Process of preparing purified aqueous indole solution

Details Process of preparing purified aqueous indole solution Process of preparing purified aqueous indole solution

1989-07-21

1992-02-04

Ceperley, Mary E.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing alpha or beta amino acid or substituted amino acid...

548469, 548497, C12P 1322, C07D20908

Patent

active

050859919

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

This invention relates to a process of preparing purified aqueous indole solution. More particularly, this invention relates to a process of preparing substantially purified aqueous indole solution from crude indole containing an organic impurities.
A number of methods are known for synthesizing useful compounds by an enzyme reaction employing indole as substrate of the enzyme. A typical example of such a method is a process of synthesizing L-tryptophan from indole and L-serine using tryptophan synthase, and this process is now industrially practiced.
In general, indole is produced by a chemical synthesis process or by fractional distillation of coal tar, and indole produced by such a process usually contains substituted indoles such as ethylindole and other aromatic compounds as impurities. Some of these impurities inhibit or even completely deactivate the enzyme if they exist in the reaction mixture. Therefore, in order to conduct the enzyme reaction fluently, these impurities should not exist in the reaction mixture and should not be accumulated in the reaction mixture.
On the other hand, for most of the enzymes which are used in enzyme reactions, water acts as an ideal solvent. Therefore, in most cases, the enzyme reaction is conducted in aqueous solution. Further, since most enzymes exhibit high activity at a temperature of 15.degree. C. to 50.degree. C., enzyme reactions are usually conducted in this temperature range.
However, indole is insoluble in water and its solubility in water at room temperature is only 3-4 g/l. Therefore, in an enzyme reaction in which indole is used as a starting material, it is not easy to prepare and supply aqueous indole solution, especially quantitatively.
Conventional methods of preparing aqueous indole solution include a method in which solid indole powder is dissolved in water or in aqueous solution which already contains indole by vigorous agitation to prepare aqueous indole solution, and the undissolved indole is recovered by separating solid from liquid, and a method in which indole solution is prepared by dissolving indole in a solvent which is miscible with water such as lower aliphatic alcohol, e.g., methanol, and the indole solution is then mixed with aqueous indole solution.
By these methods, the quantitativeness of the indole concentration in the aqueous solution is assured However, when industrially produced crude indole is used, the impurities contained therein are also dissolved in the aqueous solution and are supplied to the enzyme reaction. Thus, when crude indole is used in the above-mentioned conventional methods, purification of the indole is not performed, so that it is not preferred for use in the enzyme reaction. Further, in cases where a solvent miscible with water is used, the solvent per se denatures, inhibits or deactivates the enzyme, so that the use of such a solvent should be avoided.
As an enzyme reaction in which indole is a substrate, a method using an organic solvent in the reaction has been proposed.
That is, Japanese Patent Publication No. 45593/81 discloses to conduct an enzyme reaction in which the substrate of the enzyme is almost insoluble in the enzyme-containing solution, in the presence of an organic solvent which is immiscible with water but miscible with the substrate. However, in this method, the function of the organic solvent is to assure the substrate in substantially the same concentration as the saturated concentration thereof in the aqueous phase in which the reaction by enzyme is conducted.
Japanese Patent Disclosure (Kokai) No. 11187/84 discloses that in an enzyme reaction in which at least one substrate inhibits the enzyme activity, an organic solvent which is immiscible with water but miscible with the substrate is added so as to reduce the substrate concentration in the aqueous phase to a level at which the enzyme activity is not inhibited. In this reference, a method of enzymatically synthesize L-tryptophan using indole as a substrate is disclosed.
Further, Bang et al disclose a metho

REFERENCES:
patent: 3926726 (1975-12-01), Antonini et al.
Won-Gi Bang et al., "Production of L-Tryptophan by Escherichia coli Cells", Biotechnology and Bioengineering, vol. XXV, pp. 999-1011, (1983).
English Language Abstract of Japanese patent 59-11187, published Jan. 20, 1984.

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