JPS61108393A - Production of pyrogallol - Google Patents

Abstract

PURPOSE:Hydrolyzable tannin, gallic acid is allowed to act on a new kind of microorganism which is capable of producing pyrogallol to obtain pyrogallol which is used in medicines for external use, developing agent in photography or reagent for chemical analysis. CONSTITUTION:Aeromonas sp. 631 (FERM 7899) and Erwinia sp. (FERM 7900) is inoculated and cultured in a medium containing hydrolyzable tannin such as gallotannin and/or gallic acid to accumulate pyrogallol. Then, the pyrogallol is extracted with diethyl ether, decolored with activated carbon, concentrated and sublimed to give the objective pyrogallol.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing pyrogallol using microorganisms.

Pyrogallol is an extremely useful compound as an external medicine for skin diseases, a photographic developer, an analytical reagent, etc., or as an intermediate for industrial chemicals.

Conventionally, pyrogallol was produced using microorganisms, such as Citrobacter.
Bacteria of the genus Penicillium (Japanese Unexamined Patent Application Publication No. 67/3♂373), molds of the genus Penicillium [Biohimica, Biophysica acta (Bioch.
imicaet Biophysica Acta),
Vol. 28, p. 21 (I!) and Science, Vol. 26,
42 pages (/ri! 9')] or Klebsiella (K
levsiella) bacteria [AntonievanLeevwen
hoek), Vol. 35, p. 325 (1996)] and the like are known methods for producing cypyrogallol.

Therefore, in addition to the above, the present inventors conducted a wide search for microorganisms capable of producing pyrogallol from the natural world, and found that
Aeromonas (A6) newly isolated from forest soil.
r□monas) or Krwinia
The present invention was completed based on the knowledge that two new fungal species belonging to the genus ) produce pyrogallol extremely efficiently from hydrolyzed tannins or gallic acid.

That is, the present invention produces pyrogallol by causing a microorganism belonging to the genus Aeromonas or the genus Erwinia and having the ability to produce pyrogallol, its contents, or a treated bacterial cell to act on hydrolyzed tannin and/or gallic acid. This is a method for producing pyrogallol, which is characterized by collecting the compound.

The present invention will be specifically explained below.

First, the bacteria used in the present invention are microorganisms that belong to the genus Aeromonas or Erwinia and produce pyrogallol from hydrolyzed tannins and/or gallic acid.
Any bacteria may be used, and variants or mutant strains of these bacteria may be used.

Specific examples of the above-mentioned microorganisms include Aeromonas sp. 631 and 0: Erwini 78P and 932, respectively, and their mycological properties are shown below.

The experimental method is from "Classification and Identification of Microorganisms", edited by Takeharu Hase.
With reference to the first edition, identification was carried out using the Purge Aids Manual of Determinative Bacteriology (B
ergey+s Manual Of Deter −
This was done based on the 8th edition (/'771/L) of Minative Acteriology.

Mycological properties of Aeromonas EIp, 631 (a) Morphology Microscopic observation (cultivated in bouillon medium at 30°C for 1 hour) ■Cell shape and size: 0, j ~/, OX O, 1~4L, Ott m
It is a short bacillus.

■Presence or absence of cell pleomorphism: Not observed.

■Mobility: Has 7 to 2 polar flagella and is motile.

■Presence or absence of spores; not formed.

■Durham staining: Negative.

■Anti-acidity: Negative.

(1)) Growth status in each medium ■ Juice agar plate culture: After culturing at a temperature of 3oC for 21 hours, perfectly round, pale yellow, and translucent colonies are formed. The colony surface is smooth and shiny, but no pigment is produced.

■Juice agar slant culture: Growth is good in a spreading pattern.

■ Broth liquid culture: Grows well throughout and produces sediment.

■Meat juice gelatin puncture culture: Grows along the puncture line and is not liquefied.

■ Lidmus milk: No change is observed on the second day, but it becomes slightly alkaline on the // day.

(C) Physiological properties ■Reduction of nitrate: No reduction.

■Denitrification reaction: Negative.

■MR test: Negative.

■VP test: slightly positive.

■Indole production: Negative.

■Generation of hydrogen sulfide: Lead acetate test strip method: Positive.

TS engineering agar method: Negative.

■Hydrolysis of starch; negative.

■Use of citric acid; Christensen
-sen) and Koser medium.

■ Utilization of inorganic nitrogen sources: Use a small amount of both ammonium salts and nitrates.

[Phase] Pigment formation: Negative.

■The presence or absence of fluorescence is negative.

■Urea-9D 0 oxidase: slightly positive.

0 catalase: positive.

[Phase] Growth range: Optimal temperature: 10~3♂℃ Optimal pH: J-~ri [Strain] Attitude towards oxygen: Facultatively anaerobic.

@'O - F test: Produced fermentatively.

[Phase] Generation of acid and gas from sugars Generation of acid Generation of gas (1) L-arabinose + - (2)
D-xylose 10-(3) D-glucose 10-(4) D-mannose 10-(5) D-fusictose
10 - (6) D-galactose 10 - (7) Maltose - - (
8) Sugar − −(9) Lactose −(1
ω trehalose + −αυDnlubit − −117J D-mannit + −(13) Inosit
10 −α-leached glycerin
+ - (15) Starch
- - (L6) Adonitol - -
αη Zulsit -- kaeskurin 10 -- (L'JI
Salicin 10-(20)α-
Methyl-glucoside --I2]) β-Methyl-glucoside 10 -@melezitose
−− (c) Melibiose −− (c) Cellobiose 10 − (to)
Rhamnose - (d) Other properties■Lysine decarboxylation: Negative.

■Use of amino acids as carbon sources: L-Arginine: Positive.

L-asparagine: positive.

L-histidine: positive.

L-serine: negative.

L-alanine: positive.

L-glutamic acid: positive.

■Phenylalanine deaminase: Positive.

■Pectin degradation double negative.

■Cold 4F decomposition: Negative.

■DNase: Negative.

■Gluconic acid oxidation: Positive.

■Generation of sucrose reducing substances: Negative.

■(0//2ri) Susceptibility to Testini 2, IA-diamino-O, 7-diimpropylpteridine: Negative.

In the above Aeromonas sp., 631, the oxidase is
Although it is unclear, it has polar flagella and is a member of the Vibridae family (Vibridae).
ionaceae).

There are no curved cells or spherical cells, no fluorescence, and it grows even in broth medium without salt.
In addition, λ, 4L-diamino-O, 7-diituprovir pteridi7 (, l, 41-diamino-1
,,7-diisopropylpteridine)
It was identified as belonging to the genus Aeromonas based on its characteristics such as not being susceptible to.

Aeromonas hydrophylla grows even at a temperature of 37°C and produces acetoin fermentatively from glucose.
Although closely related to Aeromonas hydrophila), this strain differs from Aeromonas hydrophila in that it does not degrade starch, does not liquefy gelatin, is urease positive, indole negative, and grows in 73% saline. However, the Purge Aids Manual of Determinative Bacteriology, 8th edition (/711t) also does not mention a species that matches this strain, and this strain is considered to be a new species, Aeromonas J), 631. It was named.

In addition, Aeromonas 81) and 631 were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 7899 (FI
RM P-7899).

Mycological properties of Erwinia 8p, 932 (a) Morphology Microscopic observation (cultivated in bouillon medium at 30°C for an hour) ■Cell shape and size: 0, ! r −0, r x O, Otsu~2. ! It is a short bacillus of ff1m.

■Presence or absence of cell pleomorphism: Not observed.

■Motility: Has periflagella and is motile.

■Presence or absence of spores: Not formed.

■Durham staining; negative.

■Anti-acidity: Negative.

(b) Growth status in each medium - Juicy agar plate culture: By culturing at a temperature of 30°C for 2≠ hours, perfectly round, pale yellow, and translucent colonies are formed. The surface of the colony is smooth and shiny, and the medium turns pale yellow.

■Juice agar slant culture: Growth is good in a spreading pattern.

■ Broth liquid culture: Growth occurs throughout the plant, but the growth is poor and produces sediment.

■ Flesh gelatin puncture culture: Grows along the puncture line and is weakly liquefied at the mouth for ≠ weeks.

■ Lidmus milk: No change observed.

(C) Physiological properties ■Reduction of nitrate in broth medium: Although it does not reduce after 3 days of culture,! It is reduced in culture for 1 day.

Sodium succinate medium: Reducing.

■Denitrification reaction: Negative.

■MR test: Negative.

■VP test: slightly positive.

■Indole production: Negative.

■Generation of hydrogen sulfide; Lead acetate test strip method: Positive.

TS engineering agar method: Negative.

■Starch hydrolysis: Negative.

■Use of citric acid: Christensen
-8θn)- and Koser medium, both unused.

■ Utilization of inorganic nitrogen sources: Use a small amount of both ammonium salts and nitrates.

[Phase] Pigment formation: Negative.

■Presence or absence of fluorescence: Negative.

■Urease: Negative.

0 oxidase: negative.

0 catalase: positive.

[Stock] Growth range: Optimal temperature: 10-37℃ Optimal pH: J"~ri [Phase] Attitude toward oxygen: bipartite anaerobic.

oo-y' test: produced fermentatively.

■ Generation of acid and gas from sugars Generation of acid Generation of gas (1) L-arabinose + -(2
)p-xylose 10-(3)D-
Glucose 10 - (4) D-mannose 10 - (5) p-7 lactose + - (6) D-galactose
+ - (7) Maltose -- (8) Sugar -- (9) Milk
Sugar - ω-trehalose
10-(11) D-Sorvit
--(L21D-Mannit 10
-(131 Inojitsu 10 -'
I glycerin 10-α starch-(16
) Adonitol 10 - Sumi D Zurshit - (2) Aesculin 10 - (1'
J salicin + − (a) α−
Methyl-glucoside −-C◇β-methyl-glucoside 10 −@meridiose −
- @Melibiose 10 - (c) Cellobiose + + (a) Rhamnose 10 - (d) Other properties ■Lysine decarboxylation: Negative.

■Use of amino acids as carbon sources: L-Arginine: Negative.

L-asparagine: positive.

L-histidine: negative.

L-serine; negative.

L-alanine: negative.

L-glutamic acid: slightly positive.

■Phenylalanine deaminase: Positive.

■Pectine/decomposition: Negative.

■Cold 4F decomposition: Negative.

■DNase: Negative.

■Gluconic acid oxidation: Positive.

■Generation of sucrose reducing substances: Negative.

■(0//2ri) Test: λ, Hiro-Diamino-Otsu, Sensitivity to 7-diimbrovirpteridine: Negative.

The above Erwinia sp. 932 is oxidase negative,
Knterobacteridae has periflagella and produces glucose fermentatively.
ioceae).

The optimum temperature for growth is 30°C, urease negative, benzoate, oxalats and propionate.
PrO-piOnate) is not used as a carbon source,
The production of sugar gas and the ability to hydrolyze starch were negative, so it was identified as belonging to the genus Krwinia.

Additionally, there are no growth factors and the temperature is 37. Siervinia can grow even at ℃ and exhibit a pale yellow color when cultured on broth agar slants for a week. "Erwinia hsrbicola group
), but the liquefaction of gelatin is weak and the generation of acid from sugars, i.e., Inojit,
The description of a species that differs in methane, ribiose, maltose, adonitol, cellobiose, glycerin, and sorbitol and that matches this strain is listed in the Purge Aids Manual Op.
Determinative Bacteriology, 8th Edition (l
However, this strain was regarded as a new species and named Erwinia S11.932.

In addition, Elpinia 8p and 932 were submitted to the Institute of Microbiological Technology, Agency of Industrial Science and Technology, as part of the Microbiology Research Institute No. 7900 (FERM).
P-7900).

In the present invention, in order to cause the above-mentioned microorganisms, their contents, or their bacterial cell-treated products to act on hydrolyzed tannins and/or gallic acid, , inoculated with the microorganism,
A method in which pyrogallol is produced and accumulated in the culture in parallel with the cultivation of microorganisms.

(2) A method of producing pyrogallol by contacting a culture obtained by inoculating and culturing the above microorganisms in a medium, that is, a material containing the microorganisms, and hydrolyzed tannin and/or gallic acid.

(2) A method of inoculating and cultivating the above-mentioned microorganisms in a medium in advance and bringing the bacterial cells or treated bacterial cells isolated from the obtained culture into contact with hydrolyzed tannin and/or gallic acid, etc. can be mentioned.

In addition, any hydrolyzable acid may be used, such as gallic acid tannin, five-fold tannin, gallic tannin, cod tannin, and gallic acid.
It may be of any origin, manufacturing method, etc.

In the method (2) above, the culture medium may be any medium as long as it contains hydrolyzed tannins and/or gallic acid in addition to the usual carbon sources, nitrogen sources, inorganic salts, micronutrient sources, etc. Sources include glucose, calactose, fructose, mannose, arabinose, xylose,
Examples include ribose, mannitol, inosit, glycerin, etc. Nitrogen sources include peptone, yeast extract, meat extract, casamino acid, cornstarch liquor, natural nitrogen sources such as amino acids, and inorganic nitrogen sources such as ammonium salts. can be used alone or in combination.

Furthermore, as micronutrient sources, vitamins, nucleobases,
Examples include amino acids.

The initial pH of the medium is 0-1, preferably 0-6.
, 0, and the culture temperature may be any temperature at which the microorganisms used can grow, and usually /! ~37°C, preferably
The temperature is 20-30°C.

The microorganism may be cultured by a solid culture method, but it is usually advantageous to use a liquid culture method, and specifically, aeration culture, shaking culture, static culture, etc.
72 hours, preferably 2'A~g? Cultivate for hours to produce and accumulate pyrogallol in the medium.

In the method (2) above, the same medium as in (2) above is used for culturing the microorganism, and the microorganism can be grown satisfactorily even in a medium that does not contain hydrolyzed tannins and/or gallic acid. Hydrolyzable tannins and/or
Alternatively, by adding a small amount of gallic acid to the medium, it is possible to obtain bacterial cells with an increased ability to produce pyrogallol from hydrolyzed tannins and/or gallic acid.

In addition, the pH of the medium, the culture temperature, and the culture method may be the same as those described in (2) above, but the culture time is determined from the time when the growth of the fungus enters the logarithmic growth phase to indicate the ability to produce pyrogallol. The culture time may be shorter than that in (2).

In this method, hydrolyzed tannins and/or gallic acid may be added directly to the culture, or hydrolyzed tannins and/or gallic acid may be added to the cultured product after concentrating the culture. You can.

The concentration of the tannin and/or gallic acid to be added is not particularly limited, but it is usually preferable that the concentration in the culture or cultured product is 0.2 to 10% by weight. The pH of the reaction is, for example, 3.0-10, preferably lAθ~
z is 0, the reaction temperature may be any temperature as long as one reaction proceeds, and is usually about 20 to tAO°C, and pyrogallol is produced by shaking, stirring or standing still. Preferably, it is accumulated.

In the method (2) above, microorganisms are cultured in the same manner as in the method (2) above, and after completion of the culture, the microorganisms are extracted from the culture, for example,
Bacterial cells can be separated by conventional means such as centrifugation and filtration.

The bacterial cells used in the method (2) may be untreated bacterial cells after separation, and may be treated with mechanical destruction methods such as ultrasonic treatment, homogenizer, blender, Vibrogen Cell Mill, Dyno Mill, etc., or with Triton X-100, etc. Processed bacterial cells that have been destroyed by methods using surfactants, enzymes such as lysozyme, or chemical destruction methods such as freeze-thaw and autolysis may also be used.After these treatments, ordinary enzyme separation methods can be used. Enzymes and the like that have been fractionated using these methods can also be used as appropriate.

It is also possible to continuously produce pyrogallol using cells or enzymes that have been immobilized according to a conventional immobilization method.

The pyrogallol produced and accumulated in this way can be collected by a known method, for example, by extracting it with diethyl ether, decolorizing it with activated carbon, concentrating it, and sublimating it. Pyrogallol can be isolated.

As described above, according to the present invention, as the bacteria of the genus Aeromonas or the genus Erwinia, hydrolyzable tannins and/or
It has not been known to produce cypyrogallol from gallic acid, and the present invention can be produced very efficiently, so the present invention is extremely significant industrially.

The present invention will be specifically explained below with reference to Examples.

Ru.

Example 1 Meat broth bouillon medium [meat extract 0.3% (W/'l')
, peptone/% (W/V), N a CI Q, j%
C-W/V), pH 7,0:) Aeromonas Bp was dispensed into test tubes with cotton plugs.

631 strain (FIRM P-7899) and Erpinia s.
p, 932 strains (FBRM P-7900), respectively.
Platinum loops were inoculated and cultured with shaking at a temperature of 30°C for several hours. -% (W/v), phosphoric acid/ammonium 0.2%
(W/V), phosphoric acid/potassium 0.2% (W/V),
It was inoculated into 100 nl of a liquid medium containing 0.7% (W/v) magnesium sulfate and pH adjusted, and cultured with shaking at a temperature of 30°C for 30 hours. The amount of pyrogallol produced in the resulting culture was measured by high performance liquid chromatography. The results of measurements using Twinkle HPLC (manufactured by JASCO Corporation) are shown in the table below.

Table 1 Example 2 Gallic acid/, j % (W/su, poly-peptone/
% (w/v), glucose j% (w/v), yeast extract 0.2% (w/'7), phosphoric acid/potassium o: 2
s (”/v), magnesium sulfate 0.7% (W/V)
, pk13.0 liquid medium/0. The same preculture solution as in Example 1 was inoculated into Orttl, and the mixture was incubated at a temperature of 30°C for 2 hours.
The amount of pyrogallol produced in the resulting culture was measured in the same manner as in Example 1 after shaking culture for ≠ hours, and the conversion rate from gallic acid is shown in the table below.

Table 2 Example 3 Gallic acid 0. j % (W/V), gazamino acid/
% (w/v)g/L/course/, 0%(w/v
), yeast extract 0.2% (W/'7), phosphoric acid/potassium 0.2% (W/v), magnesium sulfate 0. /
% (w/v), in 100 ml of liquid medium at pH .
The culture obtained by inoculating the same preculture solution as in Example 1 and culturing at a temperature of 30°C for 70 hours was /2,000r, II
m.

-(N Morpho↓ino)ethane 5
ulfonic acid, no-n0hydrat
e) It was added to a solution containing buffer solution (1) H=j:O) and stirred gently for 6 hours at a temperature of 30°C, and the amount of pyrogallol produced in the reaction completed solution was determined in the same manner as in Example 1. The measured results are shown in the table below.

Table 3 Example 4 10jrttl of the culture medium obtained by the method of Example 2 was
Centrifugation was performed for 100 tttl at / 20 δ Or, p, m, to obtain 100 tttl of supernatant, and the pH of the supernatant was adjusted to pH 20 using hydrochloric acid, and then centrifuged twice using 100 rd diethyl ether. The ether layer was evaporated to dryness in a conventional manner, and the resulting dry powder was sublimated under reduced pressure in a conventional manner to form crystals. ! θ■ was obtained.

The melting point of the crystal thus obtained is /3/~/3'
/L℃, the Rf value of thin layer chromatography (developing solvent benzene: dioxane: acetic acid = PO: 26:≠) matched that of pyrogallol, and the R spectrum completely matched that of pyrogallol. The crystals were identified as pyrogallol.

Claims (1)

[Claims] Microorganisms belonging to the genus Aeromonas or the genus Erwinia and having the ability to produce pyrogallol, their contents or treated bacterial cells are allowed to act on hydrolyzed tannins and/or gallic acid to produce pyrogallol, and the compound is collected. Characteristic manufacturing method of pyrogallol.