CN87100250A - Preparation method of new antibiotic aureonucleomycin and agricultural bactericide - Google Patents
Preparation method of new antibiotic aureonucleomycin and agricultural bactericide Download PDFInfo
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- CN87100250A CN87100250A CN87100250.7A CN87100250A CN87100250A CN 87100250 A CN87100250 A CN 87100250A CN 87100250 A CN87100250 A CN 87100250A CN 87100250 A CN87100250 A CN 87100250A
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- antibiotic
- new antibiotic
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- aureonuclemycin
- aureonucleomycin
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Abstract
The present invention provides a new antibiotic Aureonuclemycin and its preparation method. The antibiotic has strong growth inhibiting effect on plant bacterial diseases caused by ceriferous bacteria (Xanthomonas). The invention also provides an efficient low-toxicity low-residue agricultural bactericide with the aureonucleomycin as an active ingredient. Especially has high effect on preventing and treating bacterial leaf blight of rice. The antibiotic can be prepared by culturing S.aureus Suzhou variant SP-371.
Description
The invention relates to new antibiotic aureonuclemycin (Aureonuclemycin) and its preparation method, and be the agricultural or horticultural bactericide of effective ingredient with golden caryomycin.
In recent years, microbiotic and also is widely used aspect agricultural chemicals not only aspect medical.The present inventor isolates microorganism and produces 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.
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.Thereby can develop the novel pesticide of a kind of efficient, low toxicity, low residual amt.
The microorganism of using
Being used for producing microbiotic gold caryomycin of the present invention is a kind of bacterial classification that belongs to streptomyces, and it has this antibiotic ability of production.Streptomyces aureus Suzhou mutation SP-371(Strepto-myces aureus Var Suzhouneusisn Var yen etal 1982) (being designated hereinafter simply as " SP-371 bacterial strain ") just has above-mentioned characteristic, is suitable for producing golden caryomycin of the present invention.This bacterial strain separated in the soil of Dongshan, Suzhou City, China Jiangsu Province area and gets in June, 1981, and it is numbered CGMCC № 0122 China Committee for Culture Collection of Microorganisms.
" SP-371 bacterium " has following character:
1. morphological specificity:
With the inserted sheet method on asparagine, the glucose agar medium 28 ℃ cultivate these bacterial strains after 7～10 days, with form and the spore shape that opticmicroscope and electron microscope are observed mycelia respectively, surface tissue.The result shows that the aerial hyphae of this bacterium is shape in the shape of a spiral, generally about 2～6 circles; Spore is spherical in shape or avette, and size is uneven, and spore surface is smooth.
2. the feature on various substratum:
After 28 ℃ of cultivation inoculations, observed three times respectively at the 7th, 14,30 day.The chromatogram (nineteen fifty-seven version) of publishing with Science Press is as the standard of color description.
(1) sucrose, nitrate nutrient agar
The weak thin IV 62 of aerial hyphae growth '-V 62 '-V 72 '
Vegetative hyphae IV 51 '
But lysochrome does not have
(2) asparagine glucose agar medium (ISP5)
The greyish white point of grey camel-V 72 in the aerial hyphae well-grown ' be with
The vegetative hyphae lotus seeds are white-II 42 '
But lysochrome does not have
(3) Gao Shi-number nutrient agar
Aerial hyphae well-grown V 72 '
Vegetative hyphae II 22 '-lotus seeds are white
But the little Huang of lysochrome
(4) Starch Agar substratum (ISP4)
Aerial hyphae growth medium white-II 62 '-V 72 '
The vegetative hyphae milky white
But the little Huang of lysochrome
(5) tyrosine nutrient agar (ISP7)
Grey camel-V 72 in the aerial hyphae well-grown '
Vegetative hyphae bamboo shoot skin palm fibre
But lysochrome yellowish brown
(6) calcium malate nutrient agar
Aerial hyphae well-grown V 61 '-V 71 '-V 72 '
Vegetative hyphae bamboo shoot skin palm fibre-II 33 '
But the pale brown look of lysochrome
(7) yeast glucose agar medium
Aerial hyphae grow medium shallow in grey camel-V 72 '
Vegetative hyphae IV 72 '
But lysochrome is brown brown
(8) glycerine asparagine nutrient agar
Grey camel-V 72 during the aerial hyphae well-grown is shallow '
Vegetative hyphae yellowish pink-III 65 '-bamboo shoot skin palm fibre
But the pale brown look of lysochrome
(9) oatmeal nutrient agar (ISP3)
Aerial hyphae growth difference is greyish white
The vegetative hyphae tawny
But lysochrome does not have
(10) Ke Shi-number nutrient agar
Grey camel-V 72 in the aerial hyphae well-grown '
The vegetative hyphae lotus seeds are white
But lysochrome is yellowish
(11) potato block substratum
Aerial hyphae is luxuriant, wrinkle, have globule light gray-II 51 '-IV 61 '
Vegetative hyphae
But lysochrome piece browning
3. physiological and biochemical property:
(1) gelatine liquefication+(the black brown pigments is arranged)
(2) starch water be situated between-
(3) nitrate reduction+(in Cha Shi solution)
(4) milk peptonizes+(milk becomes the transparent liquid of dark brown)
Milk solidifies-
(5) H
2The S generation+
(6) Mierocrystalline cellulose divides Jie-(not growing)
(7) pigment generation+
4. utilization of carbon source:
Fine utilization: D-glucose D-pectinose
D-wood sugar D-fructose
L-rhamnosyl N.F,USP MANNITOL
General using: D-inositol
Do not utilize or grow suspicious: sucrose, raffinose
Show that according to the streptomycete identification handbook of Institute of Microorganism, Academia Sinica and the result of above-mentioned test " SP-371 bacterial strain " belongs to the streptomyces aureus monoid, but tangible difference is arranged again with streptomyces aureus.Its main difference point is that this bacterial strain can not grow on Mierocrystalline cellulose, do not produce the active substance (Plating) that can restrain Gram-positive, negative bacteria and filamentous fungus.Through professor Yan Xunchu of Institute of Microorganism, Academia Sinica authorization, this bacterial strain named be Streptomycesaureus Var Suzhouneusisn Var yen etal 1982.
Cultivate and process for purification
With the above-mentioned production bacterium of streptomyces,, can cultivate " SP-371 bacterial strain " by the production of antibiotics method of routine, and make microbiotic gold caryomycin of the present invention.Training method both can be liquid culture, also can be solid culture.Cultivation when producing in order to help industrialness should be inoculated into the spore suspension of above-mentioned production bacterium on the substratum, and 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 golden caryomycin 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 ℃, and the component of these substratum, hydrogen ion concentration, culture temperature, agitation condition etc. all should carry out suitable adjusting according to the kind of use bacterial strain and external conditions etc. certainly, to obtain best effect.
Culture by above-mentioned gained sets out, and just can make golden caryomycin by some appropriate means, and these methods are often to be used in the method for extracting metabolite.For example can utilize golden caryomycin and impurity to extract in the difference of aspects such as solubleness, ionic bond power, absorption avidity and molecular weight.These methods can be used separately, also can suitably cooperate or recycle.Specifically, the gold caryomycin is present in the ferment filtrate, available positively charged ion, anionite-exchange resin exchange, use dilute hydrochloric acid and acetic acid desorb then, also available macroporous adsorbent resin or activated carbon adsorb, and carry out desorb with aqueous acetone then, can obtain crude extract like this, crude extract by macroporous adsorbent resin or ion exchange resin, just can make flaxen half highly finished product again.Adopt low temperature to place at last, separate out white crystals.Crystallization after separating in water recrystallization once promptly get the pure product of golden caryomycin.
As above the golden caryomycin of gained has following physics and chemistry and biological property:
1. shape: white rectangle sheet crystallization
2. fusing point: (divide Jie's browning) more than 146 ℃
3. molecular weight: 425(FAB-MS) (see accompanying drawing 1)
4. molecular formula: C
16H
19N
5O
9H
2O
Analytical value: carbon 43.41%, hydrogen 4.76%, nitrogen 15.7%, oxygen 36.6%
Theoretical value: carbon 43.43%, hydrogen 4.74%, nitrogen 15.8%, oxygen 36.1%
7. specific optical rotation: (α)
25 DIn the=+ 0.22(C=0.25 water)
8. ultra-violet absorption spectrum: λ
Max258nm(0.1N among the HCl)
260nm(0.1N among the NaOH)
In the 260nm(neutral water)
9. infrared absorption spectrum: (KBr method) (seeing accompanying drawing 2)
3400、2945、2362、1694、1643、1604、1504、1477、1416、1382、1308、1222、1176、1129、1087、1016、984、896、850、795、709、849、559、454cm
-1
10.
1H-nuclear magnetic resonance spectrum: (seeing accompanying drawing 3)
The bimodal m-multiplet of the unimodal d-of S-
11.
13C-nuclear magnetic resonance spectrum: (seeing accompanying drawing 4)
The bimodal t-triplet of the unimodal d-of s-
12. antimicrobial spectrum:
Adopt common agar plate dilution method, crystallized sample concentration 128r/ml, with multiple spot inoculator inoculated bacteria, to streptococcus aureus (Staphylococcus aureus) 6 strains, intestinal bacteria (Escherichia coli) 6 strains, citrobacter (Citro-bacter) 5 strains, Pseudomonas aeruginosa (Pseudomonas auruginosa) 7 strains, pneumobacillus (Kebsiella Pneumoniae) 6 strains, enterobacter cloacae (Enter-bacter) 6 strains, bacterium flexneri (Flexueri Shigella) 6 strains, Corynebacterium diphtheriae (Subtilis typhi) 5 strains, 9 kind of 51 strain bacterium such as sick bacillus (Scmei Shigella) 6 strains of Song Na Shi dysentery all do not have antibiotic effect.
13. acute toxicity: small white mouse is oral, observes for 2 weeks, and 10000mg/kg does not have any toxicity symptom.
14. stability: stable in acidity, unstable in alkalescence.
It is that 425 molecular formula are C that the retrievals of the two cover microbiotic robot calculator retrieval systems of compiling through Chinese Academy of Medical Sciences's institute of Antibiotics and Japanese RIKEN antibiotics research department all find no molecular weight
16H
19N
5O
9H
2The sweet class microbiotic of the nuclear of O.As from the foregoing, golden caryomycin of the present invention is a kind of new antibiotic of not seeing reported in literature, and it is similar to the weeding element (Herbicdins A.B.C.E.F.G) that Japanese Sankyo Co., Ltd delivers on chemical structure
Agricultural or horticultural bactericide
Golden caryomycin of the present invention is a kind of phytobacterial disease to causing because of wax yellow Bacillus bacteria (Xanthomonas), particularly to the effective new antibiotic of bacterial blight of rice (Xanthomonas Ory-zae).It not only has prevention and therapeutic action to the disease of crop, and have resistance of rainwater washing against, to performances such as ultraviolet light stabilized.
Making when being the disinfectant use in agriculture of effective ingredient with golden caryomycin of the present invention, can use equally known agricultural chemicals in this technical field general various carriers such as solid phase, liquid phase and emulsifying dispersant.And can be mixed with formulation arbitrarily such as granule, pulvis, emulsifying agent, wettable powder, tablet, finish, sprays, smoke substance.Simultaneously, in various preparations, generally some auxiliarys can be suitably added, but alkaline auxiliary can not be added as quaternary amine and so on.
Generally speaking, the proportioning of effective ingredient in each preparation is 10～90% in emulsion and wettable powder; In pulvis and finish etc. is 0.1-10%; Certainly, also can suitably increase and decrease according to different application targets.
Preparation of the present invention also can be used with other sterilant, weedicide, sterilant, fertilizer material, soil improvement agent etc., but must not mix use with alkaline matters such as Bordeaux mixture, liming.
Below illustrate with regard to manufacturing of the present invention, enforcement, test example.
Production Example:
Ratio with glucose 5%, groundnut meal 2%, dextrin 1%, peptone 0.2%, ammonium sulfate 0.2%, salt 0.3%, lime carbonate 0.6% is made substratum.PH=7.2-7.5 before the sterilization, (microorganism storage CGMCC No0122) is inoculated in this substratum with the Suzhou mutation of above-mentioned streptomyces aureus.The 250ml triangle shakes that loading amount is 50ml in the bottle, 28 ℃ of shaking culture 40 hours, as seed liquor.Get the 200ml seed liquor in the glass fermentor tank of 12 liters, in adorn the substratum of the above-mentioned same ratio of 9 liters, at 28 ℃ with air flow 1: 76 hours after-filtration of 0.6-0.8 stir culture, the content of golden caryomycin this moment in ferment filtrate is about 500r/ml.Get filtrate 6 liters (PN6.5) by 711 anionite-exchange resin (COO
-Type) (φ 5.5 * 60cm), use 1 liter water washing then, are situated between with the 1N acetic acid solution and inhale for post.There is the part of absorption at collection 260nm place, again by 300ml 1 * 4 Zeo-karb (H
+Type) (φ 3.5 * 20cm), inhale with 0.3N HCl 4000ml Jie after washing for post.Collecting 260nm place has the part of absorption, is adjusted to PH=3 with 2N NaOH, and again by macroporous adsorbent resin 1300-II type, the washing back is with 20% aqueous ethanolic solution Jie suction.Jie's imbibition through concentrating under reduced pressure, left standstill liquid (about 0 ℃ of low temperature), the adularescent crystallization is separated out.Filter back recrystallization in water and once get pure product 1500mg, total recovery is about 50%.
10 parts of golden caryomycin are mixed, pulverize with 5 parts of sodium laurylsulfonates, 2 parts of ditane sodium disulfonate yubans and 83 parts of potter's clay, promptly get 100 parts of preparations.
Embodiment 2 emulsions
With 8 parts of golden caryomycin and 10 parts of ethylene glycol, 20 parts of dimethyl formamides, 10 parts of alkyl xylene ammonium chlorides and 52 parts of molten Jie of methanol mixed, promptly get 100 parts of emulsions.
Embodiment 3 pulvis
0.2 part of golden caryomycin is mixed pulverizing with 0.5 part of calcium stearate, 50 parts of talcum powder and 49.3 parts of potter's clay, promptly get 100 parts of pulvis.
10 parts of golden caryomycin are mixed pulverizing with the sodium salt of 15 parts of starch, 72 parts of powdered bentonites and 3 parts of sulfuric acid dodecyl esters, promptly get 100 parts of granules.
The prophylactic tria of 1 pair of bacterial blight of rice of test
To be diluted to the concentration of being scheduled to by embodiment 1 made wettable powder, spray (kind: first rich morning) on the potted plant rice plants in tillering phase then.After air-dry,, in blade inoculation concentration the bacterial leaf spot pathogeny bacterium suspension of 1,000,000,000 sporozoite numbers with the spininess method respectively at 12,24,48 hours.In the greenhouse, cultivate 14 days (behind the spray medicine), calculate preventive effect by 5 grades of disease index methods, result such as following table:
*Opposing withered two chemical name is N, N-methylene-two (2-amino-1,3,4-thiadiazoles)
The therapeutic test of 2 pairs of bacterials blight of rice of test
Medicament, rice plant, pathogeny bacterium liquid are all with test 1.Inoculation bacterial leaf spot bacterium on plant is earlier adopted in this test, sprays medicine then after the 1st, 3,5,7 day, inoculates back 15 days and checks result of treatment, result such as following table:
Test the test of 3 resistance of rainwater washing against
Medicament, rice plant, pathogeny bacterium liquid are all with test 1.Spray medicine is earlier adopted in this test, washes away blade respectively at water after 0.5,3,24 hour then and compares with non-flushing brush, inoculates the pathogeny bacterium behind the spray medicine in 48 hours.Wash away long 65cm, wide 39cm at every turn, area is 2535cm
2, flushing time is 10 minutes, amount of flush water is 3500ml.Check its result such as following table after 15 days at the spray medicine:
Medicament, rice plant, pathogeny bacterium liquid are all with test 1.Inoculation pathogeny bacterium is earlier adopted in this test, sprays medicine after 48 hours again.Soup re-uses after shining 24,48 hours away from 1 meter through the 30W ultraviolet lamp earlier.Checked its result such as following table in 14 days behind the spray medicine:
Can prove that according to above test the medicament that is effective ingredient with golden caryomycin of the present invention is a kind of novel disinfectant use in agriculture.Medicament demonstrates remarkable effect to the control bacterial blight of rice especially like this, and toxicity and residual quantity are all very low.
Claims (3)
1, a kind of preparation method of new antibiotic aureonuclemycin is characterized in that cultivation belongs to the production bacterium of the microbiotic gold caryomycin of streptomycete (Streptomyces) genus, separation and Extraction new antibiotic aureonuclemycin from these cultures again.
2, by the described preparation method of claim 1, it is characterized in that the production bacterium of described microbiotic gold caryomycin is streptomyces aureus Suzhou mutation SP-371(Streptomyces aureus Var Suzhouneusisn Var Yen etal 1982).
3, a kind of agricultural or horticultural bactericide is characterized in that contained effective ingredient is a new antibiotic aureonuclemycin.
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| CN 87100250 CN1030470C (en) | 1987-03-18 | 1987-03-18 | Preparation method for new antibiotic aureonuclemycin and agricultural bactericide |
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|---|---|---|---|
| CN 87100250 CN1030470C (en) | 1987-03-18 | 1987-03-18 | Preparation method for new antibiotic aureonuclemycin and agricultural bactericide |
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Cited By (8)
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| CN102217613A (en) * | 2011-05-05 | 2011-10-19 | 青岛海利尔药业有限公司 | Pesticide composition containing aureonucleomycin and ethylicin |
| CN102239856A (en) * | 2011-05-05 | 2011-11-16 | 海利尔药业集团股份有限公司 | Pesticide composition containing aureonucleomycin and xinjunan |
| CN102363753A (en) * | 2011-03-07 | 2012-02-29 | 浙江师范大学 | Streptomyces termitum ACT-2 strain, its culture and application |
| CN103540603A (en) * | 2012-07-09 | 2014-01-29 | 中国科学院上海有机化学研究所 | Biosynthetic gene cluster of aureonuclemycin and application thereof |
| CN105494370A (en) * | 2015-12-09 | 2016-04-20 | 陕西麦可罗生物科技有限公司 | Pesticide composition prepared from aureonucleomycin and tetramycin |
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| CN108432782A (en) * | 2018-04-03 | 2018-08-24 | 杜桂香 | A kind of bactericidal composition |
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-
1987
- 1987-03-18 CN CN 87100250 patent/CN1030470C/en not_active Expired - Fee Related
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102363753A (en) * | 2011-03-07 | 2012-02-29 | 浙江师范大学 | Streptomyces termitum ACT-2 strain, its culture and application |
| CN102363753B (en) * | 2011-03-07 | 2012-11-21 | 浙江师范大学 | Streptomyces termitum ACT-2 strain, culture and application thereof |
| CN102217613A (en) * | 2011-05-05 | 2011-10-19 | 青岛海利尔药业有限公司 | Pesticide composition containing aureonucleomycin and ethylicin |
| CN102239856A (en) * | 2011-05-05 | 2011-11-16 | 海利尔药业集团股份有限公司 | Pesticide composition containing aureonucleomycin and xinjunan |
| CN102239856B (en) * | 2011-05-05 | 2013-03-13 | 海利尔药业集团股份有限公司 | Pesticide composition containing aureonucleomycin and xinjunan |
| CN103540603A (en) * | 2012-07-09 | 2014-01-29 | 中国科学院上海有机化学研究所 | Biosynthetic gene cluster of aureonuclemycin and application thereof |
| CN103540603B (en) * | 2012-07-09 | 2015-10-28 | 中国科学院上海有机化学研究所 | The biological synthesis gene cluster of gold alloy hydride and application thereof |
| CN105494370A (en) * | 2015-12-09 | 2016-04-20 | 陕西麦可罗生物科技有限公司 | Pesticide composition prepared from aureonucleomycin and tetramycin |
| CN107517988A (en) * | 2017-10-24 | 2017-12-29 | 惠州市无龄康态健康科技有限公司 | A kind of complex composition containing gold alloy hydride and benziothiazolinone |
| CN108432782A (en) * | 2018-04-03 | 2018-08-24 | 杜桂香 | A kind of bactericidal composition |
| CN114521566A (en) * | 2022-02-21 | 2022-05-24 | 广西壮族自治区农业科学院 | Synergistic composition for preventing and treating soft rot of taro |
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| Publication number | Publication date |
|---|---|
| CN1030470C (en) | 1995-12-06 |
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