DE3852098T2 - METHOD FOR PRODUCING A CLEANED AQUEOUS INDOLUTION. - 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

The present invention relates to a process for purifying indole. In a narrower sense, the invention relates to a process for producing a substantially purified aqueous indole solution, whereby the indole can be reacted from crude indole with organic impurities contained therein.

A number of methods are known to synthesize useful compounds by an enzymatic reaction using indole as an enzyme substrate. A typical example of such a method is the synthesis of L-tryptophan from indole and L-serine using tryptophan synthase. This method is already used on an industrial scale.

Generally, indole is obtained by a chemical synthesis process or by fractional distillation of coal tar, and the indole produced by such a process usually contains substituted indoles such as ethylindole and other aromatic compounds as impurities. Some of these impurities lead to inhibition or even complete inactivation of the enzyme if they are present in the reaction mixture. In order for the enzyme reaction to proceed smoothly, such impurities should therefore not be present in the reaction mixture and should not accumulate in the reaction mixture.

On the other hand, it must be noted that water serves as an ideal solvent for the majority of enzymes used in enzyme reactions. Therefore, in most cases the enzyme reaction is carried out in an aqueous solution. In addition, since most enzymes have a high activity at a temperature of 15ºC to 50ºC, enzyme reactions are usually carried out in this temperature range.

Indole, however, is insoluble in water and its solubility in water at room temperature is only 3 to 4 g/l. For this reason, in the case of the enzyme reaction in which indole is used from the starting substance, it is not easy to prepare an aqueous indole solution and, in particular, to make it available in large quantities.

Conventional methods for preparing an aqueous indole solution include a procedure in which solid indole powder is dissolved in water or in an aqueous solution in which indole is already contained by vigorous stirring for the purpose of preparing an aqueous indole solution, the undissolved indole being collected by solid-liquid separation, and a procedure in which the indole solution is prepared by dissolving indole in a water-miscible solvent such as lower aliphatic alcohol, e.g. methanol, and then mixing the indole solution with an aqueous indole solution.

By means of these methods, a sufficient amount of indole concentration is ensured in the aqueous solution. However, when industrially produced crude indole is used, the impurities contained therein are also dissolved in the aqueous solution and fed into the enzyme reaction. Thus, when crude indole is used according to the above-mentioned conventional methods, no purification of the indole is carried out, so that it is not recommended for use in the enzyme reaction. Furthermore, in cases where a water-miscible solvent is used, the solvent per se denatures, inhibits or inactivates the enzyme, so that the use of such a solvent should be avoided.

As an enzyme reaction using indole as a substrate, a method has been proposed in which an organic solvent is used for the reaction.

For example, Japanese Patent Publication No. 45593/81 discloses carrying out an enzyme reaction in which the substrate of the enzyme is almost insoluble in the enzyme-containing solution and in which an organic solvent is present which is not miscible with water but is miscible with the substrate. However, the function of the organic solvent in this process is to ensure substantially the same concentration of the substrate as its saturation concentration in the aqueous phase in which the reaction is carried out by the enzyme.

In Japanese Patent Disclosure (Kokai) No. 11187/84 it is stated that in an enzyme reaction in which at least one substrate hinders the enzyme activity, an organic solvent which is not miscible with water but is miscible with the substrate is added to reduce the substrate concentration in the aqueous phase to a level reduce the enzyme activity without inhibiting it. The document in question discloses a process for the enzymatic synthesis of L-tryptophan using indole as a substrate.

Furthermore, Bang et al disclose a method for the synthesis of L-tryptophan from indole and L- or DL-serine using E. coli cells, in which indole is dissolved in a water-immiscible organic solvent and the indole solution serves as an indole source in the enzyme reaction [Biotechnology and Bioengineering, Vol. XXV (1983), pp. 999 to 1011].

The above processes, which use a water-immiscible solvent, are effective methods when a substance such as indole, which is essentially insoluble in water, serves as a substrate for an enzyme. However, these processes do not deal in any way with the impurities contaminating the indole and they assume that pure indole is used. Therefore, the organic solvent in these processes only has the function of taking up the indole and introducing it at a certain concentration into the aqueous phase when an enzyme reaction is carried out.

However, in cases where the starting indole is crude indole with its organic impurities, the organic solvent should be carefully selected. The present inventors have found that o-ethylaniline or 3-methylindole, which may be contained in the crude indole, at a concentration of 200 ppm inhibits the enzyme tryptophan synthase, which synthesizes tryptophan from indole and L-serine, and that 2-ethylindole inhibits the enzyme even at a concentration as low as about 20 ppm. Some other impurities inhibit the enzyme when present at a high concentration of 1000 ppm or more.

Even if the water-immiscible organic solvent absorbs indole and ensures the introduction of indole into the aqueous phase at a constant concentration, the impurities coexist in the enzyme reaction system and inhibit the enzyme if the organic impurities are distributed in the aqueous phase to the same extent as the indole. And even if an organic solvent is used is used in which the distribution ratio of impurities is slightly lower than that of indole, the impurities accumulate in the reaction system because the substrate indole is consumed in the enzyme reaction, while the impurities are not normally consumed in the reaction. Therefore, if an organic solvent is used in which the distribution ratio of impurities in the aqueous phase is not sufficiently low, it is not easy to prevent the inhibition of the enzyme reaction or the contamination of the product of the enzyme reaction, resulting in difficulties in smoothly conducting the enzyme reaction.

However, no knowledge or consideration has been given in the literature to date to use a water-immiscible organic solvent as a countermeasure to solve the problem of contaminating impurities. However, although indole can be purified by rectification or recrystallization, the majority of the impurities contained are substituted derivatives of indole with similar physiological properties to indole, so that the purity of indole obtained by a purification process such as rectification is limited. Among the purification processes, recrystallization is a suitable method to obtain indole of high purity. However, the yield of indole is low and the process involves the problem that the impurities accumulate in the filtrate. In addition, the cost of carrying out recrystallization is high, so that a purification process without recrystallization is required for industrial-scale production.

An object of the present invention is to provide a process for preparing a substantially purified aqueous indole solution from crude indole containing organic impurities, which indole solution can be used for an enzymatic or other reaction in which indole serves as the starting material.

According to the present invention, there is provided a cleaning method comprising:

Dissolving crude indole containing an organic impurity in a water-immiscible organic solvent;

contacting the organic indole solution with water or an aqueous reaction medium to cause a reaction with the indole to produce a two-phase liquid system and to distribute indole in the aqueous phase, wherein the distribution ratio of the impurity within the organic phase is greater than that of the indole in forming the two-phase liquid system.

The inventors have discovered that impurities which limit the enzyme activity are contained in chemically synthesized crude indole. In the process of the invention, in contrast to the general prior art in which indole is isolated by means of the above-mentioned complicated purification process and fed to the reaction, crude indole is used as the starting material and a purified aqueous indole solution is prepared by a simple process so that purified indole can be introduced into an aqueous reaction system containing an enzyme for reacting with the indole.

Conventionally, for an enzyme reaction in which indole serves as a substrate of the enzyme, it was necessary to first produce purified indole by rectification or recrystallization and prepare a purified aqueous indole solution in order to be able to feed a purified indole solution into the reaction system. In contrast, with the method of the invention, not only can a purified aqueous indole solution be produced from industrially produced crude indole of low purity by using crude indole in its unaltered form as a starting substance, but also an aqueous indole solution having a desired indole concentration can be prepared in a simple manner and in large quantities.

For this reason, the process of the invention is of great importance for industrial purposes because an enzyme reaction can be carried out using crude indole, which is produced by chemical synthesis or in a similar manner, as a starting substance.

Although the process of the invention is extremely advantageous when applied to a reaction such as an enzyme reaction in which contamination by organic impurities is a problem, it should be noted that the process of the invention is not limited to such an enzyme reaction but can also be applied to other reactions for the production of dyes or various other substances in which indole is used as the starting substance.

The indole to be used for the process according to the invention includes, for example, that which is chemically synthesized by the reaction of anilines and ethyl glycols in the presence of various catalysts, that which is produced by the catalytic reaction of aniline ethanol and o-nitrotoluene or similar substances and by the dehydrogenation reaction of indoline, and that which is obtained by the fractional distillation of coal tar. The impurities in the crude indole prepared by these processes include, for example, substituted indoles such as 2-methylindole, 3-methylindole, N-methylindole, 2-ethylindole, 3-ethylindole and N-ethylindole, cyclic compounds such as 2-methylquinoline and N-phenylpyrrole, substituted anilines and derivatives such as aniline, o-ethylaniline, N-ethylaniline and acetaldehyde anil, o-toluidine and aniline ethanol.

The organic solvent for dissolving the crude indole containing such impurities is one which is immiscible with water, which forms a two-phase system when mixed with water and can dissolve the indole. In order that the organic solvent does not hinder the enzyme activity, those whose solubility in water is very low are preferable, regardless of whether it is an aliphatic or an aromatic solvent. Preferred examples of the organic solvents include saturated aliphatic hydrocarbons such as pentane, n-hexane, heptane, isooctane and nonane, cycloaliphatic hydrocarbons such as cyclohexane and methylcyclohexane, aromatic hydrocarbons such as benzene, toluene and xylene, esters such as butyl acetate, butyl citrate and ethyl citrate, and ketones such as methyl isobutyl ketone and diisobutyl ketone. Aliphatic solvents are preferable to aromatic solvents because they show less denaturation and inhibition of the enzyme.

In order to obtain a substantially purified aqueous indole solution, an organic solvent is also selected to which the impurities have a different affinity than that of the indole. This means that an organic solvent with a distribution ratio of the impurities within the organic phase which is larger than that of indole when the two-phase system is formed from the organic solvent and an aqueous medium. As already mentioned, the organic impurities in crude indole produced industrially by chemical synthesis or similar methods have a different melting point or boiling point than indole. In order to widely differentiate the distribution ratio of the impurities and indole by utilizing these differences, an organic solvent is selected which has at least a high solubility of the impurities. Therefore, solvents which have a solubility parameter of 7 to 9.5 at the reaction temperature are preferably selected. Depending on the organic impurities contained in the crude indole, an even more favorable organic solvent can be selected. Thus, in cases where the crude indole contains impurities such as 2-methylindole and 3-methylindole, both of which have a melting point and a boiling point higher than indole, solvents having a solubility parameter (SP) of about 8, such as methylcyclohexane and cyclohexane, are preferred; while in cases where the crude indole contains impurities such as 2-ethylindole and 3-ethylindole, which have a melting point lower than indole, solvents having a SP of about 7, such as n-hexane and isooctane, provide a satisfactory cleaning effect. A suitable solvent can be determined for the actual industrial process to be carried out by taking into account the influence of the solvent on the enzyme reaction, its handleability and similar issues.

The purified aqueous indole solution can be prepared by dissolving the crude indole in the organic solvent described above and then contacting the organic indole solution with water. This may be pure water without any ingredients, but the water may also contain all or part of the other substrates and/or inorganic salts that may be required to carry out the enzyme reaction. Furthermore, if the use of the organic solvent does not affect the activity of the enzyme, an enzyme source may be present in the water, i.e. an unbound enzyme or bacterial cells that produce the enzyme, as well as enzymes immobilized in a carrier substance.

The crude indole is dissolved in the organic solvent described above and the resulting solution is brought into contact with water and mixed or an aqueous solution containing a substrate or similar materials required for the reaction. Due to the difference in the partition coefficient of indole and the organic impurities between the organic and aqueous phases, almost all of the impurities are retained in the organic layer and are not transferred to the aqueous phase, so that the aqueous phase becomes a substantially purified aqueous indole solution containing substantially no impurities. In addition, if necessary, the mixed solution can be fed to the enzyme reaction after the organic phase has been separated from the aqueous phase by allowing the mixture to stand.

There are no restrictions on the method by which the organic solution phase and the aqueous phase are brought into contact and mixed. Devices such as stirred tanks or static mixers, which are otherwise used in liquid-liquid extraction, can be used. In order to accelerate the exchange rate between the organic and aqueous phases, devices in which the liquid-liquid interface is large are preferred. If necessary, the separation of the phases by settling can also be carried out in a conventional liquid-liquid extractor.

Another characteristic feature of the present invention is that aqueous indole solution having a desired indole concentration can be prepared in large quantities by appropriately selecting the concentration of crude indole dissolved in the organic solvent, the ratio of the organic phase to the aqueous phase, the temperature and the duration of contact when the organic solution is brought into contact with the aqueous medium and mixed.

Of course, additional crude indole may be introduced into the organic solvent phase after the settling separation to regulate the indole concentration, and then the organic solvent may be again subjected to contact and mixing with the aqueous medium. When the organic solvent is repeatedly used in the above-mentioned manner, organic impurities accumulate in the organic solvent, and if necessary, the indole contained in the organic solvent may be recovered by distillation or recrystallization, and the collected indole may be reused to prepare the purified aqueous indole solution according to the present invention.

In the following, the present invention is described in more detail using examples.

example 1

Ten grams of crude indole in powder form with a purity of 98.5 mass%, containing as impurities 2-methylindole, 3-methylindole, N-ethylindole and aniline, was dissolved in 50 ml of toluene (with a SP value of 8.9). The resulting solution and 50 ml of distilled water were added to a separatory funnel and the resulting mixture was shaken at 30°C for 10 minutes. The mixture was then allowed to stand to separate the organic and aqueous phases. The concentrations of indole and impurities in the aqueous phase were measured. The concentration of indole was 2.0 g/L and the percentage of indole to all organic components in the aqueous phase was 99.9 mass%. The same crude indole was dissolved in 50 ml of tert-butyl alcohol (with SP value of 11.0) in the same amount and the same operations were carried out. The result was that the indole concentration in the aqueous phase was 5.9 g/L and the percentage of indole in the aqueous phase was 98.9 mass%.

Example 2

In 500 ml of n-hexane (with an SP value of 7.3) was dissolved 16 g of crude indole with a purity of 97.5% by mass and the same impurities as in Example 1, and the resulting solution was brought into contact with 500 ml of an aqueous solution containing 7.5 g of L-serine and mixed. The resulting mixture was separated by settling. The indole concentration in the resulting aqueous phase was 1.9 g/L, and the ratio of indole to the impurities derived from indole was 99.8% by mass to 0.2% by mass. The concentration of L-serine did not change either before or after the operation. Furthermore, the concentration of n-hexane in the aqueous phase was 10 ppm.

Example 3

A given amount of 3-methylindole with a purity of 99.9 mass% was mixed with 15 g of indole used for perfume making, consisting of 99.95 mass% indole and 0.05 mass% 3-methylindole, to prepare indole with different purities. The indole thus prepared was dissolved in 90 ml of n-hexane, and the solution was contacted and mixed with 210 ml of an aqueous solution containing 2.8 g of L-serine, 1.0 g of glycine, 0.1 g of calcium chloride dihydrate and 2 mg of pyridoxal-5'-phosphate and its pH was adjusted to 8.5 with aqueous ammonia. The organic phase and the aqueous phase were separated by settling and the concentrations of indole and 3-methylindole were analyzed by high performance liquid chromatography. The mass ratio of indole to 3-methylindole in the n-hexane solution before contact with the aqueous medium and the ratio in the aqueous phase after contact are shown in Table 1.

Table 1

Organic phase before contact

Indole 3-methylindole

98.5 mass% 1.5 mass%

94.0 mass% 6.0 mass%

90.0 mass% 10.0 mass%

80.0 mass% 20.0 mass%

Aqueous phase after contact

Indole 3-methylindole

99.7 mass% 0.3 mass%

98.5 mass% 1.5 mass%

97.6 mass% 2.4 mass%

94.5 mass% 5.5 mass%

Comparison example 1

Escherichia coli MT-1 0232 (FERM BP-19), which is a tryptophan synthase-producing bacterium, was inoculated into 100 ml of the culture medium contained in a 500 ml flask having the composition shown in Table 2 and incubated at 35ºC for 24 hours. Two hundred milliliters (in two flasks) of the culture medium was inoculated into 15 ml of the culture medium having the composition shown in Table 3 contained in a 30 L fermenter and incubated at 35ºC and pH 6.8 (adjusted with 28% aqueous ammonia) for 30 hours.

After cultivation, the culture medium was centrifuged to collect 600 g of wet mass of bacterial cells. The cells were placed in a sealed container, stored in a refrigerator at 4ºC and used to prepare the source of immobilized enzymes.

Table 2

Ehrlich's meat extract 10 g

Polypeptone 10 g

NaCl 5g

dissolved in 1 l distilled water (PH value 6.8)

Table 3: Composition of the culture medium

Glucose 10g

(NH₄)₂SO₄ 1.5g

K₂HPO₄ 1 g

MgSO₄-7H₂O 1 g

Polypeptone 0.5 g

Yeast extract 0.5 g

L-tryptophan 0.15g

surfactant (Adecanol LG-805, manufactured by Asahi Denka Co., Ltd.) 5 g

dissolved in 1 l distilled water (pH 6.8)

One part of the above wet bacterial cells and one part of isotonic sodium chloride solution were mixed by stirring. In another case, 7.76 parts of distilled water and 0.24 parts of sodium alginate (NSPLL manufactured by Kibun Food Chemifa, Co., Ltd.) were mixed by stirring while adjusting the pH to 8.5 using sodium hydroxide.

Two parts of the cell suspension and eight parts of the sodium alginate solution were mixed by stirring, the resulting mixture was filled into an injection burette and dripped into a gelling solution from a needle tip with an inner diameter of 0.5 mm to 0.8 mm.

The gelling solution was 0.5 M aqueous calcium chloride dihydrate solution, adjusted to pH 8.5 with 6 N potassium hydroxide solution, stored at 10ºC and used in the amount of 10 parts.

The particles formed by instilling the mixture into the gelling solution were matured with stirring to produce a source of immobilized enzymes.

Each of the aqueous solutions with a volume of 100 ml containing the amounts of aniline, N-ethylaniline, o-ethylaniline, o-toluidine, 3-methylindole shown in Table 4 or 2-ethylindole, 0.15 g of calcium chloride dihydrate and 1 mg of pyridoxal-5'-phosphate were added and the pH of the solutions was adjusted to 8.5 with aqueous ammonia.

Into these aqueous solutions were added 5 g of the above-mentioned source of immobilized enzymes containing the tryptophan synthase-producing bacteria, and the mixtures were left to stand at 30°C. In addition, as a control solution, 100 ml of distilled water without organic ingredients was subjected to the same procedure as above, and the source of immobilized enzymes was added thereto in the manner described, after which the whole was left to stand at 30°C.

After 24 and 96 hours, 1 g of the source of immobilized enzymes was taken from each of the solutions and the enzyme activity was measured. The ratio of the activity of the enzyme left in the organic solution to the activity of the enzyme in the control solution was determined and is shown in Table 4.

The enzyme activity was measured as follows: Ten milliliters of the reaction medium containing indole, L-serine, pyridoxal-5'-phosphate (PLP) and 0.3 g of the source of immobilized enzymes were prepared and the reaction was carried out at 35ºC for 1 hour in a shaking incubator with shaking. The L-tryptophan produced was analyzed by high performance liquid chromatography. Table 4 Organic impurity Concentration ppm Activity ratio Aniline N-Ethylaniline o-Ethylaniline o-Toluidine 3-Methylindole 2-Ethylindole

Example 4

In a continuous stirred tank containing a source of immobilized enzymes, an enzyme reaction was carried out using crude indole and purified indole solution and the half-life of the enzyme activity was compared. An aqueous solution containing 10.0 g/L L-serine, 1.5 g/L calcium chloride dihydrate and 10 mg/L pyridoxal-5'-phosphate, adjusted to pH 8.5 with aqueous ammonia (hereinafter referred to as solution A) was added to the reaction. Three 500 ml glass reactors with a stirrer were each charged with 100 ml of solution A described above and 30 g of the source of immobilized enzymes containing the tryptophan synthase producing cells. The reactors were kept in warm water to maintain the temperature at 35°C.

In the first reactor, solution A was fed at a rate of 49 ml per hour. Besides, a crude indole containing 94.0 mass% of indole and 6.0 mass% of 3-methylindole was dissolved in an aqueous methanol solution of 70 vol.% to obtain an aqueous solution having an indole concentration of 75 g/l and a 3- Methylindole concentration of 4.79 g/L. This solution was continuously added to the reactor at a rate of 1 ml per hour. In addition, the reaction mixture was withdrawn from the reactor at a rate of 50 ml per hour to carry out continuous synthesis of L-tryptophan. At fixed times, an aliquot of the reaction mixture was taken and the concentration of 3-methylindole and L-tryptophan was determined by high performance liquid chromatography. 24 hours after the start of the addition of the indole solution, the concentration of 3-methylindole was 96 mg/L. The yield of L-tryptophan given in a unit time with respect to the indole fed was measured. The time from the start of the reaction to the reduction of the yield to 50%, that is, the half-life of the enzyme activity, was 615 hours.

In addition, crude indole having the same composition was dissolved in n-hexane and brought into contact with solution A and mixed as in Example 3. After separation by settling, a purified aqueous indole solution (solution B) was obtained. Solution B was fed into the second reactor at a rate of 50 ml per hour and the reaction mixture was continuously withdrawn at the same rate. The concentration of 3-methylindole in the reaction mixture was 19 mg/L and the half-life of enzyme activity was 960 hours.

Furthermore, in the third reactor, a continuous enzyme reaction was carried out in the same way as in the first reactor, with the only difference that indole was used, which is used for perfume production and consists of 99.95% by mass of indole and 0.05% by mass of 3-methylindole. The concentration of 3-methylindole in the reaction mixture was 0.7 mg/l and the half-life of the enzyme activity was 1070 hours.

The amount of indole fed to the individual reactors was uniformly 75 mg per 1 hour.

The process according to the invention can be applied to enzyme reactions or to reactions in which no enzyme is used, for the production of tryptophan, dyes and various other substances.

Claims (8)

1. Cleaning process comprising: Dissolving crude indole containing an organic impurity in an organic solvent that is not miscible with water, Contacting the organic indole solution with water or an aqueous reaction medium to react with the indole to form a two-phase liquid system and to distribute the indole in the aqueous phase, wherein the distribution ratio of the impurity within the organic phase in forming the two-phase liquid system is greater than that of indole. 2. The process of claim 1, wherein the aqueous reaction medium is capable of carrying out an enzyme reaction and contains an enzyme and/or a substrate. 3. A process according to claim 1 or 2, wherein the organic impurity is aniline, N-ethylaniline, o-ethylaniline, o-toluidine, 3-methylindole or 2-ethylindole. 4. Process according to any preceding claim, in which the organic indole solution is brought into contact with an aqueous reaction medium to carry out a reaction with the indole and in which, after formation of the two-phase liquid system, the reaction is carried out, the solubility parameter of the organic solvent at the temperature of this reaction being 7 to 9.5. 5. A process for producing tryptophan comprising the steps of: Dissolving crude indole containing an organic impurity in an organic solvent that is not miscible with water, contacting the organic indole solution with an aqueous reaction medium containing L-serine and tryptophan synthase to form a two-phase liquid system and distribute indole in the aqueous phase, and Carrying out the reaction to produce tryptophan in the aqueous phase of the two-part liquid system, wherein the distribution ratio of the impurity within the organic phase in the formation of the two-phase liquid system is greater than that of indole. 6. The process of claim 5, wherein the organic impurity is aniline, N-ethylaniline, o-ethylaniline, o-toluidine, 3-methylindole or 2-methylindole. 7. A process according to claim 5 or 6, wherein the solubility parameter of the organic solvent at the temperature of the reaction for producing tryptophan is 7 to 9.5. 8. A process according to any preceding claim, wherein the organic solvent is n-hexane.