CN1014527B - Prepn method of new antibiotic sr-1223 - Google Patents
Prepn method of new antibiotic sr-1223Info
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- CN1014527B CN1014527B CN 86104124 CN86104124A CN1014527B CN 1014527 B CN1014527 B CN 1014527B CN 86104124 CN86104124 CN 86104124 CN 86104124 A CN86104124 A CN 86104124A CN 1014527 B CN1014527 B CN 1014527B
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- antibiotic
- new antibiotic
- growth
- methyl alcohol
- bacterium
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Abstract
The present invention provides a new antibiotic SR-1223 and a preparing method thereof. The antibiotic which shows a great effect on inhibiting the growth of phytopathogenic filamentous bacteria of some plants is expected to be an agricultural antibiotic medicine or/and preparation. The antibiotic is obtained by culturing streptomycete sp. SR-1223 (Streptomycessp. SR-1223).
Description
The invention relates to new antibiotic SR-1223 and preparation method thereof.Microbiotic of the present invention is to be got by separation in the nutrient solution of the antibiotic SR-1223 production bacterium of streptomyces, is a kind of new antibiotic of not seeing bibliographical information.RS-1223 in this antibiotic SR-1223 and the corresponding Japanese patent application of the application is a same substance.
In recent years, microbiotic and also is widely used aspect agricultural chemicals not only aspect medical.The present inventor isolates microorganism and cultivates microbiotic in order to seek out the more more efficiently new antibiotic than in the past from multiple soil, however to these microbiotic make with extra care respectively, evaluation and Application and Development.
The present inventor once from Chinese soil, separated and the culture of soil actinomycete in extracted multiple new antibiotic.Successfully extracted specifically a kind of new antibiotic different again with above-mentioned microbiotic.
The object of the present invention is to provide a kind of new antibiotic, and provide a kind of and from the microorganism of streptomyces, separate, produce this antibiotic method.
The microorganism of using
The microorganism that is used for producing antibiotic SR-1223 of the present invention is a kind of bacterial classification that belongs to streptomyces, and it has the ability of producing antibiotic SR-1223.
Streptomycete SPSR-1223(Streptcmyces spSR-1223) (is designated hereinafter simply as " SR-1223 bacterial strain ") and is exactly a wherein example.This microorganism has above-mentioned characteristic, is suitable for producing antibiotic SR-1223 of the present invention, thereby can be used in effectively among the preparation method of the present invention.
Simultaneously, the nature of above-mentioned " SR-1223 bacterial strain " and artificial variation's bacterial strain thereof also all belong to the bacterial classification of streptomyces, and all microorganisms with production aftermentioned antibiotic SR-1223 ability all can be used method of the present invention.
Above-mentioned " SR-1223 bacterial strain " be from Chinese Zhejiang Province soil, separate and soil actinomycete, be deposited with China Committee for Culture Collection of Microorganisms on November 5th, 1986.Register on the books and be numbered CGMCC № 0117 in its preservation center.
" SR-1223 bacterial strain " has following character:
1. morphological specificity:
Cultivate this bacterial strain in the thalline analysis that contains glucose, yeast extract on substratum, carry out thalline then and separate dryly, use 6N hydrochloric acid again, add hot water and be situated between 20 hours in 110 ℃.When detecting the amino acid composition of cell walls, can find the L.L-diaminopimelic acid with Paper Chromatography.
This bacterial strain property in the shape of a spiral after flat board is cultivated forms the spore lock that is linked to each other by a plurality of spores.Its on starch, yeast nutrient agar, starch inorganic salt nutrient agar, aerial hyphae well-grown gray shape; On the tyrosine nutrient agar, can generate melanochrome, but the generation of soluble pigment is not remarkable.The utilization of carbon source sugar is except that sorbose, sorbyl alcohol, and is all very good.As seen from last, this bacterial strain belongs to streptomyces, and its proterties on various substratum is as follows.
2. the growth conditions on various substratum
In 27 ℃ of three weeks of cultivation, color is recorded and narrated according to " color designation is described dictionary " (Descri-ptive color names dictionary).
(1) sucrose nitrate nutrient agar
Growth is general
The medium b ash of aerial hyphae lacteous (oysten white)
2cb light beige (ivory tint) in the base
Soluble pigment does not have
(2) glucose aspargine nutrient agar
Growth is general
The a small amount of e grey of aerial hyphae (gray)
The bright orange brown of 3gc (light tan) in the base
The bright orange brown of the few 3gc of soluble pigment (light tan)
(3) glycerine aspargine nutrient agar
Well-grown
Aerial hyphae is enriched 4gc rose-colored (rose beige)
3lg fulvescent (light brown) in the base
The few 3lg fulvescent of soluble pigment (light brown)
(4) starch inorganic salt nutrient agar
Well-grown
Aerial hyphae is enriched 5fe dying embers look (ash)
The bright beige of 3ec (light beige) in the base
Soluble pigment does not have
(5) tyrosine nutrient agar
Growth is general
Aerial hyphae does not have
4Ni Chestnut (chestnut brown) in the base
Soluble pigment 4Pl Vandyke brown (deep brown)
(6) nutrient agar
Growth is general
Aerial hyphae does not have
2gc bamboo look (bamboo) in the base
Soluble pigment does not have
(7) yeast malt agar
Well-grown
Aerial hyphae does not almost have e grey (gray)
3ie light yellowish brown (cinnamon) in the base
The a small amount of 3lg fulvescent of soluble pigment (light brown)
(8) oatmeal nutrient agar
Poor growth
The a small amount of 3dc Natural color of aerial hyphae (natural)
2dc nature slice look (natural string) in the base
Soluble pigment does not have
(9) peptone yeast iron nutrient agar
Growth is general
Aerial hyphae does not have
3ie light yellowish brown (cinnamon) in the base
Soluble pigment 3ie light yellowish brown (cinnamon)
(10) starch yeast nutrient agar
Well-grown
Aerial hyphae is enriched e grey (gray)
3ie light yellowish brown (cinnamon) in the base
The a small amount of 3lg fulvescent of soluble pigment (light brown)
3. the usability of carbon source (general dagger-axe nutrient agar)
Growth conditions
L-arabinose +++
The D-wood sugar +++
D-glucose +++
D-fructose +++
Sucrose +++
Inositol +++
The L-rhamnosyl +++
Raffinose +++
D-N.F,USP MANNITOL sugar +++
Lactose ++
Melibiose ++
The D-semi-lactosi +++
The L-sorbose ±
D-maltose +++
The D-sorbyl alcohol ±
Salicin+
Contrast-
+++growth is very good
++ growth is better
+ growth
The growth of ± minute quantity
-do not grow
4. other physiological property
(1) suitable temp is 25～30 ℃
(2) the liquefaction feminine gender of gelatin
(3) solidifying of breast peptonizes the positive
(4) the starch hydrolysis positive
(5) it is positive that Mierocrystalline cellulose divides the feminine gender～plan that is situated between
(6) melanic generation is positive
(7) the nitrate reduction positive
(8) molten Jie of xanthine is positive
As mentioned above, this bacterial classification belongs to the streptomyces griseus genus.Press Bai Shi bacteriology identification handbook the 8th edition (Bergey ' s Manual of Determinative Bacteri-ology 8th edition) classification, this bacterial classification of deducibility belongs to Streptomyces griseosporeus, Streptomyces lute-ogriseus or its extremely near edge bacterium of streptomyces.
Cultivate and process for purification
With the above-mentioned production of antibiotics bacterium of streptomyces,, can cultivate " SR-1223 bacterial strain " by the production of antibiotics method of routine, and make antibiotic SR-1223 of the present invention.Training method both can be liquid culture, also can be solid culture.When industrialness is produced, in order to help cultivating, spore suspension or the nutrient solution of above-mentioned production bacterium should be inoculated on the substratum, the gas mixing that works of going forward side by side is mixed.
Nutrition source in the substratum be there is no special regulation, can make and contain carbon source, nitrogenous source and other nutrition source that is usually used in microorganism culturing in the substratum.Wherein carbon source can be starch, dextrin, glycerine, glucose, sucrose, inositol, N.F,USP MANNITOL etc.Nitrogenous source can be peptone, soyflour, meat extract, rice bran, wheat skin, urea, corn steep liquor, ammonium salt, nitrate, and other organic or inorganic nitrogenous compound.Then can suitably add some inorganic salts as for other nutrition source.The metal-salt of salt, phosphoric acid salt and potassium, calcium, zinc, manganese, iron and so on for example.Also can add in case of necessity move, plant, the mineral wet goods is as defoamer.
Culture condition such as temperature, time be there is no strict restriction, being suitable for the using growth of bacterium to be as the criterion, and to select the highest condition of SR-1223 output for well.For example, the PH scope of substratum can be 4～9, especially with near neutral for well, culture temperature should be at 25～35 ℃.Certainly the hydrogen ion concentration of the component of these cultivations, substratum, culture temperature, agitation condition etc. all should carry out suitable adjusting according to the kind of use bacterial strain and external conditions etc., to obtain best effect.
Culture by above-mentioned gained sets out, and just can make SR-1223 by some appropriate means.This is the ordinary method that is usually used in extracting meta-bolites a bit.For example, can be separately, make up or recycle SR-1223 and separate in the difference of aspects such as molten Jie's degree, ionic bond power, absorption avidity and molecular weight with impurity.Specifically: the SR-1223 major part is present in the culturing filtrate.Nutrient solution is adjusted to slant acidity, and the example example is adjusted to PH4.0, and the organic solvent with vinyl acetic monomer and so on extracts again, and the concentrated oily matter of gained is collected and the active differentiation of concentrated SR-1223 behind silica gel column chromatography (for example solvent is a chloroform-methanol).And then (for example filling agent is Nucle-osil5C to pass through high-speed liquid chromatography
18, developping agent is that methanol-water adds 1% diethylamine formic acid in mutually).Collect the active peak of SR-1223 and be adjusted to PH and be acid (as PH=4.0), concentrate the back with ethyl acetate extraction, clean, reconcentration, can obtain the coarse meal of SR-1223.At last again through high-speed liquid chromatography (as: High carbon ODS, solvent are methanol-water-1% diethylamine formic acid), collect the active peak of SR-1223, the samely concentrate, extract, clean, reconcentration, after lyophilize, get final product the pure product of SR-1223 of pale yellow powder shape.
As above its physico-chemical property of the SR-1223 of gained and biological property are as follows:
Shape: pale yellow powder
Melting point: 130 ℃ (decomposition)
Ultimate analysis: carbon 63.85%, hydrogen 8.56%
Molecular weight: 660(is according to the SIMS mass spectrum)
Specific optical rotation: (α)
25 D+ 10 ° (C=1.0 methyl alcohol)
Ultra-violet absorption spectrum:
270(579) (with reference to Fig. 1)
Infrared absorption spectrum: ν
KBr MaxCm
-13400,2900,1755,1700,1570,1450,1370,1270,1170,1030
Color reaction: aubepine-sulfuric acid reaction is positive
Molten Jie's property: be soluble in methyl alcohol, vinyl acetic monomer, acetone, chloroform, be insoluble in water, Skellysolve A, normal hexane, benzene.
Tlc analysis: (the 0.25mm silicon thin layer plate that U.S. Merck company makes)
Solvent Rf value
Chloroform: methyl alcohol=2: 1 0.64
Chloroform: methyl alcohol=10: 1 0.17
Vinyl acetic monomer: benzene=4: 1 0.10
Antimicrobial spectrum: adopt common agar plate dilution method, obtain minimum inhibitory concentration (MIC).
(fungi uses potato sucrose nutrient agar, bacterium to use broth agar culture medium) its result, SR-1223 is that the shape bacterium demonstrates very strong growth-inhibiting effect to plant-pathogenic, and conventional bacterium is not almost had the growth-inhibiting effect.
Test bacterium MIC(mcg/ml)
Botrytis cinerea bacterium 32
（Botrytis cinerea）
Thorn dish spore bacterium 62.5
（Colletotrichum lagenarium）
Pyricularia oryzae 62.5
（Piricularcia oryzae）
Snake spore chamber bacterium 62.5
（Ophiobolus niyabeanus）
Black-koji mould 62.5
（Aspergillus niger）
Alternaric bacteria 125
（Alternaria mali）
Aspergillus oryzae 125
（Aspergillus oryzae）
Point sickle spore bacterium 250
（Fusarium oxysporum）
Compare with other materials
In the UV spectrum, there is the known antibiosis of absorption peak to have cryomycin (Cry-omycin), homomycin (Homomycin), naphthyridinomycin (Naphthyridomy-cin), subliomycin (Subliomycin), tuberactinomycin (Juberac-tinomycin) etc. at the 270nm place.But these microbiotic all demonstrate germ resistance to the bacterium class, have significantly different with this microbiotic in this.And evident difference is also arranged on other physico-chemical properties, so can inference SR-1223 be a kind of new antibiotic.
SR-1223 is owing to being that the shape bacterium demonstrates very strong growth-inhibiting effect to above-mentioned plant-pathogenic, so be expected to become a kind of agricultural antibiotic medicament.
Experimental example:
Proportioning with glucose 2%, Zulkovsky starch 1%, gravy medicinal extract 0.1%, dry yeast 0.4%, soyflour 2.5%, salt 0.2%, potassium primary phosphate 0.005% is made substratum." SR-1223 bacterial strain " is inoculated in this substratum, placed 28 ℃ of shaking culture 96 hours, get 15 liters of its nutrient solutions, carry out extracting with vinyl acetic monomer under the condition of PH=4, extracting solution promptly gets 8.5g oily matter behind concentrating under reduced pressure.Be solvent with chloroform-methanol (4: 1) then, modulating a diameter is 60mm, and length is the silicagel column of 400mm.In post, launch after above-mentioned oily matter is dissolved in methyl alcohol.With every 10ml is a metering, collects active constituent in the 25th～47 metering region.
The gleanings concentrating under reduced pressure of above-mentioned active constituent is obtained the 2.2g enriched material.After this separate the crude product of producing SR-1223 with high-speed liquid chromatography.It is that 20mm, length are the Nucleosil5C of 300mm that chromatographic column is selected diameter for use
18Solvent adopts methanol-water (7.5: 2.5), and adds 1% diethylamine formic acid (PH=7.3).Flow velocity 8.0ml/ branch, pressure 170kg/cm
2The peak that SR-1223 when retention time is 16 minutes, occurs.Collect this component, adjust PH to 4 with 0.1N hydrochloric acid, concentrating under reduced pressure is till eliminating methyl alcohol.Precipitation promptly gets the Powdered crude product of 26mg SR-1223 behind ethyl acetate extraction, washing, reconcentration.
After make with extra care with high-pressure liquid phase gas spectrum again.Chromatographic column is High-Carbon ODS: diameter 10mm, column length 250mm, solvent are methanol-water-1% diethylamine: formic acid (PH7.3) (7.5: 1.5: 1), flow velocity 1.4 ml/min, pressure 150kg/cm
2, the active peak of SR-1223 appears when retention time is 20 minutes.Collect zone, active peak, adjust to PH4 with 0.1N hydrochloric acid, concentrating under reduced pressure is until eliminating methyl alcohol.Use ethyl acetate extraction, with 0.1N salt acid elution, wash again, concentrate.And then enriched material is dissolved in methyl alcohol, and add water, be evaporated to and eliminate methyl alcohol, after lyophilize, promptly get the pure product of SR-1223 of 26mg pale yellow powder shape.
4. the simple declaration of accompanying drawing
Fig. 1 is the uv absorption spectra of antibiotic SR-1223 of the present invention, and Fig. 2 is the infrared absorpting light spectra of antibiotic SR-1223.
Claims (1)
1, a kind of preparation method of new antibiotic SR-1223 is characterized in that cultivating earlier streptomycete streotomyces spCGMCC0117, separation and Extraction new antibiotic SR-1223 from these cultures again, and the following physico-chemical property of this microbiotic tool:
(1) shape: pale yellow powder
(2) melting point: 130 ℃ (decomposition)
(3) ultimate analysis: carbon 63.85% hydrogen 8.56%
(4) molecular weight: 660 (according to the SIMS mass spectrums)
(5) specific optical rotation: [d]
25 D+ 10 ° (C=1.0 methyl alcohol)
(6) ultra-violet absorption spectrum:
270 (579)
(7) infrared absorption spectrum:
3400,2900,1755,1700,1570,1450,1370,1270,1170,1030
(8) color reaction: aubepine-sulfuric acid reaction is positive
(9) solvability: be soluble in methyl alcohol, vinyl acetic monomer, acetone, chloroform, be insoluble in water, Skellysolve A, benzene.
(10) antimicrobial spectrum: the plant-pathogenic thread fungus is Johnson ﹠ Johnson head's restraining effect, conventional bacterium is not almost had the growth-inhibiting effect.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 86104124 CN1014527B (en) | 1986-12-01 | 1986-12-01 | Prepn method of new antibiotic sr-1223 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 86104124 CN1014527B (en) | 1986-12-01 | 1986-12-01 | Prepn method of new antibiotic sr-1223 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN86104124A CN86104124A (en) | 1988-08-17 |
| CN1014527B true CN1014527B (en) | 1991-10-30 |
Family
ID=4802350
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 86104124 Expired CN1014527B (en) | 1986-12-01 | 1986-12-01 | Prepn method of new antibiotic sr-1223 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1014527B (en) |
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1986
- 1986-12-01 CN CN 86104124 patent/CN1014527B/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| CN86104124A (en) | 1988-08-17 |
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