CN102363755B - Streptomyces and method thereof for preparaing adriamycin - Google Patents

Streptomyces and method thereof for preparaing adriamycin Download PDF

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CN102363755B
CN102363755B CN 201110143177 CN201110143177A CN102363755B CN 102363755 B CN102363755 B CN 102363755B CN 201110143177 CN201110143177 CN 201110143177 CN 201110143177 A CN201110143177 A CN 201110143177A CN 102363755 B CN102363755 B CN 102363755B
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zorubicin
preparation
streptomyces
streptomycete
powder
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CN102363755A (en
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陈华
叶美丽
郑玲辉
王玲萍
白骅
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Zhejiang Hisun Pharmaceutical Co Ltd
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Abstract

The invention discloses a novel Streptomyces and a method for preparaing adriamycin or analogues thereof through culturing the Streptomyces. The Streptomyces sp.H323(CGMCC4827) disclosed by the invention is a brand new strain which can produce the adriamycin or the analogues of the adriamycin through fermentation in nutrient media containing assimilable carbon resources and nitrogen sources under an oxygen introducing condition.

Description

A kind of streptomycete and the method for producing Zorubicin thereof
Technical field
The present invention relates to a kind of new streptomycete, produce the method for Zorubicin or its analogue by this bacterium of fermentation culture.
Background technology
Anthracycline antibiotics (such as Zorubicin, daunorubicin, pidorubicin) is the antitumor drug of widespread use.The structure of Zorubicin and daunorubicin basic identical (difference is only on a hydroxyl, as shown in the formula I), daunorubicin is only effective to acute leukemia (leukemia), and Zorubicin is then very wide to the therapeutic domain of malignant tumour.
Figure BSA00000507376100011
R 1 R 2 R 3
Daunorubicin C(O)CH 3 H OH
Zorubicin C(O)CH 2OH H OH
Pidorubicin C(O)CH 3OH OH H
1974, US Patent No. 3,803,124 disclose Zorubicin can be made by molecular design by the tunning daunorubicin.At present, semi-synthetic by daunorubicin is the method for suitability for industrialized production Zorubicin.Gondola Pharmacia ﹠ Upjohn S.P.A discloses a kind of method that is prepared Zorubicin by daunorubicin by enzymatic conversion method in Chinese patent CN1147835A.In addition, US Patent No. 3,590,028 discloses a strain Zorubicin produces bacterial classification Streptomyces peucetius subsp.caesius ATCCNo.27952.US Patent No. 4,012,284 disclose an other strain Zorubicin produces bacterial classification Streptomyces peucetius ATCCNo.29050.
Although there is the report Zorubicin to produce bacterial classification in the prior art, also rest on laboratory stage, do not form industrialization.Therefore, searching can produce the new microbe bacterial classification of Zorubicin and new preparation method's work still in proceeding.
Summary of the invention
One of purpose of the present invention is to provide a kind of new microbial strains that can produce Zorubicin or its analogue, a kind of new streptomycete H323, and its deposit number is CGMCC4827.
The present invention also provides new streptomycete H323 (CGMCC4827) (Streptomyces sp.H323), and it can be applied to produce Zorubicin or its analogue, perhaps is applied to produce the pharmaceutical composition that contains Zorubicin.
The present invention also provides a kind of employing streptomycete H323 (CGMCC4827) to prepare the method for Zorubicin or its analogue.The method comprises that employing streptomycete H323 (CGMCC4827) containing in the nutritional medium of assimilable carbon and nitrogen sources, carries out the process of aerobic fermentation.
Above-mentioned assimilable carbon source is selected from the combination of one of starch, dextrin, glucose, industrial molasses, glycerine, sucrose, lactose, maltose, trehalose, xylan, N.F,USP MANNITOL, sorbyl alcohol or above-mentioned substance.
Above-mentioned assimilable nitrogenous source is selected from the combination of one of yeast extract, yeast powder, beef extract, Tryptones, peptone, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, wheat bran, urea, ammonium salt or above-mentioned substance.
The temperature of described fermentation culture is 20 ℃-40 ℃, and preferred 25 ℃-30 ℃, pH is between the 6.5-7.5, preferred about 6.8; Incubation time is 144-216 hour; Oxygen-supply quantity is 0.5-1.0vvm.
Described fermentation mode is liquid submerged fermentation.
Zorubicin can detect by the following method:
Chromatographic column is the C18 post, 5 μ m, 4.6 * 250mm;
Moving phase: damping fluid: acetonitrile: methyl alcohol=500: 500: 60;
Damping fluid: get sodium lauryl sulphate 1.44g and phosphoric acid 0.68ml and be dissolved in the 500ml ultrapure water;
Flow velocity: 1.35ml/min;
Detect wavelength: 254mn;
Sample size: 10 μ l.
The Zorubicin that adopts among the present invention produces the mutant strain that bacterial classification can also obtain for the spontaneous mutant strain of streptomycete H323 (CGMCC4827), streptomycete H323 (CGMCC4827) or the mutagenesis by routine, and it is streptomycete H323 (CGMCC4827) that preferred Zorubicin produces bacterium.
Streptomycete H323 (Streptomyces sp.) microbial strains (CGMCC4827) is stored in (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on May 4th, 2011, Institute of Microorganism, Academia Sinica), preserving number is CGMCC4827, and register on the books, prove survival.
The main biological property of above-mentioned bacterial strains is: the orange colour cast of colony colour is red, and colonial morphology is than rounding, and there is the fold projection on the surface, and diameter is medium-sized (approximately about 6mm), and without spore, substrate mycelium is flourishing, and mycelia is combined closely with substratum, is difficult for provoking.
The invention describes the morphology of Streptomyces sp.H323 and the feature on the molecular level, process and known Zorubicin produce bacterium and carry out the comparison of morphology and molecular level, can assert that Streptomyces sp.H323 belongs to streptomyces, but it is different from known Zorubicin and produces bacterium Streptomyces peucetius subsp.caesius ATCC No.27952, Streptomycespeucetius ATCC No.29050, but a kind of brand-new bacterial classification.
The present invention compares with former technique and has the following advantages:
1. simplification production technique.Bacterial classification of the present invention can be produced Zorubicin or its analogue by the direct fermentation method, has saved the technique that obtains Zorubicin from daunorubicin through chemosynthesis, is conducive to reduce production costs and environmental contamination reduction.
2. raising fermentation unit.The output that bacterial classification of the present invention produces Zorubicin or its analogue has increased significantly than original strain, is conducive to realize suitability for industrialized production.
Description of drawings:
Fig. 1 is the colonial morphology of bacterial strain of the present invention;
The a:ISP2 substratum adds trace element.
The b:ISP3 substratum.
The c:ISP4 substratum.
The d:ISP2 substratum.
Fig. 2 is the mycelia form of bacterial strain of the present invention;
A: * 400 times of mycelia forms.
B: * 1000 times of mycelia forms.
Fig. 3 be the fermenting process curve of bacterial strain of the present invention in shaking flask (■-: Zorubicin output;
Figure BSA00000507376100031
Total reducing sugar;-△-: reducing sugar;--: the pH value);
Fig. 4 is the HPLC collection of illustrative plates of the Zorubicin of bacterial strain generation of the present invention;
Fig. 5 is that the ESI/MS of the Zorubicin of bacterial strain generation of the present invention analyzes collection of illustrative plates;
Fig. 6 is the Zorubicin that produces of bacterial strain of the present invention 1The H-NMR collection of illustrative plates;
Fig. 7 is the Zorubicin that produces of bacterial strain of the present invention 13The C-NMR collection of illustrative plates.
Embodiment
Embodiment 1: bacterium source
Streptomyces sp.H323 system separates the original strain that obtains from the City of Taizhou one discarded farm soil of coastal area of southeastern China, obtain Zorubicin by mutagenic and breeding and produce bacterial strain.This bacterial strain in the ISP2 slant medium 28 ℃ cultivate after 10-12 days, under aseptic condition, with the inoculation shovel mycelium is scraped, in rub oral examination tube, grind mycelium and be suspended in the sterilized water, make bacteria suspension, confession NTG (nitrosoguanidine) mutagenic treatment.
Take by weighing NTG crystal 10mg, be dissolved in the aseptic Tris damping fluid of 10ml (pH8.0), add the 1ml bacteria suspension with transfer pipet again, place oscillation treatment 30min on 28 ℃ of rotary or reciprocating type shaking tables of substratum.With the bacteria suspension processed through suitable dilution spread on the ISP2 flat board.Do not make the bacteria suspension of mutagenic treatment also through suitable dilution spread in the ISP2 flat board in contrast.Cultivate after 10 days for 28 ℃, check colony number, and the meter lethality rate.
Select through single bacterium colony 1000 strains after the NTG mutagenesis, carry out shake flask fermentation, HPLC detects Zorubicin output.The result filters out enhanced variant Streptomyces sp.H323.
Embodiment 2: strain identification
The main biological property of this bacterial strain is: the orange colour cast of colony colour is red, and colonial morphology is than rounding, and there is the fold projection on the surface, and diameter is medium-sized (approximately about 6mm), and without spore, substrate mycelium is flourishing, and mycelia is combined closely with substratum, is difficult for provoking.Concrete colonial morphology and mycelia form are as depicted in figs. 1 and 2.
Design following primer according to actinomycetes 16S rRNA conserved sequence, and give birth to worker bio-engineering corporation by Shanghai and synthesize:
Upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '
Downstream primer: 5 '-AAGGA GGTGATCCAGCCGCA-3 '
Utilize the 16S rDNA sequence of above-mentioned this bacterial strain of primer pair to carry out pcr amplification, the PCR system is:
Figure BSA00000507376100041
The PCR cycling condition: 95 ℃ of denaturation 5min, 95 ℃ of sex change 1min of loop parameter, 52 ℃ of renaturation 45s, 72 ℃ are extended 1.5min, repeat 35 circulations after, 72 ℃ are extended 10min again, last 4 ℃ of insulations.Detect the PCR product with 0.8% agarose gel electrophoresis.Select band clearly product carry out purifying.
Amplified production reclaims through gel electrophoresis, and checks order after being connected to the T carrier, has obtained the sequence of this bacterial strain 16S rDNA primary structure.By in the Genebank database, carrying out similarity searching (blast), found that the 16S rDNA sequence homology of itself and ripple plug streptomycete mutation Streptomyces peucetius subsp.caesius ATCC No.27952 is 96%.With the 16S rDNA sequence homology of another strain ripple plug streptomycete Streptomyces peucetius ATCC No.29050 be 97%.
In sum, H323 belongs to streptomyces, but it is different from known Zorubicin generation bacterium Streptomyces peucetiussubsp.caesius ATCC No.27952 and Streptomyces peucetius ATCC No.29050.Streptomyces sp.H323 is the brand-new bacterial classification of a strain.
Embodiment 3: the preparation Zorubicin
(1) the mycelial preparation in inclined-plane and cultivation
Slant pore culture medium prescription (g/L): yeast extract 4, Fructus Hordei Germinatus extract 10, glucose 4, agar 20, front pH6.5-6.8 disappears, test tube 30 * 200mm, loading amount 20mL after 20 minutes, is cooled to 50-60 ℃ of pendulum inclined-plane through 121 ℃ of sterilizations, after 10 days, mycelia is ripe through 28 ± 1 ℃ of cultivations for access one ring mycelium.
(2) preparation of seed liquor and cultivation
Seed culture based formulas (g/L): Zulkovsky starch 30, glucose 10, soybean cake powder 20, CaCO 32, NaCl 3, and the front pH6.5 that disappears, shaking flask liquid amount are the bottled 25mL of 250mL triangle, through 121 ℃ of sterilizations 20 minutes.The thalline inoculum size is 10 5-10 6C.f.u./mL, 28 ± 1 ℃ of culture temperature, 250rpm, shaking table shaking culture 48 hours, nutrient solution pH7.0-7.5 at this moment, mycelial concentration is 15-20% (volume percent).
(3) preparation of Zorubicin fermention medium and cultivation
Fermentative medium formula (g/L): W-Gum 80, yeast powder 30, CaCO 33, NaCl 3, and the front pH6.8 that disappears, shaking flask liquid amount are the bottled 25mL of 250mL triangle, through 121 ℃ of sterilizations 20 minutes.With the inoculum size access of seed liquor with 5-10% (volume percent), at 28 ± 1 ℃, 250rpm shaking table shaking culture 7 days, HPLC surveys Zorubicin unit after the fermentation ends, and the unit of recording is 200mg/L.
Embodiment 4: the preparation Zorubicin
(1) preparation of seeding tank seed liquor
(proportioning of seed culture medium is seen embodiment 3 to the seed culture medium of input 15L in the 20L seeding tank, add simultaneously 0.3% soya-bean oil and make defoamer), steam sterilizing is adopted in sterilization, lower 30 minutes of 121 ℃ of conditions, after cooling, access 200ml shake-flask seed liquid, 28 ± 1 ℃ of culture temperature, mixing speed 200rpm, air flow 1vvm, cultivated 48 hours, this moment pH7.0-7.5, mycelial concentration is 15-25%.
(2) preparation of fermentation tank culture medium and cultivation
The prescription of fermention medium is identical with previous embodiment 3, but will add 0.05%PPG as defoamer, fermentor tank 50L, the volume that feeds intake is 35L, the front pH6.8 that disappears, and rear pH7.0 disappears, in 121 ℃ of lower steam sterilizings 30 minutes, after the cooling, access approximately 3.5L seed tank culture liquid, 28 ± 1 ℃ of leavening temperatures, rotating speed of agitator 150-200rpm, air flow are 0.8-1.0vvm, fermentation culture 7 days, put tank, recording the Zorubicin fermentation unit is 300mg/L.
Embodiment 5: the preparation Zorubicin
Seed culture medium (g/L): glucose 20, Zulkovsky starch 20, yeast powder 10, yeast extract 5, corn starch 5, soybean cake powder 10, CaCO 34, MgSO 47H 2O 1, and NaCl 3, and pH6.5, shaking flask liquid amount are the 30mL/250mL triangular flask, through 121 ℃ of sterilizations 30 minutes.Dig ferfas filament access seed culture medium from the inclined-plane of embodiment 3, place 28 ℃ of cultivation 45hr on the shaking table.Then the seed culture fluid of 2.5ml accesses fermention medium (g/L): maltodextrin 100, and soybean cake powder 30, seitan powder 2.5, NaCl 2, CaCO 33, FeSO 47H 2O 0.001, MnCl 24H 2O 0.003, CoCl 26H 2O 0.01, ZnSO 47H 2O0.005, pH6.8.The fermention medium loading amount is the 25mL/250mL triangular flask, and 121 ℃ of sterilizations 30 minutes place on the shaking table 28 ℃ to cultivate 7 days.HPLC detects, and fermentation unit is 280mg/L.
Embodiment 6: the preparation Zorubicin
The preparation of seed culture fluid is carried out according to embodiment 5.Then the seed culture fluid of 2.5ml accesses fermention medium (g/L): starch 100, and yeast powder 30, soybean cake powder 3, NaCl 2, CaCO 33, FeSO 47H 2O 0.001, MnCl 24H 2O 0.003, ZnSO 47H 2O 0.005, pH6.8.The fermention medium loading amount is the 25mL/250mL triangular flask, and 121 ℃ of sterilizations 30 minutes place on the shaking table 28 ℃ to cultivate 7 days.HPLC detects, and fermentation unit is 300mg/L.
Embodiment 7: preparation Zorubicin analogue
The preparation of seed culture fluid and fermention medium and cultivation are carried out according to embodiment 3.HPLC surveys Zorubicin analogue unit after the fermentation ends, and wherein daunorubicin is 60mg/L, and two hydrogen daunorubicins are 20mg/L.

Claims (7)

1. a streptomycete (Streptomyces sp.) H323, its deposit number are CGMCC No.4827 and have the ability that produces Zorubicin.
2. the application of streptomycete H323 according to claim 1 in the preparation Zorubicin.
3. the method for a fermentative production Zorubicin is characterized in that: comprise and adopt streptomycete H323 claimed in claim 1, containing in the nutritional medium of assimilable Carbon and nitrogen sources, carry out the step of aerobic fermentation.
4. preparation method according to claim 3, it is characterized in that: described assimilable carbon source is selected from the combination of one of starch, dextrin, glucose, industrial molasses, glycerine, sucrose, lactose, maltose, trehalose, xylan, N.F,USP MANNITOL, sorbyl alcohol or above-mentioned substance.
5. preparation method according to claim 3, it is characterized in that: described assimilable nitrogenous source is selected from the combination of one of yeast extract, yeast powder, beef extract, peptone, soybean cake powder, cottonseed meal, groundnut meal, seitan powder, Dried Corn Steep Liquor Powder, wheat bran, urea, ammonium salt or above-mentioned substance.
6. preparation method according to claim 5, it is characterized in that: described peptone is Tryptones.
7. preparation method according to claim 3, it is characterized in that: the temperature of described fermentation culture is 20 ℃-40 ℃, and pH is between the 6.5-7.5, and incubation time is 144-216 hour.
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CN103087124B (en) * 2012-11-21 2016-01-13 浙江海正药业股份有限公司 A kind of method preparing Zorubicin
CN105624239A (en) * 2014-10-29 2016-06-01 浙江海正药业股份有限公司 Method for producing epirubicin
CN105861455B (en) * 2016-05-16 2018-10-26 浙江海正药业股份有限公司 DoxA protein mutants and its encoding gene and application
CN107541481B (en) * 2016-06-23 2021-07-02 上海医药工业研究院 Genetic engineering bacterium for producing epirubicin and application thereof
CN113293187A (en) * 2021-06-23 2021-08-24 道中道（菏泽）制药有限公司 Fermentation liquor pretreatment method for doxorubicin production

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CN101144085A (en) * 2006-09-15 2008-03-19 上海医药工业研究院 Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof

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YU33730B (en) * 1967-04-18 1978-02-28 Farmaceutici Italia Process for preparing a novel antibiotic substance and salts thereof

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Publication number Priority date Publication date Assignee Title
US4012284A (en) * 1962-11-16 1977-03-15 Societa' Farmaceutici Italia, S.p.A. Process of preparation of antibiotic F.I. 1762 derivatives
CN101144085A (en) * 2006-09-15 2008-03-19 上海医药工业研究院 Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof

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