JPH05153972A - Cultivation of microorganism belonging to genus pseudomonas and capable of forming indigo - Google Patents

Abstract

PURPOSE:To improve the yield of microbial cell in high efficiency and yield by cultivating an indigo-producing microbial strain belonging to genus Pseudomonas in a medium containing fumaric acid, etc. CONSTITUTION:An indigo-producing microbial strain belonging to genus Pseudomonas is cultivated in a medium containing fumaric acid or its salt, succinic acid or its salt or citric acid or its salt and having a weight ratio of nitrogen of nitrogen source to carbon of carbon source of 2-30.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing a microorganism belonging to the genus Pseudomonas having the ability to produce indigo,
More specifically, it relates to a culturing method for improving the indigo-forming ability and the cell yield of the microorganism.

[0002]

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

Among various methods for producing indigo, a method that 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, in order to industrially use the enzyme, it is necessary to obtain the enzyme-containing microorganism in a high yield without lowering the enzyme activity, and it is practical to use the microorganism having indigo-forming ability. It is desired to develop a new culture method.

It has been conventionally known that xylene, toluene or m-methylbenzyl alcohol is used as a main carbon source when culturing a microorganism belonging to the genus Pseudomonas having an indigo-producing ability (N .Mermod et
al, Bio / technology, vol.4, P.321 (1986)), xylene and toluene have a problem that they affect the indigo forming ability of microorganisms and the cell yield.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a culture method capable of improving the cell yield of a microorganism having an excellent indigo-forming ability.

[0007]

Means for Solving the Problems The present invention is a culture in which a microorganism belonging to the genus Pseudomonas having an indigo-producing ability is cultured in a medium containing fumaric acid or a salt thereof, succinic acid or a salt thereof, or citric acid or a salt thereof. Is the way. Furthermore, the present invention provides a ratio of the nitrogen weight of the nitrogen source to the carbon weight of the main carbon source added to the medium (hereinafter, C /
It is a culture method of culturing in a medium such that the N ratio) becomes 2 to 30.

The present invention will be described in detail below. INDUSTRIAL APPLICABILITY The culture method of the present invention can be used for microorganisms belonging to the genus Pseudomonas having the ability to generate indigo, and in particular, Pseudomonas 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: 10-40 ° C. (b) Growth pH: 6-8 (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 various properties are described in "Bergey's Manual of Systematic Bacteriology".
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 strain because it did not match due to the assimilation and other properties of the carbon source. Therefore, in the present invention, Pseudomonas sp. It will be referred to as MY-6. In addition, this strain was assigned to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology by the microorganism deposit number
No. (FERMP-11963).

In the present invention, fumaric acid or a salt thereof, succinic acid or a salt thereof, or citric acid or a salt thereof is used as a main carbon source. The carbon source concentration in the culture is 0.1 to 5
(W / V)%, preferably 0.5 to 3 (W / V)%.

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; organic nitrogen sources such as peptone and yeast extract can be used.

The medium used in the present invention has a C / N ratio of 2
It is preferable to use a culture medium of about 30 to 30, more preferably 5 to 28, and most preferably 8 to 25.

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

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

When the bacterial cells cultured according to the present invention are used in the present enzymatic reaction, the bacterial cells can be used as they are, but a treated product of the bacterial cells, for example, crushed by sonicating the bacterial cells. The product, or an extract obtained by further extracting a crushed product thereof 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 body or these treated products may be immobilized and used.

The reaction of indigo formation in the presence of the cells or its treated product is carried out aerobically, and 0.1M phosphoric acid containing 0.1 to 2 mM of indol is added as in the conventional enzyme reaction. 20 in buffer solution (pH 6-9) or aqueous solution (pH 6-9)
At a temperature of -50 ° C, preferably 30-40 ° C, usually 10
React for ~ 72 hours. The reaction is preferably carried out with stirring.

The addition amount of the microbial cells or a treated product thereof used in the reaction is not particularly limited, but in general,
0.5-10% (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.

[0022]

【Example】

(Example _{1) (NH 4) 2 SO} 4: 3g, KH 2 PO
₄ : 0.5 g, K ₂ HPO ₄ : 0.5 g, MgSO ₄ ·
7H ₂ O: 0.5 g, CaCl ₂ · 2H ₂ O: 10 mg,
NaCl: 0.5 g, FeSO ₄ .7H ₂ O: 10 mg,
Yeast extract 1 g and distilled water: 1000 ml (pH 7.0)
Dispense 100 ml of the medium into a 500 ml Erlenmeyer flask,
After sterilization at 120 ° C. for 15 minutes, Pseudomonas sp. MY-6 strain (FER
MP-11963) was inoculated and shake-cultured at 30 ° C. for 24 hours.

1000 ml of the same sterile medium as described above was placed in a 5000 ml Erlenmeyer flask, and m-xylene, ammonium fumarate, sodium succinate or sodium citrate was added so that the concentration of the experimental group shown in Table 1 was obtained. After inoculation, 20 ml of the above shaking culture solution was inoculated, and this was incubated at 30 °
The cells were cultured for 24 hours with shaking. The concentration of m-xylene was sequentially added so as not to exceed 0.3 (W / V)%, and the total amount thereof was added to 0.6 (W / V)%.

Then, 100 ml of the obtained culture solution was centrifuged (800 rpm, 15 minutes, 4 ° C.) to collect the cells. The wet bacterial cell weight of the collected bacterial cells was measured, and the wet bacterial cell weight relative value at each carbon source was calculated as 100 when the wet bacterial cell weight when m-xylene was added was taken as the bacterial cell yield (%). I asked. The results are shown in Table 1.

Using the bacterial cells prepared by the above method as an enzyme source,
After suspending in 100 ml of the reaction solution (100 mM phosphate buffer pH 7.0), 1 mM of indol was added, and the mixture was shaken at 30 ° C.
And reacted for 24 hours. The amount of indigo produced was determined by the conventional method [H. KEIL, CM SAINT and PA WILLIAMS, Journal o
f Bacteriology, vol.169, No.2, P.764-770 (1987)], and the relative value of the amount produced at each carbon source was calculated by converting the amount of indigo produced when m-xylene was added to 100. It was determined as the amount of indigo produced (%). The results are shown in Table 1.

[0026]

[Table 1]

[0027] (Example _{2) (NH 4) 2 SO} 4: 3g, KH 2 PO 4: 0.5g,
_{K 2 HPO 4: 0.5g, Mg} SO 4 · 7H 2 O: 0.
5 g, CaCl ₂ .2H ₂ O: 10 mg, NaCl: 0.
5 g, FeSO ₄ .7H ₂ O: 10 mg, sodium citrate (dihydrate): 10 g and distilled water: 1000 ml (pH
100 ml of the 7.0) medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and then Pseudomonas sp. MY-6 strain (FERM P-11963) was inoculated, and 4 at 30 ° C.
Culture was performed with shaking for 0 hours.

In addition, 100 ml of the same sterilizing medium as described above was added to 5 ml.
The mixture was placed in a 100 ml Erlenmeyer flask, (NH ₄ ) ₂ SO ₄ was added so that the C / N ratio of the medium was the ratio shown in the experimental section of Table 2, and 2 ml of the above shaking culture solution was inoculated. 30 ° C
The cells were cultivated with shaking for 20 hours.

Next, 100 ml of the obtained culture solution was centrifuged (800 rpm, 15 minutes, 4 ° C.) to collect the cells. The wet bacterial cell weight of the collected bacterial cells was measured, and the wet bacterial cell weight relative value at each C / N ratio was calculated by converting the wet bacterial cell weight when the C / N ratio of the medium was 4 to 100. Calculated as yield (%). The results are shown in Table 2.

Using the bacterial cells prepared by the above method as an enzyme source,
The amount of indigo produced in the same manner as in Example 1 was quantified,
The production amount relative value at each C / N ratio was calculated as the indigo production amount (%) by converting the production amount of indigo when the C / N ratio of the medium was 4 to 100. The results are shown in Table 2.

[0031]

[Table 2]

[0032]

EFFECTS OF THE INVENTION According to the method of the present invention, the cell yield of a microorganism having an excellent indigo-producing ability can be improved, so that indigo can be produced at a high yield.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location (C12P 17/16 C12R 1:38) (72) Inventor Hisashi Yamagata Tamiyacho Station, Ushiku City, Ibaraki Prefecture Section Organization 48-2 Fujiheim 2-102 (72) Inventor Hideaki Yukawa 6-23-9 Chuo, Ami Town, Inashiki District, Ibaraki Prefecture

Claims (2)

[Claims] 1. A method for culturing a microorganism belonging to the genus Pseudomonas having indigo-producing ability in a medium containing fumaric acid or a salt thereof, succinic acid or a salt thereof, or citric acid or a salt thereof. 2. A medium in which the ratio of the nitrogen weight of the nitrogen source to the carbon weight of fumaric acid or its salt, succinic acid or its salt, or citric acid or its salt (C / N ratio) is 2 to 30. The culture method according to claim 1, wherein the culture is performed.