JPS6047674A - Novel microorganism - Google Patents

Abstract

PURPOSE:To provide a novel microbial strain belonging to Micrococcus genus and capable of converting an alkyl (or alkenyl) halide to the corresponding omega- halocarboxylic acid in high efficiency. CONSTITUTION:Micrococcus sp. KSM-B-1 (FERM-P No.7002) separated from a source soil using a medium containing n-paraffin by a conventional process. The microbial strain is cultured in a medium containing an alkyl (or alkenyl) halide as a reaction substrate aerobically at about 28-35 deg.C and about 6.5-8pH for about 3-5 days. Preferably after separating the microbial cells, the omega-halocarboxylic acid is extracted from the culture liquid with an organic solvent (e.g. mixture of chloroform and methanol) under acidic condition.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism belonging to the genus Micrococcus.

Conventionally, it has been considered industrially difficult to selectively butyrate only the ω-terminus of fatty acids. For example, ω-hydroxypalmitic acid is used in the production of hexadecanolide, which is the main component of lactone-based musk (synthetic fragrance), but it is this precursor, ω-hydroxypalmitic acid, that makes musk so expensive. This is due to the difficulty in producing hydroxypalmitic acid. That is, it is difficult in terms of synthetic chemistry to selectively oxidize the ω-terminus of balmitic acid to form ω-hydroxypalmitic acid.

On the other hand, it has been reported that ω-hydroxy higher fatty acids are also obtained as a by-product when normal paraffin is assimilated by microorganisms to produce dicarboxylic acids (for example, Japanese Patent Publication No. 48-26238). Hydroxy higher fatty acids are intermediates in the metabolism of normal paraffins to dicarboxylic acids by alkane-assimilating bacteria, but it is difficult to produce them in significant quantities. The reason for this is that ω-halocarboxylic acids are useful intermediates for macrocyclic lactones, which are the main components of bene-based musk. ω-halocarboxylic acids can also be derived into various derivatives by introducing a functional group into the halogen. Regarding this ω-halocarboxylic acid, it belongs to the genus Arthrobacter, Corynebacterium, and Nocardia,
A method for producing ω-halocarboxylic acids by culturing bacteria having the ability to produce ω-halocarboxylic acids from alkyl halides has been reported (Japanese Unexamined Patent Publication No. 57-50893).

Therefore, in view of the current situation, the inventors conducted a wide search in nature for compounds that have the ability to convert alkyl (or alkenyl) halides into the corresponding ω-halocarboxylic acids. They discovered that there is something capable of this, and completed the present invention.

That is, the present invention relates to a novel Micrococcus sp. KSM-8-1 (Feikoken Bibori No. 7002), which belongs to the genus Micrococcus and has the ability to assimilate alkyl (or alkenyl) halides to produce ω-halocarboxylic acids. ).

Coccus, diameter 0.5-1 JL 2) Population: No irregular chains or clusters observed 3) Motility: None 4) Spores: Not formed 5] Gram staining: Positive 6) Anti-drunkenness: Haton (b) Growth status in each medium l) Juicy agar plate culture: Produces a perfectly circular colony with a smooth surface.

Light green, glossy, convex circular shape 2) Juice agar slant culture: Shows medium growth and produces colonies with smooth surface. It has a light green luster.

3) Meat juice liquid culture: No film is formed on the surface, it is moderately turbid, and a slight precipitate is formed.

4) Meat juice gelatin puncture culture: Bacteria grows after 3 days of culture, and only the upper part liquefies. 5) Lidomus milk: Slightly alkaline and does not peptonize. There is no redemption of Lidmus.

(C) Physiological properties 1) Nitrate reduction: No reduction 2) Denitrification reaction: Negative 3) MR test: Negative 4) VP test: Negative 5) Indole formation: Negative 6) Hydrogen sulfide formation (TSI agar) 7) Hydrolysis of starch: Negative 8) Use of citric acid Koser's medium: Not used 'Christensen's medium: Used 8) Use of inorganic nitrogen sources Nitrate: Positive Ammonium salts: Negative 10) Formation of pigments : Water-soluble green pigment 11) Urease Christensen medium: Negative SSR medium: Negative 12) Oxidase: Negative 13) Catalase: Positive 14) Growth range: 14-42°C (optimum 28-35°C) pH 5,0-8 .5 (optimal 8.5-8.0) 15) Attitude towards oxygen: Aerobic 1B) OF test: Negative t ”t ) Production of acids and gases from group a: L-arabinose: - D-xylose: - D-glucose: - D-mannose: - D-fructose: - D-galacudose: - Maltose - Sucrose - Lactose - Trehalose - D-Sorvit ° - D-Mannit ° - Inosit ° - Glycerin - Starch - 18) 5% Sodium Chloride Tolerance: Growing 18) Collection location: Isolated from the soil of Yuzawa Onsen, Niigata Prefecture.
ys Manual of De-terminat
ive Bacteriology) 8th edition (1975
As a result of the search based on the year 2012), this strain was recognized as a new strain belonging to the genus Micrococcus (formerly Crococcus).
Micrococcus sp. φKSM-B-1 (Micr
ococcus sp, KSM-B-1). This strain has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology as copy number 7002.

The bacterial strain was isolated from the source soil using a normal paraffin-containing medium using a conventional method.

The composition of the medium used for culturing this strain is such that it contains an appropriate carbon source, nitrogen source, or Consists of organic nutrients, inorganic salts, etc. Carbon sources include carbohydrates (e.g., glucose, fructose, sucrose, nrubitol'4), organic acids (e.g., citric acid), and organic nutrients include, for example, nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, and yeast extract. , meat extract, and peptone. Also, examples of inorganic salts include various phosphates,
Magnesium sulfate etc. can be used. In addition, trace amounts of heavy metal salts are used, but their addition is not necessarily required in media containing natural products. Also, when using mutant strains that require nutritional requirements.

Substances that meet its nutritional needs must be added to the medium.

For culture, after sterilizing the medium by heating etc., inoculate the bacteria,
Shaking or stirring with aeration at 35°C for 3 to 5 days is fine. pH
Good results can be obtained by adjusting the value to about 6.5 to 8. When using a carbon source that is poorly soluble in water, it is also possible to add various surfactants such as polyoxyethylene solhikne to the medium.

The culture obtained above can be used as it is as an enzyme source, but when the bacterial cells are to be separated from the culture solution, a conventional solid-liquid separation method can be used (1). The live bacterial cells isolated in this way and their processed products (freeze-dried bacterial cells, etc.) can also be used as enzyme sources.

When this strain is cultured as described above using alkyl (or alkenyl)/\lide as a reaction substrate, ω-halocarboxylic acid is produced. Collection and purification of the target substance, ω-halocarboxylic acid, from these culture solutions can be carried out in accordance with the methods used to collect and purify general organic compounds. For example, the filtrate from which bacterial cells have been removed from the culture solution or the culture solution itself is acidified and extracted with an organic solvent such as ethyl ether, ethyl acetate, or a chloroform-methanol mixture.

The ω-halocarboxylic acid can be isolated from this extract using a method such as column chromatography or recrystallization.

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

Example 1 Collected soil and soil-attached deciduous spatil 1
Suspend a glassful (about 0.5 g) in 10 ml of sterilized water, stir thoroughly, and leave to stand. 0.1 ml of the 100-fold diluted solution of the soil suspension supernatant obtained in this way was mixed into an n-hexadecane-containing separation agar medium (I) having the following composition: n-hexadecane 20 g NO4NO35 g KH, PO30, 6 g Na3 HPO4' 12 Ht O4gMg So
4φ 7H,,00,5gFeSO4・? H2030m
g 1:aG12 60mg Ca SO4・5 H@Ol 5 g gNa2B4
0710H2030pg MnCu2-4JO6011-g ZnSO4・7)120 150pLgMa2 No
04 m 2 H2030gg fermentation 1M extract l O
0mg polyoxyethylene-sorbitan monolaurate (average number of EO 20 moles) 50mg agar 20g ion-exchanged water 1-part PH7 kJ, Takahashi, stratification, 4l,
R2? ~ R33 (191 (7)) The method for preparing the separation agar medium (1) is as follows: Only the FeSO4-7H2O component is sterilized by filtration, and then sterilized in an autoclave in advance for 60 minutes.
Cool to around ℃, dilute 100 times with sterile water, and dilute this diluted solution to 0.
．． Transfer 1 ml to the above-mentioned isolation agar medium (rr) and
Culture at 0'C for 3 days. Confirm macroscopically and microscopically that the multiple colonies produced are not different from each other. In addition, the agar medium for separation (11) is the agar medium for separation (11).
Based on the composition of I), n-hexadecane and polyoxyethylene sorbicune monolaurate were removed, and a filter paper impregnated with n-hexadecane was placed on the bottom of the agar medium to supply n-hexadecane.

Ten colonies out of the above were inoculated onto a slant agar medium having the same composition as the isolation agar medium (I).
After culturing at °C for 3 days, it was confirmed that the strains on the 10 slopes and on the ground were macroscopically and microscopically the same strain, and the properties and physiological properties of these 10 strains on each medium were the same. It was confirmed that The properties and physiological properties of the above strains on each medium are as described above.

As a result of the second test, it was determined that each of the 10 cultured bacteria was a single strain isolated from nature.
After cryopreservation for 3 months, the platinum loop of axillary armpits obtained by rapid thawing was resuscitated on an ordinary agar medium, and the properties and physiological properties were determined on each medium under the same conditions as above. As a result of examining the bacteria, no changes were observed compared to before freezing.In addition, the results of similarly examining the properties and physiological properties on each culture medium of the strains that were subjected to the above freezing and thawing process five times every month. No changes were observed.

Next, an example in which ω-halocarboxylic acid was produced using this strain will be listed as a reference example.

Reference example 1 50g of cetyl chloride, 10g of ammonium phosphate
, potassium phosphate 2g, magnesium sulfate (heptahydrate)
0.2 g, ferrous sulfate (7 hydrate) 0.02 g, zinc sulfate (7 hydrate) 0.01'Og, mankan sulfate (4-6
water salt)' 0.018g, yeast extract 2ccus sp
, KsM-B-1,) was inoculated with a platinum loop and incubated at 30'O.
The culture was incubated with shaking for 6 hours.

After culturing, add 1 ml of 9N sulfuric acid to this culture solution to make the pH strongly acidic, and add chloroform-methanol (2:
l) Extracted with mixture 201. This extract was concentrated under reduced pressure and then diluted with methanol-BF.

Methylation was carried out with a catalyst, and constant IJ analysis of the product ω-chlorbalmitic acid was carried out using gas chromatography. The results are shown in Table 1.

The product was confirmed to be ω-chlorbalmitic acid by gas-mass (GC-MS). FIG. 1 shows a mass pattern of methyl ester of ω-chlorbalmitic acid (esterified for measurement).

Table 1 Indication of the case 1982 Patent Application No. 53846 2 Name of the invention Novel microorganism 3 Person making an amendment Relationship to the case Patent applicant 3-1 Kasiyaseki-chome, Chiyoda-ku, Tokyo (+14) Industrial Technology August 28, 1982 (shipping date) with one copy of the order for the 4th amendment of the 5th amendment by the director. 1 specification subject to the 5th amendment and 6 drawings? +li Correct Contents As shown in the attached sheet, the following is inserted after Table 1 on page 18 of the specification.

[4. Brief explanation of the drawings

Claims (1)

[Scope of Claims] 1. A novel Micrococcus ΦSP KSM- deposited as FIKEN Bibori No. 7002 having the following properties:
B-1 strain. (a) Morphology 1) Cell shape and size: Coccus, diameter 0.5-1IL 2) Population: Irregular chains and current status not observed 3) Motility: None (. 4) Spores: Not formed 5) Durham staining: Positive 6) Acid-fastness: Hardly observed (b) Growth status in each medium 1) Juicy agar plate culture: Produces a perfectly circular colony with a smooth surface. Light green, glossy, convex circular shape 2) Juice agar slant culture: Shows medium growth, producing colonies with smooth surface. It has a light green luster. 3) Meat juice liquid culture: No film is formed on the surface, it is moderately turbid, and a slight precipitate is formed.6 4) Meat juice gelatin puncture culture: Bacteria grows after 3 days of culture, and only the upper part liquefies.5) Litmus milk: Wasp. Shows crab alkalinity and does not convert into peptonization. There is no title for Ridmus. ]) Physiological properties 1) Nitrate reduction: No reduction 2) Denitrification reaction: Negative 3) MR test: Negative 4) VP test: Negative 5) Indole formation: Negative 8) Hydrogen sulfide formation (TSI agar): Weak positive 7) Hydrolysis of starch Negative 8) Use of citric alcohol Kose r's medium: Not used Christensen's medium: Used 8) Use of inorganic nitrogen sources Nitrate: Positive Ammonium salt: Negative 10) Pigment formation: Water soluble 11) Urease Christensen medium: Negative SSR medium: Negative 12, Oxidase: Negative 13) Catalase: Positive 14) Growth range: 14-42°C (optimal 28-35°C) pH 5,0-8. 5 (optimal 6.5-8.0) 15) Attitude towards oxygen: Aerobic IEI) OF test: Negative + 7) Production of acids and gases from l'Js: L-arabinose: - D-xylose: - D - Glucose - D-Mannose: - D-Fructose: D-Caractose - Maltose - Sucrose - Lactose - Trehalose. D-Sorbitol - D-Mannitol-Inozite Glycerin - Starch - 18) 5% Sodium Chloride Tolerance: Grows