KR20010109831A - Novel streptomyces sp. strain and method for producing oleamide therefrom - Google Patents

Abstract

The present invention is a new strain Streptomyces sp. Isolated from soil producing oleamide. KK90378 (KCTC18014P) and a method for producing oleamide by this strain. Since the new strain according to the present invention has a high production capacity of oleamide, oleamide is a physiologically active substance that induces sleep by culturing the strain to produce oleamide, a physiologically active substance that induces sleep, or by immobilizing the bacteria on a carrier. The amide can be produced efficiently.

Description

Oleamide-producing Streptomyces spp. Strain and a method for producing oleamide from the strain {Novel Streptomyces sp. Strain and Method for Producing Oleamide Therefrom}

The present invention provides a new strain producing an oleamide having a physiological activity that induces sleep.

Another object of the present invention is to provide a method for producing oleamide by culturing a strain.

Oleamide has a cis double bond in the middle of a long alkane chain, and an amide that is tightly bonded to a hydrogen bond at the end of the chain. Cis double bonds in oleamides disrupt the packing of saturated alkane compounds and cause primary amides to destroy cell membrane structures composed of lipids. Oleamide is found in the cerebrospinal fluid of cats with poor sleep and is known to be produced in small amounts in the animal brain. The physiological effects of oleamide have been reported to have effects of inducing sleep and lowering body temperature [Lerner, RA Proc. Natl. Acad. Sci ., 94, 13375-13377, 1997].

On the other hand, oleamide in the cell membrane protein assists in the operation of serotonin receptors and has a closing effect of gap junction channels. Oleamide has no effect on the action of serotonin receptors alone, but when combined with serotonin, it shows an effect of enhancing serotonin activity on serotonin receptors [Boger, DL, Proc. Natl. Acad. Sci. , 95, 4102-4107, 1998].

Oleamides have a similar effect by inducing a change in cell membrane structure, as conventional anesthetics show activity by altering the stable structure of the receptor-channel complex of the cell membrane (closure of the channel structure) in the body. Known.

Oleamide is known to be biosynthesized from acyl-glycine precursor by enzymatic reaction of peptidylglycine-amideating monooxygenase (PAM) in the body [Lerner, RA Proc. Natl. Acad. Sci ., 94, 13375-13377, 1997] The degradation of oleamide is known to be degraded by the action of a cell membrane binding enzyme, fatty acid amide hydrolase (FAAH), to convert oleamide to monovalent unsaturated fatty acid oleic acid.

In the case of anesthetics or sleeping pills that induce sleep to date, most of them are substances produced outside of cells or artificially synthesized chemically. Therefore, the mechanism by which such noncellular substances act in the body is different from the substances produced in the body and causes a number of side effects (headache, indigestion, etc.).

Soil microorganism, Streptomyces sp. We found that KK90378 (KCTC18014P) produced oleamide and completed a method to produce this functional fat by biotechnological methods.

1 is Streptomyces sp. An electron micrograph of KK90378 (KCTC18014P);

2 is a graph showing the results of thin layer chromatography used to search for oleamide producing strain represented by Formula 1;

3 is Streptomyces sp. Which is a production strain of oleamide represented by formula (1). Dry cell mass, pH, and oleamide production of KK90378 (KCTC18014P) by culture days;

4 is a TLC result of the fraction obtained by performing column chromatography on the culture solution;

5 is a mass spectrometry spectrum of the EISMS of oleamide represented by formula 1 in HP5890GC / JEOL;

FIG. 6 is a 500 MHz hydrogen nuclear magnetic resonance spectrum of oleamide represented by Formula 1 in JEOL JNM-LA400 solution; FIG.

FIG. 7 is a 125 MHz carbon nuclear magnetic resonance spectrum of oleamide represented by Formula 1 in JEOL JNM-LA400 solution.

The present invention is a novel microorganism Streptomyces sp. KK90378 (KCTC18014P) and a method for purifying and preparing the present fat from a microbial culture solution are featured.

In the present invention, a novel microorganism producing oleamide, a functional fat produced only in small amounts in the brain tissue of an animalStreptomycessp. KK90378 can be used to secure methods for mass production of this material biotechnologically.

In addition, the present invention includes a sleep inducing agent containing the oleamide represented by the formula (1) and derivatives thereof.

Referring to the present invention in more detail as follows.

1. Identification of Strains

1) Morphological characteristics

According to the International Streptomyces Project (ISP) regulations, after culturing for 14 days at 28 ° C, the morphological characteristics of the spores were spherical, and the spores of the spores were spherical. The surface was smooth and the aerial mycelia grow and branched relatively well and did not divide under normal conditions. The mycelial growth was good in the medium except glycerol-asparagine agar medium and tyrosine-agar medium, and the formation of engraftment spores was also abundant. The spores were spherical in size, 0.8-1.0 x 0.8-1.0 ㎛, usually 15-20 chains.

2) Cultural characteristics according to various media conditions

After culturing at 28 ℃ for 21 days under various ISP medium conditions, the culture characteristics of the strain were investigated, and the results are shown in Table 1. Growth of this strain was good in the medium except glycerol-asparagine agar medium and tyrosine-agar medium. The color of the mycelia was gray in most of the medium, the color of the back of the medium was brown, and the water-soluble pigment was colorless in all of the medium.

Cultural Characteristics According to Various Media Conditions Type of badge Growth and Color of Parasitic Hyphae Growth and color of substrate mycelia Color on the back of the badge Water-soluble pigment Growth color Growth color Earthy brown Colorless Yeast-malt agar medium (ISP2) Good grey Good Brown Taupe Colorless Oatmeal Agar Medium (ISP3) Good grey Good Brown Brown Colorless Inorganic salts-starch agar medium (ISP4) Good grey Good Light brown Brown Colorless Glycerol-asparagine agar medium (ISP5) Bad Yellowish white Bad Bad Bad Colorless Peptone-Yeast Agar Medium (ISP6) usually Yellowish white Bad Light yellow Light brown Colorless Tyrosine Agar Badge (ISP7) Bad Bad Bad Bad Bad Colorless Bennet Agar Badge Good grey Good Brown Brown Colorless Nutritional Agar Medium usually grey Bad Light yellow Light yellow Colorless

3) Physiological characteristics

Physiological characteristics of the strain were cultured for 21 days at 28 ° C. using Schilling-Gurtrib agar medium. The results were summarized in Table 2.

Physiological Characteristics Survey item Starch hydrolysis positivity Gelatin liquefaction voice Water soluble pigments voice Milk Peptonation positivity Nitrate reducing power positivity Melanin Pigment Generation voice Scheme Milk Hydrolysis positivity Cell Wall DAP Analysis L.L type

4) Carbon Availability

Carbon availability of this strain was investigated after incubating at 28 ° C. for 14 days using a Schilling-Gutribe agar medium. The results are shown in Table 3.

This strain used D-glucose, D-fructose, inositol, raffinose, and D-gyros among 11 carbon sources disclosed in Table 3.

Sugar availability D-glucose positivity L-Arabinose voice D-fructose positivity Galactose voice Inositol positivity Mannitol voice Raffinos positivity Rhamnos voice Sucrose voice D-gyros positivity Cellulose voice

5) Cell composition

The cell wall component of the strain was investigated by Becker's method (B. Becker et al ., Applied Microbiology 12, 421-423 (1964)) and the result was the diaminopimellic acid, a cell hydrolysis component of the strain. Was type LL.

As a result of examining the above morphological, culture, physiological properties, sugar availability, and cell components, the strain was identified as Streptomyces spis strain, and the strain was named Streptomyces spis KK90378. The inventors of the present invention were deposited on April 28, 2000 to the strain deposit institution of the Korea Institute of Science and Technology Biotechnology Center Genetic Resources Center and received the strain number of KCTC-18014P.

It is a general property that actinomycetes are not constant but easily changed naturally or artificially. As in the case of Streptomyces sp. KK90378 (KCTC18014P) strain, its properties are likely to change. Therefore, the present invention streptomyces spesis KK90378 (KCTC18014P), as well as mutant strains derived from this strain (natural mutation or mutant strain), a strain newly produced by a conjugate or genetic engineering method oleamide substance It is understood that all of these are included in the present invention.

2. Cultivation of Strains

The strain is cultured in a medium containing nutrients that can be used by ordinary microorganisms. Glucose and soluble starch are used as carbon sources, and soybean meal and yeast extracts as nitrogen sources. extract, bee extract was used. In addition, dipotassium hydrogenphosphate (dipotalssiumhydrogenphosphate) was used, and cultured using calcium carbonate (calcium calbonate) for pH control. The culture method was shaken under aerobic conditions, and the culture temperature was 28 ℃. In addition, in the case of culture period or shaking culture, the production of active substance reached the highest by culturing for 7 days.

3. Isolation and Purification of New Compounds

The active material is obtained by culturing the strain under the culture conditions as described above. Since the active substance is present only in the culture solution, the culture solution is centrifuged to separate the culture supernatant and the cells, and then the extraction and purification is efficiently performed by the method described below. That is, since the compound to be displayed is a fat-soluble substance, the culture solution is extracted by adding an organic solvent such as ethyl acetate.

After evaporating the solvent from the mixture containing the obtained active ingredient, silica gel column chromatography is performed using a solvent of chloroform: methanol = 100: 1-1: 1 (V / V). The obtained active site was again subjected to column chromatography using chloroform: methanol = 95: 5 (V / V). The obtained active site is then subjected to preparative thin layer chromatography (prep. TLC) using a solvent of chloroform: methanol = 98: 5 (V / V). Repeat the above to obtain a single product. Identification of single material was confirmed by TLC and gas chromatography.

For structural analysis, ¹ H-NMR (500 MHz), ¹³ C-NMR (125 MHz) and DEPT spectra were measured with JEOL JNM-LA400, GC-MS was HP5890GC / JEOL, and electrosprayionization mass spectroscopy (ESIMS) was JEOL JMS-AX505WA The UV spectrum (Shimazu UV-260) measured methanol as a solvent.

Oleamide produced by the Streptomyces sp. ₁₈ H ₃₅ ON was determined, and the structure was confirmed by ¹ H, ¹³ C, and heteronuclear multiple bond coherence (HMBC).

The physicochemical characteristics of the other active substances oleamides are as follows.

1) Appearance of substance: Colorless oil

2) Molecular Weight: 280

3) Molecular Formula: C ₁₈ H ₃₅ ON

4) Mass spectrometry (M + H): 280 [see FIG. 5]

5) UV absorption spectrum (UV λ max (MeOH)): 208 nm

6) nuclear magnetic resonance (NMR) absorption spectrum: a CD ₃ OD as solvent and TMS (Si (CH _{3) 4),} hydrogen nuclear magnetic resonance ⁽¹ H-NMR) spectra and carbon nuclear magnetic resonance ⁽¹³ to a standard C-NMR) spectra were examined and shown in FIGS. 6 and 7, respectively.

7) Solubility

Soluble: Acetone, Chloroform, Ethyl Acetate, Hexane, Methanol

Insoluble: water

8) Thin layer chromatography: Rf value

Adsorbent: Merck Silica Gel 60F 254

Developing solvent: CHCl ₃ : MeOH = 10: 1 (V / V)

9) Chemical Structural Formula-

In the present invention, the immobilized cells and enzymes are one of attempts to artificially improve the cells or the enzymes produced by the cells to be suitable for the purpose of use, and to have the cells or catalytically active enzymes capable of producing useful substances. It is immobilized as it is to make it insoluble in water. Immobilization of cells or enzymes increases the stability of heat, pH, organic solvents, denaturants, etc., and can be used continuously for a long time, thus reducing the cost of producing useful materials. In addition, the catalyst density is increased, the reaction speed is increased, the reaction time is shortened, and there is no need to remove cells or enzymes from the reaction product, thereby improving purity and yield.

Therefore, Streptomyces sp. Through a comprehensive method of forming a gel from the polymer dissolved state using the algaic acid, a seaweed polysaccharide as a carrier. KK90378 (KCTC18014P) cells can be immobilized.

Example 1 Microbial Culture

PH7 in medium containing 20% glucose, 10% soluble starch, 25% soybean wheat, 1% gravy, 4% yeast extract, NaCl 2%, K ₂ HPO ₄ 0.25%, CaCO ₃ as seed and production medium Used after adjusting to .2. 50 ml each of two 500 ml baffle flasks was sterilized for 20 minutes at 121 ° C., inoculated with agar piece of strain and shaken at 28 ° C. and 150 rpm for 2 days. The culture was used as the species culture of the incubator. A 2 L baffle flask containing 400 ml sterilized at 121 ° C. for 20 minutes was seeded with 2.5% of the seed culture solution and incubated at 28 ° C. at 140 rpm for 7 days. The results showing the cell growth and oleamide production by the number of culture days are shown in FIG.

Example 2 Isolation and Structure Determination of Active Substances from Culture

The culture filtrate obtained in Example 1 was centrifuged at 5,000 × g for 30 minutes to separate the culture supernatant and the cells, and the supernatant was extracted by adding the same amount of ethyl acetate. The ethyl acetate extract was concentrated and dissolved in methanol, and silica gel column chromatography was performed using a solvent of chloroform: methanol = 100: 1-1: 1 (V / V). TLC analysis with the standard material showed the same Rf value, and the active fractions 95: 5① and 95: 5② were developed in the coloring reagent dragendorf reagent [Fig. 4].

The obtained active site was prep. Using chloroform: methanol = 98: 5 (V / V) solvent. Thin layer chromatography is carried out in solvent conditions chloroform: methanol = 98: 2 (V / V). The band appeared up to ①- 이중, and the band ② and ③, in which the active ingredient is present in high concentration, were added together and the solvent condition hexane (ethyl) = ethyl acetate = 4: 1 (V / V) was prep. TLC is performed. Bands ②, ④, and ⑤ of the eight bands were found to be active, and TLC was carried out in the solvent condition chloroform: methanol = 10: 1 (V / V) together with the standard material. The same spot was shown. Oleamides also contain positively charged amino groups, which can be separated and purified from other anionic charges using ion exchange resins.

Example 3: Streptomyces sp. Immobilization of KK90378 (KCTC18014P) Cells

2.5% sodium alginate solution and Streptomyces sp. KK90378 (KCTC18014P) cell cultures were mixed at a 1: 1 ratio. In this case, 0.1M CaCl ₂ was added to give solidity to sodium alginate, and the mixture was used after being left at room temperature for 15 minutes to form a semi-transparent spherical gel. Immobilized cells were stored and used in a 4 ℃ refrigerator.

As described above, the present invention can efficiently produce oleamide by culturing the strain to produce oleamide, a bioactive substance that induces sleep, or by immobilizing the cells on a carrier.

Claims (7)

New strain producing oleamideStreptomycessp. KK90378 (KCTC18014P). Culturing the strain producing oleamide; Separating the supernatant from the culture solution; And extracting and purifying the supernatant with an organic solvent. The method of claim 2, wherein the strain that produces the oleamide isStreptomycessp. KK90378 (KCTC18014P). The method of claim 2, wherein the organic solvent is selected from the group consisting of hexane and ethyl acetate. The method of claim 2, wherein said strain is immobilized using a seaweed polysaccharide as a carrier. The method of claim 2, wherein the purification is carried out by using an ion exchange resin. The method of claim 2, wherein the purification is performed using silica gel.