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WO1989002923A1 - Procede de preparation d'une solution aqueuse purifiee d'indole - Google Patents

Procede de preparation d'une solution aqueuse purifiee d'indole Download PDF

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Publication number
WO1989002923A1
WO1989002923A1 PCT/JP1988/000968 JP8800968W WO8902923A1 WO 1989002923 A1 WO1989002923 A1 WO 1989002923A1 JP 8800968 W JP8800968 W JP 8800968W WO 8902923 A1 WO8902923 A1 WO 8902923A1
Authority
WO
WIPO (PCT)
Prior art keywords
indole
organic solvent
reaction
phase
aqueous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1988/000968
Other languages
English (en)
Japanese (ja)
Inventor
Shinji Ogawa
Seiya Iguchi
Hiroshi Kimura
Hideharu Kuwamoto
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mitsui Toatsu Chemicals Inc
Original Assignee
Mitsui Toatsu Chemicals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to BR888807219A priority Critical patent/BR8807219A/pt
Application filed by Mitsui Toatsu Chemicals Inc filed Critical Mitsui Toatsu Chemicals Inc
Priority to DE3852098T priority patent/DE3852098T2/de
Priority to EP88908359A priority patent/EP0333877B1/fr
Priority to KR1019890700919A priority patent/KR920008598B1/ko
Priority to CA000594656A priority patent/CA1335970C/fr
Publication of WO1989002923A1 publication Critical patent/WO1989002923A1/fr
Priority to DK248789A priority patent/DK169947B1/da
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • an indole produced by a chemical synthesis method contains an impurity which inhibits the activity of the enzyme
  • the crude indole containing such an impurity is known.
  • 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.
  • the purified indole solution 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the partition ratio between the organic solvent phase and the aqueous phase 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.
  • 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.
  • 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.
  • an enzyme source that is, a chick enzyme, bacterial cells, and those obtained by immobilizing these on a suitable carrier may be present.
  • 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.
  • the stationary separation after the two-phase contact may be performed using the same device as for ordinary liquid-liquid extraction.
  • 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.
  • an aqueous solution of indole having a desired concentration can be quantitatively prepared.
  • 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 283 ⁇ 4 ammonia water) for 30 hours.
  • 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.
  • 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.
  • 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 ⁇ . Material 4 ⁇ / activity ratio
  • 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 / ⁇ . Calcium chloride dihydrate 1.5 also from I ⁇ , 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.
  • 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.
  • a perfume indole consisting of 99.9% by weight of indole and 0.05% by weight of 3-methylindole was used.
  • 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.
  • 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.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Indole Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Procédé de préparation d'une solution aqueuse d'indole essentiellement pure, consistant à dissoudre un indole cru contenant des impuretés organiques dans un solvant organique ne se mélangeant pas à l'eau, lesdites impuretés présentant un coefficient de séparation supérieur à celui que présente l'indole entre ledit solvant et l'eau, et à mettre la solution en contact avec de l'eau ou un milieu de réaction aqueux pour former deux phases liquides et transférer l'indole dans la phase aqueuse par séparation.
PCT/JP1988/000968 1987-09-25 1988-09-22 Procede de preparation d'une solution aqueuse purifiee d'indole Ceased WO1989002923A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
BR888807219A BR8807219A (pt) 1987-09-25 1988-09-11 Processo para preparacao de solucao de indol aquosa purificada
DE3852098T DE3852098T2 (de) 1987-09-25 1988-09-22 Verfahren zur herstellung einer gereinigten wässrigen indollösung.
EP88908359A EP0333877B1 (fr) 1987-09-25 1988-09-22 Procede de preparation d'une solution aqueuse purifiee d'indole
KR1019890700919A KR920008598B1 (ko) 1987-09-25 1988-09-22 정제된 인돌 수용액의 제조방법
CA000594656A CA1335970C (fr) 1987-09-25 1989-03-23 Procede d'obtention de solutions aqueuses d'indole purifiees
DK248789A DK169947B1 (da) 1987-09-25 1989-05-23 Fremgangsmåde til fremstilling af L-tryptophan samt til fremstilling af vandige indolopløsninger til brug herved

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP23900187 1987-09-25
JP62/239001 1987-09-25
CA000594656A CA1335970C (fr) 1987-09-25 1989-03-23 Procede d'obtention de solutions aqueuses d'indole purifiees

Publications (1)

Publication Number Publication Date
WO1989002923A1 true WO1989002923A1 (fr) 1989-04-06

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1988/000968 Ceased WO1989002923A1 (fr) 1987-09-25 1988-09-22 Procede de preparation d'une solution aqueuse purifiee d'indole

Country Status (5)

Country Link
AT (1) ATE113988T1 (fr)
AU (1) AU615232B2 (fr)
CA (1) CA1335970C (fr)
DE (1) DE3852098T2 (fr)
WO (1) WO1989002923A1 (fr)

Also Published As

Publication number Publication date
CA1335970C (fr) 1995-06-20
DE3852098D1 (de) 1994-12-15
AU2520688A (en) 1989-04-18
ATE113988T1 (de) 1994-11-15
DE3852098T2 (de) 1995-03-23
AU615232B2 (en) 1991-09-26

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