JPH0195770A - Novel microorganism - Google Patents

Abstract

PURPOSE:To convert the alkyl groups bonding to the aromatic ring in an alkyl- substituted naphthalene into hydroxyalkyl groups utilizing a novel strain of Pseudomonas sp. SA-05B. CONSTITUTION:A novel strain of Pseudomonas sp.-SA-05B (FERM 9632), capable of oxidizing alkyl groups bonding to the naphthalene ring into hydroxyalkyl groups, can oxidize not only a monoalkylated naphthalene such as 1-methyl- naphthalene, but also dialkylated or trialkylated naphthalene such as 2, 6dimethylnaphthalene. Thus, the novel bacterium is cultured in a medium containing alkylated naphthalene to produce aromatic substituted alcohols such as naphthylmethanol which is useful as a starting substance for medicines, agrochemicals and dyes. Further, coal tar components also can be effectively utilized as a substrate.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is directed to a novel microorganism that oxidatively transforms an alkyl group on an aromatic ring of a condensed ring aromatic hydrocarbon such as naphthalene into a hydroxyalkyl group. The present invention relates to a novel bacterial strain belonging to the genus Pseudomonas, which exhibits the ability to transform, and to a method for its cultivation for producing useful substances.

(Prior Art) Conventionally, the effects of microorganisms on aromatic hydrocarbons, especially fused polycyclic aromatic compounds (e.g., naphthalene, anthracene, phenanthrene, or alkyl-substituted compounds thereof) contained in coal tar, have not been studied. From the viewpoint of preventing environmental pollution, many studies have been conducted on the decomposition of these compounds through the catabolism of microorganisms, and bacteria belonging to the genus Pseudomonas and ^lcaligenes have been investigated as bacteria that decompose aromatic hydrocarbon compounds. A large number of bacteria are known to exist.

On the other hand, if we focus on the energy-saving material conversion ability performed by microorganisms, that is, assimilation, we can use the various substances contained in coal tar as raw materials and convert them into useful substances through the assimilation of microorganisms. It is considered to be very advantageous and important in terms of effective utilization of. However, aromatic hydrocarbon compounds, especially polycyclic aromatic compounds, are less susceptible to enzymatic reactions by microorganisms than aliphatic compounds, so microbiological methods using polycyclic aromatic hydrocarbon compounds as raw materials are useful. There is not much research on the production of substances.

As a conventional technology related to the microbial assimilation of polycyclic aromatic hydrocarbon compounds, it has long been known to produce salicylic acid, a raw material for pharmaceuticals, etc. from naphthalene by microbial action (Japanese Patent Publication No. 43-20704). ). Recently, it has also been proposed to use bacteria of the genus Pseudomonas to hydroxylate biphenyl and convert it into phenylphenol.
Publication No.).

In the above-mentioned conventional microbiological assimilation of polycyclic aromatic compounds, the aromatic compounds used as raw materials do not have substituents.
It is a substance with a relatively simple structure, such as a cyclic compound.

On the other hand, the bacterial strain Pseudomonas slacchieri (Pseudomonas 5tut) that the present inventors discovered earlier
zeri) SA (Microtechnical Research Institute No. 9118)
2-39925) and Pseudomonas putida (P.
9-Ant (
Microtechnical Research Institute No. 9350) (Patent Application 1987-127591)
No.) can oxidatively assimilate alkyl-substituted naphthalenes or more complex compounds such as anthracene and phenanthrene, converting methylnaphthalene to methylsalicylic acid, for example, but both act on the aromatic ring itself; It has no effect on substituents bonded to aromatic rings.

(Problems to be Solved by the Invention) The object of the present invention is to act on alkyl-substituted naphthalenes (e.g., methylnaphthalene) such as those contained in coal tar, and convert the alkyl substituents bonded to aromatic rings into hydroxyalkyl groups. The object of the present invention is to provide a new microorganism that has the ability to convert

Another object of the present invention is to provide a microbial culture method for producing hydroxyalkyl-substituted naphthalenes, ie, naphthyl alcohols, from alkyl-substituted naphthalenes using this novel microorganism.

(Means for solving the problem) As a result of searching for microorganisms having the above-mentioned ability in the natural world, the present inventors found that some bacterial species belonging to the genus Pseudomonas have this ability. The present invention has been completed.

According to the present invention, a bacterial strain belonging to the genus Pseudomonas acts on the alkyl substituent of alkyl-substituted naphthalenes such as 1-methylnaphthalene to oxidize it to a hydroxyalkyl group, and converts it into a naphthyl alcohol such as 1-naphthylmethanol. A novel strain of Pseudomonas sp.
sp,) SA-05B (Huikoken Bacillus No. 9632)
is provided.

The mycological properties of the novel strain according to the present invention isolated by the present inventors will be described below.

Target fal of Pseudomonas sp. SA-05B
Morphology ■Cell shape and size: Bacillus, (0.5-0.6) x (1.3-1.5) (p)
■Presence or absence of motility and epiphytic status of flagella: Yes, polar flagella (1) ■Cell pleomorphism and presence or absence of spores: None ■Gram staining dinegative (BLGrowth status in various media ■Meat juice agar plate Culture: Poor growth, pale yellowish brown, circular, convex undulations in the center ■ Meat juice agar slant culture: Poor growth, pale yellow, wrinkled colonies ■ Meat liquid culture: Slightly poor growth, small flakes on the surface ■ Meat juice Gelatin puncture culture: Poor growth, growth on the surface layer, no liquefaction ■ Lidomus milk: Reduces lidmus, alkalinizes (
(C) Physiological properties ■ Nitrate reduction: Negative ■ MR test: Negative ■ VP test: Negative ■ Indole production: Negative ■ Hydrogen sulfide production: Negative (TSI medium) ■ Starch hydrolysis: Negative ■ Desorption Nitrogen reaction: negative ■ Arginine dihydrolase: negative 0 Basin: negative [phase] Lipase: negative ■ Urease: negative [phase] Catalase: positive ■ Oxidase: positive ■ Growth range temperature = 10-37"C pH: 6-9 ■Attitude towards oxygen: Aerobic [phase] O-F test: Generation of acids from oxidative OXs (Hugh-Leifson method)
: [Phase] Assimilation of carbon compounds (Iizuka-Komagata method): The present inventors have developed a bacterial strain of the present invention (SA-05B) that has the ability to oxidize an alkyl group bonded to a naphthalene ring to a hydroxyalkyl group. Based on the above mycological properties, Bergey's Manual of Systematic Hateriology
l of Systematic Bacteriol
ogy).

As a result of examining the differences with known strains according to the criteria described in Vol. 1 (1984), this strain was found to be a new bacterial strain belonging to the genus Pseudomonas. , and named Pseudomonas sp. SA-05B.

This strain has been deposited and stored at the Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 9632.

The novel strain of the present invention acts not only on monoalkyl-substituted naphthalenes such as 1-methylnaphthalene, but also on alkyl groups of dialkyl- or trialkyl-substituted naphthalenes such as 2,6-dimethylnaphthalene. , each alkyl group could be oxidized to a hydroxyalkyl group.

The medium used for culturing the bacterial strain of the present invention may have any composition as long as it allows the strain to grow well and allows the microbial reaction to proceed sufficiently using the aromatic hydrocarbon compound serving as the raw material as a substrate. Alternatively, two different types of media may be used, one for cell growth and one for reaction.

The medium used at this time may contain appropriate carbon sources, nitrogen sources, inorganic salts, etc. as medium components.

As a carbon source, in the case of a reaction medium, an alkyl-substituted naphthalene compound as a raw material can be used alone. If desired, any carbon source available to the strain of the present invention may be used in combination as an additional carbon source. Organic substances that can be used as such additional carbon sources include organic compounds such as glycerin and ethanol, carbohydrates such as glucose and fructose, and m-acids such as citric acid, as shown in the mycological properties above. An example of an organic compound suitable as an additional carbon source is glycerin. In the case of a culture medium for cell growth, not only alkyl-substituted naphthalene compounds but also one or more carbon compounds that can be used by the strain of the present invention as described above can be used as a carbon source.

The nitrogen source is not particularly limited, but inorganic nitrogen compounds such as ammonium sulfate and ammonium nitrate, and organic nitrogen sources such as peptone can be used. When an organic nitrogen compound is used, since it also contains carbon, a separate carbon source is not necessarily required in the bacterial growth medium.

As inorganic salts, various phosphates, magnesium sulfate, etc. can be used. Furthermore, the medium may contain trace amounts of heavy metal salts (eg, iron salts, manganese salts, etc.).

The culturing method may be a shaking culture method, a deep aeration agitation culture method, or the like. Culture temperature is 25-3
The temperature is 5°C, the pH is near neutral, and the number of days for culturing can be determined depending on the progress of the reaction, but 5 to 10 days is usually appropriate. At this time, it is also possible to add various surfactants such as polyoxyethylene sorbitan to the medium since the aromatic compound serving as the raw material is poorly soluble in water. Also,
If necessary, an antifoaming agent may be added.

After the completion of the culture, separation and purification of the target product from the culture solution can be carried out by appropriately combining means well-known to those skilled in the art, such as solvent extraction, column chromatography, concentration, and crystallization, in the same way as the separation and purification of general organic compounds. It can be done together. for example,
After the bacterial cells are removed from the culture solution by centrifugation, they are extracted with an organic solvent such as ethyl acetate or chloroform. The target product can be isolated from the obtained extract by methods such as column chromatography or recrystallization.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Activated sludge (approximately 1 c
J) was suspended in 100 ml of sterilized water, thoroughly stirred, and left to stand. The obtained soil suspension supernatant (1 cJ) was
S of the following composition with the addition of 0.1% 1-methylnaphthalene
In addition to the isolation medium (50C♂) consisting of N medium, 30℃
The cells were cultured with shaking for 7 days (enrichment culture). SN medium was pa
After conditioning, it was filtered and sterilized before use.

Rakushita 1111 Ammonium sulfate 5g Dipotassium hydrogen phosphate 2g Sodium dihydrogen phosphate 2g Magnesium sulfate heptahydrate 0.1g Ferrous sulfate heptahydrate 0.1g Manganese sulfate heptahydrate 0.1 g Yeast extract
io,mg□Urio! 3J ship temporary −□−−−−
--↓J-H8 The supernatant of the culture solution (ICJ) obtained by enrichment culture was
% of 1-methylnaphthalene added to the SN medium,
Culture was performed again under the same culture conditions. After diluting one platinum loop of the culture solution obtained by this enrichment culture with sterilized water, it was spread on an SCD agar medium (manufactured by Otama Nutrition@) and cultured at 30°C for 2 days. The appearance of the resulting colonies, such as their shape and color, was visually identified, and two types of bacterial strains were obtained. Each of these strains was inoculated into the same medium as the enrichment culture, and cultured at 30°C to examine their reactivity towards l-methylnaphthalene. SA-05B bacteria was obtained.

The following Example 2 shows an example of producing a useful substance using the culture of the novel strain of the present invention.

Article 5 This example illustrates the conversion of 1-methylnaphthalene to 1-naphthylmethanol utilizing the strain of the present invention.

One platinum loop of Pseudomonas sp. SA-05B on the SCD agar slant culture medium isolated in Example 1 was added to the bacterial cell growth medium 5Q ca prepared by adding 0.1% glycerin to the SN medium shown in Example 1. was inoculated and cultured with shaking at 30°C overnight. This was inoculated into a 51 capacity jar fermentor containing 0.2% SCD medium 31, and incubated at 30°C.
The culture was further carried out overnight under the conditions of pH 7.5 to 7.6, stirrer rotation speed 30 rpm, and aeration rate 311/min.

Thereafter, 6 g of 1-methylnaphthalene was added as a substrate, and the culture was continued for another 8 days under the same culture conditions as above. After the culture was completed, the bacterial cells were removed by centrifugation and concentrated to dryness under reduced pressure. After the obtained residue was extracted with chloroform, the extract was again concentrated to dryness under reduced pressure. The dry matter is n-
Recrystallization from hexane/chloroform (volume ratio 10/1) yielded 149 μl of 1-naphthylmethanol. Melting point: 59.0-60.0°C. (Melting point of standard: 58.0
~58.5°C) Elemental analysis: C++H+. O Calculated value: C83, 52%, H6, 37% Actual value: C
81,46%, H6,19%'H-NMR (CDCQ3
): δ-1,78 (11-1), 5.14 (2H
).

7.33-8.14 (711).

(Effect of the invention) A novel microorganism according to the present invention, Pseudomonas sp.
Strain A-05B, as exemplified above, can be cultured using alkyl-substituted naphthalene as a substrate, thereby producing aromatic substituted alcohols such as naphthylmethanol in a microbiological manner. Aromatic substituted alcohols such as naphthylmethanol are useful chemicals as raw materials for pharmaceutical and agrochemical products, dyes, and the like. Conventionally, methods for producing 1-naphthylmethanol, such as reduction of 1-naphthalene aldehyde or hydrolysis of 1-chloromethylnaphthalene, have been known, but these methods require expensive raw materials, so the product It also has to be expensive.

On the other hand, alkyl-substituted naphthalenes such as 1-methylnaphthalene used as raw materials in the culture method of the present invention are components contained in coal tar, and therefore can be obtained at low cost by separation from coal tar. However, it is extremely difficult to introduce a hydroxyl group into this alkyl substituent by chemical methods.

According to the method of the present invention, the desired aromatic substituted alcohols can be produced by an energy-saving microbial oxidation method using inexpensive raw materials separated from coal cour, so aromatic substituted alcohols can be produced at low cost. It becomes possible to provide In addition, this method also allows effective use of coal tar components.

Claims (1)

[Claims] A new bacterial strain, Pseudomonas sp. SA, belonging to the genus Pseudomonas, has the ability to oxidatively convert an alkyl group on a naphthalene ring to a hydroxyalkyl group, and was deposited as Microtechnical Laboratory Deposit No. 9632.
-05B bacteria.