CN103805648B - High yield ansamitocin zymotechnique - Google Patents
High yield ansamitocin zymotechnique Download PDFInfo
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Abstract
The invention discloses the zymotechnique of efficient large-scale production ansamitocin, the technique is noteworthy characterized by carries out fed-batch fermentation first, is subsequently converted to repeated fed-batch fermentation.The process for purification of high-purity ansamitocin is obtained the invention also discloses a kind of culture medium for producing ansamitocin and by zymotic fluid.
Description
Technical field
The present invention relates to the culture medium and a kind of new ansamitocin zymotechnique and process for purification of production ansamitocin, category
In bioengineering and biological technical field.
Background technology
(content is 2/10000000ths) separates first in the ovum leaf Caulis Mayteni that general money in S.M. storehouses in 1972 etc. is collected from Africa
Maytansine is obtained, its parent nucleus is 19-C Macrocyclic lactams or I type poly ketone, and precious orange synnema actinomycetes are found later
（Actinosynnema pretiosum）Also similar multi-products can be produced, it is referred to as CHROMATOGRAPHIC FRACTIONATION AND MASS antibiotic,
Under the condition of culture of restriction, ansamitocin P-3（AP-3）It is its main component.Because it is to various tumours, such as L-1210, P-388
Leukaemia, S-180, W-256, lewis lung cancer and external nasopharyngeal carcinoma have significant curative effect, so that with huge application prospect,
The clinical test that such as current AP-3 treats malignant tumour as the mode of immunotoxin bonding agent has been enter into final stage.Cause
This, future market will be substantial amounts of to AP-3 demand, so that efficiently large-scale production AP-3 turns into one of study hotspot.
Efficiently large-scale production AP-3, mainly starts with terms of three.One is to obtain AP-3 superior strains, and two be to obtain profit
The culture medium for growing and being metabolized in superior strain, three be to explore the suitable zymotechnique of application.The AP-3 Producing Strains reported at present
Strain is obtained by the means of genetic modification and mutagenesis.Bandi etc.（J.Microbiol.biotechnol.（2006）,16（9）,
1338~1346）By by starting strain ATCC 31565 asm2（AP-3 generates related gene) to delete, AP-3 contents are by 0.53
Mg/L brings up to 5.0 mg/L, and optimizing its culture medium by response surface method afterwards constitutes, and AP-3 contents bring up to 78.3
mg/L.Daniel etc.（J Ind Microbiol Biotechnol(2009 36:1345~1351)）Respectively by asm2 and asm39
Up-regulated expression so that AP-3 contents are respectively increased to 33 mg/L and 52 mg/L.Pungent West Asia Guo etc. are by by bacterial strain
ATCC31565 obtains dissociant Actinosynnema pretiosum PF4-4 through ultraviolet and chemical reagent two-wheeled mutagenesis,
Under the culture medium and fed-batch fermentation technique of its restriction, 900 L/1500 L fermentation tank level AP-3 contents reach 304mg/
L（The A2 of patent CN 101103120A, WO 03/064610）.This is also the AP-3 highests fermentation level and maximum hair reported at present
Ferment scale.
The content of the invention
The invention provides a kind of ansamitocin high-efficiency fermenting technique, it is characterised in that carries out fed-batch fermentation first,
It is subsequently converted to repeated fed-batch fermentation.In one embodiment, in the fed-batch fermentation stage, fermentation medium is used
Ferment after a period of time, isobutanol is added into zymotic fluid, continue after fermentation a period of time, stream plus glucose into zymotic fluid；
Continue after fermentation a period of time, into repeated fed-batch fermentation stage, bleed off a part for zymotic fluid, add fermentation medium
And isobutanol is added into zymotic fluid, and after a period of time, stream plus glucose into zymotic fluid.
In one embodiment, in the fed-batch fermentation stage of the zymotechnique, fermentation medium fermentation 2 is used
After ~ 3 days, 0.10 ~ 0.30% (v/v) isobutanol is added into zymotic fluid, after fermentation is carried out 4 ~ 6 days, stream plus Portugal into zymotic fluid
Grape sugar, stream rate of acceleration is 3 ~ 10 g/L/d；After fermentation is carried out 8 ~ 12 days, into repeated fed-batch fermentation stage, fermentation is bled off
The 30 ~ 50% of liquid, add fermentation medium and 0.10 ~ 0.30% (v/v) isobutanol are added into zymotic fluid, repeating feed supplement point
Criticize fermentation stage to start after 2 ~ 4 days, stream plus glucose into zymotic fluid, stream rate of acceleration are 3 ~ 10 g/L/d, repeated fed-batch
The fermentation stage duration is 7 ~ 10 days.
In one embodiment, the fermentation parameter is that inoculum concentration is the 0.3% ~ 10.0% of fermentation medium（v/
v）, 25 ~ 30 DEG C of temperature, pH 6.8 ~ 7.6, DO（Dissolved oxygen）20% ~ 60%, the MPa of pressure 0.03 ~ 0.08, the VVM of throughput 0.1 ~ 0.6
（Air volume/culture volume/min, ventilation ratio）, the rpm of mixing speed 100 ~ 300.
In a preferred embodiment, the fermentation parameter is that inoculum concentration is the 6.0% of fermentation medium（v/
v）, 28 DEG C of temperature, pH 7.3, DO 40%, the MPa of pressure 0.05, the VVM of throughput 0.4, the rpm of mixing speed 160.
In one embodiment, pH control is by auto-feeding NH in the zymotechnique3·H2O and 30% acetic acid
Come what is realized.
In one embodiment, the strain that the zymotechnique is used is precious orange synnema actinomycetes ATCC
31565.Fermentation seed is utilizes fermentation medium, from picking single bacterium colony on the flat board of precious orange synnema actinomycetes, at 28 DEG C
What 260 rpm shaking table cultures 4 ~ 6 days were obtained.
In one embodiment, the fermentation medium that the zymotechnique is used, its carbon source is lactose, maltose, sugarcane
One kind or its mixing in sugar, starch, dextrin, fructose and glucose, its nitrogen source is groundnut meal, soy meal, dusty yeast, albumen
One kind or its mixing in peptone, corn steep liquor, glycine, serine, phenylalanine, alanine, valine, arginine.Wherein, carbon
Source ratio is 0.2% ~ 6.0%, and nitrogen source ratio is 0.1% ~ 3.0%.
In a preferred embodiment, the carbon source of the fermentation medium be 0.3% ~ 4.0% glucose and 0.2% ~
The mixing of 2.0% dextrin；Nitrogen source is the mixing of 0.3% ~ 3.0% corn steep liquor, 0.1% ~ 0.5% glycine and 0.1% ~ 0.5% valine.
In a further preferred embodiment, the carbon source of the fermentation medium is 2.6% glucose and 1.5% dextrin
Mixing；Nitrogen source is the mixing of 1.0% corn steep liquor, 0.2% glycine and 0.2% valine.
In one embodiment, the fermentation medium also contains 0.1% ~ 1.0%CaCl2·2H2O、0.1%~1.0%
KH2PO4、0.1%~1.2%NH3·H2O and 0.01% ~ 0.3% defoamer.
In a preferred embodiment, CaCl in the fermentation medium2·2H2O content is 0.5%, KH2PO4Contain
Measure as 0.2%, NH3·H2O content is 0.15%, and antifoam content is 0.05%.
The invention further relates to after the fermentation has been completed, ansamitocin is purified from zymotic fluid with water-soluble extracts reagent, specifically
Ground comprises the following steps：
（1）Inactivate microorganism in zymotic fluid；
（2）50% ~ 80% extracts reagent is added into zymotic fluid, the extracting of ansamitocin is realized and removes solid content；
（3）Thalline and solid content are removed by centrifuging or filtering, fermented supernatant fluid is obtained, is then added into supernatant
0.1%~0.3%（Mass ratio）Flocculant, stand 6 ~ 16 h, to remove removal of residue.The flocculant wherein used be selected from aluminum sulfate,
One kind or its mixing in iron chloride and alum；
（4）Using silica gel column chromatography means, ansamitocin in initial gross separation zymotic fluid is realized；
（5）Take the method concentrating ansamitocins of evaporation；
（6）Take the method purified ansamitocins of high performance liquid chromatography.
In one embodiment, microorganism in zymotic fluid is inactivated by heating and/or adjusting zymotic fluid pH method.
In a preferred embodiment, by the way that zymotic fluid is heated into 60-70 DEG C and 1.0-2.0 h are maintained（Stirring
Or do not stir）To inactivate microorganism in zymotic fluid.
In a preferred embodiment, by adjusting, zymotic fluid pH is 3.0-4.0 or pH is 10.0-11.0 and maintained
1.0-2.0 h（Stirring or not）To inactivate microorganism in zymotic fluid.
In one embodiment, the extracts reagent used is one kind or its mixing in alcohols and esters of gallic acid, wherein
Alcohols is selected from methanol, ethanol, one kind of propyl alcohol or its mixing, and esters of gallic acid is selected from ethyl acetate, butyl acetate, the one of ethyl propionate
Plant or its mixing.
Brief description of the drawings
Fig. 1 is to explore different culture media composition using Design-Expert softwares to analyse 2 IV 8-3 of AP-3 fermentation influences
Because of design effect analysis figure（Half normal state figure, Half-Normal Plot）, wherein abscissa is standardization effect
（Standardized Effect）, ordinate is half normal probability paper（Half-Normal%Probability）.
Fig. 2 detects the AP-3 standard curves that AP-3 concentration is set up in zymotic fluid for use HPLC.
Fig. 3 detects the glucose standard curve that glucose is set up in zymotic fluid for use glucose determination reagent box.
Fig. 4 is using the fermentation of 15L- tanks, AP-3, pH, PCV（Cell compression volume）The conditional curve changed over time.
Fig. 5 is to carry out fed-batch fermentation, the mistake that AP-3, pH, PCV, concentration of glucose are changed over time using 150L- tanks
Journey curve.
Fig. 6 is to be combined fed-batch fermentation and repeated fed-batch fermentation using 150L- tanks, AP-3, pH, PCV, Portugal
The conditional curve of grape sugar concentration changes with time.
Fig. 7 is the data that AP-3 purity is detected using HPLC.
Embodiment
Following examples are provided, to facilitate those skilled in the art to more fully understand the present invention, the embodiment merely for
Exemplary purpose, is not intended to limit the scope of the present invention.
The plating medium formula for being used for strain line in embodiment is shown in Table 1.
The plating medium formula of table 1.
Flat board condition of culture is：26 DEG C incubated 7 ~ 10 days.
AP-3 detections use HPLC methods, take the mL of zymotic fluid 4.0, add absolute ethyl alcohol to 8mL, shake 10 ~ 15 min,
10000 rpm centrifuge 10 min, take supernatant to can detect after 0.45 μm of membrane filtration.HPLC use reverse C18 posts, 5 μm,
250 × 4.6 mm, preposition pre-filtering post, mobile phase uses ACN:Water=7:3, the μ L of 1.0 mL/min sample sizes of flow velocity 20, wavelength
252 nm.The concentration of associated AP -3 and peak area, set up AP-3 standard curves, see Fig. 2.
Glucose assays use glucose determination reagent box (Ningbo Meikang Biotechnology Co., Ltd., product standard
Numbering：YZB/ Zhejiang 2120-2012) method, concrete operations are shown in the kit specification, take zymotic fluid 8.0 mL, 10000 rpm from
The min of the heart 10, takes supernatant to can detect after 0.45 μm of membrane filtration.Concentration of glucose and OD value relations are associated, glucose is set up
Standard curve, is shown in Fig. 3.
Biomass estimation uses cell compression volume（PCV）Method, takes zymotic fluid 4.0 mL, 10000rpm to centrifuge 10 min,
It is PCV to calculate ratio of the precipitation solid content in cumulative volume.
Completed in fermentation, in inactivation zymotic fluid after microorganism, 50% ~ 80% extracts reagent is added into zymotic fluid, peace is realized
The extracting of silk rhzomorph and removal solid content, the extracts reagent used is water miscible, and extraction rate is very fast, high income, passes through
Prepared by high performance liquid chromatography, product purity is higher.
Embodiment
The influence that the different culture media composition of embodiment 1 ferments to AP-3
In view of initial medium（It is shown in Table 2）In tryptone, two kinds of composition price factors of yeast extract, with remove this two
Based on the initial medium for planting composition, fermented in shaking flask.The fermented bacterium used is tested to put for precious orange synnema
Line bacterium ATCC 31565, shake flask culture conditions are：28 DEG C, 260 rpm, and in after culture 1 day, add 0.2%（v/v）Isobutanol,
If do not illustrated, the shaking flask capacity used is 100 mL, and liquid amount is 30 mL, and adding 3 mL concentration after starting culture 3 days is
100 g/L glucose solution, later every this operation of repetition in three days, so doing purpose has two, and one is as preliminary
Sugar strategy is mended, one is that the influence to fermentation results is evaporated in eliminating incubation（Experiment proof takes this to operate after fermentation to open
The volume of zymotic fluid with the end of that begins is basically identical）, zymotic fluid is harvested after cultivating 13 days.And detect AP-3 in zymotic fluid with HPLC
Concentration, explore the influence that the culture medium of heterogeneity as shown in table 3 composition ferments to AP-3.
The initial medium of table 2.
Table 3.2Ⅳ 8-3Factorial Design factor coding schedule
This experiment uses 2Ⅳ 8-3Factorial Design, according to Design-Expert softwares（Version 7.1.6）Output result（Table
4）Tested, AP-3 testing results input program is obtained into analysis result after experiment terminates（See Fig. 1）.
Table 4.2Ⅳ 8-3Factorial Design experimental arrangement table
It is observed that there is 5 kinds of compositions from Fig. 1 effect analysis figure（A：Glucose, B：Glycine, C：Corn steep liquor, D：
Valine, E：Dextrin）AP-3 fermentations, which are produced between significant impact, and A compositions and B component and C compositions, reciprocation.
As can be seen from Table 4, AP-3 highests fermentation level can reach 270 more than mg/L in experiment, be initial medium（140 mg/L）
1.93 times, the result being more satisfied with.Screening initial incubation is replaced with the various composition combination of AP-3 highest fermentation levels
Tryptone, yeast extract in base, constitute new fermentative medium formula, wherein carbon source is 2.6% glucose and 1.5% dextrin
Mixing, nitrogen source is the mixing of 1.0% corn steep liquor, 0.2% glycine and 0.2% valine, and also containing 0.5% CaCl2·2H2O,
0.2% KH2PO4, 0.15% NH3·H2O, 0.05% defoamer.
Embodiment 2.15L- tanks AP-3 ferments（Fed-batch fermentation）
Seed culture：- 20 DEG C of glycerine are preserved into the precious orange synnema actinomycetes ATCC31565 of pipe in flat lining out point
From after 26 DEG C are cultivated 8 days, by 20 single bacterium colony pickings into the shaking flask equipped with fermentation medium, culture 5 days merges and obtained
About 0.5L nutrient solutions are seed.
Use 15L fermentation tanks（Upper ocean lattice biological plant Co., Ltd,）Culture medium is prepared according to fermentative medium formula
10L, 121 DEG C, sterilize 30min, is cooled to 28 DEG C, above-mentioned seed asepsis is accessed.Condition of culture, 28 DEG C of temperature, pH 7.3 leads to
Tank pressure 0.03 ~ 0.08MPa, 0.1 ~ 0.6VVM of throughput, 100 ~ 300rpm of rotating speed are overregulated, by DO controls more than 40%.Culture
0.2% is added two days later（v/v）Isobutanol, culture starts to add the Glucose Liquid containing 450g/L after carrying out three days, mends sugared speed
For 0.1L/d, terminate until cultivating.AP-3 contents are 223mg/L in zymotic fluid when HPLC measures fermentation ends, fermentation process
Fig. 4 is shown in pH and PCV changes.
Embodiment 3.150L- tanks AP-3 ferments（Fed-batch fermentation）
First order seed culture：- 20 DEG C of glycerine are preserved into the precious orange synnema actinomycetes ATCC31565 of pipe on flat board strokes
Line is separated, after 26 DEG C are cultivated 8 days, by 20 single bacterium colony pickings into the shaking flask equipped with fermentation medium, is cultivated 5 days, is merged
It is seed to obtain about 0.5L nutrient solutions.
Secondary seed culture：Use 15L fermentation tanks（Upper ocean lattice generating apparatus Co., Ltd,）According to fermentative medium formula
Culture medium 10L is prepared, 121 DEG C, sterilize 30min, is cooled to 28 DEG C, above-mentioned seed asepsis is accessed.Condition of culture, temperature 28
DEG C, pH 7.3 presses 0.03 ~ 0.08MPa, 0.1 ~ 0.6VVM of throughput, 100 ~ 300rpm of rotating speed by adjusting tank, and DO controls are existed
More than 40%.Culture starts to add the Glucose Liquid containing 450g/L after carrying out three days, and it is 0.1L/d to mend sugared speed, until culture
Terminate.Incubation time is 5 days.
Use 150L fermentation tanks（Upper ocean lattice generating apparatus Co., Ltd）Culture medium is prepared according to fermentative medium formula
100L, 121 DEG C, sterilized 30min, is cooled to 28 DEG C, and above-mentioned 5.0L seed asepsis is accessed.Condition of culture, 28 DEG C of temperature, pH
7.3, by adjust tank press 0.03 ~ 0.08MPa, 0.1 ~ 0.6VVM of throughput, 100 ~ 300rpm of rotating speed, by DO control 40% with
On.Culture adds 0.2% two days later（v/v）Isobutanol, culture starts to add the Glucose Liquid containing 450g/L after carrying out three days,
It is 1.0L/d to mend sugared speed, until culture terminates.AP-3 contents are 290mg/L, hair in zymotic fluid when HPLC measures fermentation ends
Fig. 5 is shown in pH, concentration of glucose and the PCV changes of ferment process.
The 150L- tanks AP-3 of embodiment 4 ferments（Fed-batch fermentation and repeated fed-batch fermentation are combined）
First order seed culture：- 20 DEG C of glycerine are preserved into the precious orange synnema actinomycetes ATCC31565 of pipe on flat board strokes
Line is separated, after 26 DEG C are cultivated 8 days, by 20 single bacterium colony pickings in equipped with the shaking flask of fermentation medium described in embodiment 1, training
Support 5 days, it is seed to merge about 0.5 L nutrient solutions of acquisition.
Secondary seed culture：Use 15 L fermentation tanks（Upper ocean lattice generating apparatus Co., Ltd）According to fermentative medium formula
Culture medium 10 L, 121 DEG C of 30 min of sterilizing are prepared, 28 DEG C is cooled to, above-mentioned seed asepsis is accessed.Condition of culture is, temperature
28 DEG C, pH 7.3, tank pressure 0.03 ~ 0.08 MPa, the VVM of throughput 0.1 ~ 0.6, the rpm of rotating speed 100 ~ 300, by DO controls 40%
More than.Culture starts to add the Glucose Liquid that concentration is 450 g/L after carrying out three days, and it is 0.1 L/d to mend sugared speed, until culture
Terminate.Incubation time is 5 days.
The fed-batch fermentation stage：Use 150 L fermentation tanks（Upper ocean lattice generating apparatus Co., Ltd）According to the institute of embodiment 1
State fermentative medium formula and prepare culture medium 100 L, 121 DEG C of sterilizing 30min, be cooled to 28 DEG C, above-mentioned seed asepsis is accessed.
Condition of culture is, 28 DEG C of temperature, pH 7.3, and tank presses 0.03 ~ 0.08 MPa, the VVM of throughput 0.1 ~ 0.6, rotating speed 100 ~
300rpm, by DO controls more than 40%.Culture adds 0.2% two days later（v/v）Isobutanol, culture starts to add after carrying out three days
Concentration is 450 g/L Glucose Liquid, and it is 1.0L/d to mend sugared speed.
Repeated fed-batch fermentation stage：Fed-batch fermentation is carried out after 280 h, bleeds off 33 L zymotic fluids（Now AP-3
Content is 320 mg/L）, use 15 L fermentation tanks（Upper ocean lattice generating apparatus Co., Ltd）According to fermented and cultured described in embodiment 1
Based formulas prepares the L of culture medium 30（In three times）, 121 DEG C sterilizing 30 min, be cooled to 28 DEG C, by it is sterile be transferred to 150L hair
Fermentation tank, and 0.10% is added simultaneously（v/v）Isobutanol, continues to cultivate, condition of culture is ibid.HPLC ferments when measuring fermentation ends
AP-3 contents are 410 mg/L in liquid, hence it is evident that divided higher than feed supplement is only carried out in embodiment 3 using identical strain and technological parameter
The 150L- tanks of wholesale ferment（290mg/L）.Fig. 6 is shown in pH, concentration of glucose and the PCV changes of fermentation process.
The processing of the zymotic fluid of embodiment 5 and ansamitocin extracting
By the fermentation culture in embodiment 4（105L）65 DEG C are heated in fermentation tank, 120rpm stirrings maintain 1.0
h.Then the propyl alcohol with 0.6 times of fermentating liquid volume is mixed, under normal temperature, and 120 rpm stir 2.0 h, and supernatant is collected in centrifugation.Mycelia
Precipitation is added under the propyl alcohol of 0.5 times of thalline volume, normal temperature again, and 120 rpm stir 2.0 h, and supernatant is collected in centrifugation.Repeating should
Step, until collected supernatant is free of or ansamitocin containing small amount.0.1% is added into the above-mentioned supernatant handled well
（Mass ratio）Aluminum sulfate, stands overnight.After residue is flocculated out, supernatant is collected by centrifugation.
The ansamitocin initial gross separation of embodiment 6 and refined
By the supernatant finally obtained in embodiment 5 by the chromatographic column equipped with 3kg silica gel, ansamitocin is set to be adsorbed onto silicon
On glue.Then 80% acetonitrile solution with 2 column volumes of restriction, ansamitocin is eluted.Eluent is passed through into rotary evaporation
Instrument evaporates excess of solvent, and final solution volume is between 2-4L.Solution after above-mentioned concentration is passed through into 0.45 μm of organic filter membrane
Filtering, Varian Prostar218 preparative high performance liquid chromatography posts are loaded to by filtered fluid, collect pure ansamitocin, and
Excess of solvent is evaporated by Rotary Evaporators.Freeze-drying 40h is eventually passed, ansamitocin P-3 41.7g are obtained, with efficient
Its purity of liquid chromatographic detection is more than 98%, is specifically shown in Fig. 7（Area percentage is purity）, it is about 95% to adjust yield.
Bibliography：
1.Bandi Srinivasulu,Yoonjung Kim,Yongkeun Chang,et al.Construction of
asm2 Deletion Mutant of Actinosynnema pretiosum and Medium Optimization for
Ansamitocin P-3 Production Using Statistical Approach [J].J Microbiol
Biotechnol,2006,16(9):1338-1346.
2.Daniel Ng·Hing Kah Chin·Victor Vai Tak Wong Constitutive
overexpression of asm2 and asm39 increase AP-3 production in the actionmycete
Actionsynnema pretiosum[J].J Ind Microbiol Biotechnol.2009,36(11):1345~1351.
3. pungent West Asia Guo, Graham Sodd method of investing S guest, the Wei grace C gloomy in Vad are used for the method for preparing ansamitocin
Chinese invention patent publication number:101103120. patent application day 2005-12-12.METHODS FOR THE PRODUCTION
OF ANSAMITOCINS.US20050170475A1.
4.J CHUNG.Mutant Actinosynnema pretiosum strain with increased
maytansinoid production.US Patent App.10/057,561,2002.US Patent 7,192,750,
2007.WO Patent WO/2003/064,610,2003.。
Claims (11)
1. a kind of ansamitocin high-efficiency fermenting technique, it is characterised in that carry out fed-batch fermentation first, is subsequently converted to repeat
Fed-batch fermentation, wherein, in the fed-batch fermentation stage, fermented using fermentation medium after a period of time, into zymotic fluid
Isobutanol is added, is continued after fermentation a period of time, stream plus glucose into zymotic fluid；Continue after fermentation a period of time, into weight
In the multiple fed-batch fermentation stage, a part for zymotic fluid is bled off, fermentation medium is added and isobutanol, one is added into zymotic fluid
After the section time, stream plus glucose into zymotic fluid.
2. zymotechnique as claimed in claim 1, it is characterised in that in the fed-batch fermentation stage, use fermentation medium
After fermentation 2~3 days, 0.10~0.30% (v/v) isobutanol is added into zymotic fluid, after fermentation is carried out 4~6 days, to fermentation
Stream plus glucose in liquid, stream rate of acceleration are 3~10g/L/d；After fermentation is carried out 8~12 days, into repeated fed-batch fermentation rank
Section, bleeds off the 30~50% of zymotic fluid, adds fermentation medium and 0.10~0.30% (v/v) isobutyl is added into zymotic fluid
Alcohol, after repeated fed-batch fermentation stage starts 2~4 days, stream plus glucose into zymotic fluid, stream rate of acceleration are 3~10g/
L/d, the repeated fed-batch fermentation stage duration is 7~10 days.
3. zymotechnique as claimed in claim 1, its parameter is that inoculum concentration is 0.3%~10.0% (v/ of fermentation medium
V), 25~30 DEG C of temperature, pH 6.8~7.6, DO (dissolved oxygen) 40%~60%, 0.03~0.08MPa of pressure, throughput 0.1~
0.6VVM (air volume/culture volume/min, ventilation ratio), 100~300rpm of mixing speed.
4. zymotechnique as claimed in claim 3, its parameter is that inoculum concentration is 6.0% (v/v) of fermentation medium, temperature
28 DEG C, pH 7.3, DO 40%, pressure 0.05MPa, throughput 0.4VVM, mixing speed 160rpm.
5. zymotechnique as claimed in claim 1, it is characterised in that the fermented bacterium used is precious orange synnema actinomycetes
(Actinosynnema Pretiosum) ATCC 31565 and its derivative strain.
6. zymotechnique as claimed in claim 1, it is additionally included in after the completion of fermentation, with water-soluble extracts reagent from zymotic fluid
Middle purifying ansamitocin, specifically comprises the following steps：
(1) microorganism in inactivation zymotic fluid；
(2) 50%~80% extracts reagent is added into zymotic fluid, the extracting of ansamitocin is realized and removes solid content；
(3) thalline and solid content are removed by centrifuging or filtering, obtains fermented supernatant fluid, 0.1% is then added into supernatant
The flocculant of~0.3% (mass ratio), stands 6~16h, to remove removal of residue, wherein the flocculant used is selected from aluminum sulfate, chlorine
Change iron and one kind in alum or its mixing；
(4) silica gel column chromatography means are used, ansamitocin in initial gross separation zymotic fluid is realized；
(5) the method concentrating ansamitocins of evaporation are taken；
(6) the method purified ansamitocins of high performance liquid chromatography are taken.
7. zymotechnique as claimed in claim 6, the extracts reagent is one kind or its mixing in alcohols and esters of gallic acid, its
Middle alcohols is selected from methanol, ethanol, one kind of propyl alcohol or its mixing, and esters of gallic acid is selected from ethyl acetate, butyl acetate, ethyl propionate
A kind of or its mixing.
8. the zymotechnique as described in claim any one of 1-7, wherein in the fermentation medium, carbon source is 0.3%~
The mixing of 4.0% glucose and 0.2%~2.0% dextrin；Nitrogen source is 0.3%~3.0% corn steep liquor, 0.1%~0.5% sweet ammonia
The mixing of acid and 0.1%~0.5% valine.
9. zymotechnique as claimed in claim 8, wherein in the fermentation medium, the carbon source is 2.6% glucose
With the mixing of 1.5% dextrin；The nitrogen source is the mixing of 1.0% corn steep liquor, 0.2% glycine and 0.2% valine.
10. zymotechnique as claimed in claim 8, wherein the fermentation medium also contains 0.1%~1.0%CaCl2·
2H2O, 0.1%~1.0%KH2PO4, 0.1%~1.2%NH3·H2O and 0.01%~0.3% defoamer.
11. zymotechnique as claimed in claim 10, wherein CaCl2·2H2O content is 0.5%, KH2PO4Content is 0.2%,
NH3·H2O content is 0.15%, and antifoam content is 0.05%.
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| CN104894183B (en) * | 2015-06-25 | 2018-04-27 | 齐鲁制药有限公司 | A kind of method that ansamitocin P-3 is prepared with precious orange synnema actinomycetes |
| CN106066398B (en) * | 2016-05-25 | 2018-08-21 | 山东农业大学 | A kind of indirect ELISA detection method of A types clostridium perfringens toxoid antibody |
| CN105907681B (en) * | 2016-05-30 | 2019-11-19 | 苏州康聚生物科技有限公司 | The mutant strain of high yield ansamitocin P-3 a kind of and the preparation method of ansamitocin P-3 |
| CN108048503B (en) * | 2018-01-23 | 2022-03-08 | 上海交通大学 | Method for improving ansamitocin P-3production |
| CN110117626A (en) * | 2018-02-06 | 2019-08-13 | 上海键合医药科技有限公司 | A kind of zymotechnique preparing Ansamitocins P3 |
| CN112630368A (en) * | 2020-12-18 | 2021-04-09 | 卓和药业集团有限公司 | High performance liquid chromatography detection and analysis method for ansamitocin content |
| CN115960976A (en) * | 2021-10-13 | 2023-04-14 | 杭州中美华东制药有限公司 | Fermentation method of ansamitocin P-3 |
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