CN1173045C - High-yield fermentation method for producing boanmycin - Google Patents
High-yield fermentation method for producing boanmycin Download PDFInfo
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- CN1173045C CN1173045C CNB991110536A CN99111053A CN1173045C CN 1173045 C CN1173045 C CN 1173045C CN B991110536 A CNB991110536 A CN B991110536A CN 99111053 A CN99111053 A CN 99111053A CN 1173045 C CN1173045 C CN 1173045C
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- China
- Prior art keywords
- spermine
- substratum
- zhengguangmycin
- salt
- bleomycin
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- 108700035333 bleomycin A6 Proteins 0.000 title claims abstract description 40
- FOUFFVYWFNBHHH-YNGSZULRSA-N [(2r,3s,4s,5r,6r)-2-[(2r,3s,4s,5s,6s)-2-[(1r,2s)-2-[[6-amino-2-[(1s)-3-amino-1-[[(2s)-2,3-diamino-3-oxopropyl]amino]-3-oxopropyl]-5-methylpyrimidine-4-carbonyl]amino]-3-[[(2r,3s,4s)-5-[[(2s,3r)-1-[2-[4-[4-[3-[4-(3-aminopropylamino)butylamino]propylcarbamo Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCNCCCCNCCCN)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1NC=NC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C FOUFFVYWFNBHHH-YNGSZULRSA-N 0.000 title claims abstract description 34
- 238000000855 fermentation Methods 0.000 title claims abstract description 23
- 230000004151 fermentation Effects 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 28
- 241000187747 Streptomyces Species 0.000 claims abstract description 21
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical class NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 claims description 21
- 230000035772 mutation Effects 0.000 claims description 18
- 240000008042 Zea mays Species 0.000 claims description 13
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 13
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 13
- 235000005822 corn Nutrition 0.000 claims description 13
- KBDDIZRDKLGWGW-UHFFFAOYSA-N 3-[4-(3-aminopropylamino)butylamino]propylazanium;chloride Chemical group [Cl-].NCCCNCCCCNCCC[NH3+] KBDDIZRDKLGWGW-UHFFFAOYSA-N 0.000 claims description 11
- 241001147844 Streptomyces verticillus Species 0.000 claims description 6
- 229940063675 spermine Drugs 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 239000012043 crude product Substances 0.000 claims description 2
- 235000015097 nutrients Nutrition 0.000 claims description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 abstract description 21
- 108010006654 Bleomycin Proteins 0.000 abstract description 19
- 229960001561 bleomycin Drugs 0.000 abstract description 19
- 150000001412 amines Chemical group 0.000 abstract description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 239000011347 resin Substances 0.000 description 27
- 229920005989 resin Polymers 0.000 description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 24
- 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 description 18
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 18
- 229920002472 Starch Polymers 0.000 description 18
- 239000008103 glucose Substances 0.000 description 18
- 239000000843 powder Substances 0.000 description 18
- 239000008107 starch Substances 0.000 description 18
- 235000019698 starch Nutrition 0.000 description 18
- 239000003463 adsorbent Substances 0.000 description 15
- 238000003756 stirring Methods 0.000 description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 244000068988 Glycine max Species 0.000 description 12
- 235000010469 Glycine max Nutrition 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical class Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 239000000306 component Substances 0.000 description 12
- 238000010612 desalination reaction Methods 0.000 description 12
- 238000011010 flushing procedure Methods 0.000 description 12
- 239000002054 inoculum Substances 0.000 description 12
- 210000000582 semen Anatomy 0.000 description 12
- 239000011780 sodium chloride Substances 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- 238000004659 sterilization and disinfection Methods 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 11
- 230000003115 biocidal effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000012266 salt solution Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 7
- 238000010828 elution Methods 0.000 description 7
- -1 organic acid salt Chemical class 0.000 description 7
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 229960000583 acetic acid Drugs 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 description 6
- 230000001186 cumulative effect Effects 0.000 description 6
- 239000012362 glacial acetic acid Substances 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 235000006408 oxalic acid Nutrition 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000011218 seed culture Methods 0.000 description 6
- QWSZRRAAFHGKCH-UHFFFAOYSA-M sodium;hexane-1-sulfonate Chemical compound [Na+].CCCCCCS([O-])(=O)=O QWSZRRAAFHGKCH-UHFFFAOYSA-M 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 244000005700 microbiome Species 0.000 description 5
- 239000003456 ion exchange resin Substances 0.000 description 4
- 229920003303 ion-exchange polymer Polymers 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 206010061924 Pulmonary toxicity Diseases 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 231100000374 pneumotoxicity Toxicity 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000003729 cation exchange resin Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 229910017053 inorganic salt Inorganic materials 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 108010057150 Peplomycin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108700004675 bleomycetin Proteins 0.000 description 1
- QYOAUOAXCQAEMW-UTXKDXHTSA-N bleomycin A5 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCNCCCCN)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C QYOAUOAXCQAEMW-UTXKDXHTSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- QIMGFXOHTOXMQP-GFAGFCTOSA-N peplomycin Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCCN[C@@H](C)C=1C=CC=CC=1)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1NC=NC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C QIMGFXOHTOXMQP-GFAGFCTOSA-N 0.000 description 1
- 229950003180 peplomycin Drugs 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 208000005069 pulmonary fibrosis Diseases 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 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 present invention relates to a high-yield method for producing bleomycin A6, which comprises the following steps: a terminal amine body of the bleomycin is added in a fermentation cultivation medium; Streptomyces verticillatus capable of generating the bleomycin is cultivated by fermentation, and the bleomycin is recovered from cultivation solution. Compared with the existing method, the content ratio of the bleomycin in a fermentation product of the high-yield method for producing bleomycin A6 can be enhanced by from 2 to 7 times.
Description
The present invention relates to a kind of high yield and produce the method for Zhengguangmycin A6.
Zhengguangmycin A6 is a known microbiotic, belong to bleomycin (Bleomycin, BLM) family, i.e. bleomycin A6 (Bleomycin A6), it has the structure shown in the following formula:
The microbiotic majority of bleomycin family all has anti-tumor activity, and that use at first clinically is bleomycin (A
2+ B
2Be main), be mainly used in the treatment squamous cell carcinoma, but its outstanding shortcoming is that lung toxicity is big, can cause irreversible pulmonary fibrosis.Be used in succession subsequently clinical have a Lay mycin (Pepleomycin, PEP) and Zhengguangmycin A5 (BLM A
5), but its kind is also few, active and toxicity with regard to it still lacks clinically some tumour is had specific inhibitory effect and toxic side effect is little, especially the little improved seeds of lung toxicity.Clinical study proof Zhengguangmycin A6 has good antitumor action, incidence cancer, malignant lymphoma, mammary cancer, esophagus cancer are all had curative effect preferably, especially liver cancer is also had satisfied curative effect, and lung toxicity is extremely low, also do not find its toxicity to the conscience kidney, potential applicability in clinical practice is fine.Because the bleomycin family antibiotic has quite similar structure, in microbial fermentation, multiple bleomycin family antibiotic often produces simultaneously, and the shared therein ratio of Zhengguangmycin A6 is not high.(Acta Pharmaceutica Sinica such as Xu Hongzhang, V23 (9): 667-671,1988) reported that wherein Zhengguangmycin A6 only accounts for about 10% of bleomycin family antibiotic total amount by weight through fermentation culture streptoverticillium Pingyang mutation (Streptomyces verticillus var.pingyangensis n.var.) 72 a series of bleomycin family antibiotics that produced.In order to reduce production costs, obtain higher Zhengguangmycin A6 productive rate, press for the method that a kind of high productivity is produced Zhengguangmycin A6.
The purpose of this invention is to provide a kind of high productivity and produce the method for Zhengguangmycin A6.
For this reason, the contriver is by further investigation, found a kind of method of high yield production Zhengguangmycin A6, this method comprises the terminal amine body that adds Zhengguangmycin A6 in fermention medium, fermentation culture can produce the microorganism that belongs to streptoverticillium (Streptomyces verticillus) of Zhengguangmycin A6 then, and reclaims Zhengguangmycin A6 from nutrient solution.The content ratio of Zhengguangmycin A6 accounts for the 20-70% of bleomycin family antibiotic total amount by weight in the fermentation culture of this method.
All microorganisms that belong to streptoverticillium that can produce Zhengguangmycin A6 are all at the present invention's row.Being used for microorganism preferably of the present invention is streptoverticillium Pingyang mutation (Streptomycesverticillus var.pingyangensis n.var.), wherein preferred bacterial strain is streptoverticillium Pingyang mutation (Streptomyces verticillus var.pingyangensis n.var) 72, streptoverticillium Pingyang mutation Q 835.Streptoverticillium Pingyang mutation 72 is known bacterial strain, about existing document records such as its morphological specificity, cultural characteristic, physiological property, utilization of carbon source and antimicrobial spectrum (Zhao Yiying etc., microorganism journal, 19 (4): 361-364,1979).
Be used for cultivating the substratum of mentioned microorganism, can adopt to be carbon source, nitrogenous source, inorganic salt, organic salt and can be by the trace nutrition of its utilization by the nutrition source of its utilization, these compositions can give and adding once in the substratum earlier, perhaps intermittently or add in the substratum continuously.
Training method can adopt static cultivation, sways cultivation, stir culture or alternate manner, but concerning mass production, preferred mode is submerged culture.
Culture condition (time, temperature, pH etc.) is different and different, also different with the variation of medium component with bacterial classification, and those skilled in the art can select as the case may be.
The terminal amine body that adds in fermention medium is meant that spermine salt is (as the spermine hydrochloride, spermine vitriol) or its crude product, (these materials can be plant origins perhaps to contain the material of spermine salt, as corn steep liquor, the corn steep liquor that is adopted can be to handle the corn steep liquor that has improved the spermine salts contg through the whole bag of tricks).The add-on of terminal amine body is that per 1000 liters of substratum add 0.1-1 kilogram (by the spermine hydrochloride), and preferred add-on is that per 1000 liters of substratum add 0.4-0.6 kilogram (by the spermine hydrochloride).
Stop when reaching peak concentration cultivating when Zhengguangmycin A6 accumulates in substratum, adopt method well known in the art to carry out separation and purification according to its character.Zhengguangmycin A6 can the free form obtain separating, form (example hydrochloric acid salt, vitriol or organic acid salt that also can pharmacy acceptable salt, especially the form of hydrochloride) obtain separating, if desired, can the salt form of Zhengguangmycin A6 be changed into free form with ordinary method.
Any can be from culture the method for separation and purification Zhengguangmycin A6 all at the present invention's row.Generally can use absorption, wash-out, column chromatography, cold method such as do, these methods can be used separately in each separating step, also can be used in combination, and can also reuse in whole sepn process.
Adsorption method comprises with charcoal absorption, macroporous resin adsorption or ion exchange resin absorption etc.Macroporous adsorbent resin can be a nonpolar macroporous adsorption resin, also can be the macroporous adsorbent resin of middle polarity, can also be high polar macroporous adsorbent resin.Ion exchange resin can adopt weakly acidic cation-exchange resin or storng-acid cation exchange resin.
Elution process comprises the mixed solvent wash-out of neutral salt solution wash-out, single solvent wash-out, mixed solvent wash-out or solvent and the inorganic salt of using acidic solution wash-out, different concns etc.
Column chromatography comprises adsorpting column chromatography, ion-exchange chromatography, ion exclusion column chromatography, affinity column chromatography, size-exclusion column chromatography and reversed phase column chromatography etc.
The following example is used for further specifying method of the present invention.
Embodiment 1
(pH 7.2-7.5) is slant medium with glucose 1.0%, peptone 0.5%, starch 1.0%, NaCl 0.5% and agar 2.0%, sterilized 30 minutes down for 120 ℃, bevel, place 37 ℃ of thermostatic chambers 3 days dried slightly to surface-moisture, during no varied bacteria growing, inoculation streptoverticillium Pingyang mutation 72 bacterial classification spores were cultivated 8 days in 28 ℃.(substratum is formed: starch 2.5%, glucose 0.5%, soybean cake powder 3.5%, KH to be respectively charged into the 100ml substratum in a plurality of 500ml Erlenmeyer flasks
2PO
40.1%, ZnSO
40.05%, CuSO
40.01%, pH6.0-6.5)., inoculate after 30 minutes in 120 ℃ of sterilizations, 28 ℃ of shaking culture 48 hours, with this culture as seed.Prepare the 500L fermentor tank, volume 200L feeds intake, substratum is formed identical with seed culture medium, sterilizes 30 minutes for 120 ℃, and inoculum size is 0.5%, at 1: 1 air flow, 29 ℃, and 180～200 rev/mins of stir culture 48 hours, transferred species to 2 ton fermentor tank, the volume that feeds intake of substratum is 1000L, adds 0.6 kilogram of spermine hydrochloride.Its substratum consists of starch 2.5%, glucose 0.5%, Semen Maydis powder 2.0%, soybean cake powder 3.2%, corn steep liquor 3.0%, NaCl 0.5%, KH
2PO
40.015%, ZnSO
40.05%, CuSO
40.01%, Semen Maydis oil 0.3%, pH6.0-6.5 inoculate behind 130 ℃ of-150 ℃ of continuous sterilizations, and inoculum size is 15-20%.29 ℃ of jar temperature, air flow 1: 1 to 1: 1.5,145 rev/mins of stirring velocitys were cultivated 7 days, and to pH more than 7.5, tiring stops when peaking cultivating.Detect (chromatographic column: YWG C 1810 μ 3.9 * 300mm through high performance liquid chromatography; Moving phase: A methyl alcohol, B 5mM sodium hexanesulfonate adds 0.5% Glacial acetic acid, transfers pH to 4.3, A: B=32: 68 with strong aqua; Flow velocity: 1ml/min), the content of Zhengguangmycin A6 accounts for 70% of bleomycin family antibiotic total amount by weight in the fermentation culture.
800 liters of fermentation cultures are transferred pH to 3.0 with oxalic acid, transfer pH to 6.5 with 5N NaOH again.Add Zeo-karb 122 (H
+Type) 13 liters, stirred 3 hours.Collect resin, with usefulness 0.3N HCl wash-out in the post of after the no salt solution flushing resin being packed into, collection active component.Transfer pH to 6.5 with 5N sodium hydroxide, living solution is passed through macroporous adsorbent resin 4006 posts (10 liters of post beds) desalination, with 10% acetone (containing 0.01N HCl) wash-out.Collect active component, concentrating under reduced pressure.Concentrated solution is transferred pH to 6.5 with ammoniacal liquor, and it is passed through CM-Sephadex-C-25 (NH
4 +Type) post (2.5 liters of post beds), the NH of 0.05N of elder generation
4After the Cl flushing, use 0.1-1.0N NH again
4Cl gradient elution, elutriant cumulative volume are equivalent to 15 times of column volume approximately.Collect by peak position, merge, after macroporous adsorbent resin 4006 posts carry out the desalination operation, lyophilize.Obtain 10.5 gram blue Zhengguangmycin A6 hydrochlorides (cupric product).
Embodiment 2
(pH 7.2-7.5) is slant medium with glucose 1.0%, peptone 0.5%, starch 1.0%, NaCl 0.5% and agar 2.0%, sterilized 30 minutes for 120 ℃, bevel, place 37 ℃ of thermostatic chambers 3 days dried slightly to surface-moisture, during no varied bacteria growing, inoculation streptoverticillium Pingyang mutation Q835 bacterial classification spore was cultivated 8 days in 28 ℃.(substratum is formed: starch 2.5%, glucose 0.5%, soybean cake powder 3.5%, KH to be respectively charged into the 100ml substratum in a plurality of 500ml Erlenmeyer flasks
2PO
40.1%, ZnSO
40.05%, CuSO
40.01%, pH6.0-6.5)., inoculate after 30 minutes in 120 ℃ of sterilizations, 28 ℃ of shaking culture 48 hours, with this culture as seed.Prepare the 500L fermentor tank, volume 200L feeds intake, substratum is formed identical with seed culture medium, sterilizes 30 minutes for 120 ℃, and inoculum size is 0.5%, at 1: 1 air flow, 29 ℃, and 180～200 rev/mins of stir culture 48 hours, transferred species to 2 ton fermentor tank, the substratum volume that feeds intake is 1000L, adds 0.4 kilogram of spermine hydrochloride.Its substratum consists of starch 2.5%, glucose 0.5%, Semen Maydis powder 2.0%, soybean cake powder 3.2%, corn steep liquor 3.0%, NaCl 0.5%, KH
2PO
40.015%, ZnSO
40.05%, CuSO
40.01%, Semen Maydis oil 0.3%, pH6.0-6.5 inoculate behind 130 ℃ of-150 ℃ of continuous sterilizations, and inoculum size is 15-20%.29 ℃ of jar temperature, air flow 1: 1 to 1: 1.5,145 rev/mins of stirring velocitys were cultivated 7 days, and to pH more than 7.5, tiring stops when peaking cultivating.Detect (chromatographic column: YWG C1810 μ 3.9 * 300mm through high performance liquid chromatography; Moving phase: A methyl alcohol, B 5mM sodium hexanesulfonate adds 0.5% Glacial acetic acid, transfers pH to 4.3, A: B=32: 68 with strong aqua; Flow velocity: 1ml/min), the content of Zhengguangmycin A6 accounts for 60% of bleomycin family antibiotic total amount by weight in the fermentation culture.
Fermentation culture is transferred pH to 3.0 with oxalic acid, transfer pH to 6.5 with 5N NaOH again, filter.800 liters of filtrates are passed through ion exchange resin D151 (H
+Type) post (10 liters of post beds) with no salt solution flushing, is used 0.3N HCl wash-out again, collects active component, transfers PH to 6.5, and it is passed through macroporous adsorbent resin 4006 post desalinations (10 liters of post beds).With 10% acetone (containing 0.01NHCl) wash-out resin column.Collect active component, be evaporated to small volume, concentrated solution is transferred pH to 6.5, last CM-Sephadex-C-25 (NH with ammoniacal liquor
4 +Type) post (2.5 liters of post beds).With no salt solution flushing back 0.1-1.0N NH
4Cl gradient elution, elutriant cumulative volume are equivalent to 15 times of column volume approximately.Collect by peak position, merge.After macroporous adsorbent resin 4006 post desalinations, lyophilize.Get 10 gram Zhengguangmycin A6 hydrochlorides (cupric product).
Embodiment 3
(pH 7.2-7.5) is slant medium with glucose 1.0%, peptone 0.5%, starch 1.0%, NaCl 0.5% and agar 2.0%, sterilized 30 minutes for 120 ℃, bevel, place 37 ℃ of thermostatic chambers 3 days dried slightly to surface-moisture, during no varied bacteria growing, inoculation streptoverticillium Pingyang mutation 72 bacterial classification spores were cultivated 8 days in 28 ℃.(substratum is formed: starch 2.5%, glucose 0.5%, soybean cake powder 3.5%, KH to be respectively charged into the 100ml substratum in a plurality of 500ml Erlenmeyer flasks
2PO
40.1%, ZnSO
40.05%, CuSO
40.01%, pH6.0-6.5)., inoculate after 30 minutes in 120 ℃ of sterilizations, 28 ℃ of shaking culture 48 hours, with this culture as seed.Prepare the 500L fermentor tank, volume 200L feeds intake, substratum is formed identical with seed culture medium, sterilizes 30 minutes for 120 ℃, and inoculum size is 0.5%, at 1: 1 air flow, 29 ℃, and 180～200 rev/mins of stir culture 48 hours, transferred species to 2 ton fermentor tank, the volume that feeds intake of substratum is 1000L, adds 20 kilograms of corn steep liquors.Substratum consists of starch 2.5%, glucose 0.5%, Semen Maydis powder 2.0%, soybean cake powder 3.2%, corn steep liquor 3.0%, NaCl 0.5%, KH
2PO
40.015%, ZnSO
40.05%, CuSO
40.01%, Semen Maydis oil 0.3%, pH6.0-6.5 inoculate behind 130 ℃ of-150 ℃ of continuous sterilizations, and inoculum size is 15-20%.29 ℃ of jar temperature, air flow 1: 1 to 1: 1.5, stirring velocity was cultivated 7 days for 145 rev/mins, in culturing process, added a corn steep liquor every 8 hours, addition is 20 kilograms, (sum total of the corn steep liquor that is added when feeding intake is 300 kilograms to add 14 times altogether, be equivalent to add 0.15 kilogram of spermine hydrochloride), to pH more than 7.5, tiring stops when peaking cultivating.Detect (chromatographic column: YWG C1810 μ 3.9 * 300mm through high performance liquid chromatography; Moving phase: A methyl alcohol, B 5mM sodium hexanesulfonate adds 0.5% Glacial acetic acid, transfers pH to 4.3, A: B=32: 68 with strong aqua; Flow velocity: 1ml/min), the content of Zhengguangmycin A6 accounts for 20% of bleomycin family antibiotic total amount by weight in the fermentation culture.
800 liters of fermentation cultures are transferred pH to 3.0 with oxalic acid, transfer pH to 6.5 with 5N NaOH again.Add Zeo-karb 122 H
+20 liters, stirred 3 hours.Collect resin, with usefulness 0.3N HCl wash-out in the post of after the no salt solution flushing resin being packed into, collection active component.Transfer pH to 6.5 with 5N sodium hydroxide, by macroporous adsorbent resin 4006 posts (10 liters of post beds) desalination, with 10% acetone (containing 0.01N HCl) wash-out.Collect active component, concentrating under reduced pressure.Concentrated solution is transferred pH to 6.5 with ammoniacal liquor, and it is passed through CM-Sephadex-C-25 (NH
4 +Type) post (2.5 liters of post beds), the NH of 0.05N of elder generation
4After the Cl flushing, use 0.1-1.0N NH
4Cl gradient elution, elutriant cumulative volume are equivalent to 15 times of column volume approximately.Collect by peak position, merge, after macroporous adsorbent resin 4006 posts carry out the desalination operation, concentrate lyophilize.Obtain 3.1 gram blue Zhengguangmycin A6 hydrochlorides (cupric product).
Embodiment 4
(pH 7.2-7.5) is slant medium with glucose 1.0%, peptone 0.5%, starch 1.0%, NaCl 0.5% and agar 2.0%, sterilized 30 minutes for 120 ℃, bevel, place 37 ℃ of thermostatic chambers 3 days dried slightly to surface-moisture, during no varied bacteria growing, inoculation streptoverticillium Pingyang mutation Q835 bacterial classification spore was cultivated 8 days in 28 ℃.(substratum is formed: starch 2.5%, glucose 0.5%, soybean cake powder 3.5%, KH to be respectively charged into the 100ml substratum in a plurality of 500ml Erlenmeyer flasks
2PO
40.1%, ZnSO
40.05%, CuSO
40.01%, pH6.0-6.5)., inoculate after 30 minutes in 120 ℃ of sterilizations, 28 ℃ of shaking culture 48 hours, with this culture as seed.Prepare the 500L fermentor tank, volume 200L feeds intake, substratum is formed identical with seed culture medium, sterilizes 30 minutes for 120 ℃, and inoculum size is 0.5%, at 1: 1 air flow, 29 ℃, and 180～200 rev/mins of stir culture 48 hours, transferred species to 2 ton fermentor tank, the substratum volume that feeds intake is 1000L, adds 0.4 kilogram of spermine vitriol.Its substratum consists of starch 2.5%, glucose 0.5%, Semen Maydis powder 2.0%, soybean cake powder 3.2%, corn steep liquor 3.0%, NaCl 0.5%, KH
2PO
40.015%, ZnSO
40.05%, CuSO
40.01%, Semen Maydis oil 0.3%, pH6.0-6.5 inoculate behind 130 ℃ of-150 ℃ of continuous sterilizations, and inoculum size is 15-20%.29 ℃ of jar temperature, air flow 1: 1 to 1: 1.5,145 rev/mins of stirring velocitys were cultivated 7 days, and to pH more than 7.5, tiring stops when peaking cultivating.Detect (chromatographic column: YWG C1810 μ 3.9 * 300mm through high performance liquid chromatography; Moving phase: A methyl alcohol, B 5mM sodium hexanesulfonate adds 0.5% Glacial acetic acid, transfers pH to 4.3, A: B=32: 68 with strong aqua; Flow velocity: 1ml/min), the content of Zhengguangmycin A6 accounts for 50% of bleomycin family antibiotic total amount by weight in the fermentation culture.
Fermentation culture is transferred pH to 3.0 with oxalic acid, transfer pH to 6.5 with 5N NaOH again, filter.800 liters of filtrates are passed through ion exchange resin D151 (H
+Type) post (10 liters of post beds) with no salt solution flushing, is used 0.3N HCl wash-out again, collects active component, transfers PH to 6.5, and it is passed through macroporous adsorbent resin 4006 post desalinations (10 liters of post beds).With 10% acetone (containing 0.01NHCl) wash-out resin column.Collect active component, be evaporated to small volume, concentrated solution is transferred pH to 6.5 with ammoniacal liquor, carries out CM-Sephadex-C-25 (NH
4 +Type) column chromatography (2.5 liters of post beds).With no salt solution flushing back 0.1-1.0N NH
4Cl gradient elution, elutriant cumulative volume are equivalent to 15 times of column volume approximately.Collect by peak position, merge.After macroporous adsorbent resin 4006 post desalinations, lyophilize.Get 9.1 gram blue Zhengguangmycin A6 hydrochlorides (cupric product).
Embodiment 5-8
Fermentation condition and extracting method just adopt other bacterial strain with embodiment 1, and the terminal amine body that is added is different, as following table:
| Embodiment | Bacterial strain | Terminal amine body and consumption | Zhengguangmycin A6 content |
| 5 | Streptoverticillium Pingyang mutation Q841 | 1 kilogram of spermine hydrochloride | 49％ |
| 6 | Streptoverticillium Pingyang mutation 7058 | 0.1 kilogram of spermine hydrochloride | 20％ |
| 7 | Streptoverticillium Pingyang mutation Q841 | 0.5 kilogram in spermine vitriol | 52％ |
| 8 | Streptoverticillium Pingyang mutation 7058 | 0.8 kilogram in spermine vitriol | 38％ |
Embodiment 9 (comparative examples)
(pH 7.2-7.5) is slant medium with glucose 1.0%, peptone 0.5%, starch 1.0%, NaCl 0.5% and agar 2.0%, sterilized 30 minutes down for 120 ℃, bevel, place 37 ℃ of thermostatic chambers 3 days dried slightly to surface-moisture, during no varied bacteria growing, inoculation streptoverticillium Pingyang mutation 72 bacterial classification spores were cultivated 8 days in 28 ℃.(substratum is formed: starch 2.5%, glucose 0.5%, soybean cake powder 3.5%, KH to be respectively charged into the 100ml substratum in a plurality of 500ml Erlenmeyer flasks
2PO
40.1%, ZnSO
40.05%, CuSO
40.01%, pH6.0-6.5)., inoculate after 30 minutes in 120 ℃ of sterilizations, 28 ℃ of shaking culture 48 hours, with this culture as seed.Prepare the 500L fermentor tank, the volume 200L that feeds intake, substratum is formed identical with seed culture medium, sterilizes 30 minutes for 120 ℃, inoculum size is 0.5%, at 1: 1 air flow, and 29 ℃, and 180～200 rev/mins of stir culture 48 hours, transferred species to 2 ton fermentor tank, the volume that feeds intake of substratum is 1000L.Its substratum consists of starch 2.5%, glucose 0.5%, Semen Maydis powder 2.0%, soybean cake powder 3.2%, corn steep liquor 3.0%, NaCl 0.5%, KH
2PO
40.015%, ZnSO
40.05%, CuSO
40.01%, Semen Maydis oil 0.3%, pH6.0-6.5 inoculate behind 130 ℃ of-150 ℃ of continuous sterilizations, and inoculum size is 15-20%.29 ℃ of jar temperature, air flow 1: 1 to 1: 1.5,145 rev/mins of stirring velocitys were cultivated 7 days, and to pH more than 7.5, tiring stops when peaking cultivating.Detect (chromatographic column: YWG C1810 μ 3.9 * 300mm through high performance liquid chromatography; Moving phase: A methyl alcohol, B 5mM sodium hexanesulfonate adds 0.5% Glacial acetic acid, transfers pH to 4.3, A: B=32: 68 with strong aqua; Flow velocity: 1ml/min), the content of Zhengguangmycin A6 accounts for 10.1% of bleomycin family antibiotic total amount by weight in the fermentation culture.
800 liters of fermentation cultures are transferred pH to 3.0 with oxalic acid, transfer pH to 6.5 with 5N NaOH again.Add Zeo-karb 122 (H
+Type) 13 liters, stirred 3 hours.Collect resin, with usefulness 0.3N HCl wash-out in the post of after the no salt solution flushing resin being packed into, collection active component.Transfer pH to 6.5 with 5N sodium hydroxide, living solution is passed through macroporous adsorbent resin 4006 posts (10 liters of post beds) desalination, with 10% acetone (containing 0.01N HCl) wash-out.Collect active component, concentrating under reduced pressure.Concentrated solution is transferred pH to 6.5 with ammoniacal liquor, and it is passed through CM-Sephadex-C-25 (NH
4 +Type) post (2.5 liters of post beds), the NH of 0.05N of elder generation
4After the Cl flushing, use 0.1-1.0N NH again
4Cl gradient elution, elutriant cumulative volume are equivalent to 15 times of column volume approximately.Collect by peak position, merge, after macroporous adsorbent resin 4006 posts carry out the desalination operation, lyophilize.Obtain 1.6 gram blue Zhengguangmycin A6 hydrochlorides (cupric product).
Embodiment 10 (comparative examples)
(pH 7.2-7.5) is slant medium with glucose 1.0%, peptone 0.5%, starch 1.0%, NaCl 0.5% and agar 2.0%, sterilized 30 minutes down for 120 ℃, bevel, place 37 ℃ of thermostatic chambers 3 days dried slightly to surface-moisture, during no varied bacteria growing, inoculation streptoverticillium Pingyang mutation Q835 bacterial classification spore was cultivated 8 days in 28 ℃.(substratum is formed: starch 2.5%, glucose 0.5%, soybean cake powder 3.5%, KH to be respectively charged into the 100ml substratum in a plurality of 500ml Erlenmeyer flasks
2PO
40.1%, ZnSO
40.05%, CuSO
40.01%, pH6.0-6.5)., inoculate after 30 minutes in 120 ℃ of sterilizations, 28 ℃ of shaking culture 48 hours, with this culture as seed.Prepare the 500L fermentor tank, the volume 200L that feeds intake, substratum is formed identical with seed culture medium, sterilizes 30 minutes for 120 ℃, inoculum size is 0.5%, at 1: 1 air flow, and 29 ℃, and 180～200 rev/mins of stir culture 48 hours, transferred species to 2 ton fermentor tank, the volume that feeds intake of substratum is 1000L.Its substratum consists of starch 2.5%, glucose 0.5%, Semen Maydis powder 2.0%, soybean cake powder 3.2%, corn steep liquor 3.0%, NaCl 0.5%, KH
2PO
40.015%, ZnSO
40.05%, CuSO
40.01%, Semen Maydis oil 0.3%, pH6.0-6.5 inoculate behind 130 ℃ of-150 ℃ of continuous sterilizations, and inoculum size is 15-20%.29 ℃ of jar temperature, air flow 1: 1 to 1: 1.5,145 rev/mins of stirring velocitys were cultivated 7 days, and to pH more than 7.5, tiring stops when peaking cultivating.Detect (chromatographic column: YWG C1810 μ 3.9 * 300mm through high performance liquid chromatography; Moving phase: A methyl alcohol, B 5mM sodium hexanesulfonate adds 0.5% Glacial acetic acid, transfers pH to 4.3, A: B=32: 68 with strong aqua; Flow velocity: 1ml/min), the content of Zhengguangmycin A6 accounts for 9.8% of bleomycin family antibiotic total amount by weight in the fermentation culture.
800 liters of fermentation cultures are transferred pH to 3.0 with oxalic acid, transfer pH to 6.5 with 5N NaOH again.Add Zeo-karb 122 (H
+Type) 13 liters, stirred 3 hours.Collect resin, with usefulness 0.3N HCl wash-out in the post of after the no salt solution flushing resin being packed into, collection active component.Transfer pH to 6.5 with 5N sodium hydroxide, living solution is passed through macroporous adsorbent resin 4006 posts (10 liters of post beds) desalination, with 10% acetone (containing 0.01N HCl) wash-out.Collect active component, concentrating under reduced pressure.Concentrated solution is transferred pH to 6.5 with ammoniacal liquor, and it is passed through CM-Sephadex-C-25 (NH
4 +Type) post (2.5 liters of post beds), the NH of 0.05N of elder generation
4After the Cl flushing, use 0.1-1.0N NH again
4Cl gradient elution, elutriant cumulative volume are equivalent to 15 times of column volume approximately.Collect by peak position, merge, after macroporous adsorbent resin 4006 posts carry out the desalination operation, lyophilize.Obtain 1.5 gram blue Zhengguangmycin A6 hydrochlorides (cupric product).
Claims (7)
1, a kind of high yield is produced the method for Zhengguangmycin A6 (bleomycin A6), this method comprises the material that adds spermine salt or contain spermine salt in fermention medium, fermentation culture streptoverticillium Pingyang mutation then (Streptomyces verticillus var.Pingyangensis n.var), and from nutrient solution, reclaim said Zhengguangmycin A6.
2, according to the process of claim 1 wherein that Pingyang mutation of said streptoverticillium is streptoverticillium Pingyang mutation (Streptomyces verticillus var.Pingyangensis n.var) 72.
3, according to the process of claim 1 wherein that said spermine salt is spermine hydrochloride or spermine vitriol.
4, according to the process of claim 1 wherein that said spermine salt is the crude product of spermine salt.
5, according to the process of claim 1 wherein that the said material that contains spermine salt is a corn steep liquor.
6, according to the process of claim 1 wherein that the add-on of said spermine salt counts per 1000 liters of substratum by the spermine hydrochloride and add the 0.1-1 kilogram.
7, according to the method for claim 6, the add-on of wherein said spermine salt is counted per 1000 liters of substratum by the spermine hydrochloride and is added the 0.4-0.6 kilogram.
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| CN101463331B (en) * | 2007-12-21 | 2011-06-15 | 上海医药工业研究院 | Fermentation medium and fermentation method for bleocin |
| CN101607986B (en) * | 2008-06-20 | 2011-12-28 | 中国医学科学院医药生物技术研究所 | Method for preparing boningmycin by chemical semisynthesis |
| CN101608200B (en) * | 2008-06-20 | 2012-07-04 | 中国医学科学院医药生物技术研究所 | Preparation method capable of improving boningmycin yield |
| CN104004804B (en) * | 2013-02-26 | 2016-12-28 | 长沙天赐生物医药科技有限公司 | A kind of fermentable preparation method of bleomycin race derivant |
| CN106916866A (en) * | 2017-03-10 | 2017-07-04 | 江西师范大学 | A kind of microbial fermentation preparation method of bleomycin race derivative |
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