CN101597578A - A kind of enramycin producing strain and utilize the method for macroporous resin extraction - Google Patents
A kind of enramycin producing strain and utilize the method for macroporous resin extraction Download PDFInfo
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- CN101597578A CN101597578A CNA2009100323401A CN200910032340A CN101597578A CN 101597578 A CN101597578 A CN 101597578A CN A2009100323401 A CNA2009100323401 A CN A2009100323401A CN 200910032340 A CN200910032340 A CN 200910032340A CN 101597578 A CN101597578 A CN 101597578A
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- enramycin
- macroporous resin
- hydrochloric acid
- methyl alcohol
- extraction
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- NJCUSQKMYNTYOW-MWUYRYRWSA-N enramicina Chemical compound O.N1C(=O)NC(=O)C(C=2C=C(Cl)C(O)=C(Cl)C=2)NC(=O)C(CO)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(CC2N=C(N)NC2)NC(=O)C(CCCNC(N)=O)NC(=O)C(C(C)O)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C(C)O)NC(=O)N(CCCCN)C(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)/C=C/C=C/CCCCC(C)CC)C(C)OC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C)NC(=O)C1CC1CNC(N)=N1 NJCUSQKMYNTYOW-MWUYRYRWSA-N 0.000 title claims abstract description 84
- 229950003984 enramycin Drugs 0.000 title claims abstract description 84
- 108700041171 enramycin Proteins 0.000 title claims abstract description 84
- 238000000605 extraction Methods 0.000 title claims abstract description 30
- 229920005989 resin Polymers 0.000 title claims abstract description 29
- 239000011347 resin Substances 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000000855 fermentation Methods 0.000 claims abstract description 14
- 230000004151 fermentation Effects 0.000 claims abstract description 14
- 238000010828 elution Methods 0.000 claims abstract description 10
- 238000010521 absorption reaction Methods 0.000 claims abstract description 8
- 238000013467 fragmentation Methods 0.000 claims abstract description 7
- 238000006062 fragmentation reaction Methods 0.000 claims abstract description 7
- 230000003068 static effect Effects 0.000 claims abstract description 7
- 239000012141 concentrate Substances 0.000 claims abstract description 3
- 238000001035 drying Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 156
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 40
- 239000007864 aqueous solution Substances 0.000 claims description 29
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 26
- 239000008103 glucose Substances 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
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- 239000000047 product Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 15
- 239000002609 medium Substances 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
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- 244000005700 microbiome Species 0.000 claims description 8
- 235000016709 nutrition Nutrition 0.000 claims description 8
- 229910052700 potassium Inorganic materials 0.000 claims description 8
- 229940041514 candida albicans extract Drugs 0.000 claims description 7
- 239000012138 yeast extract Substances 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 244000068988 Glycine max Species 0.000 claims description 6
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- 229910019142 PO4 Inorganic materials 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 6
- 241000276851 Streptomyces sp. NJWGY3665 Species 0.000 claims description 6
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 claims description 6
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 229920001429 chelating resin Polymers 0.000 claims description 6
- 229960004756 ethanol Drugs 0.000 claims description 6
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 6
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- 235000019698 starch Nutrition 0.000 claims description 6
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- FUKUFMFMCZIRNT-UHFFFAOYSA-N hydron;methanol;chloride Chemical compound Cl.OC FUKUFMFMCZIRNT-UHFFFAOYSA-N 0.000 claims description 4
- -1 sodium-chlor methanol Chemical compound 0.000 claims description 4
- 238000012262 fermentative production Methods 0.000 claims description 3
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
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- 229940049954 penicillin Drugs 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
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- 238000000926 separation method Methods 0.000 description 2
- HGUFODBRKLSHSI-UHFFFAOYSA-N 2,3,7,8-tetrachloro-dibenzo-p-dioxin Chemical compound O1C2=CC(Cl)=C(Cl)C=C2OC2=C1C=C(Cl)C(Cl)=C2 HGUFODBRKLSHSI-UHFFFAOYSA-N 0.000 description 1
- ACTOXUHEUCPTEW-BWHGAVFKSA-N 2-[(4r,5s,6s,7r,9r,10r,11e,13e,16r)-6-[(2s,3r,4r,5s,6r)-5-[(2s,4r,5s,6s)-4,5-dihydroxy-4,6-dimethyloxan-2-yl]oxy-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy-10-[(2s,5s,6r)-5-(dimethylamino)-6-methyloxan-2-yl]oxy-4-hydroxy-5-methoxy-9,16-dimethyl-2-o Chemical compound O([C@H]1/C=C/C=C/C[C@@H](C)OC(=O)C[C@@H](O)[C@@H]([C@H]([C@@H](CC=O)C[C@H]1C)O[C@H]1[C@@H]([C@H]([C@H](O[C@@H]2O[C@@H](C)[C@H](O)[C@](C)(O)C2)[C@@H](C)O1)N(C)C)O)OC)[C@@H]1CC[C@H](N(C)C)[C@@H](C)O1 ACTOXUHEUCPTEW-BWHGAVFKSA-N 0.000 description 1
- 241000186361 Actinobacteria <class> Species 0.000 description 1
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- STUSTWKEFDQFFZ-UHFFFAOYSA-N Chlordimeform Chemical compound CN(C)C=NC1=CC=C(Cl)C=C1C STUSTWKEFDQFFZ-UHFFFAOYSA-N 0.000 description 1
- ZDPIZLCVJAAHHR-UHFFFAOYSA-N Clopidol Chemical compound CC1=NC(C)=C(Cl)C(O)=C1Cl ZDPIZLCVJAAHHR-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- MVTQIFVKRXBCHS-SMMNFGSLSA-N N-[(3S,6S,12R,15S,16R,19S,22S)-3-benzyl-12-ethyl-4,16-dimethyl-2,5,11,14,18,21,24-heptaoxo-19-phenyl-17-oxa-1,4,10,13,20-pentazatricyclo[20.4.0.06,10]hexacosan-15-yl]-3-hydroxypyridine-2-carboxamide (10R,11R,12E,17E,19E,21S)-21-hydroxy-11,19-dimethyl-10-propan-2-yl-9,26-dioxa-3,15,28-triazatricyclo[23.2.1.03,7]octacosa-1(27),6,12,17,19,25(28)-hexaene-2,8,14,23-tetrone Chemical compound CC(C)[C@H]1OC(=O)C2=CCCN2C(=O)c2coc(CC(=O)C[C@H](O)\C=C(/C)\C=C\CNC(=O)\C=C\[C@H]1C)n2.CC[C@H]1NC(=O)[C@@H](NC(=O)c2ncccc2O)[C@@H](C)OC(=O)[C@@H](NC(=O)[C@@H]2CC(=O)CCN2C(=O)[C@H](Cc2ccccc2)N(C)C(=O)[C@@H]2CCCN2C1=O)c1ccccc1 MVTQIFVKRXBCHS-SMMNFGSLSA-N 0.000 description 1
- 239000004187 Spiramycin Substances 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000971005 Streptomyces fungicidicus Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000004182 Tylosin Substances 0.000 description 1
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- 108010080702 Virginiamycin Proteins 0.000 description 1
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- 239000006030 antibiotic growth promoter Substances 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
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- UCRLQOPRDMGYOA-DFTDUNEMSA-L zinc;(4r)-4-[[(2s)-2-[[(4r)-2-[(1s,2s)-1-amino-2-methylbutyl]-4,5-dihydro-1,3-thiazole-4-carbonyl]amino]-4-methylpentanoyl]amino]-5-[[(2s,3s)-1-[[(3s,6r,9s,12r,15s,18r,21s)-3-(2-amino-2-oxoethyl)-18-(3-aminopropyl)-12-benzyl-15-[(2s)-butan-2-yl]-6-(carbox Chemical compound [Zn+2].C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC([O-])=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2NC=NC=2)C(=O)N[C@H](CC([O-])=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 UCRLQOPRDMGYOA-DFTDUNEMSA-L 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of enramycin producing strain and utilize the method for macroporous resin extraction enramycin.Belong to the technology of extracting enramycin by fermentation back mycelium.This method obtains enramycin by fermentation method, is raw material with the mycelium after the fermentation, and the process of preparation, macroporous resin absorption, gradient elution and the concentrate drying of process cytoclasis, enramycin crude extract obtains enramycin.The preparation that it is characterized in that the enramycin crude extract is that isothermal vibration after the mycelium fragmentation is extracted, and extracting solution concentrating under reduced pressure under vacuum gets crude extract, and crude extract adsorbs with macroporous resin; Static absorption back gradient elution obtains product through concentrating under reduced pressure again.Technology of the present invention is simple, and is with short production cycle, and production cost is low, is suitable for suitability for industrialized production, has certain social, economic benefit.
Description
Technical field
The invention belongs to the separation engineering technical field, relate to a kind of enramycin producing strain and utilize the method for macroporous resin extraction.
Background technology
From now various antibiotic being extensive use of of being found to of penicillin in 1945, microbiotic is not only being brought into play enormous function in the treatment of human diseases and prevention, critical role is also arranged in agricultural especially livestock industry.China is a large agricultural country, and livestock industry is rapid as the mainstay industry development of agricultural, and (2006) livestock industry output value accounted for agriculture proportion and reaches 35% in recent years.Livestock industry is very big to antibiotic dependency, present domestic annual microbiotic spends about 500,000,000 dollars, and along with new microbiotic kind constantly is developed exploitation, the consumption of microbiotic in mixed feed enlarges gradually, and China has the microbiotic of about 6000t every year on average as fodder additives at present.
Yet, be extensive use of along with antibiotic, engender drug residue and resistance problem, when the mankind are edible when containing meat product, the milk preparation of antibiotic residues, hidden danger of bringing and drawback are extremely serious, eliminate the germ of existing penicillin resistant, xacin-series, need the mankind to reach and thoroughly do not re-use these two kinds of medicines about 100 years, the appearance of endurance strain will be dwindled following antibiotic use range.In addition, owing to the meat food safety problem that feed causes,, also make us so far having a lingering fear as incidents such as the pollution of mad cow disease, dioxin, French sewage feeds.Thereby European Union forbids adding microbiotic such as Spiramycin Base, virginiamycin, Zinc-bacitracin and tylosin in feed.Simultaneously, antibiotic remains exceeds standard and seriously restricts the outlet of China's livestock product, and the chicken clopidol is residual to exceed standard, and bee product " Chlorphenamidine " and residual chloromycetin exceed standard, and are closed down and require to destroy, and cause financial loss to China.For this reason, the use of antibiotic feed additive produces serious negative impact to livestock industry and human health.Yet relevant report shows, forbids that the country that adds microbiotic or other antibiotic growth promoters in feed can cause the increase of livestock industry production cost, profit to reduce, and also might cause such as the problems such as trade war between ecotope and the country simultaneously.Face current severe situation, develop have growth promoting function, low drug residue and the new antibiotic kind of not having a cross resistance have become instant thing please.
Enramycin is a kind of wide spectrum, efficient, safe novel fodder additive, and as fodder additives, enramycin has following advantage: the enramycin that (1) only adds trace to feed just can promote the weightening finish to livestock and poultry.(2) have gram-positive microorganism intensive antimicrobial acivity.(3) can not residue in the livestock and poultry body.(4) and between existing clinically microbiotic or the antimicrobial drug there is not cross resistance.Sensitive organism produces resistance to enramycin hardly, and under experiment condition, chemical sproof generation is also very slow, even occur, resistance is also unstable, and very easily loses.(5) can keep stability in the feed the inside.In addition, enramycin also has hepatitis B virus (HBV) antigen and hepatitis B virus e antigen (HBeAg) and suppresses effect preferably.Just because of enramycin have efficiently, advantage such as safety, noresidue, in the short period of time of China's listing, the market share rises rapidly, become a line products of antibiotic feed additive for promoting growth, but at present domestic relevant report of not seeing that as yet enramycin is produced, seminar has determined the optimal separation extraction process of enramycin by a large amount of experimental studies.
Summary of the invention
The purpose of this invention is to provide the process of a kind of enramycin producing strain (streptomycete), fermentative production enramycin and the method for application macroporous resin extraction enramycin.
Technical scheme of the present invention: a kind of streptomycete, its classification called after streptomycete, the Latin formal name used at school of bacterial classification is Streptomyces sp.NJYWG3665, and preservation date is on March 21st, 2008, and registering on the books and number in the preservation center is CGMCC NO.2410.
The present invention also provides a kind of method with the macroporous resin extraction enramycin, concrete steps are as follows: the process of A, fermentative Production enramycin is: (1) slant culture: with streptomycete Streptomyces sp.NJWGY3665 bacterial classification, the microbial strains preservation center numbering CGMCC No.2410 of the Chinese Academy of Sciences, receive in the glucose nutritional medium, carry out slant culture, the weight percent proportioning of glucose nutritional medium is glucose 0.5～1%, extractum carnis 0.5～1%, peptone 0.5～1%, NaCl0.3～0.5% or K
2HPO
40.1～0.2%, agar 1.5～2%, water surplus, pH 6.0～9.0,28～37 ℃ of culture temperature, incubation time 2 days～6 days; (2) seed culture: with sterilized water the spore of cultivating on the inclined-plane is made monospore suspension, and be inoculated in the 50ml seed culture medium and cultivate, the weight percent proportioning of seed culture medium is glucose 1～3.0%, corn steep liquor 1～3%, CaCO
31～2%, yeast powder 0.5%, NaCl 0.1～0.8%, water surplus, pH is 6.0～9.0, and temperature is 28～32 ℃, and rotating speed is 200rpm, cultivates 2 days～6 days; (3) fermentation culture: seed liquor is inoculated in the fermention medium cultivates, the weight percent proportioning of fermention medium is glucose 2.0～5.0%, starch 0.5～2.0%, corn steep liquor 0.5～3.0%, yeast extract paste 0.1～0.5%, soybean cake powder 0.5～2%, NaCl0.5～1.0%, CaCO
31～2%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water surplus, temperature is controlled at 28～37 ℃, and pH is controlled at 6.0～9.0, and rotating speed 180～220rpm fermented 5～9 days; B, the enramycin producing strain after will fermenting carry out the microorganism cells fragmentation with KQ-100 type ultrasonic machine, obtain containing the mixture of enramycin; C, to be diluted with water to volume fraction be that the vibration of 10%～100% extraction agent was extracted the mixture described in the B 0.5～4 hour, and the pH value of system is controlled between 4.0～8.5, and acquisition contains the extracting solution of enramycin; D, the extracting solution that will contain enramycin are centrifugal, collect the supernatant liquor that contains enramycin, and supernatant liquor is evaporated to absence of liquid under 30～40 ℃, obtain enramycin and slightly carry product; After E, the macroporous resin pre-treatment, the enramycin after concentrating is slightly put forward the product dissolution with solvents, after 2～5 hours, carry out obtaining the pure product of enramycin behind gradient elution, the concentrate drying with eluent through the static absorption of macroporous resin.
Above-mentioned extraction agent is that extraction agent is the methanol aqueous solution of ethanol, methyl alcohol, hydrochloric acid, sodium-chlor, the methanol aqueous solution of hydrochloric acid, wherein said ethanol is that dehydrated alcohol, methyl alcohol are analytical pure, the volumetric molar concentration of hydrochloric acid is 1～5mol/L, the sodium-chlor volumetric molar concentration is that 0.03～0.09mol/L, methyl alcohol volume fraction are 40%～70% in the sodium-chlor methanol aqueous solution, and the hydrochloric acid volumetric molar concentration is that 0.003～0.009mol/L, methyl alcohol volume fraction are 40%～70% in the hydrochloric acid methanol aqueous solution.
The volume fraction of extraction agent is 30%～100%, and the time of utilizing extraction agent to extract is 0.5～2.5 hour.
The mycelium of enramycin producing strain quality/extraction agent volume is 1/10～1/150 among the above-mentioned steps C.
The pH of said extracted system is 5.0～7.5.
Above-mentioned selected macroporous resin is macroporous resin D101, Amberlite XAD-2, Amberlite XAD-4 or Amberlite XAD-16.
During gradient elution, eluent be methanol aqueous solution, the sodium-chlor volumetric molar concentration of methyl alcohol volume fraction 30%～90% be 0.03～0.09mol/L and methyl alcohol volume fraction be 40%～70% sodium-chlor methanol aqueous solution, hydrochloric acid volumetric molar concentration be 0.003～0.009mol/L and methyl alcohol volume fraction be 40%～70% the hydrochloric acid methanol aqueous solution or volumetric molar concentration be 1～3mol/L hydrochloric acid in any two kinds; The volume ratio of two kinds of eluents is 1: 9～5: 5.
Above-mentioned streptomycete Streptomyces sp.NJYWG3665 be by this laboratory from the soil of school district, Nanjing University of Technology Jiangpu, primary dcreening operation obtains the actinomycetes that 47 strains can suppress the bacillus subtilis bacteria growing; Through shaking the multiple sieve of bottle, detect acquisition one strain enramycin producing strain with HPLC (high performance liquid chromatography), analyze by physio-biochemical characteristics experiment and 16S rDNA sequence similarity, the called after streptomycete is identified in classification, and be preserved in Chinese common micro-organisms culture presevation administrative center, it abbreviates CGMCC as, and the numbering of registering on the books is CGMCCNo.2410, and preservation date is: on March 21st, 2008.With this bacterial classification as producing bacterial strain.
CGMCC No.2410 bacterial strain has following character:
1, morphological specificity:
The bacterial strain moderate of on the Gause I substratum, growing, aerial hyphae growth moderate, it is curved that fibrillae of spores is ripple, and spore becomes catenation, spore ovalization under electron microscope.
2, cultural characteristic:
Cultivated 7-14 days for 28 ℃, observe the feature of bacterial strain on following various substratum.
The cultural characteristic of table 1CGMCC No.2410 bacterial strain
3, Physiology and biochemistry character:
The Physiology and biochemistry character of table 2CGMCC No.2410 and S.fungicidicus NO.B5477 bacterial strain relatively
+++: enrichment and growth; ++: the appropriateness growth; +: growth;-: do not grow
4,16S rDNA sequential analysis
Extract genomic dna with the bacteriolyze enzyme process from new fresh thalli, adopt the streptomycete universal primer to carry out the 16SrDNA amplification, the PCR product after testing, transfer to TAKARA company behind the purifying and check order.The 16SrDNA sequence of being surveyed after check and correction, splicing with the GenBank database in the sequence of relevant kind carry out BLAST and learn that relatively bacterial strain Streptomyces sp.NJYWG3665 (CGMCC No.2410) belongs to streptomyces.
Record the most of sequence 1650bp of 16S rDNA, specific as follows:
5’-ATTGAGTTTATCCTGGCTCAGGGTTTCTTCGGTACGCTGAGTCGAAACACCCACTTCGACGATATAGTTGCGGCTGGATCACCTCCTTAGAGTTTGATCCTGGCTCAGTGCGGCTAGATCACCTCCTTAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGAACCACTTCGGTGGGGATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGACCCGCCTGGGCATCCAGGCGGTTCGAAAGCTCCGGCGGTGCAGGGTGAGCCCGCGGCCTATCAGCTTGTTGGTGAGGTAACGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATCGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCGCGTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGAGCCATTACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGAACTAGGTGTTGGCGACATTCCACGTCGTCGGTGCCGCAGCTAACGCATTAAGTTCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCAGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACACCGGAAAGCATTAGAGATAGTGCCCCCCTTGTGGTCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCACAACCCTTGTCCTGTGTTGCCAGCATGCCCTTCGGGGTGATGGGGACTCACAGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGGCCGGTACAATGAGCTGCGAAACCGTGAGGTGGAGCGAATCTCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTTGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTTGGTAACACCCGAAGCCGGTGGCCCAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGGACGAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGATCACCTCCTTAAT-3’
Beneficial effect: utilize the technology of this bacterium production enramycin simple, the unit volume fermented liquid enramycin height of tiring, utilize this method to extract enramycin, have characteristics such as extraction yield height, simple to operate and cost be low, be easy to realize industrialization, the production of adding enramycin for feed provides feasible extraction process.
Embodiment
The mycelium that following all examples are selected is: Streptomyces sp.NJWGY3665, the microbial strains preservation center numbering CGMCC No.2410 of the Chinese Academy of Sciences
Embodiment 1
Streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received glucose 0.5%, extractum carnis 0.5%, peptone 0.5%, NaCl0.5%, agar 1.5%, water 96.5% are in the glucose nutritional medium of pH6.0, carry out slant culture, cultivated 2 days down at 28 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed, make spore suspension, get the 0.5ml spore suspension and be inoculated into and contain 40ml glucose 1.0%, corn steep liquor 3%, CaCO with transfering loop
31%, yeast extract paste 0.5%, NaCl0.1%, water 94.4% were cultivated 3 days in the liquid seed culture medium of pH7.0, and culture temperature is 28 ℃, and rotating speed is 200rpm.The 5ml seed liquor being linked into liquid amount is that 100ml contains glucose 4.0% again, starch 2.0%, corn steep liquor 2.0%, yeast extract paste 0.1%, soybean cake powder 2%, NaCl 0.5%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, the fermentation culture of water 86.9% is concentrated, at 28 ℃, 180rpm, shaker fermentation was cultivated 5 days under the condition of pH6.0, acquisition contains the mycelium of enramycin, with the mycelium after the 1g fermentation, use washed with de-ionized water, be suspended in the 30mL ethanol, the alcoholic acid volume fraction is 70%, pH is 4.5, carry out the broken 15min of microorganism cells with KQ-100 type ultrasonic machine, obtain containing the mixture of enramycin, the mixture after the fragmentation is shaken bottle cabinet with HYG-II a type vibrate that (the adjusting temperature and the rotating speed that shake bottle cabinet is respectively 15 ℃, 180rpm) extracted 1 hour, obtain containing the extracting solution of enramycin, will contain extracting solution centrifugal 15min under the rotating speed of 1500r/min of enramycin then, collect the supernatant liquor that contains enramycin, supernatant liquor is evaporated to absence of liquid under 30 ℃, obtains enramycin and slightly carry product.After macroporous resin carried out pre-treatment according to conventional treatment method, the glass pillar of packing into and having the constant temperature protective sleeve was that to be 70% methanol aqueous solution pump into pillar with the speed of 60mL/h finishes balance for 0.09mol/L and methyl alcohol volume fraction with sodium chloride concentration.The product of slightly carrying of enramycin are dissolved in the 10mL ethanol, be placed on the top of pillar, static absorption 2 hours, with the eluent of 0.5L (methyl alcohol/sodium chloride concentration is that 0.09mol/L and methyl alcohol volume fraction are that the volume ratio of 70% methanol aqueous solution is 1: 9) gradient elution, with elutriant rotary vacuum evaporator concentrating under reduced pressure evaporate to dryness, productive rate reaches 60%.
Embodiment 2
Streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received glucose 0.5%, extractum carnis 0.8%, peptone 0.8%, K
2HPO
40.1%, agar 1.8%, water 96% in the glucose nutritional medium of pH7.0, carry out slant culture, cultivate 3 days down at 37 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed with transfering loop, make spore suspension, get the 0.5ml spore suspension and be inoculated into spore suspension and be inoculated into and contain 40ml glucose 2.0%, corn steep liquor 2.0%, CaCO
31.5%, yeast powder 0.5%, NaCl0.5%, water 93.5% were cultivated 4 days in the liquid seed culture medium of pH7.0, and culture temperature is 30 ℃, and rotating speed is 200rpm.The 10ml seed liquor being linked into liquid amount is that 100ml contains glucose 2.0% again, starch 1.5%, corn steep liquor 0.5%, yeast extract paste 0.3%, soybean cake powder 1.5%, NaCl0.8%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, in the fermention medium of water 90.9%, at 32 ℃, 220rpm, shaker fermentation was cultivated 6 days under the condition of pH7.0, acquisition contains the mycelium of enramycin, with the mycelium after the 1g fermentation, use washed with de-ionized water, be suspended in the 90mL hydrochloric acid, the volume fraction of hydrochloric acid is 50%, pH is 5.0, carry out the broken 15min of microorganism cells with KQ-100 type ultrasonic machine, obtain containing the mixture of enramycin, the mixture after the fragmentation is shaken bottle cabinet with HYG-II a type vibrate that (the adjusting temperature and the rotating speed that shake bottle cabinet is respectively 22 ℃, 200rpm) extracted 4 hours, obtain containing the extracting solution of enramycin, will contain extracting solution centrifugal 20min under the rotating speed of 1800r/min of enramycin then, collect the supernatant liquor that contains enramycin, supernatant liquor is evaporated to absence of liquid under 40 ℃, obtains enramycin and slightly carry product.After macroporous resin carried out pre-treatment according to conventional treatment method, the glass pillar of packing into and having the constant temperature protective sleeve was that to be 50% methanol aqueous solution pump into pillar with the speed of 55mL/h finishes balance for 0.09mol/L and methyl alcohol volume fraction with sodium chloride concentration.The product of slightly carrying of enramycin are dissolved in the 15mL methyl alcohol, be placed on the top of pillar, static absorption 4 hours, with the eluent of 1.0L (volume fraction is that methanol aqueous solution/concentration of hydrochloric acid of 70% is that 0.006mol/L and methyl alcohol volume fraction are that the volume ratio of 70% methanol aqueous solution is 3: 7) gradient elution, with elutriant rotary vacuum evaporator concentrating under reduced pressure evaporate to dryness, productive rate reaches 63%.
Embodiment 3
Adopt broth culture streptomycete Steptomyces sp.NJWGY3665 bacterial classification to be received glucose 1%, extractum carnis 1%, peptone 1%, NaCl 0.5%, and agar 2%, water 94.5% are in the glucose nutritional medium of pH9.0, carry out slant culture, cultivated 5 days down at 32 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed, make spore suspension, get the 1ml spore suspension and be inoculated into and contain 50ml glucose 3.0%, corn steep liquor 1%, CaCO with transfering loop
32%, yeast powder 0.5%, NaCl 0.8%, water 92.7% were cultivated 4 days in the liquid seed culture medium of pH 9.0, and culture temperature is 32 ℃, and rotating speed is 200rpm.Again just the 50ml seed liquor to be inoculated into liquid amount be that 1.5L contains glucose 5.0%, starch 0.5%, corn steep liquor 3.0%, yeast extract paste 0.5%, soybean cake powder 0.5%, NaCl1.5%, CaCO
31.5%, NH
4In the 2L fermentor tank of the fermention medium of Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water 95%, at 37 ℃, air is that 1.0L/min flows into the overall flow rate, and mixes with 400rpm, keeps oxyty (DO) and is 60-100%.With 20% (w/v) NaOH the pH value is maintained 8.5-9.0, appropriate supplement sugar, when cell concn optical density(OD) (O.D.) when level reaches 32, change little oxygen over to, keep oxyty (DO) and be 5-10%, fermentation culture 9 days, acquisition contains the mycelium of enramycin, with the mycelium after the 1g fermentation, use washed with de-ionized water, be suspended in the 130mL concentration of hydrochloric acid and be 0.006mol/L and methyl alcohol volume fraction and be in 50% the methanol aqueous solution, concentration of hydrochloric acid is that 0.006mol/L and methyl alcohol volume fraction are in 50% the methanol aqueous solution, pH is 6.5, carry out the broken 15min of microorganism cells with KQ-100 type ultrasonic machine, obtain containing the mixture of enramycin, mixture after the fragmentation is shaken the vibration of bottle cabinet with HYG-II a type, and (the adjusting temperature and the rotating speed that shake bottle cabinet is respectively 25 ℃, 230rpm) extracted 2 hours, obtain containing the extracting solution of enramycin, extracting solution centrifugal 25min under the rotating speed of 2500r/min that will contain enramycin then, collection contains the supernatant liquor of enramycin, supernatant liquor is evaporated to absence of liquid under 38 ℃, obtains enramycin and slightly carry product.After macroporous resin carried out pre-treatment according to conventional treatment method, the glass pillar of packing into and having the constant temperature protective sleeve was that to be 50% methanol aqueous solution pump into pillar with the speed of 65mL/h finishes balance for 0.09mol/L and methyl alcohol volume fraction with sodium chloride concentration.It is that 0.006mol/L and methyl alcohol volume fraction are in 50% the methanol aqueous solution that the product of slightly carrying of enramycin are dissolved in the 20mL concentration of hydrochloric acid, be placed on the top of pillar, static absorption 3 hours, with the eluent of 2.0L (volume fraction is that methanol aqueous solution/concentration of hydrochloric acid of 70% is that 0.006mol/L and methyl alcohol volume fraction are that the volume ratio of 50% methanol aqueous solution is 5: 5) gradient elution, with elutriant rotary vacuum evaporator concentrating under reduced pressure evaporate to dryness, productive rate reaches 68%.
Embodiment 4
Streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received glucose 0.5%, extractum carnis 0.8%, peptone 0.8%, K
2HPO
40.1%, agar 1.8%, water 96% in the glucose nutritional medium of pH7.0, carry out slant culture, cultivate 3 days down at 37 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed with transfering loop, make spore suspension, get the 0.5ml spore suspension and be inoculated into spore suspension and be inoculated into and contain 40ml glucose 2.0%, corn steep liquor 2.0%, CaCO
31.5%, yeast powder 0.5%, NaCl0.5%, water 93.5% were cultivated 4 days in the liquid seed culture medium of pH7.0, and culture temperature is 30 ℃, and rotating speed is 200rpm.The 10ml seed liquor being linked into liquid amount is that 100ml contains glucose 2.0% again, starch 1.5%, corn steep liquor 0.5%, yeast extract paste 0.3%, soybean cake powder 1.5%, NaCl0.8%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, in the fermention medium of water 90.9%, at 32 ℃, 220rpm, shaker fermentation was cultivated 6 days under the condition of pH7.0, acquisition contains the mycelium of enramycin, with the mycelium after the 1g fermentation, use washed with de-ionized water, be suspended in the 50mL methyl alcohol, the volume fraction of methyl alcohol is 80%, pH is 5.5, carry out the broken 15min of microorganism cells with KQ-100 type ultrasonic machine, obtain containing the mixture of enramycin, the mixture after the fragmentation is shaken bottle cabinet with HYG-II a type vibrate that (the adjusting temperature and the rotating speed that shake bottle cabinet is respectively 18 ℃, 230rpm) extracted 1.5 hours, obtain containing the extracting solution of enramycin, will contain extracting solution centrifugal 20min under the rotating speed of 2000r/min of enramycin then, collect the supernatant liquor that contains enramycin, supernatant liquor is evaporated to absence of liquid under 35 ℃, obtains enramycin and slightly carry product.After macroporous resin carried out pre-treatment according to conventional treatment method, the glass pillar of packing into and having the constant temperature protective sleeve was that to be 50% methanol aqueous solution pump into pillar with the speed of 60mL/h finishes balance for 0.09mol/L and methyl alcohol volume fraction with sodium chloride concentration.It is that 0.09mol/L and methyl alcohol volume fraction are in 50% the methanol aqueous solution that the product of slightly carrying of enramycin are dissolved in the 20mL sodium chloride concentration, be placed on the top of pillar, static absorption 5 hours, with the eluent of 1.0L (sodium chloride concentration is that 0.09mol/L and methyl alcohol volume fraction are that methanol aqueous solution/concentration of hydrochloric acid of 50% is that 0.006mol/L and methyl alcohol volume fraction are that the volume ratio of 50% methanol aqueous solution is 8: 2) gradient elution, with elutriant rotary vacuum evaporator concentrating under reduced pressure evaporate to dryness, productive rate reaches 71%.
＜110〉Nanjing University of Technology
＜120〉a kind of enramycin producing strain and utilize the method for macroporous resin extraction
<160>1
<210>1
<211>1650
<212>DNA
＜213〉streptomycete Streptomyces sp.
<400>1
attgagttta?tcctggctca?gggtttcttc?ggtacgctga?gtcgaaacac?ccacttcgac 60
gatatagttg?cggctggatc?acctccttag?agtttgatcc?tggctcagtg?cggctagatc 120
acctccttag?agtttgatcc?tggctcagga?cgaacgctgg?cggcgtgctt?aacacatgca 180
agtcgaacga?tgaaccactt?cggtggggat?tagtggcgaa?cgggtgagta?acacgtgggc 240
aatctgccct?gcactctggg?acaagccctg?gaaacggggt?ctaataccgg?atactgaccc 300
gcctgggcat?ccaggcggtt?cgaaagctcc?ggcggtgcag?ggtgagcccg?cggcctatca 360
gcttgttggt?gaggtaacgg?ctcaccaagg?cgacgacggg?tagccggcct?gagagggcga 420
ccggccacac?tgggactgag?acacggccca?gactcctacg?ggaggcagca?gtggggaata 480
tcgcacaatg?ggcgaaagcc?tgatgcagcg?acgccgcgtg?agggatgacg?gccttcgggt 540
tgtaaacctc?tttcagcagg?gaagaagcga?aagtgacggt?acctgcagaa?gaagcgccgg 600
ctaactacgt?gccagcagcc?gcggtaatac?gtagggcgcg?agcgttgtcc?ggaattattg 660
ggcgtaaaga?gctcgtaggc?ggcttgtcgc?gtcggttgtg?aaagcccggg?gcttaacccc 720
gggtctgcag?tcgatacggg?caggctagag?ttcggtaggg?gagatcggaa?ttcctggtgt 780
agcggtgaaa?tgcgcagata?tcaggaggaa?caccggtggc?gaaggcggat?ctctgagcca 840
ttactgacgc?tgaggagcga?aagcgtgggg?agcgaacagg?attagatacc?ctggtagtcc 900
acgccgtaaa?cggtgggaac?taggtgttgg?cgacattcca?cgtcgtcggt?gccgcagcta 960
acgcattaag?ttccccgcct?ggggagtacg?gccgcaaggc?taaaactcaa?aggaattgac 1020
gggggcccgc?acaagcagcg?gagcatgtgg?cttaattcga?cgcaacgcga?agaaccttac 1080
caaggcttga?catacaccgg?aaagcattag?agatagtgcc?ccccttgtgg?tcggtgtaca 1140
ggtggtgcat?ggctgtcgtc?agctcgtgtc?gtgagatgtt?gggttaagtc?ccgcaacgag 1200
cacaaccctt?gtcctgtgtt?gccagcatgc?ccttcggggt?gatggggact?cacaggagac 1260
cgccggggtc?aactcggagg?aaggtgggga?cgacgtcaag?tcatcatgcc?ccttatgtct 1320
tgggctgcac?acgtgctaca?atggccggta?caatgagctg?cgaaaccgtg?aggtggagcg 1380
aatctcaaaa?agccggtctc?agttcggatt?ggggtctgca?actcgacccc?atgaagtcgg 1440
agttgctagt?aatcgcagat?cagcattgct?gcggtgaata?cgttcccggg?ccttgtacac 1500
accgcccgtc?acgtcacgaa?agttggtaac?acccgaagcc?ggtggcccaa?ccccttgtgg 1560
gagggagctg?tcgaaggtgg?gactggcgat?tgggacgaag?tcgtaacaag?gtagccgtac 1620
cggaaggtgc?ggctggatca?cctccttaat 1650
Claims (9)
1, a kind of streptomycete, its classification called after streptomycete, the Latin formal name used at school of bacterial classification is Streptomycessp.NJYWG3665, and preservation date is on March 21st, 2008, and registering on the books and number in the preservation center is CGMCC NO.2410.
2, a kind of method with the macroporous resin extraction enramycin, concrete steps are as follows: the process of A, fermentative Production enramycin is: (1) slant culture: with streptomycete Streptomyces sp.NJWGY3665 bacterial classification, the microbial strains preservation center numbering CGMCC No.2410 of the Chinese Academy of Sciences, receive in the glucose nutritional medium, carry out slant culture, the weight percent proportioning of glucose nutritional medium is glucose 0.5～1%, extractum carnis 0.5～1%, peptone 0.5～1%, NaCl0.3～0.5% or K
2HPO
40.1～0.2%, agar 1.5～2%, water surplus, pH 6.0～9.0,28～37 ℃ of culture temperature, incubation time 2 days～6 days; (2) seed culture: with sterilized water the spore of cultivating on the inclined-plane is made monospore suspension, and be inoculated in the 50ml seed culture medium and cultivate, the weight percent proportioning of seed culture medium is glucose 1～3.0%, corn steep liquor 1～3%, CaCO
31～2%, yeast powder 0.5%, NaCl 0.1～0.8%, water surplus, pH is 6.0～9.0, and temperature is 28～32 ℃, and rotating speed is 200rpm, cultivates 2 days～6 days; (3) fermentation culture: seed liquor is inoculated in the fermention medium cultivates, the weight percent proportioning of fermention medium is glucose 2.0～5.0%, starch 0.5～2.0%, corn steep liquor 0.5～3.0%, yeast extract paste 0.1～0.5%, soybean cake powder 0.5～2%, NaCl 0.5～1.0%, CaCO
31～2%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water surplus, temperature is controlled at 28～37 ℃, and pH is controlled at 6.0～9.0, and rotating speed 180～220rpm fermented 5～9 days; B, the enramycin producing strain after will fermenting carry out the microorganism cells fragmentation with KQ-100 type ultrasonic machine, obtain containing the mixture of enramycin; C, to be diluted with water to volume fraction be that the vibration of 10%～100% extraction agent was extracted the mixture described in the B 0.5～4 hour, and the pH value of system is controlled between 4.0～8.5, and acquisition contains the extracting solution of enramycin; D, the extracting solution that will contain enramycin are centrifugal, collect the supernatant liquor that contains enramycin, and supernatant liquor is evaporated to absence of liquid under 30～40 ℃, obtain enramycin and slightly carry product; After E, the macroporous resin pre-treatment, the enramycin after concentrating is slightly put forward the product dissolution with solvents, after 2～5 hours, carry out obtaining the pure product of enramycin behind gradient elution, the concentrate drying with eluent through the static absorption of macroporous resin.
3, by the described method with the macroporous resin extraction enramycin of claim 2, it is characterized in that: the extraction agent among the step C is the methanol aqueous solution of ethanol, methyl alcohol, hydrochloric acid, sodium-chlor, the methanol aqueous solution of hydrochloric acid.
4, by the described method of claim 2 with the macroporous resin extraction enramycin, it is characterized in that: the ethanol in the extraction agent is that dehydrated alcohol, methyl alcohol are analytical pure, the volumetric molar concentration of hydrochloric acid is 1～5mol/L, the sodium-chlor volumetric molar concentration is that 0.03～0.09mol/L, methyl alcohol volume fraction are 40%～70% in the sodium-chlor methanol aqueous solution, and the hydrochloric acid volumetric molar concentration is that 0.003～0.009mol/L, methyl alcohol volume fraction are 40%～70% in the hydrochloric acid methanol aqueous solution.
5, by the described method with the macroporous resin extraction enramycin of claim 2, it is characterized in that: the volume fraction of extraction agent is 30%～100%, and the time of utilizing extraction agent to extract is 0.5～2.5 hour.
6, by the described method with the macroporous resin extraction enramycin of claim 2, it is characterized in that: the mycelium of enramycin producing strain quality/extraction agent volume is 1/10～1/150 among the step C.
7, by the described method with the macroporous resin extraction enramycin of claim 2, it is characterized in that: the pH that extracts system is 5.0～7.5.
8, by the described method with the macroporous resin extraction enramycin of claim 2, it is characterized in that: the macroporous resin of selecting for use is macroporous resin D101, Amberlite XAD-2, Amberlite XAD-4 or Amberlite XAD-16.
9, by the described method of claim 2 with the macroporous resin extraction enramycin, it is characterized in that: during gradient elution, eluent be methanol aqueous solution, the sodium-chlor volumetric molar concentration of methyl alcohol volume fraction 30%～90% be 0.03～0.09mol/L and methyl alcohol volume fraction be 40%～70% sodium-chlor methanol aqueous solution, hydrochloric acid volumetric molar concentration be 0.003～0.009mol/L and methyl alcohol volume fraction be 40%～70% the hydrochloric acid methanol aqueous solution or volumetric molar concentration be 1～3mol/L hydrochloric acid in any two kinds; The volume ratio of two kinds of eluents is 1: 9～5: 5.
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| CN101974469A (en) * | 2010-10-22 | 2011-02-16 | 安徽丰原发酵技术工程研究有限公司 | Streptomyces fungicidious mutant strain and application thereof |
| CN102154419A (en) * | 2010-12-20 | 2011-08-17 | 安徽丰原发酵技术工程研究有限公司 | Fermentation medium for improving enramycin yield |
| CN102250993A (en) * | 2011-07-18 | 2011-11-23 | 南京工业大学 | Optimized process for producing enramycin through fermenting streptomyces |
| CN102311486A (en) * | 2010-06-30 | 2012-01-11 | 中华人民共和国四川出入境检验检疫局 | Method for separating and extracting enramycin by using macroporous weakly-acidic cationic resin |
| CN102391363A (en) * | 2011-09-26 | 2012-03-28 | 浙江升华拜克生物股份有限公司 | Method for extracting enramycin |
| CN102408435A (en) * | 2011-07-18 | 2012-04-11 | 南京工业大学 | Method for purification of ascomycin from streptomyces fermentation liquid |
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Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JPS6038120B2 (en) * | 1980-10-31 | 1985-08-30 | 武田薬品工業株式会社 | Production method of the antibiotic enzyuracydin |
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