CN104450580A - Preparation method of actinomycin D and application thereof - Google Patents

Preparation method of actinomycin D and application thereof Download PDF

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CN104450580A
CN104450580A CN201410756563.3A CN201410756563A CN104450580A CN 104450580 A CN104450580 A CN 104450580A CN 201410756563 A CN201410756563 A CN 201410756563A CN 104450580 A CN104450580 A CN 104450580A
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dactinomycin
fermented liquid
actinomycin
ethanolic soln
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张利莉
万传星
罗晓霞
吕玲玲
仼晓镤
夏占峰
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Tarim University
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Abstract

The invention discloses a novel actinomycin strain which generates actinomycin D: Streptomycesmutabilis TRM45540, preserved in U. S. Department of Agriculture Agricultural Research Service (ABS) Culture Collection on 14th, April, 2014 with the preservation number of NRRL B-59117. Meanwhile, the invention further discloses a seed medium which efficiently produces actinomycin D by using the strain, a fermentation medium and a preparation process of extracting and separating actinomycin D from fermentation liquor. An antibacterial activity test shows that the strain and the actinomycin D generated by fermentation of the strain have broad-spectrum and efficient antagonistic activity on pathogenic bacteria such as cotton verticillium and fusarium wilts, walnut pythium ultimum, staphylococcus aureus, staphylococcus epidermidis, candida albicans and the like and have an important application value and a very good application and development prospect on safe production of crops such as cotton, walnuts and the like and in preventing and controlling diseases such as bovine mastitis, women's vaginitis and the like.

Description

The preparation method of dactinomycin and application thereof
Technical field
The present invention relates to agricultural and field of medicaments, be specifically related to dactinomycin novelty teabag and a kind of can produce dactinomycin new strains and utilize this bacterial strain to produce the preparation method of dactinomycin.
Background technology
This two strains bacterial strain of Smelanochromogenes No.1779 and Streptomyces parvullus can produce actinomycetes D, its main mechanism is hinder the function of RNA polymerase, suppress the synthesis of RNA particularly mRNA, thus hinder protein synthesis and Tumor suppression growth, can also share with radiotherapy simultaneously, improve tumour to radiocurable susceptibility, but the effect of dactinomycin to the pathogenic bacterium Candida albicans of cotton spoting verticillium wilt, the rotten mildew phytopathogen of walnut and female vagina have not been reported.
Cotton wilt and cotton verticillium wilt are the main diseases of harm Cotton Production, and cotton wilt and cotton verticillium wilt are on the rise in China cotton region in recent years.But, also do not prevent and treat the beneficial agents of cotton wilt and cotton verticillium wilt at present.From Microbial resources, find biological pesticide is one of effective way, and the agricultural antibiotic that actinomycetes produce is most important source wherein.The development of the biological pesticides such as jingganmycin, Avrmectin and Qinlingmycin also promotes reasonableness and the superiority that sufficient proof screens economical and efficient agricultural antibiotic from actinomycetic meta-bolites.The microbiotic of the withered and yellow bacterium that withers of control cotton of current registration have not been reported.
Therefore, be main purpose of the present invention by screening control cotton wilt and the efficient microbiotic of cotton verticillium wilt from actinomycetes.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of actinomycetes producing dactinomycin, it is variable streptomycete (Streptomyces mutabilis) TRM45540, U.S.Department ofAgriculture Agricultural Research Service (ARS) Culture Collection (address: U.S.Departmentof Agriculture is preserved on April 14th, 2014, Peoria, IL 61604USA), preserving number is NRRL B-59117, survives after testing.
The application of described actinomycetes in antibacterial, it is for preventing and treating cotton spoting verticillium wilt, the rotten mildew phytopathogen of walnut, or for preventing and treating as the streptococcus aureus of pathogens from dairy cattle affected smastitis, staphylococcus epidermidis, or for preventing and treating the pathogenic bacterium Candida albicans of female vagina.
The application of described actinomycetes in the dactinomycin produced.Described application, is characterized in that comprising the steps: to cultivate described actinomycetes under suitable conditions, obtains fermented liquid; Separation and purification dactinomycin from fermented liquid.
Described application, it cultivates described actinomycetic culture medium prescription and culture condition is: Semen Maydis powder 15g/L (boil 20min, four layers of filtered through gauze get juice), peptone 5g/L, glucose 5g/L, CaCO 31g/L, NaCl 40g/L, adjustment pH is 7.2-7.4; Inoculum size by 6% is inoculated in fermention medium, 28 DEG C, shaking table cultivates 7 days under 180rpm condition.
From fermented liquid, the method for separation and purification dactinomycin is specially: after fermented liquid filters, wet method is loaded to D101 or AB-8 macroporous resin chromatography macroporous resin; Then the clear water of 2 times of column volumes, 10% ethanolic soln, 30% ethanolic soln, 50% ethanolic soln, 70% ethanolic soln, 95% ethanolic soln wash-out is used respectively; Merge 70-95% ethanol eluate, concentrating under reduced pressure obtains the fermented liquid 70-95% ethanol extract being rich in dactinomycin after removing solvent.
From fermented liquid, the method for separation and purification dactinomycin comprises the steps: first to use methyl alcohol normal temperature ultrasonic dissolution by after the oven dry of fermented liquid 70-95% ethanol extract further, methanol soluble part uses acetone solution again, acetone soluble part obtains orange powder after reclaiming acetone solvent, and the content of dactinomycin reaches more than 90%.
From fermented liquid, the method for separation and purification dactinomycin crosses Sephadex LH-20 chromatographic column after comprising the steps: the orange powder dissolve with methanol obtained by acetone extract further, collect and merge brown color ~ orange position, after reclaiming methanol solvate, obtaining the orange dactinomycin sterling of purity more than 98%.
The present invention has following beneficial effect: variable streptomycete TRM45540 and main metabolites dactinomycin thereof have wide spectrum and antagonistic activity efficiently to pathogenic bacterias such as cotton spoting verticillium wilt bacterium, walnut rotten mildew fungus, streptococcus aureus, staphylococcus epidermidis, Candida albicanss, to the safety in production of the farm crop such as cotton, walnut and the prevention and control of the disease such as mammitis of cow and female vagina, there is significant application value, may be used for exploitation high-performance bio agricultural chemicals and biological medicine.
Accompanying drawing explanation
The taxonomy of the variable streptomycete TRM45540 of Fig. 1 and phylogenetic tree;
The electromicroscopic photograph (showing spirrillum fibrillae of spores and elliptic spore) of the variable streptomycete TRM45540 of Fig. 2;
The chemical structure of Fig. 3 dactinomycin;
Fig. 4 dactinomycin 1hNMR spectrogram;
Fig. 5 dactinomycin 13cNMR spectrogram;
The hsqc spectrum figure of Fig. 6 dactinomycin;
The HMBC spectrogram of Fig. 7 dactinomycin.
Embodiment
Detailed description below by embodiment illustrates the present invention further, but is not limitation of the present invention, only does example explanation.
The source of the variable streptomycete TRM45540 of embodiment one and separation and Culture
From ancient city, Lou Lan, Xinjiang (height above sea level 1400m, 89 ° 22 ' 22, footpath, east "; in north latitude 40 ° 29 ' 55 ") 0-30cm pedotheque, adopt spread plate method, by 8 kinds of substratum containing 5%, 10%, 15%, 20% and 25% sodium-chlor, 28 DEG C of constant temperature culture, isolate 7 actinomycetes 35 strains belonged to altogether, sieved again by flat board face-off method primary dcreening operation and cup-plate method, final Isolation and screening has gone out the best biocontrol microorganisms TRM45540 of fungistatic effect.
The qualification of the variable streptomycete TRM45540 of embodiment two
For determining the taxonomy of biocontrol microorganisms TRM45540 further, the present invention adopts conventional sorting methods and molecular classification method to carry out taxonomic identification to this bacterial strain.
1 morphologic observation substratum
Inorganic salt Starch Agar (ISP4) substratum: starch 10gL -1, (NH 4) 2sO 44gL -1, MgSO 42gL -1, K 2hPO 42gL -1, CaCO 31gL -1, NaCl 40gL -1, agar 18gL -1, pH 7.2-7.4.
2 primers
Actinomycetes universal primer is adopted to be synthesized by AudioCodes biotechnology (Beijing) company limited.Its sequence is respectively: 27F (5 '-AGAGTTTGATCCTGGCTC-3 ') and 1492R (5 '-CGGCTACCTTGTTACGACTT-3 ')
3 test methods
3.1 morphological observation
Simple microscope is observed: adopt inserted sheet method, cultivates biocontrol microorganisms TRM45540 for 28 DEG C.With observation by light microscope record hypha form, aerial hyphae and substrate mycelium growing state, whether mycelium produces arrangement mode, the shape of fibrillae of spores and fibrillae of spores; Spore shapes and size; The presence or absence of spore, shape, size and generation type etc.
The molecular biology identification of 3.2 biocontrol microorganisms TRM45540
(A) extraction of biocontrol microorganisms TRM45540 genomic dna
The TRM45540 thalline collected on culture plate puts into the sterile centrifugation tube of 1.5mL, adds 480 μ L's
1 × TE damping fluid.Add 20 μ L N,O-Diacetylmuramidase (50mgmL -1), put into 37 DEG C of shaking tables, 200rpm shaken overnight.Often pipe adds the SDS of 50 μ L 20%, adds 5 μ L 20mgmL -1proteinase K, put into 60 DEG C of shaking tables, 200r/min vibrates 1h.Add the phenol of 550 μ L: chloroform: primary isoamyl alcohol (25:24:1), the centrifugal 10min of 12000rpm, get supernatant and move in another centrifuge tube, extracting 2-3 time repeatedly.Get supernatant, add isopyknic dehydrated alcohol, add the sodium acetate (3molL of 0.1 times of volume -1), put into 4 DEG C of refrigerators and precipitate DNA and be about 0.5h.The centrifugal 10min of 12000rpm, abandons supernatant.With the centrifugal product of 70% ethanol purge 2 times of 200 μ L, the centrifugal 5min of 12000rpm, abandons supernatant, by ethanol volatilization completely.Fully dissolve the DNA of bottom with the aseptic ultrapure water of 50 μ L, the agarose gel electrophoresis of 1% detects DNA extraction quality, the DNA of extraction is put into-20 DEG C of refrigerators and saves backup.
(B) amplification of biocontrol microorganisms TRM4554016S rDNA gene
By the 16S rDNA gene fragment in actinomycetes 16S rDNA gene universal primer 27F (5 '-AGAGTTTGATCCTGGCTC-3 ') and 1492R (5 '-CGGCTACCTTGTTACGACTT-3 ') amplification actinomycetes genomic dna.
The PCR reaction system of 25 μ L is: ddH 2(damping fluid is containing Mg for O 20.4 μ L, 10 × Buffer 2+) 2.5 μ L, dNTPs0.5 μ L, primer 2 7F (10 μm of olL -1) 0.5 μ L, primer 1492R (10 μm of olL -1) 0.5 μ L, Taq archaeal dna polymerase 0.1 μ L, template DNA 0.5 μ L.
PCR reaction conditions is: denaturation 94 DEG C of 4min; Sex change 94 DEG C of 1min, anneal 56 DEG C of 1min, extends 72 DEG C of 2min, 30 circulations; Total elongation 72 DEG C of 8min.React rear use 1% agarose gel electrophoresis to detect.Qualified PCR primer carries out sequencing.
(C) compare of analysis of sequencing result
Sequencing result DNAMAN5.2 software splices, sequence is compared by the strain sequence of the effective publication in BLAST and GenBank database, download the 16S rDNA gene order of the higher bacterial strain of effective publication of similarity, enter to build the row structure of phylogenetic tree with MEGA5.0 software to sequence, determine actinomycetic taxonomy.
4 test-results
The morphological observations of 4.1 biocontrol microorganisms TRM45540
Biocontrol microorganisms TRM45540 is well-grown on ISP4 substratum, and bacterium colony circle is flat, surface drying, and colony edge is neat, and aerial hyphae is enriched, sorus white, and substrate mycelium is yellow, has yellow soluble pigment to produce.
Biocontrol microorganisms TRM45540 gramstaining is positive, optimum growth temperature 37 DEG C, the most suitable growth pH is 7.2, the suitableeest NaCl (W/V) is 5%, on ISP4 substratum, 37 DEG C of inserted sheets cultivate 7d, are found by observation by light microscope: the substrate mycelium branch of biocontrol microorganisms TRM45540 is many, without tabula; Aerial hyphae is enriched, and aerial mycelium forms spore chain different in size, and spore bunchiness grows, and fibrillae of spores is more straight.Measure according to biocontrol microorganisms TRM45540 bacterium colony, thalli morphology and physiological characteristic, determine that biocontrol microorganisms TRM45540 belongs to streptomyces (Streptomyces).
The molecular biology identification result of 4.2 biocontrol microorganisms TRM45540
4.2.1 the 16S rDNA sequencing results of biocontrol microorganisms TRM45540
Sequencing result DNAMAN5.2 software splicing, determine that this fragment is by 1404 based compositions, the 16S rDNA sequence obtaining biocontrol microorganisms TRM45540 is:
AATCGCCAGTCCCACCTTCGACAGCTCCCTCCCACAAGGGGTTGGGCCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACTAGCGACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGAGACCGGCTTTTTGAGATTCGCTCCACCTCGCGGTATCGCAGCTCATTGTACCGGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGACTTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTCCCGTGAGTCCCCAGCACCACAAGGGCCTGCTGGCAACACGGGACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTACACCGACCACAAGGGGGACCCTGTCTCCAGGGTTTTCCGGTGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAGCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGGCACTTAATGCGTTAGCTGCGGCACGGACAACGTGGAATGTTGCCCACACCTAGTGCCCACCGTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTATCGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCCTGATATCTGCGCATTTCACCGCTACACCAGGAATTCCGATCTCCCCTACCGAACTCTAGCCTGCCCGTATCGACTGCAGACCCGGGGTTAAGCCCCGGGCTTTCACAACCGACGTGACAAGCCGCCTACGAGCTCTTTACGCCCAATAATTCCGGACAACGCTTGCGCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGGCGCTTCTTCTGCAGGTACCGTCACTTTCGCTTCTTCCCTGCTGAAAGAGGTTTACAACCCGAAGGCCGTCATCCCTCACGCGGCGTCGCTGCATCAGGCTTTCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGGTCGCCCTCTCAGGCCGGCTACCCGTCGTCGCCTTGGTGAGCCATTACCTCACCAACTAGCTGATAGGCCGCGGGCTCATCCTGCACCGCCGGAGCTTTCGAACCTCGCAGATGCCTGCGAGGGTCAGTATCCGGTATTAGACCCCGTTTCCAGGGCTTGTCCCAGAGTGCAGGGCAGATTGCCCACGTGTTACTCACCCGTTCGCCACTAATCCCCACCCGAAGGTGGTTCATCG
4.2.2 homology evolutionary tree builds
By BLAST comparison, in GenBank database, download the sequence nearer with actinomycetes 16S rDNA gene order similarity to be measured, with MEGA5.0 software, Multiple sequence alignments is carried out, phylogenetic tree construction to actinomycetes 16S rDNA gene order.Biocontrol microorganisms TRM45540 evolutionary tree sees Fig. 1-1, the 16S rDNA gene order (consistence is 99.87%) of biocontrol microorganisms TRM45540 and variable streptomycete (Streptomyces mutabilis) is gathered in same phyletic evolution branch, therefore this biocontrol microorganisms is accredited as variable streptomycete (Streptomyces mutabilis) TRM45540.
Embodiment three utilizes variable streptomycete TRM45540 ferment and prepare dactinomycin
Inoculate a small amount of mycelium in ISP4 substratum, cultivate 4d for 28 DEG C, obtain the seed liquor of variable streptomycete TRM45540, get 2mL (inoculum size of 8%) seed liquor and be inoculated in fermentation culture in fermentation of corn starch substratum, fermentation condition is: rotating speed 180rpm, initial pH 7.2-7.4, liquid amount 250mL, leavening temperature 28 DEG C, fermentation 7d.Collect fermented liquid, with four layers of filtered through gauze fermented liquid, wet method is loaded to macroporous resin chromatography (the every 100L of ferment filtrate loads D101 or AB-8 macroporous resin 2.0kg); Then the clear water of 2 times of column volumes, 10% ethanolic soln, 30% ethanolic soln, 50% ethanolic soln, 70% ethanolic soln, 95% ethanolic soln wash-out is used respectively; Merge 70-95% ethanol eluate, concentrating under reduced pressure obtains the fermented liquid 70-95% ethanol extract being rich in dactinomycin after removing solvent, fermented liquid 70-95% ethanol extract first uses methyl alcohol normal temperature ultrasonic dissolution after drying, methanol soluble part uses acetone solution again, acetone soluble part obtains orange powder after reclaiming acetone solvent, and the content of dactinomycin reaches more than 90%.Cross Sephadex LH-20 chromatographic column after the orange powder dissolve with methanol that acetone extract obtains, collect and merge brown color-orange position, after reclaiming methanol solvate, obtain the orange dactinomycin sterling of purity more than 98%.
The quantitative analysis of embodiment four dactinomycin and structural characterization
(1) quantitative analysis method of dactinomycin
High performance liquid chromatography is adopted to carry out quantitative analysis to dactinomycin.Chromatographic condition is: 4.6mm × 150mm C18 chromatographic column (0.5 μm), and 80% methanol-water is as moving phase, and flow velocity is 1.0mLmin -1, determined wavelength is 254nm, 280nm, 330nm and 445nm.
(2) structural characterization of dactinomycin
Table 1 dactinomycin 1h NMR and 13c NMR spectral data
The Determination of Antibacterial Activity of embodiment five variable streptomycete TRM45540 fermented liquid and dactinomycin
(1) by cultured pathogenic fungi spore sterilized water from wash-out culture plate, make the spore suspension of 106-107 spore/mL, draw the spore suspension of 100 μ L on PDA flat board, with sterilized spreading rod by even for bacteria suspension coating, Bechtop dries up a little.At the equidistant placement Oxford cup on flat board that carries disease germs, the dactinomycin of 200 μ L purifying is added in the cup of each Oxford, in same flat board, Oxford cup adds according to the concentration gradient of the dactinomycin of purifying, with the solvent acetone of dactinomycin (not having restraining effect to pathogenic bacteria) for contrast, cultivate 4-5d for 28 DEG C, right-angled intersection method measures antibacterial circle diameter, often processes repetition 3 times.Plant pathogenic fungi (cotton spoting verticillium wilt bacterium, grape mould germ, cucumber fusarium axysporum, walnut rotten mildew fungus) carries out bacteriostatic experiment on PDA substratum, the pathogenic bacterium (streptococcus aureus, staphylococcus epidermidis) of mammitis of cow carry out bacteriostatic experiment in LB culture medium culturing, and the pathogenic bacterium (Candida albicans) of female vagina carry out bacteriostatic experiment on SDA substratum.
(2) dactinomycin is to the antagonistic action of streptococcus aureus and staphylococcus epidermidis
The single bacterium colony of picking pathogenic bacteria is in LB substratum, activated overnight, by bacterium liquid with 1/100 concentration transfer in fresh LB substratum, putting 37 DEG C, to be cultured to OD value be the bacteria suspension that namely 0.4-0.6 makes pathogenic bacteria, draw the bacteria suspension of 100 μ L on LB flat board, with sterilized spreading rod by even for bacteria suspension coating, Bechtop dries up a little.At the equidistant placement Oxford cup on flat board that carries disease germs, add the dactinomycin of 200 μ L purifying in the cup of each Oxford successively, concentration is respectively: 10mgmL -1, 1mgmL -1, 0.1mgmL -1, 0.01mgmL -1, take fermention medium as contrast, 37 DEG C of overnight incubation, right-angled intersection method measures antibacterial circle diameter, often processes repetition 3 times.
(3) dactinomycin is to the antagonistic action of Candida albicans
By cultured pathogenic fungi spore sterilized water from wash-out culture plate, make the spore suspension of 106-107 spore/mL, the spore suspension drawing 100 μ L, on SDA flat board, with sterilized spreading rod by even for bacteria suspension coating, Bechtop dries up a little.At the equidistant placement Oxford cup on flat board that carries disease germs, add the dactinomycin of 200 μ L purifying of different concns gradient in the cup of each Oxford successively, concentration is respectively: 10mgmL -1, 1mgmL -1, 0.1mgmL -1, 0.01mgmL -1, take fermention medium as contrast, 28 DEG C of overnight incubation, right-angled intersection method measures antibacterial circle diameter, often processes repetition 3 times.
Above-mentioned experimental result, visible table 2.
The anti-microbial effect of table 2 dactinomycin

Claims (8)

1. produce actinomycetes for dactinomycin, for variable streptomycete ( streptomyces mutabilis) TRM45540, be preserved in U.S. Department of Agriculture Agricultural Research Service (ARS) Culture Collection on April 14th, 2014, preserving number is NRRL B-59117.
2. according to the purposes of actinomycetes according to claim 1 in anti-microbial effect, it is characterized in that it is for preventing and treating cotton spoting verticillium wilt, the rotten mildew phytopathogen of walnut, or for preventing and treating as the streptococcus aureus of pathogens from dairy cattle affected smastitis, staphylococcus epidermidis, or for preventing and treating the pathogenic bacterium Candida albicans of female vagina.
3. producing the application in dactinomycin according to actinomycetes according to claim 1.
4. apply as claimed in claim 3, it is characterized in that comprising the steps: to cultivate described actinomycetes under suitable conditions, obtain fermented liquid; Separation and purification dactinomycin from fermented liquid.
5. apply as claimed in claim 4, it is characterized in that: cultivate described actinomycetic culture medium prescription and culture condition is: Semen Maydis powder 15g/L, peptone 5g/L, glucose 5g/L, CaCO 31g/L, NaCl 40 g/L, adjustment pH is 7.2-7.4, and wherein Semen Maydis powder boils 20min, and four layers of filtered through gauze are got juice and used; Inoculum size by 6% is inoculated in fermention medium, 28 DEG C, shaking table cultivates 7 days under 180rpm condition.
6. apply as claimed in claim 4, it is characterized in that: the method for separation and purification dactinomycin is specially from fermented liquid:
After fermented liquid filters, wet method is loaded to D101 or AB-8 macroporous resin chromatography macroporous resin; Then the clear water of 2 times of column volumes, 10% ethanolic soln, 30% ethanolic soln, 50% ethanolic soln, 70% ethanolic soln, 95% ethanolic soln wash-out is used respectively; Merge 70-95% ethanol eluate, concentrating under reduced pressure obtains the fermented liquid 70-95% ethanol extract being rich in dactinomycin after removing solvent.
7. apply as claimed in claim 6, it is characterized in that: from fermented liquid, the method for separation and purification dactinomycin comprises the steps: first to use methyl alcohol normal temperature ultrasonic dissolution by after the oven dry of fermented liquid 70-95% ethanol extract further, methanol soluble part uses acetone solution again, acetone soluble part obtains orange powder after reclaiming acetone solvent, and the content of dactinomycin reaches more than 90%.
8. apply as claimed in claim 7, it is characterized in that: from fermented liquid, the method for separation and purification dactinomycin crosses Sephadex LH-20 chromatographic column after comprising the steps: the orange powder dissolve with methanol obtained by acetone extract further, collect and merge brown color ~ orange position, after reclaiming methanol solvate, obtaining the orange dactinomycin sterling of purity more than 98%.
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CN108715604A (en) * 2018-05-08 2018-10-30 上海交通大学医学院附属仁济医院 D actinomycin D class compound a ctinomycins D1-D4 and preparation method thereof and purposes
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CN113528388A (en) * 2021-07-19 2021-10-22 海南大学 Streptomyces corallini symbiosis, method for producing actinomycin D by fermentation and application
CN114525216A (en) * 2021-11-15 2022-05-24 塔里木大学 Actinoplanes targeted separation method

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CN105254712A (en) * 2015-09-23 2016-01-20 福建省微生物研究所 Purifying method of highly pure actinomycin D
CN105254712B (en) * 2015-09-23 2018-09-28 福建省微生物研究所 A kind of method of purification of high-purity actinomycin D
CN105255788A (en) * 2015-11-19 2016-01-20 塔里木大学 Streptomyces mutabilis trehalose synthase gene and application thereof
CN108715604A (en) * 2018-05-08 2018-10-30 上海交通大学医学院附属仁济医院 D actinomycin D class compound a ctinomycins D1-D4 and preparation method thereof and purposes
CN108715604B (en) * 2018-05-08 2021-07-23 上海交通大学医学院附属仁济医院 Actinomycin compound D1-D4, preparation method and application thereof
CN113308523A (en) * 2021-06-08 2021-08-27 塔里木大学 Bacterial line for detecting pear pollen branch blight and construction method thereof
CN113528388A (en) * 2021-07-19 2021-10-22 海南大学 Streptomyces corallini symbiosis, method for producing actinomycin D by fermentation and application
CN114525216A (en) * 2021-11-15 2022-05-24 塔里木大学 Actinoplanes targeted separation method
CN114525216B (en) * 2021-11-15 2024-01-05 塔里木大学 Target separation method for actinoplanes

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