of this culture (2 flasks) was inoculated into 15 £ of a medium having the composition shown in Table 3 in a 30 £ jar fermenter, and incubated at 35 ° C and pH6. .8 (adjusted with 28¾ ammonia water) for 30 hours.

After completion of the culture, the culture was collected by centrifugation to obtain 600 g of wet cells. Put Re this in a sealed container, and stored in a refrigerator at 4 ^e C, was used in making the immobilized enzyme source.

 Table 2 Ale Riu Beef Extract 10 s Polyton 10 S

NaCl 5 S

Dilute to 1 £ with distilled water and use (ρΗ6.8)

Table 3 Composition of growth medium

Glucose 1 0 S

(NH ₄ ) ₂ S (1.5 g

K _Z HP0 ₄ 1 S

MgSO ^ · 7Η ₂ 0 1 S port re-peptidic t 0. 5 g yeast Ekisu 0. 5 g

L Tributane 0.15 g Surfactant (ADEKA NOL LG-805) 5 S

 (Made by Asahi Denka)

 Diluted to 1 £ with distilled water (使用 6.8)

One part of the wet cells and 1 part of a physiological saline solution were mixed with stirring. Separately, 7.76 parts of distilled water and 0.24 parts of sodium alginate (NSPLL, manufactured by Ekibun Food Chemifa) were stirred and mixed to adjust the pH to 8-5 with potassium hydroxide. 2 parts of the bacterial cell suspension and 8 parts of the above-mentioned sodium alginate solution are mixed with stirring, filled into a syringe, and dripped into the gelling solution from the tip of a syringe needle having an inner diameter of about 0.5 to 0.8 mm. I

For the gelling solution, adjust the pH of a 0.5 molar aqueous solution of calcium chloride dihydrate to 8.5 with a 6N aqueous solution of potassium hydroxide, and use 10 parts of the solution kept at 10 ° C. Was. The particles formed by dropping into the gelling solution are allowed The mixture was aged while stirring to obtain an immobilized enzyme.

 Table 4 for each of aniline, N—ethylaniline, o—ethylaniline, o—triluijin, 3—methyliline dolele, and 2—ethylilaindol. Prepare 100 ml of each of the aqueous solutions with the concentrations shown, add 0.15 g of calcium chloride dihydrate and 1 mg of pyridoxal-5'-phosphate to each, and add ammonia water. The pH was adjusted to 8.5.

 To these aqueous solutions, 5 g of an immobilized enzyme source containing the Trib-to-Fan synthase-producing bacterium prepared by the method described above was inserted, and left at 30 ° C while maintaining the temperature. . The same adjustment was performed in 100 ml of distilled water containing no organic matter as a reference solution, and the immobilized enzyme source was similarly inserted and kept at 30 ° C.

 24 hours and 96 hours after standing, 1 g of each immobilized enzyme source was removed from each solution, the activity of the enzyme was measured, and the immobilized enzyme was left in each organic aqueous solution. The ratio of the activity of the enzyme source to the activity when left in the reference solution was determined and is shown in Table 4.

 Enzyme activity was measured as follows.

A reaction solution containing indole, L-serine, pyridoxal-5'-phosphoric acid (PLP) and 0.3 g of immobilized enzyme source is mixed, and the mixture is shaken in a shaking incubator. The reaction was carried out by shaking at ° C for 1 hour, and the resulting L-tributerfan was analyzed by high-performance liquid chromatography. Table 4 Machines! , Silk ΨΏ. Material 4ί / activity ratio

 ppm After 24 hours After 96 hours

Π Q Q

 Aniline 200 U. O O 丄 1, n U 丄 1

1000 1.00 U. O

ェ -ethylamine 200 1.04

 1000 1.00 n D n

 U. O

0-ethylamine 200 0.90 n 7

1000 0.71 n c n

 O C

 0-Toluidine 200 0.97 1.06

 1000 0.80

 3-methylene 200 0.94 0.89

 2-ethyl ethyl 20 0.93 0.71

 50 0.73 0.54

200 0.68 0, 48 Example 4 An enzymatic reaction using a crude indole and a crude indole aqueous solution was carried out in a continuous flow stirring reactor in which the immobilized enzyme source was suspended, and the enzyme activity was reduced by half. Comparison of the periods was performed. L-Serine 10.0 g / Ά. Calcium chloride dihydrate 1.5 also from IΆ, pyridoxal-5'-phosphate 10 mg / £ Then, a solution adjusted to pH 8.5 with ammonia water was prepared and used as a reaction feed solution. Prepare three glass reactors with a stirrer with an internal volume of 500 ml, and in each reactor, supply 100 nil of the reaction supply solution and a tributary filter prepared by the same method as in Reference Example 1. After inserting 30 g of the immobilized enzyme source containing the insynthase-producing bacteria, the reactor was kept in a warm water bath and kept at a temperature of 35 ° C.

The first reactor was continuously supplied with the reaction feed at a rate of 49 ml / hour. Simultaneously, a crude indole consisting of 94.0% by weight of indole and 6.0% by weight of 3-methylindole was dissolved in 70% by volume aqueous solution of methanol to obtain an indole concentration. An aqueous solution having a concentration of 75 g / pound and a concentration of 4.79 g / pound of 3-methylindole was prepared, and the reactor was continuously supplied at a rate of 1 ml / h. Further, a continuous synthesis reaction of L-tributtophan was performed in the reactor by draining the solution at a rate of 5 Oml / hour. Samples were collected from the reaction solution at regular intervals, and the concentrations of 3-methylindole and L-tributophan were analyzed by high-performance liquid chromatography. . After 24 hours from the start of the supply of the indole solution, the concentration of 3-methylindole showed a value of 96 mg / · β. When the yield of L-tributyphan produced was determined based on the amount of indole supplied to the reactor per unit time, the yield was 50%. The time from the start of the reaction, ie, the half-life of the enzyme activity was 615 hours. On the other hand, a crude indole having the same composition as above was dissolved in n-hexane in the same manner as in Example 3, contacted with the reaction feed solution and mixed, and then allowed to stand still for purification. A reaction feed solution B containing the obtained indole was obtained. The reaction supply liquid B was continuously supplied to the second reactor at a rate of 50 ml / h, and continuously discharged at the same flow rate. At this time, the concentration of 3-methylindole in the reaction solution showed a value of 19 mg / £. The half-life of the enzyme activity was 960 hours.

 In addition, in the third reactor, a perfume indole consisting of 99.9% by weight of indole and 0.05% by weight of 3-methylindole was used. When the enzymatic reaction was carried out continuously in the same manner as in the first reactor, the concentration of 3-methylindole in the reaction product was 0.7 mg /, and the enzyme activity was reduced by half. The period was 1070 hours.

 The amount of indole supplied to the first to third reactors per hour was 75 mg, which was the same.

 [Industrial applicability]

 The method of the present invention can be applied to enzymatic reactions for producing various substances using indole as a raw material, for example, tributane, dyes and the like, and reactions not using enzymes. Wear.

Claims

The scope of the claims  1. The crude indole containing organic impurities is separated from the organic solvent phase, which is an immiscible organic solvent and the organic solvent phase and the aqueous phase, with the organic solvent phase. Is dissolved in an organic solvent larger than the partition ratio of the indole to the organic solvent phase, and then the solution is brought into contact with water or an aqueous reaction medium for carrying out the reaction to form a two-liquid phase. A method for preparing a substantially purified aqueous solution of indole, characterized in that indole is obtained by partitioning and transferring indol to an aqueous phase.  2. The method according to claim 1, wherein the aqueous reaction medium is for performing an enzymatic reaction, and contains an enzyme and / or a substrate.  3. The method according to claim 1, wherein the organic impurity is aniline, N-ethylaniline, o-ethylaniline, o-trisolidine, 3-methylindole, or 2-ethylindole. Item 1 or 2.  4. The method according to claim 1, wherein the solubility parameter of the organic solvent at the reaction temperature is 7 to 9.5. 5. Distributing the crude indole containing organic impurities to an organic solvent phase which is an organic solvent immiscible with water and between the organic solvent phase and the aqueous phase. Contains L-serine and tributafincinase after pre-dissolving in an organic solvent larger than the partition ratio of indole to the organic solvent phase Substantially characterized in that it is brought into contact with an aqueous solution to form two liquid phases, and the indole is supplied to the enzyme reaction system. For preparing a highly purified aqueous solution of indole.  6. The method according to claim 5, wherein the organic impurity is aniline, N-ethylaniline, o-ethylaniline, 0-toluidine, 3-methylindole or 2-ethylethylindole. .  7. The method according to claim 5, wherein the solubility parameter of the organic solvent at a temperature at which the reaction is carried out is 7 to 9.5.  8. The method according to claim 5, wherein the organic solvent is n-hexane.