JPH0638782A - Production of indigo by enzymatic process - Google Patents

Abstract

PURPOSE:To produce indigo useful as an industrial dye on an industrial scale at a low cost by treating an indole-containing aqueous solution with cells (or its treated product) of an indigo-producing microbial strain belonging to the genus Pseudomonas in the presence of a nonionic surfactant. CONSTITUTION:An indigo-producing mirobial cell belonging to the genus Pseudomonas [e.g. Pseudomonas sp. MY-6 (FERM P-11963)] or its treated product is suspended in an aqueous solution containing indole. A nonionic surfactant [e.g. polyoxyethylene(10) octylphenyl ether] is added to the suspension in an amount of 0.5-10%(wt/vol) and the components are made to react with other at 30 deg.C for 10hr under aeration and agitation. The bacterial cells are removed e.g. by centrifugal separation, the obtained reaction liquid is extracted with a solvent such as chloroform and the solvent is removed from the extracted liquid. Indigo widely utilized as an industrial coating can be produced in high efficiency and yield by this process.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for efficiently producing indigo from indole by an enzymatic method.

[0002]

2. Description of the Related Art Conventionally, indigo is produced by a chemical synthesis method and is widely used as an industrial dye. However, the chemical synthesis method has drawbacks such that the reaction is multistage and the yield is poor, and there are many by-products due to chemical decomposition.

Further, among various methods for producing indigo, a method which is promising is a method for producing from indole by using a microorganism containing xylene oxygenase or naphthalene dioxygenase as an enzyme catalyst. Known (Burt D. Ensley, Barry J. Ratzkin, Timthy D.
Osslund and Mary J. Simon; Science, vol.222, P.167-16
9 (1983)). However, a practical manufacturing technique has not been established yet, and development of a method for efficiently manufacturing indigo is desired.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to produce indigo efficiently and in high yield by an enzymatic method.

[0005]

Means for Solving the Problems In order to establish a method for producing indigo by an enzymatic method using a bacterium having an indigo-forming ability or a treated product thereof, the present inventors have conducted intensive studies on reaction conditions and the like, It was found that the indigo-forming ability was caused to be significantly inhibited by indole in the reaction solution, and in order to solve this, improvement was made by adding a nonionic surfactant to the reaction solution and allowing it to act. I knew it would be done.

The present invention is a method for enzymatically producing indigo by allowing a bacterial cell belonging to the genus Pseudomonas having the ability to produce indigo or a treated product thereof to act on an aqueous solution containing indole to enzymatically produce indigo. It is a method for producing indigo by an enzymatic method, which comprises adding a surfactant to act.

The present invention will be described in detail below. In the present invention, strains belonging to the genus Pseudomonas and having the ability to generate indigo are used.
omonas) -sp. The MY-6 strain is preferably used. The mycological and taxonomic properties of this strain are as follows.

I. Microscopic properties (a) Cell shape and size: bacillus, 1 × 2-4 μm (b) Polymorphism: No (c) Motility: Yes (d) Flagella epidemic state: polar flagella (e ) Presence or absence of spores: None (f) Gram stain: Negative

II. Culture properties (a) Growth on broth agar: Yes (b) Assimilable carbon source: glucose, acetic acid, succinic acid,
Fumaric acid, L-malic acid, lactic acid, citric acid, glycerol, L-valine, β-alanine, DL-arginine

III. Growth conditions (a) Growth temperature: 10 to 40 ° C. (b) Growth pH: 6 to 8 (c) Oxygen requirement: Aerobic

IV. Physiological properties (a) Oxidase: positive (b) Catalase: positive (c) Content of guanine and cytosine (GC) in DNA: 61
% (D) Starch hydrolysis: Negative (e) Protocatechuic acid degradation: Positive

The above-mentioned various properties are described in "Bergey's Manual of Systematic Bacteriology (Bergey's
Manual of Systematic Bacteriology) "Volume 2 (198
6 years). As a result, this bacterium is Pseudomonas
(Pseudomonus) was identified as a strain belonging to the genus,
The species was considered to be a new species because there are some disagreements from the assimilation and other characteristics of the carbon source. Therefore, in the present invention, Pseudomonas sp. It will be called MY-6. In addition, this strain was sent to the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology, Micro Engineering Research Institute No. 11963 (FERM P
-11963).

As the carbon source of the medium used for culturing the microorganism, carbohydrates such as glucose, xylene, toluene and the like can be used, and among them, m-xylene and p-xylene are preferable.

As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate and ammonium phosphate; nitrates such as sodium nitrate, potassium nitrate and ammonium nitrate; ammonia and the like can be used.

As the inorganic substance, potassium phosphate, magnesium nitrate, iron, manganese, zinc or the like can be used. If necessary, nutritional sources such as vitamins and amino acids can be added.

The culture temperature is 20 ° C. to 50 ° C., preferably 30 ° C. to 40 ° C., and the pH of the medium during the culture is 6 to 9, preferably 7 to 8. Culture is performed aerobically.

When the cultured bacterial cells are used in the enzymatic reaction, the bacterial cells can be used as they are, but a treated product of the bacterial cells, for example, a crushed product obtained by crushing the bacterial cells by ultrasonication or the like, or The crushed product may be used in the form of an extract obtained by further extracting with water or the like, or a crudely purified product obtained by further treating the extract with ammonium sulfate or the like to precipitate the enzyme component. If necessary, the treated product may be immobilized and used.

The reaction of the cells or the treated product thereof with indole is carried out in the same manner as a usual enzymatic reaction, for example, 0.1 M.
Phosphate buffer (pH 6-9) or aqueous solution (pH 6-9)
In this, the reaction is carried out at a temperature of 20 to 50 ° C., preferably 30 to 40 ° C. for usually 10 to 72 hours. The reaction is preferably carried out with stirring.

Nonionic surfactants that can be added to the reaction solution include, for example, t-octylphenol,
Compounds obtained by polymerizing addition of ethylene oxide to alkylphenols such as isooctylphenol and nonylphenol (Triton type); Compounds obtained by polymerizing addition of ethylene oxide to higher fatty acid esters of polyhydric alcohols such as sorbitan (Tween type); Lauryl alcohol, stearyl alcohol, oleyl A compound obtained by polymerizing addition of ethylene oxide to a higher alcohol such as alcohol (Brij
System) and the like.

Further, preferred specific examples of the nonionic surfactant include polyoxyethylene (10) octyl phenyl ether (Triton X100) and polyoxyethylene (2
Examples thereof include 0) sorbitan monolaurate (Tween 20) and polyoxyethylene (20) sorbitan monooleate (Tween 80).

The concentration of the nonionic surfactant added to the reaction solution is not particularly limited, but is generally 0.5.
-10 (wt / vol) is used.

The amount of the microbial cells or the treated product thereof is not particularly limited, but is generally 0.5 to 10%.
(Wt / vol) is used. After the reaction, indigo can be separated and purified from the reaction solution by a method known per se, for example, a method such as solvent (chloroform) extraction.

[0023]

【Example】

(Reference _{Example) (NH 4) 2 SO 4} : 3g, KH 2 PO 4: 0.5g,
K ₂ HPO ₄ : 0.5 g, MgSO ₄ / 7H ₂ O: 0.
5 g, CaCl ₂ .2H ₂ O: 10 mg, NaCl: 0.
_{5g, FeSO 4 · 7H 2 O} : 10mg, yeast extract 1g
And distilled water: 100 ml of medium of 1000 ml (pH 7.0)
Was dispensed into a 500 ml Erlenmeyer flask and heated at 120 ° C for 15
After sterilizing for 1 minute, 1 ml of m-xylene was added, and Pseudomonas sp. The MY-6 strain was inoculated and cultured with shaking at 30 ° C. for 24 hours.

1000 ml of the same sterilized medium as described above was placed in a 5 liter Erlenmeyer flask and 10 ml of m-xylene was added.
After adding ml, inoculate 20 ml of the above shaking culture solution,
The cells were cultured at 0 ° C for 24 hours with shaking. 10 of the obtained culture solution
Centrifuge (800 rpm, 15 minutes, 4 ° C.) of 00 ml to collect the cells was used as an enzyme source.

(Example) Using the bacterial cells prepared by the above method,
Reaction solution (Indol 2 mM, 100 mM phosphate buffer, pH
7.0) After suspending in 1000 ml, put in a 3 liter aeration stirrer and add 1 (w) of the nonionic surfactant shown in Table 1.
/ v)% so that the mixture was reacted at 30 ° C. for 10 hours.

The amount of indigo produced was measured by the conventional method [H. KEIL,
CM SAINT and PA WILLIAMS, Journal of Bacteriol
ogy, 169, No. 2, P. 764-770 (1987)].
The result is shown as a relative value with 100 when the surfactant was not added.

[0027]

[Table 1]

[0028]

According to the method of the present invention, the enzymatic method is used.
Indigo can be produced efficiently and in high yield.

 ─────────────────────────────────────────────────── ─── Continued front page (72) Inventor Hisashi Yamagata 48-2 Fujimiya 2-102 Tamiyacho Station Ushiku City, Ibaraki Prefecture (72) Inventor Hideaki Yukawa 6-23-9 Chuo, Ami Town, Inashiki-gun, Ibaraki Prefecture

Claims (1)

[Claims] 1. A method for enzymatically producing indigo by allowing a bacterial cell belonging to the genus Pseudomonas having the ability to produce indigo or a treated product thereof to act on an aqueous solution containing indole to produce a nonionic surfactant in the reaction solution. A method for producing indigo by an enzymatic method, which comprises adding an agent to act.