CN105153073A - Antibiotic difluostatin A as well as preparation method and application thereof to preparation of antibacterial drug - Google Patents
Antibiotic difluostatin A as well as preparation method and application thereof to preparation of antibacterial drug Download PDFInfo
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Abstract
The invention discloses antibiotic difluostatin? A and preparation method thereof and the application in preparation antibacterials. Compound difluostatin? A, shown in structural formula such as formula (I), does is it by streptomyces coelicolor (Streptomyces? coelicolor.) YF11-3 fermentation generates, contain novel fluostatin-prefluostatin dimeric structure, and to Klebsiella Pneumoniae, Aeromonas hydrophila and staphylococcus aureus have growth inhibitory activity, and bioengineering transformation is carried out to it or modifying for chemical structure is expected to be developed into antibacterial new drug
.
Description
Technical field:
The invention belongs to industrial microorganism field, be specifically related to new microbiotic difluostatinA and preparation method thereof and the application in preparation antibacterials.
Background technology:
Fluostatins compounds various structures and there is multiple biology, pharmacologically active.Some compounds have very strong cell toxicant or bacteriostatic activity, have good application prospect.
Summary of the invention:
First object of the present invention is to provide a kind of fluostatin class dimer compound difluostatinA with anti-microbial activity newly.
A kind of new fluostatin class dimer compound difluostatinA of the present invention, its structure is such as formula shown in (I):
Second object of the present invention is to provide the preparation method of fluostatin class dimer compound difluostatinA.
Compound difluostatinA of the present invention is that preparative separation obtains from the fermenting culture of streptomyces coelicolor (Streptomycescoelicolor.) YF11-3.
The present invention prepares compound difluostatinA preferably by following methods from the fermenting culture of streptomyces coelicolor (Streptomycescoelicolor.) YF11-3, and concrete steps are as follows:
A, prepare the fermenting culture of streptomyces coelicolor (Streptomycescoelicolor.) YF11-3, the fermented liquid of this fermenting culture and mycelium are separated, fermentation liquor macroporous resin adsorption, after use acetone wash-out macroporous resin, after elutriant reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate, and ethyl acetate layer obtains extractum A after distillation and concentration; Mycelium first uses acetone extraction, and after leaching liquid reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate again, and ethyl acetate layer obtains medicinal extract B after distillation and concentration;
B, crude extract extractum A and medicinal extract B merged is through silica gel column chromatography, by chloroform/methanol as eluent, gradient elution is carried out from volume ratio 100:0 ~ 0:100, collect the cut Fr.2 that chloroform/methanol volume ratio 95:5 gradient elution gets off, by the anti-phase medium pressure liquid chromatography of ODS, with water/methyl alcohol as eluent, gradient elution is carried out from volume ratio 100:0 ~ 0:100, collect the cut Fr.2-7 that water/methyl alcohol volume ratio 20:80 gradient elution gets off, after LH-20 gel column, using chloroform/methanol volume ratio 1:1 as moving phase wash-out, eluting fraction is separated through Thin Layer Chromatography again, obtain compound difluostatinA.
The fermenting culture preparing streptomyces coelicolor (Streptomycescoelicolor.) YF11-3 of described a) step is prepared preferably by following methods: streptomyces coelicolor (Streptomycescoelicolor.) YF11-3 of activation is accessed seed culture medium, 28 DEG C, 200rpm, cultivate 48h and obtain seed liquor, by seed liquor with 10% inoculum size be linked in fermention medium, 28 DEG C, 200rpm, shaking culture 120h, and obtained fermenting culture, the formula of described seed culture medium contains in often liter of substratum: starch 10g, yeast powder 5g, peptone 4g, CaCO
32g, thick sea salt 30g, surplus is water, pH7.2, the formula of described fermention medium is containing starch 10g, fish peptone 1g, Semen Maydis powder 6g, bacto peptone 2g in often liter of substratum, glycerine 5ml, CaCO
32g, thick sea salt 30g, surplus is water, pH7.0.
3rd object of the present invention is to provide streptomyces coelicolor (Streptomycescoelicolor.) YF11-3 and is preparing the application in compound difluostatinA, described streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3 is by replacing on the cosmid of the biological synthesis gene cluster containing fluostatins by carrier pSET152 fragment, then be transformed in streptomyces coelicolor (Streptomyces.coelicolor.) YF11 and obtain, the nucleotide sequence of the biological synthesis gene cluster of described fluostatins is as shown in SEQIDNO.1.
The present inventor found through experiments, compound difluostatinA is to KlebsiellapneumoniaATCC13883, AeromonashydrophilaATCC7966, StaphylococcusaureusATCC29213 and EnterococcusfaecalisATCC29212 has growth inhibitory activity, wherein to KlebsiellapneumoniaATCC13883, the MIC value of AeromonashydrophilaATCC7966, StaphylococcusaureusATCC29213 is 4 μ gmL respectively
-1, 4 μ gmL
-1with 8 μ gmL
-1.Therefore, the 4th object of the present invention is to provide the application of compound difluostatinA in preparation antibacterials.
Described antibacterials are preferably the medicine of anti-Klebsiella Pneumoniae, Aeromonas hydrophila or streptococcus aureus.
5th object of the present invention is to provide a kind of antibacterials, it is characterized in that, includes the compound difluostatinA of effective amount as active ingredient.
Described antibacterials are preferably the medicine of anti-Klebsiella Pneumoniae, Aeromonas hydrophila or streptococcus aureus.
The present invention is separated from streptomyces coelicolor (Streptomycescoelicolor.) YF11-3 and obtains 1 new fluostatin compounds difluostatinA with anti-microbial activity, they can for the preparation of antibacterials, and being expected exploitation by biotechnology or chemically modified becomes antibacterial new drug.
Described streptomyces coelicolor (Streptomycescoelicolor.) YF11-3 is prepared by the following method:
One, the extraction of Fluostatins and related compound producing strains micromonospora SCSION160 genomic dna thereof
By the mycelium of fresh micromonospora SCSION160 according to 5% inoculum size be inoculated in 1 of 50mL
#in substratum (starch 10g, yeast powder 4g, bacteriological peptone 2g, sea salt 10g, adds water and be settled to 1L, pH7.2-7.4), 28-30 DEG C, shaking culture is about 2-3 days, and 4000rpm collects mycelium in centrifugal 10 minutes.Mycelium STE solution (NaCl75mM, EDTA25mM, Tris-Cl20mM) washes twice, the N,O-Diacetylmuramidase of 30mLSTE solution and final concentration 3mg/mL is added in the mycelium after washing, vortex is even, and 37 DEG C of temperature are bathed 3 hours, are added to the Proteinase K of final concentration 0.1-0.2mg/mL, mixing, 37 DEG C of temperature are bathed 10 minutes, are added to the SDS of final concentration 1-2%, mixing, put into 55 DEG C of water-baths about 1 hour, period puts upside down for several times.Add isopyknic phenol-chloroform-primary isoamyl alcohol (V/V/V=25:24:1), mix, be placed in cooled on ice 30 minutes.12000rpm, 4 DEG C centrifugal 10 minutes, then carefully draws supernatant in new centrifuge tube with the big bore head cut, and uses the same method and repeatedly process 3 times, then centrifugal 10 minutes with isopyknic chloroform twice, 12000rpm, 4 DEG C.With the big bore head cut, aqueous phase sucking-off is transferred to new centrifuge tube, adds 1/10 volume 3mol/LNaAc (pH5.2), add isopyknic Virahol again after mixing, place on ice after mixing, precipitation DNA.With glass stick, DNA fiber group is transferred in new centrifuge tube carefully, by 70% washing with alcohol twice, liquid is inclined to, slightly dry at 37 DEG C, add 5mLTE and dissolve, and add the RNA enzyme of 3-5U, obtain micromonospora SCSION160 genomic dna thus.
Two, Fluostatins produces the foundation of bacterium micromonospora SCSION160 genomic library
First the consumption of restriction endonuclease Sau3AI is determined by a series of dilution experiment, in 20 μ L systems, micromonospora SCSION160 genomic dna containing 17 μ L, 10 × reaction buffer and the different dilution Sau3AI of 1 μ L of 2 μ L, its termination reaction is 4 μ L0.5mol/LEDTA and suitable sample-loading buffer.Proper by the Mei Huo unit groping to determine 0.025-0.05U.Pass through the partially digested genomic DNA fragment obtaining 30 ~ 42kb in a large number on this basis, spend Starch phosphorylase and carry out dephosphorylation process.
Carrier S uperCosl plasmid for building library first cuts in the middle of two cos sequences with restriction endonuclease XbaI, then carries out dephosphorylation process, then cuts from multiple clone site restriction endonuclease BamHI, obtain two arms.Carrier after process is connected with the genomic DNA fragment of the 30 partially digested ~ 42kb prepared before and spends the night, linked system is 10 μ L, SuperCos1 plasmid after the genomic DNA fragment prepared containing 1.25 μ g and 0.5 μ g process, the 10 × Buffer of 1 μ L, the ligase enzyme of 0.3U.Connect product in 65 DEG C of process 15 minutes, make ligase enzyme inactivation.From-80 DEG C of refrigerators, take out a tube packaging mixture (50 μ L) is placed on ice, packing mixt is melted rapidly between finger, careful absorption half packing mixt (25 μ L) is in a new centrifuge tube, add the connection product after 10 μ L thermal treatments, all the other packing mixts are in-80 DEG C of preservations.Careful mixing, 30 DEG C of temperature are bathed 90 minutes, add other half packing mixt (25 μ L), and 30 DEG C of temperature bathe continuation 90 minutes.Add 500 μ L phage dilution buffer (100mmol/LNaCl, 10mmol/LMgCl
2, 10mmol/LpH8.3Tris-HCl), then add 25 μ L chloroforms, mix gently, in 4 DEG C of preservations.
The frozen bacterial strain E.coliLM392MP in-80 DEG C is coated on LB substratum and recovers.Packaging reacts the day before yesterday, and picking mono-clonal is inoculated in LB substratum and (adds 0.2% maltose and 10mMMgSO
4), 37 DEG C of shaking culture are spent the night, and packaging reacts the same day, and the bacterium liquid getting 5mL incubated overnight joins in the fresh LB substratum of 50mL and (adds 0.2% maltose and 10mMMgSO
4), 37 DEG C, 200rpm vibration is to culture OD
600when reaching 0.8-1,4 DEG C save backup.The packaging liquid getting Host Strains liquid that 100 μ L as above process and the dilution of 100 μ L appropriateness mixes gently, in 37 DEG C of temperature baths 15 minutes, then coats on the LB flat board containing 100 μ g/mL penbritins and 50 μ g/mL kantlex, 37 DEG C of overnight incubation.By longer single clone, with sterile toothpick dibbling on 96 orifice plates that 30 pieces contain the above antibiotic LB substratum, 37 DEG C of overnight incubation, add the glycerine that final concentration is 20%, mix, and are placed in-80 DEG C of preservations.Obtain Fluostatins thus and produce bacterium micromonospora SCSION160 genomic library.
Three, positive colony of the biological gene of screening containing fluostatins synthesis bacterium micromonospora SCSION160 genomic library is produced from fluostatins
Nanfang Research Centre, State Human Gene Group is sent to carry out genome-wide screening and annotation micromonospora SCSION160 genomic dna, according to the result scanned and annotate, pass through bioinformatic analysis, primer flsGEF and flsGER (primer sequence is as shown in table 1) is screened with the sequences Design PCR being positioned at the flsG monooxygenase gene in the middle of fluostatins main body gene cluster, screening from genomic library 3072 clone of micromonospora SCSION160, obtain 13 positive colonies, afterwards with being positioned at the upper of main body gene cluster, downstream gene design PCR screens primer: flsF carboxyltransferase gene primer flsFDTF and flsFDTR, flsS adenylosuccinate lyase gene primer flsSEF and flsSER and bordering gene orf2 ~ 3-phosphorus shikimic acid-1-carboxyl vinyl transferase gene primer ORF2EF/ORF2ER (primer sequence is as shown in table 1) carry out multiple sieve to above-mentioned sieved positive colony and in conjunction with positive colony restriction analysis and terminal sequencing results, determine the wherein relative position of 4 cosmids in gene cluster, and have 1 cosmid (pCSG5003) to contain the biological synthesis gene cluster of whole fluostatins, other 3 cosmid (pCSG5000, pCSG5001, pCSG5002) portion gene bunch is comprised.The nucleotide sequence of the insertion sequence fragment pCSG5003 that described cosmid (pCSG5003) comprises is as shown in SEQIDNO.1, and this fragment pCSG5003 contains the biological synthesis gene cluster (its nucleotide sequence is as shown in SEQIDNO.1) of complete fluostatins.
Primer and sequence needed for table 1 library screening and heterogenous expression host strains
Then fragment pCSG5003 (biological synthesis gene cluster of fluostatins) is inserted in integrating vector pSET152, structure obtains mutational vector pCSG5033, then mutational vector pCSG5033 heterologous transformant pattern streptomycete StreptomycescoelicolorYF11 is obtained transformant S.YF11-3, be streptomyces coelicolor (Streptomycescoelicolor.) YF11-3.
Streptomycete StreptomycescoelicolorYF11 of the present invention is disclosed in document: Zhou, H.; Wang, Y.; Yu, Y.; Bai, T.; Chen, L.; Liu, P.; Guo, H.; Zhu, C.; Tao, M.; Deng, Z.Curr.Microbiol.2012,64,185-190..This bacterial strain the applicant also hold, and ensure provided to the public in 20 years.
Micromonospora (Micromonosporarosaria) SCSION160 of marine source of the present invention, this bacterium is preserved in China typical culture collection center (CCTCC) on October 08th, 2012, address: Wuhan University of Wuhan, China city, its deposit number is: CCTCCNO:M2012392; It is disclosed in the patent No.: ZL201210467946.X, and denomination of invention is in the patent of " a kind of micromonospora and utilize this bacterium to prepare the method for Multiple Classes of Antibiotics ".
Accompanying drawing illustrates:
Fig. 1 is the extract-extractum A of supernatant liquor and the high-efficient liquid phase chromatogram of mycelial extract-medicinal extract B;
High performance liquid chromatography (HPLC) condition: chromatographic column is phenomex150 × 4.6mm (SphereCloneSAX), moving phase comprises A phase and B phase, mobile phase A phase: the formic acid of the acetonitrile+0.08% (volume fraction) of 10% (volume fraction), solvent is water, flowing B phase: the acetonitrile of 90% (volume fraction), solvent is water; Injection procedure: 0-20min, mobile phase ratio is A phase/B phase (volume ratio): 95:5-0:100,20-21min, mobile phase ratio is A phase/B phase (volume ratio): 0:100,21-22min, mobile phase ratio is A phase/B phase (volume ratio): 0:100-95:5,22-30min, mobile phase ratio is A phase/B phase (volume ratio): 95:5, determined wavelength 254nm, flow velocity 1ml/min, wherein 1 representation compound 1.
Fig. 2 is the HMBC of the key of compound 1,
1h–
1hCOSY with NOE is relevant.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1: the separation of active metabolite difluostatinA and preparation
1, substratum: (1) seed culture medium: often liter contains starch 10g, yeast powder 5g, peptone 4g, CaCO
32g, thick sea salt 30g, surplus is water, pH7.2.Mix by above-mentioned component and content thereof, 121 DEG C, sterilizing 30min, obtains seed culture medium; (2) fermention medium: often liter contains starch 10g, fish peptone 1g, Semen Maydis powder 6g, bacto peptone 2g, glycerine 5ml, CaCO
32g, thick sea salt 30g, surplus is water, and pH7.0 mixes by above-mentioned component and content thereof, 121 DEG C, and sterilizing 30min, obtains fermention medium.
2, ferment
2.1, seed culture: single bacterium colony of the streptomyces coelicolor that culture dish activates (Streptomycescoelicolor.) YF11-3 is accessed 18 bottles respectively, in the taper culturing bottle of every bottle of 250mL containing 50mL seed culture medium, 28 DEG C, 200rmin
-1, cultivate 48h, obtained seed liquor 900mL.
2.2, fermentation culture: by seed liquor with 10% inoculum size (volume percent) be linked into 9L fermention medium (be placed in the taper culturing bottle of 250mL, every bottle containing 50ml fermention medium, totally 180 bottles) in, 28 DEG C, 200rmin
-1, shaking culture 120h, obtains 9L fermenting culture.
3, extract: fermenting culture first carries out centrifugation (3500rmin
-1, 8min), obtain supernatant liquor (fermented liquid) and the mycelium of 9L volume.Fermentation liquor macroporous resin XAD16 Solid-Phase Extraction, after use acetone wash-out macroporous resin 3 times, after elutriant reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate 3 times, and ethyl acetate layer obtains supernatant liquor extract-extractum A (2.4g) after distillation and concentration; Mycelium 2L acetone at room temperature lixiviate 3 times, each 3 hours, united extraction liquid, remained water mixed liquid 6L extraction into ethyl acetate after reclaim under reduced pressure acetone, and ethyl acetate layer underpressure distillation obtains mycelium extract-medicinal extract B (0.5g).
4, the extraction and isolation of active compound and qualification
The extraction and isolation of 4.1 compound difluostatin and qualification: the sample of the extractum A that takes a morsel respectively and medicinal extract B is dissolved in 100 μ l methyl alcohol, centrifuging and taking supernatant 10 μ l, through HPLC sample detection, show, containing compound 1 in extractum A, containing compound 1 (Fig. 1) in medicinal extract B, extractum A and medicinal extract B are merged.By the crude extract of this merging through silicagel column (300-400 order) chromatography, by chloroform/methanol as eluent, gradient elution is carried out from volume ratio 100:0 ~ 0:100, collect the cut Fr.2 (1.21g) that chloroform/methanol volume ratio 95:5 gradient elution gets off, evaporate to dryness, through the anti-phase medium pressure liquid chromatography of ODS (YMC*GELODS-A ball-shaped filling material, 120A aperture, 50um particle diameter, 240 × 4.0cmI.D.), flow velocity is 25ml/min, with water/methyl alcohol as eluent, gradient elution is carried out from volume ratio 100:0 ~ 0:100, collect the cut Fr.2-7 (30mg) that water/methyl alcohol volume ratio 20:80 gradient elution gets off, after LH-20 gel column (2.5*100cm), using chloroform/methanol volume ratio 1:1 as moving phase wash-out, every 50ml collects and merges into portion, order obtains Fr.2-7-A-F five increment product, Fr.2-7-B (7mg) is prepared with thin-layer chromatography, collecting prepared by thin layer plate is 8:2 by petrol ether/ethyl acetate volume ratio is the cut of 0.8 as Rf value during developping agent, be compound 1 (difluostatinA) (2.7mg).Pass through structural analysis, identify it a compound-compound 1 (corresponding compound difluostatinA) (formula (I)) prepared from the fermenting culture of streptomyces coelicolor (Streptomycescoelicolor.) YF11-3 of the present invention, its qualification result is as follows:
Compound 1 (difluostatinA): dark red powder,
1hNMR (500MHz, CDCl
3) and
13cNMR (125MHz, CDCl
3) data, in table 2; HRESIMSm/z [M-H]
-597.1191 (calcdforC
36h
22o
9).
It is m/z [M-H] that the positive source high resolution electrospray ionization mass spectrum figure of compound 1 shows its quasi-molecular ion peak
-597.1191
+, infer that its molecular formula is C
36h
22o
9(degree of unsaturation is 26).Analyze
1h,
13c and HSQC (table 2) data show that this compound contains 1 methyl [δ
h1.93 (3H, s, H-12), δ
c18.1], 9 sp
2the methyne of hydridization, 2 sp
3methyne [the δ of hydridization
h6.17 (1H, d, J=4.0, H-1), δ
c34.6; δ
h4.84 (1H, d, J=4.0, H-2), δ
c69.0], 21 sp
2the quaternary carbon of hydridization and 1 sp
3quaternary carbon [the δ of hydridization
c78.9 (C-3)].Pass through
1h-
1hCOSY collection of illustrative plates (Fig. 2) is analyzed display compound and is contained two ABC coupling system [((δ
h7.07 (1H, dd, J=7.5,2.0Hz), (δ
h7.34 (1H, dd, J=7.5,7.0Hz), (δ
h7.33 (1H, brd, J=7.0, Hz)); ((δ
h7.02 (1H, d, J=8.5Hz), (δ
h7.22 (1H, dd, J=8.5,7.0Hz), (δ
h(7.18 1H, d, J=7.0, Hz))], in conjunction with the constitutional features of Fluostatin compounds, analysis of compounds 1 is containing two Fluostatin monomers.Two molecular ion peak m/z291,306 of the MS/MS acquisition of compound 1, this is consistent with inferring the molecular weight of segment, further provides compound 1 for dimeric evidence.Carefully Analysis for CO SY, HMBC spectrum (Fig. 2), belong to dimeric two monomers of composition.From H-1 to C-4a ' and/C-5 ', H-2toC-5 ' relevant with the HMBC of H-4 ' to H-4a '/H-5 ', and the NOESY of H-1 with H-4 ' is relevant, provides the evidence that two dimers are connected by C-1 with C-5 '.Coupling constant values H-1/H-2 in compound 1 between H-1 and H-2 (
3j
h1-H24.0Hz) advise being configured as trans between H-1/H-2.Its absolute configuration is speculated as 1S, 2S and 3S by the biosynthetic pathway according to compound 1.
According to above information, the structure of deterministic compound 1 such as formula shown in (I), called after: difluostatinA.
Table 2. compound difluostatinA's
1hNMR (500MHz) nuclear magnetic data belongs to
DatawererecordedonaBrukerAvance500NMRspectrometer,chemicalshift(δ)aregiveninppm.
Embodiment 2: the mensuration of the bacteriostatic activity of compound difluostatinA
Compound difluostatinA is for four kinds of pathogenic strainss: Klebsiella Pneumoniae KlebsiellapneumoniaATCC13883, Aeromonas hydrophila AeromonashydrophilaATCC7966, streptococcus aureus StaphylococcusaureusATCC29213 and enterococcus faecalis EnterococcusfaecalisATCC29212 has carried out determination of activity, experimental technique reference [ZhangW, LiuZ, LS, ChenY, Zhangh, ZhangG, ZhuY, ZhangG, ZhangW, LiuJ and ZhangC, FluostatinsI-KfromthesouthChinasea-derivedMicromonospora rosariaSCSION160.J.Nat.Prod, 2012, 75, 1937-1943].Positive control is made with penbritin, show through test determination, compound difluostatinA is to Klebsiella Pneumoniae KlebsiellapneumoniaATCC13883, and Aeromonas hydrophila AeromonashydrophilaATCC7966, the MIC value of streptococcus aureus is 4 μ gmL respectively
-1, 4 μ gmL
-1with 8 μ gmL
-1; Positive control be 0.25 μ gmL
-1, 0.25 μ gmL
-1with 4 μ gmL
-1.Result shows that compound difluostatinA is to Klebsiella Pneumoniae KlebsiellapneumoniaATCC13883, Aeromonas hydrophila AeromonashydrophilaATCC7966, and the MIC value of streptococcus aureus is 4 μ gmL respectively
-1, 4 μ gmL
-1with 8 μ gmL
-1; Compound difluostatinA has growth inhibitory activity to surveyed three kinds of pathogenic bacterium performances, and being expected exploitation by biotechnology or chemically modified becomes antibacterial new drug.
Claims (9)
1. compound difluostatinA, its structure is such as formula shown in (I):
2. the preparation method of a compound difluostatinA according to claim 1, it is characterized in that, described compound difluostatinA is that preparative separation obtains from the fermenting culture of streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3.
3. preparation method according to claim 2, is characterized in that, concrete steps are as follows:
A, prepare the fermenting culture of streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3, the fermented liquid of this fermenting culture and mycelium are separated, fermentation liquor macroporous resin adsorption, after use acetone wash-out macroporous resin, after elutriant reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate, and ethyl acetate layer obtains extractum A after distillation and concentration; Mycelium first uses acetone extraction, and after leaching liquid reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate again, and ethyl acetate layer obtains medicinal extract B after distillation and concentration;
B, crude extract extractum A and medicinal extract B merged is through silica gel column chromatography, by chloroform/methanol as eluent, gradient elution is carried out from volume ratio 100:0 ~ 0:100, collect the cut Fr.2 that chloroform/methanol volume ratio 95:5 gradient elution gets off, by the anti-phase medium pressure liquid chromatography of ODS, with water/methyl alcohol as eluent, gradient elution is carried out from volume ratio 100:0 ~ 0:100, collect the cut Fr.2-7 that water/methyl alcohol volume ratio 20:80 gradient elution gets off, after LH-20 gel column, using chloroform/methanol volume ratio 1:1 as moving phase wash-out, eluting fraction is separated through Thin Layer Chromatography again, obtain compound difluostatinA.
4. preparation method according to claim 3, it is characterized in that, the fermenting culture preparing streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3 of described a step prepares by the following method: streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3 of activation is accessed seed culture medium, 28 DEG C, 200rpm, cultivate 48h and obtain seed liquor, by seed liquor with 10% inoculum size be linked in fermention medium, 28 DEG C, 200rpm, shaking culture 120h, and obtained fermenting culture, the formula of described seed culture medium contains in often liter of substratum: starch 10g, yeast powder 5g, peptone 4g, CaCO
32g, thick sea salt 30g, surplus is water, pH7.2, and the formula of described fermention medium is containing starch 10g, fish peptone 1g, Semen Maydis powder 6g, bacto peptone 2g in often liter of substratum, glycerine 5ml, CaCO
32g, thick sea salt 30g, surplus is water, pH7.0.
5. the application of streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3 in preparation compound difluostatinA according to claim 1, described streptomyces coelicolor (Streptomyces.coelicolor.) YF11-3 is by replacing on the cosmid of the biological synthesis gene cluster containing fluostatins by carrier pSET152 fragment, then be transformed in streptomyces coelicolor (Streptomyces.coelicolor.) YF11 and obtain, the nucleotide sequence of the biological synthesis gene cluster of described fluostatins is as shown in SEQIDNO.1.
6. the application of compound difluostatinA according to claim 1 in preparation bacterium medicine.
7. application according to claim 6, is characterized in that, described antibacterials are the medicine of anti-Klebsiella Pneumoniae, Aeromonas hydrophila or streptococcus aureus.
8. antibacterials, is characterized in that, include the compound difluostatinA according to claim 1 of effective amount as active ingredient.
9. antibacterials according to claim 8, is characterized in that, described antibacterials are the medicine of anti-Klebsiella Pneumoniae, Aeromonas hydrophila or streptococcus aureus.
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| CN108383806A (en) * | 2018-04-13 | 2018-08-10 | 中国科学院南海海洋研究所 | A kind of preparation method of dimerization fluostatins class antibiotic |
| CN117024611A (en) * | 2023-03-01 | 2023-11-10 | 中国科学院南海海洋研究所 | Construction and activity application of oligosaccharide antibiotic everninomicin high-yield strain |
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| CN102168044A (en) * | 2010-12-21 | 2011-08-31 | 中国科学院南海海洋研究所 | Streptomyces sp. and process for preparing various antibiotics by using same |
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| CN101974464A (en) * | 2010-10-18 | 2011-02-16 | 中国科学院南海海洋研究所 | Streptomyces and process for preparing antimycin antibiotics by fermentation using same |
| CN102168044A (en) * | 2010-12-21 | 2011-08-31 | 中国科学院南海海洋研究所 | Streptomyces sp. and process for preparing various antibiotics by using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108383806A (en) * | 2018-04-13 | 2018-08-10 | 中国科学院南海海洋研究所 | A kind of preparation method of dimerization fluostatins class antibiotic |
| CN108383806B (en) * | 2018-04-13 | 2020-06-30 | 中国科学院南海海洋研究所 | Preparation method of dimeric fluorostatins antibiotics |
| CN117024611A (en) * | 2023-03-01 | 2023-11-10 | 中国科学院南海海洋研究所 | Construction and activity application of oligosaccharide antibiotic everninomicin high-yield strain |
| CN117024611B (en) * | 2023-03-01 | 2024-05-17 | 中国科学院南海海洋研究所 | Construction and activity application of oligosaccharide antibiotics everninomicin high-yield strain |
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