CN1022765C - Preparation method of cell variety mycomycin of new antibiotic - Google Patents
Preparation method of cell variety mycomycin of new antibiotic Download PDFInfo
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- CN1022765C CN1022765C CN 86100600 CN86100600A CN1022765C CN 1022765 C CN1022765 C CN 1022765C CN 86100600 CN86100600 CN 86100600 CN 86100600 A CN86100600 A CN 86100600A CN 1022765 C CN1022765 C CN 1022765C
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- mycomycin
- cell variety
- microbiotic
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- new antibiotic
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Abstract
The present invention provides cell variety mycomycin as a new antibiotic and a preparation method thereof. After flavobacterium as an antibiotic is developed, the cell variety mycomycin is another new active substance prepared by culturing streptomyces SP. SR-44. The cell variety mycomycin has specific strong growth inhibiting action on the bacteria of xanthomonas, and thus, the cell variety mycomycin can be used as a new antibiotic for agriculture.
Description
The present invention relates to cell variety mycomycin of new antibiotic (Blebomycin, code name SR-44B) and preparation method thereof.Microbiotic of the present invention is to be got by separation in the nutrient solution of the microbiotic cell variety mycomycin production bacterium of streptomyces, is a kind of new antibiotic of not seeing bibliographical information.Blebomycin(code name RS-44B in the corresponding Japanese patent application of this microbiotic cell variety mycomycin and the application) be 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.Then to these microbiotic make with extra care respectively, evaluation and Application and Development.
Consequently the present inventor is at first from Streptomycin sulphate SR-44(streptomyces spSR-44) culture successfully separate and make with extra care out new antibiotic manufacture-yellow liver rhzomorph.Through further research, from the culture of above-mentioned SR-44 bacterial strain, successfully separate again and make with extra care out cell variety mycomycin of new antibiotic.Cell variety mycomycin all has on physics and chemistry and biology and the diverse character of manufacture-yellow liver rhzomorph.
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.
Below the present invention will be described in detail.
The microorganism of using
The microorganism that is used for producing microbiotic cell variety mycomycin of the present invention is a kind of bacterial classification that belongs to streptomyces, and it has the ability of producing the microbiotic cell variety mycomycin.
Streptomycete SPSR-44(Streptomyces SPSR-44) (the preserving number CGMCC-0092 at China Committee for Culture Collection of Microorganisms common micro-organisms preservation center) (being designated hereinafter simply as " SR-44 bacterial strain ") is exactly an example wherein.
This microorganism has above-mentioned characteristic, is suitable for producing microbiotic SR-44B 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-44 bacterial strain " and artificial variation's bacterial strain thereof also all belong to the bacterial classification of streptomyces.All microorganisms with production aftermentioned microbiotic cell variety mycomycin ability all can be used method of the present invention.
Above-mentioned " SR-44 bacterial strain " is to separate from the soil of Jiangsu Province, China and get, and it has following character:
1. morphological specificity:
Cultivating this bacterial strain " SR-44 bacterial strain " with the thalline analysis that contains glucose, yeast extract on substratum, carrying out thalline then and separate dryly, using 6N hydrochloric acid again 110 ℃, heating hydrolysis 20 hours.When detecting the amino acid composition of cell walls, can find L, L-diaminopimelic acid with Paper Chromatography.
In addition, the aerial hyphae that grows on agar plate presents spirrillum, and this as can be seen spiral hyphae is made up of the dozens of spore, but but can't see the spore of sporocyst and mobility.
Can illustrate that more than this bacterial strain belongs to streptomyces.The form of spore is garden column, surface smoothing.
2. the growth conditions on various substratum (in 27 ℃ of three weeks of cultivation, record and narrate according to " color designation is described dictionary " (Descriptive color names diotionary) by color.
(1) sucrose nitrate nutrient agar
Grow
(2) glucose-l-asparagine nutrient agar
Grow
Aerial hyphae trace b ash lacteous (oyster white)
B ash lacteous (oyster white) in the base
Pigment does not have
(3) glycerine-l-asparagine nutrient agar
It is common to grow
Aerial hyphae trace b ash lacteous (oyster white)
B ash lacteous (oyster white) in the base
Pigment does not have
(4) starch-inorganic salt nutrient agar
Well-grown
Aerial hyphae volume 2ge Bai Shi look (Bergey)
The light mustard look of 2ie (light mustard) in the base
Pigment does not have
(5) tyrosine nutrient agar
Well-grown
Aerial hyphae volume 3dc Natural color (natural)
2le mustard look (mustard) in the base
Pigment 2ea glassy yellow (light maize)
(6) nutrient agar
Well-grown
Aerial hyphae does not have
1 1/2 Ca lacteous (cream) in the base
Pigment does not have
(7) yeast-malt agar
Well-grown
Aerial hyphae volume 5fe grey (ash)
2lg mustard tawny (mustard tan) in the base
Pigment does not have
(8) oatmeal nutrient agar
Poor growth
Aerial hyphae does not have
Not clear in the base
Pigment does not have
(9) peptone-yeast-iron nutrient agar
Well-grown
Aerial hyphae does not have
1 1/2 ea faint yellow (light yellow) in the base
Pigment does not have
3. the usability of carbon source (general dagger-axe nutrient agar)
L-arabinose-
The D-wood sugar+
D-glucose+
D-fructose-
Sucrose-
Inositol-
The L-rhamnosyl-
Raffinose-
D-N.F,USP MANNITOL-
+ ... growth-... do not grow
4. other physiological property
(1) suitable temp is 25～30 ℃
(2) liquefaction of gelatin liquefaction
(glucose-peptone-gelatine culture)
(3) starch hydrolysis hydrolysis
(starch-inorganic salt nutrient agar)
(4) skimming milk solidifies
(5) liquefaction of skimming milk liquefaction
(6) melanic generation
(tyrosine nutrient agar) do not generate
(peptone-yeast iron nutrient agar) do not generate
Streptomyces bacterial classification with above-mentioned each character is by Bai Shi bacteriology identification handbook the 8th edition (Bergey ' s Manual of Determinative Bacteriology 8 th ed.) classification, this bacterial classification should belong to WISIC-ISM group, from tone, spore shape, nearest edge cocoa streptomycete.Can infer that thus this bacterial classification belongs to cocoa streptomycete or its extremely near edge bacterium.
The producing and purifying of the cultivation of SR-44 bacterial strain and cell variety mycomycin
With the above-mentioned production of antibiotics bacterium of streptomyces,, can cultivate " SR-44 bacterial strain " by the production of antibiotics method of routine and make microbiotic cell variety mycomycin of the present invention, training method both can be liquid culture and also can be solid culture.But the cultivation when producing in order to help industrialness, the spore suspension of above-mentioned production bacterium or nutrient solution should being inoculated into goes forward side by side on the substratum, and cultivation is mixed in the gas mixing that works.
Nutrition source in the substratum be there is no special regulation, can contain the carbon source that is usually used in microorganism culturing, nitrogenous source and other nutrition source.Wherein carbon source can be starch, dextrin, glycerine, glucose, sucrose, lactose, 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.For other nutrition source, can suitably add some inorganic salts, 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 the temperature when cultivating, time 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 cell variety mycomycin output for well.For example, the PH scope of substratum can be 4～9, with near neutral for well.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 this is the ordinary method that is usually used in extracting meta-bolites a bit just to make cell variety mycomycin by some appropriate means.For example, can utilize cell variety mycomycin and impurity to extract in the difference of aspects such as solubleness, ionic bond power, absorption avidity and molecular weight, specifically, the cell variety mycomycin major part is present in the culturing filtrate.Nutrient solution is adjusted to slant acidity, for example adjust to PH4.0, organic solvent with vinyl acetic monomer and so on extracts again, after the concentrated oily matter of gained is made with extra care by combinations such as adsorption chromatography, silica gel column chromatography, liquid chromatography (LC), ion exchange chromatography, gel chromatographies, can obtain containing the component of cell variety mycomycin and other activeconstituents.For example, oily matter is carried out in chloroform-methanol, launch to obtain active constituent behind the silica gel column chromatography.(for example, filling agent is selected Nucleosi15C for use by high speed liquid chromatography then
18, developping agent is methanol-water (8: 2)) and collect the active part of SR-44B, carry out recrystallization with methyl alcohol then, can obtain the colourless crystallization of cell variety mycomycin.
So the cell variety mycomycin of new antibiotic of being produced has following physico-chemical property and biological property.
The physico-chemical property of microbiotic cell variety mycomycin and biological property
Shape: colourless crystallization
Fusing point: 157～160 ℃
Ultimate analysis: carbon 58.14%, hydrogen 6.77%, nitrogen 10.70%, oxygen 24.13%
Molecular weight: 895(presses the FD mass spectrum)
Specific optical rotation: (α)
25 D-76 ° (C=0.5 methyl alcohol)
Ultra-violet absorption spectrum: λ
(E
1% 1cm) 230(90), 265 acromions (7.3), 268 acromions (8.1), 274(8.5), 282(7.1) (with reference to Fig. 1)
Infrared absorption spectrum: ν
KBr MaxCm
-13300,2950,1725,1630,1525,1500,1450,1400,1295,1245,1195,1080,1050,1020,825,745,695(is with reference to Fig. 2)
Acidic hydrolysis: acidic hydrolysis can get Serine, Threonine, glycine, tyrosine and other amino acid.
Color reaction: be positive with Dragendolf and RydonSmith
Solvability: be soluble in methyl alcohol, vinyl acetic monomer, chloroform, be insoluble in water, pentane, hexane, benzene.
Tlc analysis: (the 0.25mm silicon thin layer plate that U.S. Merck company makes)
Solvent Rf value
Chloroform: methyl alcohol=10: 1 0.57
Vinyl acetic monomer: benzene=4: 1 0.13
Antimicrobial spectrum: adopt common agar plate dilution method, obtain minimum inhibitory concentration (MIC).(fungi is used potato, sucrose nutrient agar, and bacterium is used broth agar culture medium.) result, cell variety mycomycin demonstrates special Johnson ﹠ Johnson head's restraining effect to the bacterium that Xanthomonas campestris (Xantho-monas) belongs to, and common bacterium, mould are not almost had the growth-inhibiting effect.
Test bacterium MIC(mcg/ml)
Rice Xanthomonas 2
（Xanthomonas oryzae）
The withered and yellow Zymomonas mobilis 4 of mandarin orange
（Xanthomonas citri）
Colon bacillus＞1000
（Escherichia coli）
Salmonella typhimurium＞1000
（Salmonella typhimurium）
Gold-coloured staphylococci 209P＞1000
（Staphylococcus aureus 209P）
Bacillus subtilus＞1000
（Bacillus subtilis）
Cell variety mycomycin belongs to peptide antibiotics from its physico-chemical property.Owing to do not find to have in the peptide antibiotics bacterium of pair xanthomonas to have the inhibiting material of special Johnson ﹠ Johnson head as yet before this, can infer thus that cell variety mycomycin was a kind of new antibiotic.
In sum, cell variety mycomycin demonstrates the long inhibit feature of Johnson ﹠ Johnson because of the bacterium to xanthomonas, so will be expected to be used as the agricultural microbiotic.
Production 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, above-mentioned SR-44 bacterial strain (CGMCC-0092) is inoculated in this substratum, placed 28 ℃ of shaking culture 96 hours.Get 25 liters of its nutrient solutions, under the condition of PH=4, carry out extracting with vinyl acetic monomer.Extracting solution promptly gets 4.8g oily matter behind concentrating under reduced pressure, then this oily matter is placed 200ml methyl alcohol, be warming up to 60 ℃ make its dissolving after, reduce to 0 ℃ again and make its crystallization, after filtration, methanol wash, can obtain 400mg colourless crystallization body.
Above-mentioned xln is made with extra care with high-speed liquid chromatography.Chromatographic column is nucleosides 5C
18, diameter is 20mm, length is 250mm.Solvent is selected methyl alcohol for use: water=8: 2, flow velocity are the 6.0ml/ branch, and pressure is 180Kg/cm
2, the active peak of cell variety mycomycin appears when retention time is 12 minutes.Collect above-mentioned active peak, concentrating under reduced pressure is till eliminating methyl alcohol.Cross the filter cake recrystallizing methanol, can obtain the pure product of colourless crystallization shape of 150mg cell variety mycomycin.
Description of drawings
Fig. 1 is the uv absorption spectra of microbiotic cell variety mycomycin of the present invention.Fig. 2 is the infrared absorpting light spectra of microbiotic cell variety mycomycin.
Claims (1)
1, a kind of cultivation preparation method of cell variety mycomycin of new antibiotic, this microbiotic has following physico-chemical property:
Shape: colourless crystallization
Fusing point: 157～160 ℃
Ultimate analysis: carbon 58.14%, hydrogen 6.77%, nitrogen 10.70%, oxygen 24.13%
Molecular weight: 895 (pressing the FD mass spectrum)
Specific optical rotation: [α]
25 D=-76 ° (C=0.5 methyl alcohol)
Ultra-violet absorption spectrum: λ
CH30H Maxnm(E
1% 1cm) 230 (90), 265 acromions (7.3),
268 acromions (8.1), 274 (8.5), 282 (7.1)
Infrared absorption spectrum: υ
KBr MaxCm
-13300,2950,1725,1630,1525,
1500、1450、1400、1295、1245、1195、
1080、1050、1020、825、745、695
Acidic hydrolysis: acidic hydrolysis can get Serine, Threonine, glycine, tyrosine
And other amino acid.
Color reaction: be positive with Dragendolf and Rydon.Smith
Solvability: be soluble in methyl alcohol, vinyl acetic monomer, chloroform, be insoluble in water, pentane, hexane, benzene,
It is characterized in that this microbiotic by streptomycete spSR-44 (preserving number CGMCC-0092) strain culturing meta-bolites make through acidifying, organic solvent extraction, chromatography.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 86100600 CN1022765C (en) | 1986-06-20 | 1986-06-20 | Preparation method of cell variety mycomycin of new antibiotic |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 86100600 CN1022765C (en) | 1986-06-20 | 1986-06-20 | Preparation method of cell variety mycomycin of new antibiotic |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN86100600A CN86100600A (en) | 1988-07-20 |
| CN1022765C true CN1022765C (en) | 1993-11-17 |
Family
ID=4801124
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 86100600 Expired - Fee Related CN1022765C (en) | 1986-06-20 | 1986-06-20 | Preparation method of cell variety mycomycin of new antibiotic |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1022765C (en) |
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1986
- 1986-06-20 CN CN 86100600 patent/CN1022765C/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| CN86100600A (en) | 1988-07-20 |
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