JP2769996B2 - Production method and use of thiotroposin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing thiotroposin using a microorganism and a use of thiotroposin. More specifically, a method for producing thiotroposin by culturing a microorganism belonging to the genus Caulobacter , isolating and collecting thiotroposin from the obtained culture, and a fish disease antibacterial agent containing thiotroposin as an active ingredient,
It relates to an anti-microalgae agent or an anti-allergic agent.

[0002]

2. Description of the Related Art Thiotroposin is a 7-membered hetero compound containing a sulfur element, and is extremely rare among various antibiotics produced by microorganisms, and is a substance that can be expected to have various biological activities in addition to antibacterial activity. . Conventionally, thiotroposin-producing microorganisms have been used in soil bacteria Pseudomonas
Pseudomonas troposulfenii PB-5
020) (JP-A-59-205994), Pseudomonas bacteria
( Pseudomonas sp.CB-104) (K.Kintani, H.Ono, S.Tsub
otai, S. Harada, H. Okazaki: J. Antibiotics, 37 , 1294
-1300 (1984) and S. Tsubotani, Y. Wada, K. Kamiya,
H.Okazaki, S.Harada: Tetrahedron Letters, 25 , 419-4
22 (1984)).

[0003] However, it is not known at all whether or not thiotroposin is produced in other bacteria, actinomycetes, molds, and the like, and no biological activity of thiotroposin other than the antibacterial activity is known.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for producing thiotroposin using a microorganism belonging to the genus Caulobacter and having thiotroposin-producing ability, and to provide a novel use of thiotroposin.

[0005]

Means for Solving the Problems The present inventors have focused on marine microorganisms as new microbial resources, especially those that coexist with unexplored marine microalgae, and have separated many microalgae from the coastal waters of Japan. We isolated a large number of bacteria coexisting in the microalgae and worked diligently to search for and develop new microorganisms capable of producing thiotroposin. As a result, a novel microorganism having a thiotroposin-producing ability was found among the bacteria coexisting with these microalgae, and thiotroposin was successfully collected by culturing the microorganism. Further, the inventors have paid attention to the unusual chemical structure of thiotroposin, and have found that thiotroposin has a novel biological activity of a fish disease antibacterial activity, an anti-microalgae activity and an anti-allergic activity, thereby completing the present invention.

That is, the present invention is a method for producing thiotroposin, comprising culturing a microorganism belonging to the genus Caulobacter and having the ability to produce thiotropins in a medium, and collecting thiotroposin from the resulting culture. Here, microorganisms belonging to the genus Caulobacter and having the ability to produce thiotropin include Caulobacter sp.
K654 strain.

Further, the present invention relates to a Caulobacter sp. PK654 strain having a thiotroposin-producing ability. Furthermore, the present invention is a fish disease antibacterial agent, an anti-microalgae agent or an anti-allergic agent containing thiotroposin as an active ingredient. Hereinafter, the present invention will be described in detail.

[0008] The thiotroposin of the present invention can be obtained by culturing a microorganism belonging to the genus Caulobacter in a medium and collecting from the resulting culture. The microorganism used in the production method of the present invention may be any microorganism as long as it belongs to the genus Caulobacter and has a thiotropin-producing ability. For example, a PK654 strain isolated from the natural world by the present inventors is exemplified. can do.

The PK654 strain is a microorganism isolated by the present inventors from seawater in the coastal sea area of Saiki City, Oita Prefecture as a bacterium coexisting with microalgae. The bacteriological properties of the marine bacterium PK654 strain capable of producing thiotroposin are as follows.

[0010] 1. Morphological properties (1) Cell morphology (see Fig. 1) Bacillus (2) Gram stainability-(3) Stalk (see Fig. 2) + Formation position pole number 1 (4) Spores-(5) Motility + (6) Flagella (see Fig. 3) Physiological properties (1) Attitude to oxygen Aerobic (2) Oxidase + (3) Catalase + (4) OF test-(5) NaCl-requiring growth on medium without NaCl-Growth on medium with 0.05% NaCl- Growth on medium supplemented with 2% NaCl + Growth on medium supplemented with 4% NaCl + (6) Reduction of nitrate-(7) Starch degradation-(8) Formation of water-soluble pigment + (black-brown) (9) Carbon compound 2. Assimilation L-arabinose-ribose + glucose + galactose + mannose + lactose-L-proline + acetate weak butyrate + sodium pyruvate + casamino acid + (10) quinone Q-10 3. GC content of intracellular DNA (mol%) 58 4. Isolation source The marine bacterium PK654 was selected and isolated from bacteria coexisting with microalgae isolated from seawater along the coast of Saiki City, Oita Prefecture.

The tests for the above-mentioned morphological properties and physiological properties were carried out by the following methods. (1) Cell morphology, spores, Gram stain, motility, flagella, stalk
For morphological properties such as (stalk) and physiological properties such as attitude to oxygen, formation of oxidase, catalase and water-soluble dye, use Marine Agar 2216 (manufactured by Difco) at 25 ° C.
For a certain period of time, and performed according to a conventional method (mainly Kazuo Komagata: classification and identification of microorganisms (below), (revised edition, edited by Takeharu Hasegawa), Gakkai Shuppan Center, P.99-161 (1985)). did).

(2) The OF test was performed using MOF (E. Leifson: J. Bact
eriol., 85 , 1183 (1963)). (3) The NaCl requirement is determined by JSPoindexter: Bacteriol. Rev.,
28 , 231 (1964). However, the medium is Hutne
r's mineral base (Coen-Bazire G., WRSistrom, RYS
tanier: J.Cell.Comp.Physiol., 49 , 25 (1957)) 20ml
Was used.

(4) Nitrate reduction is performed by JSPoindexter: Bacte
riol. Rev., 28 , 231 (1964). However,
The medium to which Hutner's mineral base 20 ml was added was used. (5) Starch degradation was performed by streaking the test bacteria on Marine Agar2216 (manufactured by Difco) plate medium supplemented with 1% soluble starch, culturing at 25 ° C for 5-7 days, pouring Lugol's solution over the entire surface of the plate, Those that produced a zona pellucida just under or around the moss were judged as positive.

(6) The assimilation property of the carbon compound is determined by JSPoindexte
r: Bacteriol. Rev., 28 , 231 (1964), using the following modified medium. Utilization test medium Peptone (Difco) 0.05 g Yeast extract 0.05 g Agar 10.0 g Hutner's mineral base ^* 20 ml 100% artificial seawater 980 ml pH-free (8.2) Culture conditions: 25 ° C, 14 days ^* Composition of Hutner's mineral base Nitrilotriacetic acid _{10.0 g MgSO 4 · 7H 2 O} 14.45g CaCl 2 · 2H 2 O 3.335g (NH 4) 6 Mo 7 O 24 · 4H 2 O 9.25mg FeSO 4 · 7H 2 O 99.0mg Metals "44" 50.0ml (7) GC content of quinones and intracellular DNA Hideko Yabuuchi et al .: Bacterial identification method accompanying new taxonomy (1987)
(Year) (Nane Publishing).

The above mycological properties are described in Bergey's Manual of
Systematic Bacteriology, Vol.1 (NRKieg, JG Holt
Ed., Willams & Wilkins, Baltimor (1984)) and Berge
y's Manual of Determinative Bacteriology (9th edition,
JGHolt, NR Kieg, PHA Sneath, JTStaley, S.
Edited by T. Williams, Williams & Wilkins, Baltimor (199
As a result of comparison with 4)), the marine bacterial strain PK654 was identified as a bacterium belonging to the genus Caulobacter . In addition, we attempted to identify Caulobacter species. As a result, the marine bacterium PK654 is similar to Caulobacter maris in terms of NaCl requirement and 4% NaCl resistance, but differs in sugar, amino acid, butyrate, sodium pyruvate assimilation, pigment production, and the like. The GC content of the intracellular DNA was 58 mol%, which was 62% of the genus Caulobacter.
Because it is smaller than ~ 67 mol%, this PK654
The strain is considered a new species of Caulobacter bacteria,
Caulobacter sp. PK6
54.

This marine bacterium, Caulobacter sp. PK654, has been deposited with the National Institute of Bioscience and Biotechnology, Accession No. FERM BP-5080 (April 19, 1995). . Further, the microorganism used in the method for producing thiotroposin of the present invention is not limited to the Caulobacter sp. All can be used in the present invention.

The method for culturing the microorganism can be performed in principle according to the method for culturing the microorganism. The medium used for the culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Caulobacter, and any of various synthetic media, semi-synthetic media, and natural media can be used. As the medium used for the culture, any of known media can be used. For example, glucose, galactose, mannose, ribose, pyruvate, peptone, casamino acid, organic acids and the like can be used alone or in combination as a carbon source. Nitrogen sources include yeast extract, peptone, meat extract, casein,
Organic nitrogen sources such as yeast extract, amino acid solution and urea or inorganic nitrogen sources such as ammonium sulfate, ammonium chloride and sodium nitrate can be used alone or in combination. As the inorganic salt, magnesium sulfate, magnesium chloride, sodium phosphate, potassium phosphate, potassium chloride, sodium chloride, calcium chloride and the like can be used in an appropriate combination. The pH of the medium is preferably in the range of 7.4 to 7.6, but may be around neutral. The culture temperature of the microorganism is 10 to 35 ° C, preferably 15 to 25 ° C for 5 to 7 days,
It is performed by aeration stirring, shaking culture or static culture. The thiotroposin of interest is produced and accumulated by the above culture. The above various culture conditions can be appropriately changed according to the type and characteristics of the microorganism to be used, external conditions, and the like, and the optimal conditions are selected and adjusted from the above range according to each.

Isolation of thiotroposin produced by the above-mentioned cultivation is carried out according to a general method for collecting fermentation products when the accumulation of the substance is maximized, by the difference in solubility between thiotroposin and other impurities, Any of means utilizing differences in adsorption affinity, differences in molecular weight, and the like can be used, and these methods can be performed alone or in appropriate combination, and in some cases, repeated use.

For example, as a method for separating a culture solution and cells from a culture obtained by culturing, conventional methods such as centrifugation and filtration can be used, but centrifugation is preferred. After the culture solution obtained by centrifugation is filtered through a glass filter, separation is repeated by appropriately combining gel filtration, adsorption chromatography, hydrophobic chromatography, ion exchange chromatography, etc., and reconstituted from an organic solvent such as methanol. Thereby, thiotroposine can be isolated.

Also, a separation method based on the solubility of a substance, that is, a method of extracting with an organic solvent using a difference in solubility of the substance due to pH is suitable. The centrifuged culture solution is acidified with an acid, and extracted with an organic solvent such as chloroform. After extracting this extract with a basic buffer, the basic extract is acidified again with an acid and extracted with an organic solvent such as chloroform. The extract is concentrated to dryness under reduced pressure to obtain crude crystals of thiotroposin. Similarly to the above, purified thiotroposin can be obtained by reconstitution with an organic solvent such as methanol.

In general, it is known that thiotroposin exhibits an antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, yeast, and filamentous fungi (see J. Antibio, supra).
ics, 37 , 1294-1300, (1984) and JP-A-59-205994). However, no other biological activity is known. The present inventors have paid attention to the unusual chemical structure of thiotroposin, and as a result of intensive studies on new physiological activities of thiotroposin other than those described above, Table 1, Table 2, and FIG.
As shown in the above, it has been found that it has an antibacterial activity against fish disease, an anti-microalgal activity, an anti-allergic activity and the like.

Regarding the antibacterial activity and the anti-microalgal activity of fish disease,
As test bacteria, for example, Staphylococcus aureus , Escherichia coli ( Esche
richiacoli ), Pseudomonas aeruginosa ( Pseudo)
monas aeruginosa ) and other fish pathogenic bacteria such as hamachi and eel (Enterococcus seerioliisida ( En)
terococcus seriolicida )), diatoms ( Skeletonema costatum ), which are red tide forming microalgae, and raffid algae ( Heterosigma
akashiho ), Dinoflagellate ( Gymnodinium nagasakiense )
These microorganisms can be examined by measuring the minimum growth inhibitory concentration (MIC) of thiotroposin. The measurement of MIC may use a known method.

The antiallergic activity of thiotroposin can be examined by measuring mast cell degranulation inhibitory activity. That is, after the test solution is added to the IgE-sensitized mast cell suspension and incubated, a degranulation-inducing agent is further added, the amount of histamine contained in the sample solution is quantified, and this is converted into the histamine release inhibition rate. Can be obtained by

When thiotroposin is administered as a fish disease antibacterial agent, diseases caused by various fish disease fungi are prevented or treated,
It can be aimed at reducing mortality. For example, Streptococci caused by pathogenic bacteria of the genus Streptococcus such as cultured yellowtail, yellowtail, and eel, and Enterococcus seriolicida ( Richi Kusuda, Kenji Kawai, Toshio Toyoshima, Isao Komatsu: Japan) Water Journal, 42 , 1345-1352 (1976), R. Kusuda, K. Kawa
i, F. Salati, CRBanner, JLFryer: Int. J. Syst.
Bacteriol., 41 , 406-409 (1991)).

The method of administration is such that lactose or the like as the base material of the preparation contains 5 to 10% of thiotroposin to form a powder or granules, and these preparations are mixed at a feed rate of 0.1 to 0.5 g / kg of fish body weight / day, The mixture is uniformly mixed with a feed such as a consolidation bait and usually administered for 5 to 7 days. When thiotroposin is used as an anti-microalgal agent, thiotroposin may be used directly as it is, but in general, it is dissolved or dispersed in a suitable liquid carrier, or mixed or adsorbed with a suitable powder carrier, if necessary. Further add emulsifiers, dispersants, stabilizers, suspending agents, spreading agents, penetrants, wetting agents, stabilizers, etc. to these,
It is used as a formulation for emulsions, oils, wettable powders, powders and the like.

The concentration of the active ingredient is generally selected from emulsion, wettable powder,
For oils, 0.01 to 10% is appropriate, and for granules and powders, 0.1 to 10% is appropriate, but these concentrations may be appropriately adjusted depending on the purpose of use. As a liquid carrier used in the preparation, for example, water is usually used as a base, but alcohols such as methanol and ethanol, acetone, ketones such as methyl ethyl ketone, dioxane, ethers such as ethylene glycol, benzene , Solvents such as aromatic hydrocarbons such as toluene, xylene, solvent naphtha, and methylnaphthalene, and acid amides such as dimethylformamide, and one or a mixture of two or more of these can be used. .

As the powder carrier, mineral powders such as talc, kaolin, bentonite, zeolite, slaked lime, diatomaceous earth, acid clay, alumina, silica gel and the like are used, and one or more of these may be mixed. To use. Examples of the surfactant used as a spreading agent, an emulsifier, a penetrant, a dispersant, a solubilizing agent, and the like include a higher alcohol sulfate ester and an alkylene oxide surfactant.

When thiotroposin is administered as an antiallergic agent, the subject to which it is administered is not particularly limited. For example, it can be used for the purpose of preventing or treating each allergic disease (for example, bronchial asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, etc.). The method of administration may be oral or parenteral, and oral administration includes sublingual administration. For parenteral administration,
Injections (eg, intravenous injection, subcutaneous injection, intramuscular injection, infusion, etc.), suppositories, creams, cataplasms and the like are included. In addition, the dose varies depending on whether the animal or human, the age, the route of administration, and the number of administrations, and can be widely varied. In this case, the effective amount of thiotroposine and the effective amount to be administered as a composition of a suitable diluent and a pharmacologically usable carrier are 0.1 μg to 50 mg / day / kg, and may be from once to several times a day. It is administered separately.

When the antiallergic agent of the present invention is orally administered, tablets, granules, fine granules, powders, capsules and the like applied thereto are usually used in their compositions as binders generally used in pharmaceutical preparations. , Including excipients, excipients, lubricants, disintegrants, wetting agents. In addition, the liquid preparation for oral use may be in any form such as a solution for internal use, a suspension, an emulsion syrup and the like, and may be a dry product to be redissolved when used. Further, the composition comprises
It may contain either additives or preservatives.

When the antiallergic agent of the present invention is administered parenterally, it contains additives such as a stabilizer, a buffer, a preservative, and an isotonic agent, and usually contains a unit dose ampule or a multidose container or tube. Provided in a state. The composition comprises:
The powder may be reconstituted with a suitable carrier for use, for example, sterile water containing no heat.

[0031]

The present invention will be described below in detail with reference to examples. However, the present invention is not limited to these examples. [Example 1] Production of thiotroposin (1) Marine broth (Di) from a slant culture medium of Caulobacter sp. PK654 strain (Marine Agar 2216, manufactured by Difco)
fco) L-shaped test tube (outer diameter 18 mm, containing 20 ml of a medium (pH 7.4) composed of 3.74%, glucose 1%, and distilled water
One loopful was inoculated into a sample (length: 180 mm, height: 95 mm) and shake-cultured at 20 rpm at 45 rpm to obtain a seed culture. 0.1 ml of this seed culture was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the above medium, and cultured at 20 ° C. for 5 to 7 days.

[0052] 52 L of the culture in the flask cultured by the above method was collected, centrifuged at 8,000 rpm at 4 ° C., and the supernatant of the culture was filtered through Whatman GF / C (pore diameter: 0.45 m, diameter: 4.7 cm). Thereafter, the filtrate was separated with Sephadex LH-20 (filler 5
(100 g, manufactured by Pharmacia), and eluted with distilled water. Then, a black-brown eluate was fractionated. Further, the eluate was adjusted to pH 2 with a 1N-HCl aqueous solution, and then Sephadex LH-20 (filler 500
g, manufactured by Pharmacia) to obtain 54 L of a yellow eluate. The yellow eluate was applied to Seppak Bag _C18 (filler 5 g, Waters), eluted with methanol, and concentrated to dryness under reduced pressure to obtain 0.42 g of a red precipitate. Again, this precipitate was dissolved in distilled water and adjusted to pH2.
-20 mg (200 g, manufactured by Pharmacia) and Seppak Bag C ₁₈ (filler: 5 g, manufactured by Waters), followed by recrystallization from methanol to isolate 351 mg of yellow needle crystals.

[Example 2] Production of thiotroposin (2) In the same manner as in Example 1, 20 L of the culture supernatant obtained by centrifugation was distributed with 10 L of ethyl acetate, and the aqueous phase was diluted with 1N-HCl aqueous solution.
After adjusting to H2, the mixture was extracted with ethyl acetate. After the ethyl acetate extract was partitioned with a 2% aqueous sodium carbonate solution, the aqueous phase was again adjusted to pH 2 with a 1N aqueous HCl solution and extracted with ethyl acetate. The ethyl acetate extract was dehydrated with anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and then reconstituted with methanol to obtain 112 mg of yellow needle crystals.

The physicochemical properties of the yellow needle-like crystals obtained as described above are shown below. Mass spectrum (m / z): 212 [M] ⁺ , 184 [M-CO] ⁺ , 168 [M-
CO ₂ ], 140 [M-CO ₂ -CO] ⁺ , 96 [M-CO ₂ -CO-CS] ⁺ Elemental analysis: C, 45.33; H, 1.96; O, 22.64; S,
30.02 Melting point: 222-224 ° C Ultraviolet absorption spectrum: λ (MeOH) 216 (ε25400), 245 (11
300), 307 (16300), 356 (6200), 452 (2100) nm Infrared absorption spectrum: IR (KBr) 3061, 2926, 2857, 163
3, 1597, 1537, 1460,1373, 1313, 1242, 1109, 1072,
1014, 889, 827, 692, 652, 578,470cm -1 1 HNMR nuclear magnetic resonance spectrum (270 MHz, external standard TMS,
Solvent CHCl ₃ ): δ 7.12 (1H, d, 9Hz), 7.44 (1H, d, 12H
z), 7.45 (1H, dd, 9, 12), 16.7 (1H, s) ppm ¹³ C NMR nuclear magnetic resonance spectrum (67.5 MHz, external standard TM
S, solvent DMSO): δ 182.5, 170.6, 167.7, 150.1, 137.6,
137.5, 133.7, 120.0 ppm Color reaction: Positive in 2% ferric chloride and iodine-azide reaction test.

As a result of determining the structural formula from the above physicochemical properties, the structural formula was consistent with thiotroposin produced by Pseudomonas bacteria (S. Tsubotani, Y. Wada, K. Kamiya, H. Okaza)
ki, S.Harada: Tetrahedron Letters, 25 , 419-422 (198
Four) ) . From the above results, Kaurobacter SP P
The yellow needles of Examples 1 and 2 produced by strain K654 were identified as thiotroposin.

Example 3 Antibacterial Activity of Fish Disease The minimum inhibitory concentration (MIC) was measured according to Chemotherapy, 29 , 76-79 (1981) of the Japan Society of Chemotherapy.
Staphylococcus aureus
s aureus ) IFO 12732, Escheric
hia coli ) IFO14237, Pseudomonas aeruginosa IFO 12582 culture is Muller
-A medium (pH 6.0) obtained by adding 1.7% agar to Hinton Broth (manufactured by Difco), and a strain of Enterococcus seriolicida ( Enterococcus seriolicida ; strain YT-3: same strain as ATCC 49456) is used as Brain. Heart Infusion Broth (Difco)
And a medium (pH 6.0) supplemented with 1.5% agar.

The concentration of thiotroposin was adjusted as follows. After dissolving 5.0 mg of thiotroposin in 6 ml of dimethylsulfoxide, sterilizing by filtration through a membrane filter of Dimex 25 (pore size: 0.2 μm, diameter: 25 mm, manufactured by Millipore), and adding 5 ml of sterilized distilled water to the drug. It was used as a stock solution, which was further diluted with sterile distilled water to prepare a drug diluent by a two-fold dilution method.

The determination of MIC was carried out using Staphylococcus aureus IFO12732, Escherichia coli IFO 14237, and Pseudomonas aeruginosa IFO.
At 12582, after 18 hours of culture at 37 ° C, enterococcus serioliciida ( Enterococcus seriolici)
da ) For YT-3 strain (ATCC 49456), the cultivation was performed at 25 ° C. for 3 days.

The results of examining the antibacterial activity of thiotroposin by the above method are shown in Table 1. As shown in Table 1, thiotroposin exhibited strong antibacterial activity. Among them, a new biological activity of thiotroposin is the fish pathogenic bacterium Enterococcus seriolisisida ( Enterococcus ser
iolicida ; YT-3 strain: the same strain as ATCC 49456) was found to have strong antibacterial activity for the first time.

[0040]

[Table 1]

Example 4 Antimicroalgae Activity The inoculated amount of the test microalgae (see Table 2) was determined in the microalgae medium.
It was prepared to be 30 cells / ml. The microalgal medium is f
/ 2 medium (RRLGuillard and JHRyther: Can.J.Micro
Biol., 8 , 229-239 (1962)) and thiotroposine 1
5 times at 10-fold dilution with the drug concentration of the 00 μg / ml medium as the upper limit.
An f / 2 medium with different drug concentrations was prepared. MIC judgment
The culture was performed after culturing for 7 days at 20 ° C., 12 hours light-12 hours dark. For the determination, the growth of microalgae was measured using a fluorometer (Siteflow 2300).
Fluorometer, manufactured by Perceptive Limited of Japan), the excitation wavelength is 460nm, and microalga chlorophyll a
The measurement was performed by measuring the fluorescence intensity at a wavelength of 645 nm based on the above.

The results of examining the anti-microalgal activity of thiotroposin by the above method are shown in Table 2. As shown in Table 2,
Antimicroalgal activity was found as a new biological activity of thiotroposin.

[0043]

[Table 2]

Example 5 Antiallergic Activity Rat peritoneal mast cells were prepared by the method of Nakagomi (Nakagomi
et al .: J. Antibiotics, 43 , 5, 462-469 (1990)). Wistar rats intraperitoneally, 20 ml of Tyrode solution
And ascites was removed with a pipette. The collected ascites was centrifuged at 4 ° C. and 100 × g for 12 minutes, and the precipitated cells were collected. The cells were suspended in 2 ml of Tyrode solution and the specific gravity was 1.06.
8 bovine serum albumin (BSA) saline 4
After centrifugation at 4 ° C. and 100 × g for 12 minutes, precipitated mast cells were collected. IgE was bound to mast cells by reacting with an anti-DNP mouse monoclonal IgE antibody at 37 ° C. for 1 hour, and the cells were sensitized. After washing several times with Tyrode solution, mast cells were found in Tyrode solution containing 0.2% BSA.
The suspension was suspended at 10 ⁶ / ml to obtain an IgE-sensitized mast cell suspension.

The measurement of mast cell degranulation inhibitory activity was performed at 1 × 10
A test solution was added to the IgE-sensitized mast cell suspension prepared at ⁶ cells / ml, and the mixture was incubated at 37 ° C for 5 minutes. Then, antigen (DNP-BSA) (200 ng / ml) + phosphatidylserine was used as a degranulation-inducing agent. (10 μg / ml) PBS (−) was added and incubated again at 37 ° C. for 10 minutes. The amount of histamine contained in the centrifuged supernatant at 1500 × g for 5 minutes was quantified by HPLC by post-column labeling with orthophthalaldehyde (OPA).

The histamine release inhibitory activity is expressed as an inhibitory ratio to the amount of histamine released by the degranulation-inducing agent, and was determined by the formula (1). Histamine release inhibition rate (%) = {1- (Hs-Hb) / (Hi-Hb)} × 100 (1) Hb; amount of histamine released when cells are incubated only with PBS Hi; The amount of histamine released when incubated with a degranulation-inducing agent in the absence of Hs; the amount of histamine released when cells are incubated with a degranulation-inducing agent in the presence of a test solution

The results of examining the activity of thiotroposin to inhibit mast cell degranulation by the above method are shown in FIG. It was revealed that thiotroposin has strong mast cell degranulation inhibitory activity. As shown in FIG. 4, antiallergic activity was found as a new biological activity of thiotroposin.

[0048]

According to the present invention, thiotroposine can be obtained. Thiotroposine has an antibacterial activity against fish pathogenic bacteria and an extremely strong antialgal activity against microalgae, and thiotroposin also has an antiallergic activity. Accordingly, thiotroposin can be used as a drug, a marine drug, a red tide inhibitor or a conversion material thereof, and is extremely useful in industry.

[Brief description of the drawings]

FIG. 1 is an optical micrograph (morphology of organism) showing the cell morphology of Caulobacter sp. PK654. (Magnification: 2,
000 times, one scale is about 1μm)

FIG. 2 is an optical micrograph (organism) showing the cell morphology of Caulobacter sp. PK654 having a stalk. The upper and lower photographs are from different fields of view.

FIG. 3. Caulobacter sp. PK with polar flagella
Light micrograph showing the morphology of 654 cells (organism morphology). (Magnification: 2,000 times, one scale is about 1μm)

FIG. 4 shows the mast cell degranulation inhibitory activity of thiotroposin.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C07D 327/04 C07D 327/04 (C12N 1/20 C12R 1:01) (C12P 17/14 C12R 1:01) (72) Invention Person Yoshikawa Nakawa 1-3-1 Higashi, Tsukuba City, Ibaraki Pref.Institute of Biotechnology and Industrial Technology, Institute of Industrial Technology (72) Inventor Shuichi Oka 1-3-1 Higashi 1-chome, Tsukuba, Ibaraki Pref. 72) Inventor Masahiro Asada 1-3-1 Higashi, Tsukuba City, Ibaraki Pref., Institute of Biotechnology, Industrial Technology Institute (72) Inventor Mami Inoue 960-144 Okami-cho, Ushiku-shi, Ibaraki Pref. (72) Inventor Kazuya Nakagomi Ibaraki 1-3-3 Higashi, Tsukuba City, Institute of Biotechnology, Industrial Technology Institute (56) References AM. CHEM. SOC. 114, 8479-8483 (1992) (58) Fields investigated (Int. Cl. ⁶ , DB name) C12P 17/00-17/18 C12N 1/20 BIOSIS (DIALOG) WPI (DIALOG)

Claims (3)

(57) [Claims] 1. A method for producing thiotroposin, comprising culturing a microorganism belonging to the genus Caulobacter and having the ability to produce thiotroposin in a medium, and collecting thiotroposin from the resulting culture. 2. A microorganism belonging to the genus Caulobacter and having thiotroposin-producing ability is a Caulobacter sp.
The method for producing thiotroposine according to claim 1, which is a K654 strain. 3. A Caulobacter sp. PK654 strain capable of producing thiotroposin.